A pharmaceutical composition

Abstract

A pharmaceutical composition comprising Amphotericin B and Lipoate useful to fungal infections. An injectable pharmaceutical formulation further comprising cholesterol and oleate for parenteral use. The process to elaborate them and a kit for the preparation of said injectable pharmaceutical formulation.

Description

[0001] A PHARMACEUTICAL COMPOSITION

[0002] Field of the Invention

[0003] The present invention relates to pharmaceutical compositions for the treatment of invasive fungal infections. In particular, it relates to stable pharmaceutical compositions of Amphotericin B, providing good fungicidal activity.

[0004] State of the Art

[0005] Amphotericin B (AmB) is an effective fungicidal drug used in the treatment of invasive fungal infections since it forms complexes with ergosterol in fungal cell membranes, forming trans-membrane pores, leading to an increase in membrane permeability, and causing osmotic imbalance and ultimately cell death. Due to the insolubility of AmB in water, it is necessary to associate the drug with nanoparticles to keep it soluble in aqueous media. In one of the first known AmB formulations, AmB was solubilized in deoxycholate, a formulation known as AmB- DOC, or Fungizone'* (Amfostat - AFS in Argentina). However, its clinical use is limited because its toxicity produces many side effects. (Bekersky, I., R. M. Fielding, D. Buell, and I. Lawrence. 1999. Lipid-based amphotericin B formulations: from animals to man. Pharm. Sci. Technol. Today 2:230-236).

[0006] To avoid the inherent toxicity associated with AmB, other formulations of AmB were further developed, most of them using liposomes as carriers for drug delivery. In this vein, different AmB formulations in liposomal nanoparticles can be found in the state of the art, one of them is AmBisome®, a liposomal formulation of AmB in small uni-lamellar vesicles. This liposomal formulation was significantly less toxic than the conventional AmB-DOC formulation. (Bekersky, L, D. Buell, M. Tomishima, K. Maki, I. Lawrence, and R. M. Fielding. 1999. New approaches to systemic antifungal therapy: case studies of AmBisome and FK463. Recent Res. Dev. Antimicrob. Agents Chemother. 3:407-413 - Boswell, G. W., D. Buell, and I. Bekersky. 1998. AmBisome (liposomal amphotericin B): a comparative review. J. Clin. Pharmacol. 38:583-592. - Walsh, T. J., R. W. Finberg, C. Arndt, J. Hiemenz, C. Schwartz, D. Bodensteiner, P. Pappas, N. Seibel, R. N. Greenberg, S. Dummer, M. Schuster, and J. S. Holcenberg. 1999. Liposomal amphotericin B for empirical therapy in patients with persistent fever and neutropenia. N. Engl. J. Med. 340:764-771).

[0007] More recently other new formulations of liposomal AmB such as Phosome, AmBiL, Ambihope, Lambin, Lipholyn, and Ampholip (Comparison between liposomal formulations of amphotericin B., Jill P Adler-Moorel, Jean-Pierre Gangneux and Peter G Pappas, Medical Mycology, 2016, 54, 223-231) have been described in the literature. These different liposomal amphotericin B formulations show very similar lipidic constituents.

[0008] The reduction in toxicity has been attributed to diverse factors such as a different interaction with cell membranes, larger particle sizes, different pharmacokinetics, and modified drug release, among other variables. These formulations are based on expensive technologies such as the preparation of liposomes which translate into a higher cost of therapy. In addition, in many cases, the reduction of AmB toxicity was accompanied by a considerable reduction in AmB activity (M.S. Espuelas, P. Legrand, M.A. Campanero, M. Appel, M. Cheron, C. Gamazo, G. Barratt, J.M. Irache (2003) Polymeric carriers for amphotericin B: in vitro activity, toxicity and therapeutic efficacy against systemic candidiasis in neutropenic mice. J. Antimicrob. Chemother. 52 (3), 419-27. Doi:10.1093 / jac / dkg351).

[0009] In this context, achieving an AmB formulation that provides good fungicidal activity while maintaining low cytotoxicity is one of the most sought-after goals in pharmacotherapy.

[0010] Various efforts in the state of the art have been proposed to address the challenge of improving both the safety and therapeutic profile of AmB, yet with limited success. An example is the work by Franzini Cristina Maria et al.: "Structural Properties Induced by the Composition of Biocompatible Phospholipid-Based Microemulsion and Amphotericin B Association," JOURNAL OF BIOMEDICAL NANOTECHNOLOGY, vol. 8, no. 2, April 1, 2012 (2012-04-01), pages 350-359, XP093188294, US ISSN: 1550-7033, DOI: 10.1166 / jbn.2012.1435. This document proposes the use of soybean phospholipids, specifically soybean phosphatidylcholine, and a surfactant such as Tween 20 to stabilize and fluidize the emulsion. It is well known that nonionic surfactants, such as Tween 20, can be released from formulation and cause hemolysis, a critical issue in the toxicity of AmB formulations. In contrast, the present invention proposes a combination of compounds that does not require liposomal encapsulation or the use of surfactants to achieve high stability of AmB while maintaining its antifungal effect and avoiding hemolysis. The present invention provides a composition and a formulation that, through simple agitation, generates stable nanostructures in the absence of liposomes, phospholipids and non-ionic surfactants. The present invention provides an antifungal AmB-based composition that has proven an outstanding capability to treat fungal infections. In addition, the AmB-based formulation has excellent solubility and stability properties.

[0011] The present invention also provides nanoparticles with particle size less than 200 nm that allow the stability of the AmB composition of the invention. This fact ensures that the composition and formulations of the present invention can be sterilized by filtration at 0.22 microns. This fact represents a great advantage over liposomal formulations that can be filtered by a 0.45 micron filter, which does not imply sterilization.

[0012] The present invention also provides a pharmaceutical composition that dramatically reduces the hemolysis of AmB when is combined with lipoate.

[0013] The present invention also provides a non-liposomal formulation of AmB that achieves the same toxicity profile than Ambisome and is obtainable with simple mixing with minimum organic solvents, without high energy processes like turrax or high pressure homogeneization.

[0014] The present invention also provides a kit to prepare an injectable antifungal formulation with high stability, low adverse effects, and less harmful for health.

[0015] The present invention also provides a low-cost process to elaborate said injectable antifungal formulation.

[0016] Brief Description of the Invention

[0017] A pharmaceutical composition, which is the main object of the present invention, comprises Amphotericin B and Lipoate, wherein the lipoate:AmB mass ratio is at least 0.1:1; preferably at least 0.15:1; more preferably at least 0.35:1; and even more preferably at least 0.7:1. In another embodiment of the invention said lipoate:AmB mass ratio is at least 1.4:1; more preferably at least 16.5:1. In a preferred embodiment of the present invention, said pharmaceutical composition comprising Amphotericin B and Lipoate comprises a lipoate:AmB mass ratio from 0.1:1 to 22:1.

[0018] In a preferred embodiment of the present invention, the pharmaceutical composition comprising Amphotericin B and Lipoate (Lipoate-AmB) comprises Lipoate in its acid form or as a salt comprising as a counterion the monovalent cation selected from the group comprising sodium, potassium, lithium, and a combination thereof. And wherein said pharmaceutical composition comprises a state selected from the group comprising liquid as aqueous solution, powder, and lyophilized state.

[0019] In a preferred embodiment of the present invention said Lipoate comprises a concentration from 0.02 mg / ml to 176 mg / mL in an aqueous medium; preferably from 0.2 mg / ml to 16 mg / mL; more preferably from 1.0 mg / ml to 13.6 mg / mL in an aqueous medium. And said Amphotericin B is in a concentration from 0.1 to 8 mg-mL-1, in an aqueous medium.

[0020] In a preferred embodiment of the present invention said pharmaceutical composition comprising Amphotericin B and Lipoate further comprises cholesterol and a compound selected from oleate, laureate, myristate, palmitate and stearate; preferably oleate, in a concentration from 0.2 to 2.5 mg-mL-1; and with a cholesterokAmB molar ratio less than 2:1, preferebly from about 1:1 to 2:1.

[0021] Another object of the present invention is an injectable pharmaceutical formulation comprising said pharmaceutical composition comprising Amphotericin B, Lipoate, Cholesterol and Oleate, further comprising glucose in a concentration from 1 % w / v to 10 % w / v, preferably about 5 % w / v, in an aqueous medium. Where said pharmaceutical formulation of the invention comprises said pharmaceutical composition comprising Amphotericin B and Lipoate and a second composition comprising cholesterol, oleate and glucose.

[0022] Another object of the present invention is a kit for the preparation of said injectable pharmaceutical formulation comprising the pharmaceutical composition comprising Amphotericin B and Lipoate and a second pharmaceutical composition comprising cholesterol, oleate and glucose, wherein both compositions are mixed prior to injection. Wherein an alternative of said kit comprises a composition comprising Amphotericin B, Lipoate with glucose and a second pharmaceutical composition comprising cholesterol and oleate, wherein both compositions are mixed prior to injection. Said pharmaceutical kit is useful for the treatment of severe systemic mycoses, fungal infections, invasive mucormycosis infections, cryptococcal meningitis, aspergillus and Candida infections.

[0023] In a preferred embodiment of the present invention, said pharmaceutical formulation comprising Amphotericin B, Lipoate, Cholesterol, Oleate and Glucose (Lipoate-AmB-Oleate- Chol-Glu) has a concentration of Amphotericin B ranging from 0.1 to 8 mg-mL-1; a concentration of oleate ranging from 0.2 to 2.7 mg-mL-1; wherein the cholesterokAmB molar ratio is lower than 2:1, preferably from 1:1 to 2:1; and said glucose has a concentration from 1 % w / v to 10 % w / v; preferably 5%. Wherein said pharmaceutical formulation of the invention is formulated for intravenously administrable form and is useful in the treatment of systemic mycosis, fungal infections, severe systemic mycoses, invasive mucormycosis infections, cryptococcal meningitis, aspergillus and Candida infections. And its pH in an aqueous medium is from 7 to 8.5. Wherein Lipoate-AmB-Oleate-Chol-Glu formulation comprises nanoparticles less than 200 nm, preferably about 100 nm; is in absence of liposomes, polymerized lipoate, phospholipid and non-ionic surfactant.

[0024] Another object of the present invention is a process to obtain said Lipoate-AmB composition, comprising the following steps: a. adding amphotericin B powder to dimethyl sulfoxide (DMSO) solution at 30 mg / ml and stirring until complete dissolution; b. adding CO3HNa or NaOH solution to Lipoic acid powder and stirring until complete dissolution in order to obtain a Lipoate solution; c. adding the solution of amphotericin B in DMSO of step "a" to the Lipoate solution of step "b"; d. stirring the mixture prepared in step "c" for at least 10 minutes in the dark; e. sterilizing the mixture of step "d"

[0025] Wherein in a preferred embodiment of the invention, said process further comprises the following step: dialyzing the mixture of step "d" to remove all DMSO. And it comprises the following step: lyophilizing the sterilized mixture of step e. Another object of the present invention is a process to obtain said Lipoate-AmB-Oleate- Chol-Glu formulation, comprising the following steps: a. adding amphotericin B powder to dimethyl sulfoxide (DMSO) solution and stirring until complete dissolution;. b. adding CO3HNa or NaOH aqueous solution to Lipoic acid powder and stirring until complete dissolution in order to obtain a Lipoate solution; c. adding the solution of amphotericin B in DMSO of step "a" to the Lipoate solution of step "b"; d. stirring the mixture prepared in step "c" for at least 10 minutes in the dark to obtain Lipoate-AmB composition in aquous solution; e. sterilizing the mixture of step "d"; f. preparing Cholesterol in ethanol solution; g. adding to the solution of step "f" an oleate aqueous solution; h. stirring the mixture of step "g" for at least 5 minutes; i. adding glucose; j. sterilizing the solution obtained in step "i", obtaining Chol-Oleate-Glucose solution; k. mixing lyophilized mixture of step "e" with solution of step "j".

[0026] Wherein, in a preferred embodiment of the invention, before said step e, the mixture of step "d" is dialyzing to remove all DMSO. And wherein after said step "e" said mixture of step "e" is liophilizing to obtain lyophilized Lipoate:AmB composition. And wherein said step "h" further comprises heating the mixture. And wherein before or after step "i" further comprises a removal of ethanol by extraction, performed by dialyzing the mixture with bi-disti lied water.

[0027] Brief description of the figures Figure 1. (a) Size distribution of Lipoate nanoparticles (NPs). (b) Zeta potential ( ) of Lipoate NPs.

[0028] Figure 2. (a) Size distribution of Lipoate-AmB composition NPs. (b) Zeta potential ( ) of Lipoate-AmB NPs.

[0029] Figure 3. (a) Transmission electron micrographs of Lipoate NPs without associated AmB detected at magnification of 150 K. (b) Transmission electron micrographs of Lipoate-AmB composition NPs with Lipoate 20 mM and AmB 4 mg-mL1at magnification of 150 K. (c) Transmission electron micrographs of Lipoate-AmB composition NPs with Lipoate 20 mM and AmB 4 mg-mL1at magnification of 300 K.

[0030] Figure 4. X-ray diffraction patterns of: (A) Lipoate NPs powder, (B) AmB powder, (C) Lyophilized Lipoate-AmB NPs with Lipoate 40 mM and AmB 4 mg-mL1, and (D) Lipoate-AmB NPs with Lipoate 20 mM and AmB 4 mg-mL-1.

[0031] Figure 5. (a) UV-visible absorption spectra of AmB in methanol solutions, (b) UV-visible absorption spectra of Lipoate-AmB NPs.

[0032] Figure 6. (a) Size distribution of Chol-Oleate-Glucose solution.

[0033] Figure 7. (a) Size distribution of Lipoate-AmB-Oleate-Chol-glu.

[0034] Figure 8. Zeta potential ( ) of Lipoate-AmB-Oleate-Chol-Glu formulation NPs

[0035] Figure 9. Evolution over time of the particle size distribution for the Lipoate-AmB- Oleate-Chol-Glu formulation for a 2 mg / ml of AmB.

[0036] Figure 10. UV-visible absorption spectra of Lipoate-AmB-Oleate-Chol-Glu formulation formulation from 300 to 450 nm.

[0037] Figure 11. In-vitro cytotoxic effect of Lipoate-AmB composition and Lipoate-AmB- Oleate-Chol-Glu formulation compared with commercial formulations, Ambisome, and Amfostat on Hep-2 cell cultures.

[0038] Figure 12. The antifungal effect of Lipoate-AmB composition and Lipoate-AmB-Oleate- Chol-Glu formulation compared with the previously used commercial formulations, AmBisome and Amfostat (AFS) was evaluated on Candida Albicans.

[0039] Figure 13. Evolution of animal weights over time. Figure 14. Effect of concentration of Lipoate in the hemolysis of Lipoate-AmB composition of the invention in 6 minutes.

[0040] Figure 15. Effect of concentration of Lipoate in the hemolysis of Lipoate-AmB composition of the invention in 60 minutes.

[0041] Detailed Description of the Invention

[0042] Lipoic acid (LA) (l,2-dithiolane-3-pentanoic acid) is a sulfur-containing antioxidant with metal-chelating and antiglycation capabilities. It is water-soluble and has a low molecular weight. Unlike many antioxidants which are active only in lipids or aqueous phase, LA is active in both lipids and aqueous phases. Lipoate is obtained mixing LA with NaOH or bicarbonate in an aqueous medium up to pH between 7 to 9.

[0043] The pharmaceutical composition according to one or more embodiments of the present invention comprises Amphotericin B (AmB) and lipoate. The present invention surprisingly achieves that lipoate is capable of solubilizing said AmB in water, achieving a nanometric structure composition with outstanding stability.

[0044] Another surprising effect of the composition of the invention is that lipoate delays and reduces hemolysis caused by AmB. This fact demonstrates a technical effect that has never been disclosed or even hinted at before in the state of the art.

[0045] The composition of the present invention is an antifungal pharmaceutical composition to be used for the treatment of patients with severe systemic mycoses or fungal infections such as invasive mucormycosis infections, cryptococcal meningitis, and aspergillus and Candida infections.

[0046] The antifungal pharmaceutical composition, of the present invention, comprises lipoate and AmB with a lipoate / Amphotericin B mass ratio from 0.1 / 1, preferably from 0.15 / 1, more preferably from 0.35 / 1, more preferably from 0.7 / 1, more preferably from 1.4 / 1, more preferably from 5.5 / 1. And the upper limit of this mass ratio has been tested up to 22 / 1, but it is suspected that it could be higher.

[0047] This composition of the invention could be an aqueous composition or a lyophilized powder. When said Lipoate-AmB composition of the invention is an aqueous composition, AmB comprises a concentration from 0.1 to 8 mg-mL1and lipoate comprises a concentration from 0.02 to 176 mg / mL. Preferably said AmB is in a concentration of 2 mg-mL-1, and said lipoate is in a concentration from 0.2 to 16 mg / mL.

[0048] Said lipoate is the alkaline ion of lipoic acid (l,2-dithiolane-3-pentanoic acid) and can be added in its acid form or as a salt comprising as a counterion the monovalent cation selected from the group consisting of sodium, potassium, lithium, and combinations thereof. Preferably, said lipoic acid is neutralized by dissolving it into an aqueous solution of sodium hydroxide or sodium bicarbonate and pH is adjusted to a value from 6.8 to 8.5, preferably to pH 8, in order to generate a Lipoate-Na solution (lipoate). This solution is capable of solubilizing and stabilizing the AmB molecule producing a mixture in the form of nanoparticles called in this document Lipoate-AmB Np wherein these nanoparticles are a complex.

[0049] Furthermore, said Lipoate-AmB composition may be embodied either as a liquid, or as a powder or lyophilized formulation, obtained by drying or lyophilization of the aqueous solution. Both the drying process and the lyophilization of pharmaceutical compositions are well known in the prior art, therefore providing further details on the subject is not considered necessary.

[0050] The manufacturing process of said Lipoate-AmB composition of the invention comprises the following steps: a. adding lipoic acid powder to a CChHNa or NaOH solution and stirring until complete dissolution in order to obtain a Lipoate solution with a pH from 6.8 to 8.5, preferably from 7.5 to 8.5, and lipoate concentrations from 0.02 to 176 mg / mL b. adding Amphotericin B powder to dimethylsulfoxide (DMSO) and stirring until complete dissolution; c. mixing and stirring solutions from Lipoate solution (a) and AmB-DMSO dissolution (b) for at least 10 minutes and subsequently incubating it in the dark for at least 12 h; d. sterilizing the mixture by filtration through a 0.22-micron pore filter.

[0051] In a preferred embodiment, in step c adding the solution of AmB in DMSO of step "a" to the Lipoate solution of step "b".

[0052] In a preferred embodiment, dialyzing the mixture of step "c" to remove all DMSO. In a preferred embodiment, said Lipoate-AmB composition is in a lyophilized state in order to be better preserved until the moment of its administration. In this case, the manufacturing process of said Lipoate-AmB composition, further comprises the following step: lyophilizing the sterilized mixture of step d.

[0053] The Lipoate-AmB composition of the invention resulted in a very stable and simple formulation with an outstanding antifungal effect and resulted in a composition with reduced hemolysis compared to AmB-Deoxycholate.

[0054] It is provided, in the present invention, a composition that comprises Lipoate, AmB, and "supplement compounds" that, surprisingly, achieve reduced or negligible hemolytic and cytotoxic effects, while maintaining good antifungal properties.

[0055] In a preferred embodiment, said supplement compounds are selected from the group that comprises, but is not limited to: oleic acid, oleate, linoleic acid, linoleate, lauric acid, laureate, myristic acid, myristate, palmitic acid, palmitate, stearic acid, cholesterol, glucose, fructose, stearate and combinations thereof.

[0056] In a preferred embodiment, the pharmaceutical composition of the invention further comprises cholesterol and oleate. According to this embodiment, the pharmaceutical composition of the invention comprises cholesterol and oleate containing Amphotericin B and lipoate in an aqueous medium. This preferred embodiment, preferably, comprises nanoparticles.

[0057] Since AmB can bind to cholesterol, although with lower affinity than with ergosterol, the addition of cholesterol to the formulation achieves that, once the pharmaceutical composition has been introduced into the body of a subject, the AmB binds selectively with the ergosterol present in fungal cell membranes, while hindering its interaction with endogenous cholesterol molecules. Theoretically, one molecule of AmB interacts with a single molecule of cholesterol. In consequence, formulations with CholesterokAmB molar ratios lower than 1:1 are feasible although cholesterol deficiency will cause the formulation to have greater adverse effects. Formulations with a CholesterokAmB molar ratio higher of 2:1 resulted in high turbidity mixtures where cholesterol formed agglomerates, preventing its correct sterilization by filtering through a 0.22 micron filter. In a preferred embodiment of the invention, the cholesterokAmB molar ratio in the pharmaceutical composition is lower than 2:1, more preferably 1:1; and the concentration of the oleate is within a range from 0.2 to 2.5, preferably from 0.5 to 1.33 mg-mL1, more preferably about 1.33 mg-mL1.

[0058] In more preferred embodiment, said pharmaceutical composition of Lipoate, AmB, Cholesterol and Oleate, further comprises glucose and / or fructose, acting as an injectable formulation stabilizer and it will be mentioned in the present document as the pharmaceutical formulation of present invention or Lipoate-AmB-Oleate-Chol-glu. More preferably, said pharmaceutical formulation comprises glucose in a concentration from 1 % w / v to 10 % w / v. Even more preferably, said glucose is in a concentration of about 5 % w / v.

[0059] When the Lipoate-AmB composition is provided in a powder or lyophilized state, it can be resuspended in an appropriate medium before being administered.

[0060] An embodiment of the present invention is a kit comprising a container with said Lipoate-AmB composition in liquid or powder or lyophilized state and another container with an aqueous solution of cholesterol and oleate, preferably with glucose. Wherein the contents of both containers are mixed before use to prepare a fungicidal injectable pharmaceutical formulation.

[0061] Another embodiment of the present invention is a kit comprising a container with said Lipoate-AmB composition with glucose in liquid or powder or lyophilized state and another container with an aqueous solution of cholesterol and oleate. Wherein the contents of both containers are mixed before use to prepare a fungicidal injectable pharmaceutical formulation.

[0062] In a preferred embodiment, said Lipoate-AmB composition in a powder or lyophilized state is resuspended in an aqueous solution containing nanoparticles of cholesterol and oleate. In a more preferred embodiment, said aqueous solution further comprises glucose, and the aqueous solution containing glucose and nanoparticles of cholesterol and oleate is called Chol- Oleate-Glucose composition. According to this embodiment, the Lipoate-AmB composition resuspended in Chol-Oleate-Glu composition comprises nanoparticles of cholesterol and oleate containing Amphotericin B and lipoate in an aqueous medium with glucose (Lipoate-AmB- Oleate-Chol-glu); wherein the CholesterokAmB molar ratio is lower than 2:1, preferably is 1:1; wherein the concentration of the oleate is within a range from about 0.5 to about 1.33 mg-mL"1, preferably about 1.33 mg-mL-1; and wherein said glucose in a concentration from 1 % w / v to 10 % w / v, more preferably about 5 % w / v.

[0063] Said oleate is the alkaline ion of oleic acid and can be added in its acid form or as a salt, comprising as a counterion the monovalent cation selected from the group consisting of sodium, potassium, lithium, and combinations thereof.

[0064] It has been shown in laboratory tests that laureate, myristate, palmitate and stearate have the same effects as oleate for the present invention. Therefore, when oleate is mentioned in this document, it could be replaced by laureate, myristate, palmitate and stearate interchangeably.

[0065] Whether said Lipoate-AmB composition is previously in a lyophilized state or not, after the addition of cholesterol, oleate, and glucose is called Lipoate-AmB-Oleate-Chol-Glu or pharmaceutical formulation of the invention. The nanostructures present in the Lipoate-AmB- Oleate-Chol-Glu formulation are nanoparticles (Np) with a nanometric diameter and a highly ordered nanostructure that shows less toxicity than the simpler Lipoate-AmB composition. In this way, the antifungal pharmaceutical formulation of the present invention based on AmB can be used for the treatment of patients with severe systemic mycoses or fungal infections, producing very little side effects to the treated subject.

[0066] Surprisingly, the pharmaceutical formulation of the present invention could be sterilized by filtration of 0.22 micron in each step of the elaboration. Nanoparticles of the Lipoate-AmB composition, Chol-Oleate-Glucose composition Np and Lipoate-AmB-Oleate-Chol-Glu Np are lower than 220 nm, and they could be sterilized by filtration. More surprisingly is the fact that the formulation of the invention is not a liposome, then high energy for elaborate of liposomes (by turrax, e.g.) is not needed. The formulation of the present invention is very simple to elaborate, is not a liposome and is very stable in time in aqueous suspension. The present invention is a pharmaceutical formulation in absence of phospholipids and non-ionic surfactants.

[0067] Another object of the invention is a process of manufacturing said Chol-Oleate-Glucose composition that comprises the following steps: a. preparing a Cholesterol in ethanol solution, wherein Cholesterol is in a concentration from 0.1 to 50 mg-mL-1; b. adding an oleate aqueous solution with an oleate concentration from 0.5 to about 1.33 mg-mL-1; c. stirring the mixture of step b. for at least 5 minutes and incubating the mixture; d. removing the ethanol from the mixture of step c. by extraction; e. adding glucose in an amount enough to reach a final concentration from 1 to 10 % (w / v); f. sterilizing the solution obtained in step e.

[0068] In a preferred embodiment, said step c. further comprises heating the mixture to 50 - 60 °C during stirring in order to facilitate cholesterol dissolution, and the removal of ethanol by extraction of step d. is performed by dialyzing the mixture of step c. with bi-disti I led water.

[0069] Alternatively, the removal of ethanol can be performed after the addition of glucose to the mixture. In this case, the solvent used to perform the dialysis can be an aqueous glucose solution with a glucose concentration that matches the glucose concentration in the Chol- Oleate-Glucose solution in order to avoid glucose extraction.

[0070] In a preferred embodiment of the present invention said sterilization is made by filtration by 0.22 micron.

[0071] In turn, said oleate can be prepared by alkaline saponification of an oleic acid source, wherein said oleic acid source can be any high-oleic natural oil, commercial oleic acid, or mixtures thereof. Said high-oleic natural oil can be selected from the group comprising, but not limited to, olive oil, canola oil, high-oleic sunflower oil, avocado oil, purified high-oleic peanut oil, and mixtures thereof. The procedure followed to prepare said oleate is detailed hereafter: a. taking a volume of an oleic acid source; b. adding the same volume of pure ethanol to said oleic acid source; c. adding the same volume of 5 N NaOH solution to the mixture of step b); d. agitating the mixture obtained in step c. for at least 5 minutes, letting it settle for at least 15 minutes, and repeating these operations twice; e. letting the mixture settle until it forms a single phase wherein the oil has been saponified in such a way that it mixes completely with the alcohol and NaOH solution.

[0072] The antifungal pharmaceutical composition of the invention is used in the treatment of systemic mycosis. In one embodiment of the invention, the pharmaceutical composition is in intravenously administrable form. In another embodiment of the invention, the pharmaceutical is formulated for nebulization, inhalation, or topical administration.

[0073] Examples

[0074] Example 1 - Preparation of Lipoate solution

[0075] Stock solutions of Lipoate 0.5 M were prepared by dissolving 10.32 g of lipoic acid in 100 mL of a sodium bicarbonate aqueous solution of 5 % w / v and adjusted to pH 8.

[0076] Subsequently, dilutions of Lipoate stock solution, i.e.: 5, 10, 20, 25, 40, 50, and 60 mM, were prepared to be used in the following examples. The pH of said dilutions was kept at 8 in order to avoid precipitation of the lipoate. Lipoate nanoparticles (NPs) are formed by their simple preparation in an aqueous medium as described above.

[0077] Example 2 - Structural Characterization of Lipoate solutions.

[0078] Dynamic Light Scattering analysis of Lipoate NPs

[0079] Lipoate NPs were prepared by the direct dissolution of lipoic acid in an aqueous solution of pH 8, as described in Example 1. Different dilutions of the Lipoate NPs were subjected to DLS analyses in order to determine the size distribution and zeta potential (?).

[0080] The size distribution and zeta potential ( ) measurements of the present Example 1 and subsequent Examples were run using a Malvern Zetasizer Nano ZS instrument at a fixed scattering angle of 173°.

[0081] The DLS technique revealed the presence of different sized structures with high polydispersity indices that vary considerably depending on the concentration of Lipoate (Figure la). In particular, with lipoate concentrations between 5 and 10 mM, structures of around 150 nm are formed, while at higher lipoate concentrations the structures are smaller, around 50 nm for concentrations of 25 mM and 50 mM. Since the suspensions are sterilized by filtration through a 0.22-micron filter, lower particle sizes are more desirable.

[0082] Regarding the zeta potential measurements, negative values were obtained, in general, near -40 mV (Figure lb). The negative charge stabilizes the NPs and prevents their aggregation by creating electrostatic repulsions between particles providing good stability to the suspension.

[0083] Example 3 - Preparation of Lipoate-AmB composition of the invention

[0084] Stock solution of Amphotericin B (AmB) was prepared by dissolving the drug in dimethylsulfoxide (DMSO) at a final concentration of 30 mg-mL-1. Said solution was prepared in darkness and in a nitrogen atmosphere to protect it from oxidation.

[0085] Then, a stock solution of AmB was slowly added to different dilutions of Lipoate stock solution of Example 1, i.e.: 10, 20, 25, 40, and 60 mM, at room temperature (20 ^c). The volume of AmB stock solutions added was enough to reach a final AmB concentration of 2 or 4 mg-mL"1(4.33 mM) in all samples. The pH of the mixtures was adjusted to 7.5 - 8.5 in order to ensure the stability of the Lipoate-AmB composition. Said lipoate-AmB composition shows nanoparticles (NPs). After magnetic stirring at 600 rpm for 10 min, these mixtures were incubated at 4 - 8 °C for 24 h in the darkness. Finally, the samples were dialyzed for 24 h in the dark at 4 °C using SpectraPor dialysis membranes with a MW cut-off of 10,000 Da in order to remove the DMSO.

[0086] Example 4 - Determination of AmB Concentration

[0087] AmB concentrations in the different compositions were measured spectrophotometrically from its absorption at 405 nm after complete monomerization of the drug by dilution in methanol, as described by Gaboriau et al. (Heat-induced superaggregation of amphotericin B reduces its in vitro toxicity: a new way to improve its therapeutic index. Gaboriau Fl, Cheron M, Petit C, Bolard JAntimicrob Agents Chemother. 1997 Nov; 41(11) :2345- 51 https; / / www. cbj. im. jh.gov / pubmed / 9371331). In this way, the measurements become independent of the aggregation state of AmB.

[0088] The spectroscopic determinations were obtained using a Perkin Elmer lambda 25 spectrophotometer. The sample concentration was then obtained from a calibration curve from 1 to 10 pg-mL1of a 50 % v / v AmB in methanol solution. Example 5 - Structural Characterization of Lipoate-AmB composition. a- Determination of Size Distribution and Zeta Potential ( by DLS

[0089] The size distribution and zeta potential of Lipoate-AmB composition NPs of Example 3 were determined using dynamic light scattering (DLS) Malvern Instrument NANO ZS at a fixed scattering angle of 173°.

[0090] Figure 2a exhibits the size distribution of Lipoate-AmB composition NPs synthesized with AmB concentrations of 4 mg-mL1and lipoate concentrations of 10, 20, 40, and 60 mM (2.28, 4.56, 9.12, 13.68 mg / ml). DLS technique revealed particles that are mostly smaller than 100 nm.

[0091] The results suggest that, as the concentration of lipoate increases, the size of the nanoparticles decreases, covering modal sizes between 25 and 70 nm, similar to what is observed with pure lipoate nanoparticles. Regarding their polydispersity indexes, they decrease with increasing lipoate concentrations.

[0092] As can be observed from Figure 2b, the zeta potential measurements gave values around -40 mV, indicating good stability of the Lipoate-AmB composition nanoparticles in the suspension. b- Transmission Electron Microscopy

[0093] In orderto analyze their morphological characteristics, the Lipoate NPs with and without associated amphotericin B (i.e. Lipoate NPs of Example 1 and Lipoate-AmB composition of Example 3) were inspected using a JEOL JEM-1200 EX II transmission electron microscope at magnification of 150 K and 300 K.

[0094] Samples were prepared by loading 50 pL of each solution in a carbon grid, letting them stand for 5 min, and drying them. Subsequently, the sample excess was washed out with distilled water. Finally, carbon grids were incubated with 50 pL of 10% uranyl acetate solution for 1 min and then dried.

[0095] The micrographs of pure Lipoate NPs showed a population with an average diameter of 40 nm (Figure 3a). Upon incorporation of AmB (Figures 3b and 3c), a single population of Lipoate-AmB NPs with an average diameter of 25 nm was observed. It should be noted that these values are in good agreement similar to those obtained using the DLS technique. c- X-ray diffraction

[0096] The effect on the crystalline structure of loading of AmB into Lipoate NPs was explored. For this purpose, two samples of Lipoate-AmB composition NPs were lyophilized and then analyzed in a Bruker D8-Advance with Cu Anode X-ray diffractometer. Likewise, the X-ray diffraction patterns of pure Amphotericin B and Lipoic acid powder samples were used as controls.

[0097] Figure 4 exhibits the diffractograms of the samples analyzed. As can be observed, lyophilized lipoate powder (sample A) revealed several peaks indicating the presence of crystallinity. Similarly, crystalline domains were observed in the AmB powder (sample B). In contrast, the diffractograms corresponding to Lipoate-AmB composition NPs (samples C and D) show an absence of diffraction peaks, indicating that the incorporation of AmB in the Lipoate NPs abolishes the crystalline structure found in the pure samples.

[0098] Example 6 - Stability of Lipoate-AmB composition of the invention.

[0099] The stability of Lipoate-AmB composition of Example 2 in solution or after different treatment conditions was studied. a- Effect of Lipoate concentrations in the stability of Lipoate-AmB composition

[0100] Table 1 displays the particle size and zeta potential measurements of various Lipoate- AMB compositions obtained by DLS 5, 20, and 30 days after their preparation. On the one hand, it was observed that Lipoate-AmB compositions concentrations of lipoate of 10, 20, and 40 mM remained very stable for at least 30 days at 4 °C, maintaining their sizes and polydispersity index as well as their zeta potential values.

[0101] On the other hand, those formulations with higher Lipoate contents (60 mM) became more unstable over time, increasing their size considerably as well as their polydispersion. Table 1. Stability over time of Lipoate AmB complexes b- Stability of Lipoate-AmB composition after centrifugation

[0102] The Lipoate-AmB composition, stored for 30 days at 4 °C, was then submitted to centrifugation. Particle size measurements were performed before and after centrifugation (called pre-centrif and post-centrif, respectively) and the results are exhibited in Table 2. It was found that the nanoparticles were able to withstand centrifugation processes of 10 min at 15000 rpm without undergoing aggregation or modifications of their size and zeta potential, proving to be very stable.

[0103] Table 2. Particle size of Lipoate-AmB composition after being submitted to centrifugation. c- Aggregation state of Lipoate-AmB composition Nps after lyophilization and resuspension

[0104] It has been described that AmB has different aggregation states (Lavasanifar, A.; Samuel,

[0105] J.; Sattari, S.; Kwon, G.S. Block copolymer micelles for the encapsulation and delivery of amphotericin B. Pharm. Res.2002, 19, 418-422), which can influence in the unwanted toxic effect of the drug.

[0106] In order to assess the stability of Lipoate-AmB composition regarding their aggregation state, different Lipoate-AmB compositions were prepared according to the process described in Example 3. Then, the compositions were frozen at -80 °C for 24 hours before being subjected to the lyophilization process. Subsequently, they were resuspended with bi-distil led water in a volume enough to reach concentrations of AmB of 4 mg-mL-1.

[0107] After resuspension, the recovered amount was estimated by measuring the amount of AmB according to the technique described in Example 4. For all samples, the AmB recovery was greater than 95 %.

[0108] The results of the spectrophotometric studies showed that when AmB interacts with Lipoate NPs, that help it to remain soluble in an aqueous medium, it is mostly in an aggregated state, with its characteristic absorption peak at 333 nm (Figure 5). Also it is observed that in the lyophilized samples the prevalence of the aggregate state of AmB is maintained.

[0109] Example 7 - Preparation of Chol-Oleate-Glucose composition from commercial oleic acid

[0110] Stock Cholesterol in ethanol solutions were prepared in a concentration of 20 mg-mL-1.

[0111] Oleate stock solutions were prepared by alkaline saponification of commercial oleic acid. For this purpose, 99% purity oleic acid (from Sigma-Aldrich) was used, the procedure followed to prepare said oleate stock solutions is detailed hereafter: a. taking 5 mL of commercial oleic acid; b. adding 5 mL of pure ethanol to said oleic acid; c. adding 5 mL of 5 N NaOH solution to the mixture from step b); d. agitating the mixture obtained in step c) for 5 minutes, letting it settle for at least 15 minutes, and repeat these operations twice; e. letting the mixture settle until it forms a single phase wherein the oil has been saponified in such a way that it mixes completely with the alcohol and NaOH solution. Afterward, Oleate and Cholesterol stock solutions were mixed and stirred at 600 rpm for 10 minutes. After stirring, the mixture was heated and kept at 50-60 °C for 10 min and then was incubated at 4 - 8 ^c for 24 h. Subsequently, the mixture was dialyzed with bi-disti I led water to remove the ethanol. Glucose was then added in an amount enough to reach a final concentration of 5 % (w / v); and finally, the obtained solution was sterile filtered using a 0.22 micron filter.

[0112] The detailed process has been made with several fatty acids as lauric acid (Dodecanoic acid, C12:0), myristic acid (Tetradecanoic acid, C14:0), palmitic acid (Hexadecanoic acid, C16:0), and stearic acid (Octadecanoic acid, C18:0) to obtain laureate, myristate, palmitate, and stearate respectively. With all of them the present invention has been made with excellent results.

[0113] Example 8 - Preparation of Chol-Oleate-Glucose composition from peanut oil

[0114] Stock Cholesterol in ethanol solutions were prepared in a concentration of 20 mg-mL-1.

[0115] Oleate stock solutions were prepared by alkaline saponification of peanut oil. For this purpose peanut oil was used, the procedure followed to prepare said oleate stock solutions is detailed hereafter: f. taking 5 mL of peanut oil; g. adding 5 mL of pure ethanol to said oleic acid; h. adding 5 mL of 5 N NaOH solution to the mixture from step b); i. agitating the mixture obtained in step c) for 5 minutes, letting it settle for at least 15 minutes, and repeat these operations twice; j. letting the mixture settle until it forms a single phase wherein the oil has been saponified in such a way that it mixes completely with the alcohol and NaOH solution.

[0116] Afterward, Oleate and Cholesterol stock solutions were mixed and stirred at 600 rpm for 10 minutes. After stirring, the mixture was heated and kept at 50-60 °C for 10 min and then was incubated at 4 - 8 ^c for 24 h. Subsequently, the mixture was dialyzed with bi-disti I led water to remove the ethanol. Glucose was then added in an amount enough to reach a final concentration of 5 % (w / v); and finally, the obtained solution was sterile filtered using a 0.22 micron filter. Example 9 - Preparation of Chol-Oleate-Glucose composition from Sodium Oleate

[0117] Stock Cholesterol in ethanol solutions were prepared in a concentration of 20 mg-mL ~1.

[0118] Stock oleate solutions were prepared by diluting > 99 % purity sodium oleate (from Sigma-Aldrich) in bi-disti I led water in order to obtain an oleate concentration of 1.33 mg-mL-1.

[0119] Afterward, Oleate and Cholesterol stock solutions were mixed and stirred at 600 rpm for 10 minutes. After stirring, the mixture was heated and kept at 50-60 °C for 10 min and then was incubated at 4 - 8 ^c for 24 h. Subsequently, the mixture was dialyzed with bi-disti I led water to remove the ethanol. Glucose was then added in an amount enough to reach a final concentration of 5 % (w / v); and finally, the solution obtained was sterile filtered using a 0.22 micron filter.

[0120] Example 10 - Structural Characterization of Chol-Oleate-Glucose composition.

[0121] A sample of Chol-Oleate-Glucose composition, prepared as described in Example 7, was analyzed in order to determine the size distribution and zeta potential of the structures present in Chol-Oleate-Glucose composition. For this purpose, a dynamic light scattering (DLS) Malvern Instrument NANO ZS was used at a fixed scattering angle of 173°.

[0122] The analysis revealed a population of particles with an average size of about 100 nm. Figure 6.

[0123] The zeta potential is around -30 mV, suggesting that the formulation has good stability.

[0124] Example 11 - Preparation of Lipoate-AmB-Chol-Oleate-Glu formulation

[0125] The Lipoate-AmB compositions of Example 3 were frozen at -80 °C for 24 hours and subsequently subjected to the lyophilization process.

[0126] Afterwards, the lyophilized Lipoate-AmB composition was resuspended with the mixture Chol-Oleate-Glucose composition of Example 7, the volume added was enough to reach AmB concentrations of 2 mg-mL-1. Example 12 Kit for the preparation of an antifungal pharmaceutical formulation of the invention.

[0127] Preparation of Lipoate-AmB composition

[0128] Solution of Lipoate 0.5 M were prepared in a sodium bicarbonate solution by dissolving 10.32 g of lipoic acid in an aqueous sodium bicarbonate solution with a concentration of 5.2 % w / v (0.62 M) and pH 8.

[0129] Stock solutions of AmB were prepared in dimethylsulfoxide (DMSO) at a final concentration of 30 mg.mL1under light-protected and nitrogen-sealed conditions.

[0130] Solubilization of AmB into Lipoate: The solution of AmB was slowly introduced into Lipoate solution, to obtain a 5 mg / ml of Lipoate at room temperature (202C) and with a final AmB concentration of 4 mg.mL-1. After 10 min stirring, these mixtures were incubated at 4-82C for 24 h in the dark. Subsequently, the samples underwent a 24-hour dialysis process at 42C in the dark using SpectraPor dialysis membranes with a molecular weight cut-off of 10,000 Da to remove DMSO. Finally, this composition was sterilized filtered through a 0.22-micron filter.

[0131] Then, the Lipoate-AmB composition were frozen at -80 °C for 24 hours before being subjected to the lyophilization process. A lyophilized powder is obtained in a glass vial as container 1 with 25 mg of AmB each.

[0132] Preparation of Oleate-Chol-glucose composition

[0133] Solution of Cholesterol (Choi) was prepared in ethanol at a concentration of 20mg.mL-1.

[0134] Oleic acid (Oleate) stock solution was prepared by alkaline saponification of oleic acid.

[0135] A solution of Cholesterol 0.835 mg.mL1, in Oleate 0.665 mg.mL1was prepared to achieve an AmB-Chol molar ratio of 1:1. After stirring at 600 rpm for 10 minutes, these mixture was incubated at 4-8°C for 24 hours. The resulting composition was then dialyzed for 4 hours using SpectraPor dialysis membranes with a molecular weight cutoff of 10,000 Da to remove ethanol. A glucose solution was added to reach a final concentration of 5%. Finally, this composition was sterilized filtered through a 0.22-micron filter. Other glass vial, container 2 with 12.5 ml of Oleate-Chol-glucose composition are obtained.

[0136] Preparation of Lipoate-AmB-Oleate-Chol-glucose formulation Lyophilized Lipoate-AmB composition in container 1 is re-suspended with said Oleate- Chol-glucose composition of container 2 to reach AmB concentrations of 2 mg.mL ~1. Lipoate- AmB-Oleate-Chol-Glu formulation was then obtained, and it was sterilized filtered through a 0.22-micron filter. This formulation is injected in 100 ml of perfusion solution (dextrose 4.5%) for perfusion with a concentration of AmB about 0.25 mg / mL.

[0137] Said perfusion solution with AmB about 0.25 mg / ml is useful for injection to mammals, particularly human for the treatment of systemic mycosis or fungal infections.

[0138] Example 13 Kit for the preparation of an antifungal pharmaceutical formulation of the invention.

[0139] Preparation of Lipoate-AmB composition

[0140] Solution of Lipoate 0.5 M was prepared in a sodium bicarbonate solution by dissolving 10.32 g of lipoic acid in an aqueous sodium bicarbonate solution up pH 8.

[0141] Stock solutions of AmB were prepared in dimethylsulfoxide (DMSO) at a final concentration of 30 mg.mL1under light-protected and nitrogen-sealed conditions.

[0142] Solubilization of AmB into Lipoate: The solution of AmB was slowly introduced into Lipoate solution, to obtain a 5 mg / ml of Lipoate at room temperature (202C) and with a final AmB concentration of 4 mg.mL-1. After 10 min stirring, this mixture was incubated at 4-82C for 24 hours in the dark. Finally, this composition was sterilized filtered through a 0.22-micron filter

[0143] Then, the Lipoate-AmB composition was frozen at -80 °C for 24 hours before being subjected to the lyophilization process. A lyophilized powder is obtained in a glass vial as container 1 with 50 mg of AmB each.

[0144] Preparation of Oleate-Chol-glucose composition

[0145] Solution of Cholesterol (Choi) was prepared in ethanol at a concentration of 20mg.mL-1.

[0146] Oleic acid (Oleate) stock solution was prepared by alkaline saponification of oleic acid.

[0147] A solution of Cholesterol 0.835 mg.mL1, in Oleate 0.665 mg.mL1was prepared to achieve an AmB-Chol molar ratio of 1:1. After stirring at 600 rpm for 10 minutes, this mixture was incubated at 4-8°C for 24 hours. The resulting composition was then dialyzed for 4 hours using SpectraPor dialysis membranes with a molecular weight cutoff of 10,000 Da to remove ethanol. A glucose solution was added to reach a final concentration of 4.5%. Finally, this composition was sterilized filtered through a 0.22-micron filter. Other glass vial, container 2 with 25 ml of Oleate-Chol-glucose composition are obtained.

[0148] Preparation of Lipoate-AmB-Oleate-Chol-glucose formulation

[0149] Lyophilized Lipoate-AmB composition in container 1 is re-suspended with said Oleate- Chol-glucose composition of container 2 to reach AmB concentrations of 2 mg.mL-1. Lipoate- AmB-Oleate-Chol-glucose formulation is then obtained, and it was sterilized filtered through a 0.22-micron filter. This Lipoate-AmB-Oleate-Chol-glucose formulation was injected in a perfusion solution (Dextrose 5%) of 250 ml and the concentration of AmB is about 0.2 mg / mL.

[0150] Said perfusion solution is useful for injection to mammals, particularly human for the treatment of systemic mycosis or fungal infections.

[0151] Example 14 Kit for the preparation of an antifungal pharmaceutical formulation of the invention.

[0152] Preparation of Lipoate-AmB composition

[0153] Solutions of Lipoate 0.5 M was prepared in a sodium bicarbonate solution by dissolving 10.32 g of lipoic acid in an aqueous sodium bicarbonate solution up pH 8.

[0154] Stock solution of AmB was prepared in dimethylsulfoxide (DMSO) at a final concentration of 30 mg.mL1under light-protected and nitrogen-sealed conditions.

[0155] Solubilization of AmB into Lipoate: The solution of AmB was slowly introduced into Lipoate solution, to obtain a 7 mg / ml of Lipoate at room temperature (202C) and with a final AmB concentration of 4 mg.mL-1. After 10 min stirring, these mixtures were incubated at 4-82C for 24 h in the dark, then Lipoate-AmB composition is obtained. Finally, this composition was sterilized filtered through a 0.22-micron filter.

[0156] Then, 25 ml of Lipoate-AmB composition was transferred into a glass vial as container 1 with 100 mg of AmB each.

[0157] Preparation of Oleate-Chol-glucose composition Solution of Cholesterol (Choi) was prepared in ethanol at a concentration of 20mg.mL ~1.

[0158] Oleic acid (Oleate) stock solution was prepared by alkaline saponification of oleic acid.

[0159] A solution of Cholesterol 0.835 mg.mL-1, in Oleate 0.665 mg.mL-1 was prepared to achieve an AmB-Chol molar ratio of 1:1. After stirring at 600 rpm for 10 minutes, this mixture was incubated at 4-8°C for 24 hours. A glucose solution was added to reach a final concentration of 5%. Finally, this composition was sterilized filtered through a 0.22-micron filter. Another glass vial, container 2 with 25 ml of Oleate-Chol-glucose composition was obtained.

[0160] Preparation of Lipoate-AmB-Oleate-Chol-glucose formulation

[0161] Lipoate-AmB composition in container 1 is mixed with said Oleate-Chol-glucose composition of container 2 to reach AmB concentrations of 2 mg.mL-1. Lipoate-AmB-Oleate- Chol-glucose formulation is then obtained, and it was sterilized filtered through a 0.22-micron filter. This formulation is injected into a 500 ml perfusion solution of dextrose 5% for perfusion, wherein the concentration of AmB is about 0.2 mg / mL.

[0162] Said perfusion solution with AmB is useful for injection to mammals, particularly human for the treatment of systemic mycosis or fungal infections.

[0163] Example 15 - DLS analyses of Lipoate-AmB-Oleate-Chol-Glu formulation

[0164] A Lipoate-AmB-Oleate-Chol-Glu formulation prepared according to Example 11 was subjected to DLS analyses in order to determine its structure and stability. Figure 7 displays the size distribution and Figure 8 Zeta potential of Lipoate-AmB-Oleate-Chol-Glu formulation containing 2 mg / MI of AmB. After the reconstitution of the lyophilized Lipoate-AmB composition with the Chol-Oleate-Glucose composition, a main population of particles with an average size of 120 nm was observed. On the other hand, a smaller population of particles around 5000 or 6000 nm could also be detected. The stability of the Lipoate-AmB-Oleate-Chol- Glu Np was very good, with zeta potential values around -30 mV.

[0165] Example 16 - Stability of Lipoate-AmB-Oleate-Chol-Glu formulation The stability of the Lipoate-AmB-Oleate-Chol-Glu formulation of Example 12 in solution over time was studied. a - Stability of Lipoate-AmB-Oleate-Chol-Glu formulation over time

[0166] As shown in Figure 9, after 21 days at 4 °C, a slight reduction in particle size and their polydispersity index was observed, not only indicating that the Lipoate-AmB-Oleate-Chol-Glu formulation remained stable but also that further stabilization of the particles occurred during this period. This formulation of the invention could be sterilized by filtration of 0.22. In all cases, the zeta potential remained around - 30 mV.

[0167] In Figure 9:

[0168] A Lipoate-AmB-Oleato-Chol-Glu 0.2 mg / ml of AmB

[0169] B Lipoate-AmB-Oleato-Chol-Glu post 5d 4C 0.2 mg / ml of Amb

[0170] C Lipoate-AmB-Oleato-Chol-Glu post 12d 4C0.2 mg / ml of Amb

[0171] D Lipoate-AmB-Oleato-Chol-Glu post 21d 4C0.2 mg / ml of Amb

[0172] Example 17 - Aggregation state of AmB in Lipoate-AmB-Oleate-Chol-Glu formulation

[0173] The results of the spectrophotometric studies show that the AmB in the Lipoate-AmB- Oleate-Chol-Glu formulation (concentrations: AmB 4 mg-mL1, Lipoate 5 mg / mL, Cholesterol 1.67 mg-mL1, Oleate 1.33 mg-mL1, Glucose 5 % w / v) is found in a large percentage in a selfaggregated state with its characteristic absorption peak at 333 nm, however, the presence of the drug in other aggregation states such as trimers, dimers and monomers is observed. These states are represented by the absorption peaks at longer wavelengths (see Figure 10).

[0174] Example 18 - In Vitro Studies a - Hemolysis

[0175] Blood was extracted from healthy human volunteers and collected in tubes with EDTA before being washed twice with 9 volumes of 0.9 % NaCI saline solution. After each washing, cells were pelleted by centrifugation at 1,500 xg for 10 min and the supernatant was discarded. The final pellet was adjusted to 4 x 107cells per mL. Hemolytic activity of Lipoate-AmB-Oleate- Chol-Glu formulation (concentrations: AmB 4 mg-mL1, Lipoate 5 mg / mL, Cholesterol 1.67 mg-mL1, Oleate 1.33 mg-mL1, Glucose 5 % w / v) and two commercial formulations (AmB in deoxycholate micelles Amfostat (AFS) and liposomal-AmB formulations AmBisome) were studied by adding 1 volume of red blood cell suspension to 1 volume of the several formulations containing AmB at a final concentration of 0.2 mg-mL-1. 100 % hemolysis control was obtained by incubating 1 volume of the red blood cells with 1 volume of SDS solution. Samples were incubated for 1 h at 372C in a shaker and then centrifuged at 1,500 xg during 10 min. The supernatants were collected for the analysis of the percentage of hemolysis by reading the absorption of free hemoglobin at 540 nm. Hemolysis percentages of the different samples were calculated based on the comparison of their absorbance value with the 100 % hemolysis control.

[0176] The results showed that the Lipoate-AmB-Oleate-Chol-Glu formulation of the invention produced a lytic effect lower than 2 %, therefore the hematological toxicity of this formulation is considered negligible. b - Cytotoxic effect on cell cultures

[0177] The in-vitro cytotoxic effects of Lipoate-AmB compositions (AmB 4 mg-mL-1, Lipoate 5 mg / mL. Lipoate / AmB= 1.25 / 1) and Lipoate-AmB-Oleate-Chol-Glu formulation (concentrations: AmB 4 mg-mL-1, Lipoate 5 mg / mL Cholesterol 1.67 mg-mL-1, Oleate 1.33 mg-mL-1, Glucose 5 % w / v) compared with commercial formulations, AmBisome (liposomal AmB), and Amfostat (AmB in deoxycholate micelles) on Hep-2 cell cultures, were studied.

[0178] Hep2 (Human epithelial carcinoma of the larynx) cells were maintained at 37 °C with 5 % CO? in minimal essential medium (MEM) supplemented with 10 % heat-inactivated fetal bovine serum (FBS) (NATOCOR, Cordoba, Argentina), 2 mM L-glutamine and 10 pg-mL1gentamicin (all culture reagents were from Invitrogen).

[0179] For the experiments, cells were seeded into the wells of a 96-well plate and after 24 h of incubation, 100 pL of serial dilutions from 1 to 100 pg-mL1of Lipoate-AmB composition and Lipoate-AmB-Oleate-Chol-Glu composition NPs, AmB in deoxycholate micelles (AFS) and liposomal-AmB formulation (AmBisome) were added to the wells and cultured for 24 h at 37 2C. The cytotoxicity test was determined by an MTT assay.

[0180] Briefly, the reaction medium was removed from each well and 100 pL of medium without phenol red and 20 pL of the MTT reagent were added to each well, the cells were cultured for an additional 4 h at 37 °C. Then the medium was carefully removed and 150 pL of MTT solvent (4 mM HCI in isopropanol) was gently added to the wells. The solubilized formazan was measured at 595 nm using a microplate spectrophotometer (Anthos reader 2010). For each sample tested, the cell viability percentage was determined by comparing its absorbance after the MTT assay with the absorbance of the normal cell growth control. Each treatment was analyzed in triplicate.

[0181] Figure 11 clearly shows that the of Lipoate-AmB-Oleate-Chol-Glu formulation of the invention drastically reduces AmB cytotoxic effect, making the cytotoxic effect of Lipoate-AmB- Oleate-Chol-Glu formulation even lower than that of the standard AmBisome formulation. The AFS formulation was the one that showed the most cytotoxic effect in the trial. c - Effect of AmB on Candida Albicans

[0182] The antifungal effect of Lipoate-AmB composition (Lipoate 5 mg / mL , AmB 4 mg-mL-1) and Lipoate-AmB-Oleate-Chol-Glu formulation (concentrations: Lipoate 5 mg / mL, AmB 4 mg-mL1, Cholesterol 1.67 mg-mL1, Oleate 1.33 mg-mL1, Glucose 5 % w / v) against Candida albicans was then evaluated and compared with that produced by the previously used commercial formulations, AmBisome and Amfostat.

[0183] The minimum inhibitory concentrations (MIC) of AmB on C. albicans were determined by a modified version of the M27-A method fungicidal concentrations (MFCs) (Wayne, P. Reference method for broth dilution antifungal susceptibility testing of yeasts. Approved standard M27-A. National Committee for Clinical Laboratory Standards, 1997).

[0184] Briefly, 100 pL of yeast inoculum at a concentration of 0.5 x 103to 2.5 x 103CFU.mL1were added to plates containing 100 pL of serial dilutions of AmB from the different formulations (Lipoate-AmB, Lipoate-AmB-Oleate-Chol-glu, AFS, and AmBisome). After 24 h of incubation, the optical density of each microplate well was measured with a microplate spectrophotometer set at a wavelength of 620 nm (Anthos reader 2010). MIC values are defined as the lowest concentration that prevents visible fungal growth, compared with the growth of control. The results of Figure 12 show that Lipoate-AmB composition and Lipoate-AmB-Oleate- Chol-Glu formulation have an antifungal effect higher than both commercial formulations AmBisome and Amfostat.

[0185] Example 19 - In vivo Studies.

[0186] Ten (10) healthy animals (C57BL76 mice) were divided randomly into 2 groups containing 5 animals each. Animals of group 1 were administered into the tail vein with 100 mg-kg1doses of AmBisome and animals of group 2 with 100 mg-kg1doses of Lipoate-AmB- Oleate-Chol-Glu formulation of the invention (concentrations: AmB 4 mg-mL ~1, Lipoate 5 mg / mL , Cholesterol 1.67 mg-mL-1, Oleate 1.33 mg-mL-1, Glucose 5 % w / v). The animals were monitored for 14 days for mortality, weight, clinical and behavioral symptoms, and any adverse reactions.

[0187] No inconveniences were observed at the time of injecting the formulations into the tail vein of the animals.

[0188] The animals, in both groups, remained alive and with regular weights, indicating the non-toxicity of the formulations studied (See Figure 13).

[0189] Example 20

[0190] Kinetic Monitoring of Hemolysis Induced by Amphotericin in the Presence of Increasing Amounts of Sodium Lipoate at pH 8.5 Over 6 and 60 Minutes

[0191] The technique is based on the decrease in light scattering produced by red blood cells as they are lysed. As hemolysis progresses, light scattering diminishes, leading to a decrease in absorbance.

[0192] The Kinetic monitoring of Hemolysis was conducted in to different time record, 6 minutes for de lower Lipoate / AmB ratio ( 0.1 to 1.4 mg / ml) and 60 minutes for Lipoate / AmB (5.5 to 22 mg / ml).

[0193] Stabilized sheep red blood cells (RBCs) are used, diluted to 250 pL in 50 mL of phosphate- buffered saline (PBS). A 5 mg / mL solution of Amphotericin is prepared by dissolving the contents of one vial of Amphotericin Deoxycholate in 10 mL of PBS, this was the blank solution ("a" in Fig 13 and 14) that result in a completely hemolyzed sample without lipoic acid.

[0194] A reference solution of 5mg / ml of Amphotericin B in DMSO (AmB ref) is prepared by dissolving 50mg of Amphotericin B in 10ml of DMSO. Lipoate is prepared as a 0.5 M aqueous solution of Lipoic acid, adjusted to pH 8.5 with sodium hydroxide.

[0195] Solutions: PBS, lipoate (from 0 mg / ml to 4,4 mg / ml), RBCs, and reference solution of Amphotericin B were added directly in spectrophotometer cuvettes and mixed, with readings taken at 580 nm. The reading was started immediately upon placing the cuvette in the spectrophotometer.

[0196] The hemolysis curves in Figure 14, display sigmoidal shapes, clearly show a delay in the onset of the hemolytic process as the concentration of lipoate increases, as well as a reduction in the rate of hemolysis.

[0197] In Figure 14:

[0198] .a: AmB Deoxycholate 0.2 mg / ml; Inhibition of hemolysis at 360 seg.: 0%

[0199] .b: AmB ref 0.2 mg / ml; Lipoate / AmB=0.1; Inhibition of hemolysis (360seg.): 3.3 %

[0200] .c: AmB ref 0.2 mg / ml; Lipoate / AmB=0.15; Inhibition of hemolysis (360seg.): 15%

[0201] .d: AmB ref 0.2 mg / ml; Lipoate / AmB=0.35; Inhibition of hemolysis (360seg.): 40%

[0202] .e: AmB ref 0.2 mg / ml; Lipoate / AmB=0.7; Inhibition of hemolysis (360seg.): 59%

[0203] .f: AmB ref 0.2 mg / ml; Lipoate / AmB=1.4; Inhibition of hemolysis (360seg.):79%

[0204] While in Figure 15:

[0205] .a: AmB Deoxycholate 0.2 mg / ml

[0206] .g: AmB ref 0.2 mg / ml; Lipoate / AmB=5.5

[0207] .h: AmB ref 0.2 mg / ml; Lipoate / AmB=ll

[0208] .i: AmB ref 0.2 mg / ml; Lipoate / AmB=16.5

[0209] .j: AmB ref 0.2 mg / ml; Lipoate / AmB=22 A reference solution of 5 mg / ml Amphotericin B in DMSO was tested under identical conditions to the amphotericin deoxycholate blank solution and the curves were identical.

[0210] The hemolysis curves in Figure 15, display sigmoidal shapes too, clearly show a delay in the onset of the hemolytic process as the concentration of lipoate increases, as well as a reduction in the rate of hemolysis. In this figure, it can be observed, surprisingly, that with sufficient lipoate, hemolysis can be delayed by one-hour, sufficient time to change the pharmacokinetics of the AmB. This fact demonstrates a technical effect that has never been disclosed or even hinted at before in the state of the art. These results show that Lipoate inhibits hemolysis produced by AmB, starting from a

[0211] Lipoate / AmB ratio of 0.1!

[0212] The assay is conducted at a constant Amphotericin B concentration of 0.2 mg / mL, corresponding to maximum recommended infusion dilution for AmB deoxicolate.

Claims

CLAIMS1. A pharmaceutical composition comprising Amphotericin B and Lipoate.

2. The pharmaceutical composition of claim 1 wherein the lipoate:AmB mass ratio is at least 0.1:1.

3. The pharmaceutical composition of claim 1 wherein the lipoate:AmB mass ratio is at least 0.15:1.

4. The pharmaceutical composition of claim 1 wherein the lipoate:AmB mass ratio is at least 0.35:1.

5. The pharmaceutical composition of claim 1 wherein the lipoate:AmB mass ratio is at least 0.7:

16. The pharmaceutical composition of claim 1 wherein the lipoate:AmB mass ratio is at least 1.4:

17. The pharmaceutical composition of claim 1 wherein the lipoate:AmB mass ratio is at least 16.5:

18. The pharmaceutical composition of claim 1 wherein the lipoate:AmB mass ratio is from0.1:1 to 22:1.

9. The pharmaceutical composition of claim 1 wherein said lipoate is in its acid form or as a salt comprising as a counterion the monovalent cation selected from the group comprising sodium, potassium, lithium, and a combination thereof.

10. The pharmaceutical composition of claim 1 wherein said pharmaceutical composition comprises a state selected from the group comprising liquid as aqueous medium, powder, and lyophilized state.

11. The pharmaceutical composition of claim 1 wherein said Lipoate comprises a concentration from 0.02 mg / ml to 176 mg / mL in an aqueous medium.

12. The pharmaceutical composition of claim 1 wherein said lipoate comprises a concentration from 0.2 mg / ml to 16 mg / mL in an aqueous medium.

13. The pharmaceutical composition of claim 1 wherein said lipoate comprises a concentration from 1.0 mg / ml to 13.6 mg / mL in an aqueous medium.

14. The pharmaceutical composition of claim 1 wherein said Amphotericin B comprises a concentration from 0.1 to 8 mg-mL-1, in an aqueous medium.

15. The pharmaceutical composition of claim 1 further comprising cholesterol and a compound selected from oleate, laureate, myristate, palmitate and stearate.

16. The pharmaceutical composition of claim 1 further comprising cholesterol and oleate.

17. The pharmaceutical composition of claim 16 wherein said oleate comprises a concentration from 0.2 to 2.7 mg-mL-1.

18. The pharmaceutical composition of claim 16 wherein the cholesterokAmB molar ratio is about from 1:1 to 2:1.

19. An injectable pharmaceutical formulation comprising the pharmaceutical composition of claim 16 and glucose in a concentration from 1 % w / v to 10 % w / v, in an aqueous medium.

20. The injectable pharmaceutical formulation of claim 19 wherein said glucose comprises a concentration of about 5 % w / v.

21. An injectable pharmaceutical formulation comprising the pharmaceutical composition of claim 1 and a second composition comprising cholesterol, oleate and glucose.

22. A kit for the preparation of an injectable pharmaceutical formulation of claim 19 comprising the pharmaceutical composition of claim 1 and a second pharmaceutical composition comprising cholesterol, oleate and glucose, wherein both compositions are mixed prior to injection.

23. A kit for the preparation of an injectable pharmaceutical formulation of claim 19 comprising the pharmaceutical composition of claim 1 with glucose and a second pharmaceutical composition comprising cholesterol and oleate, wherein both compositions are mixed prior to injection.

24. The injectable pharmaceutical formulation of claim 19, wherein said amphotericin B is in a concentration from 0.1 to 8 mg-mL-1.

25. The injectable pharmaceutical formulation of claim 19, wherein said oleate is in a concentration from 0.2 to 2.7 mg-mL-1.

26. The injectable pharmaceutical formulation of claim 19 wherein the cholesterokAmB molar ratio is lower than 2:1.

27. The injectable pharmaceutical formulation of claim 19 wherein said glucose is in a concentration from 1 % w / v to 10 % w / v.

28. The pharmaceutical formulation of claim 19 wherein it is useful in the treatment of systemic mycosis or fungal infections.

29. The pharmaceutical formulation of claim 19, wherein is formulated for intravenously administrable form.

30. The pharmaceutical formulation of claim 19 wherein its pH in an aqueous medium is from 7 to 8.5.

31. The pharmaceutical formulation of claim 19 wherein is non-liposomal.

32. A process to obtain the composition of claim 1 comprising the following steps: a. adding amphotericin B powder to dimethyl sulfoxide (DMSO) solution at 30 mg / ml and stirring until complete dissolution; b. adding CChHNa or NaOH solution to Lipoic acid powder and stirring until complete dissolution in order to obtain a Lipoate solution; c. adding the solution of amphotericin B in DMSO of step "a" to the Lipoate solution of step "b"; d. stirring the mixture prepared in step "c" for at least 10 minutes in the dark;e. sterilizing the mixture of step "d33. The process of claim 32 further comprises the following step: dialyzing the mixture of step "d" to remove all DMSO.

34. The process of claim 32 further comprises the following step: lyophilizing the sterilized mixture of step e.

35. A process to obtain the injectable pharmaceutical formulation of claim 19 comprising the following steps: a. adding amphotericin B powderto dimethyl sulfoxide (DMSO) solution and stirring until complete dissolution; b. adding CChHNa or NaOH aqueous solution to Lipoic acid powder and stirring until complete dissolution in order to obtain a Lipoate solution; c. adding the solution of amphotericin B in DMSO of step "a" to the Lipoate solution of step "b"; d. stirring the mixture prepared in step "c" for at least 10 minutes in the dark to obtain Lipoate-AmB composition in aquous solution; e. sterilizing the mixture of step "d"; f. preparing Cholesterol in ethanol solution; g. adding to the solution of step "f" an oleate aqueous solution; h. stirring the mixture of step "g" for at least 5 minutes; i. adding glucose; j. sterilizing the solution obtained in step "i", obtaining Chol-Oleate-Glucose solution; k. mixing lyophilized mixture of step "e" with solution of step "j".

36. The process of claim 35, wherein before said step e, the mixture of step "d" is dialyzing to remove all DMSO;37. The process of claim 35, wherein after said step e said mixture of step "e" is liophilizing to obtain lyophilized Lipoate:AmB composition.

38. The process of claim 35, wherein said step "h" further comprises heating the mixture.

39. The process of claim 35, wherein before or after step "i" further comprises a removal of ethanol by extraction, performed by dialyzing the mixture with bi-distil led water.

40. A pharmaceutical kit comprising the lyophilized composition of claim 1 and a reconstitution solvent comprising Cholesterol, Oleate and Glucose in an aqueous medium.

41. The pharmaceutical kit of claim 39 wherein it is useful for the treatment of severe systemic mycoses; fungal infections; invasive mucormycosis infections: cryptococcal meningitis: or aspergillus and Candida infections.

42. Use of pharmaceutical composition of claim 1 in the manufacture of a medicament for the therapeutic of severe systemic mycoses, fungal infections, invasive mucormycosis infections, cryptococcal meningitis, or aspergillus and Candida infections.

43. The pharmaceutical composition of claim 1 wherein said composition comprises nanoparticles.

44. The pharmaceutical composition of claim 1 wherein said composition comprises nanoparticles size less than 200 nm.

45. The pharmaceutical composition of claim 1 wherein said composition comprises nanoparticles size less than 100 nm.

46. The injectable pharmaceutical formulation of claim 19, wherein it comprises nanoparticles size less than 200 nm.

47. The injectable pharmaceutical formulation of claim 19, wherein said formulation is in absence of liposomes, phospholipid and non-ionic surfactant.

48. The injectable pharmaceutical formulation of claim 1, wherein said formulation is in absence of polymerized lipoate.

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