Gallium-apramycin complexes, methods of preparation thereof and their use in the treatment of bacterial infections

Abstract

The present invention relates to novel complexes of gallium and apramycin. The complexes according to the invention are useful in the treatment of bacterial infections. The complexes are particularly suitable for administration by inhalation when they are provided in the form of spray-dried particles.

Description

DescriptionTitle: Gallium-apramycin complexes, methods of preparation thereof and their use in the treatment of bacterial infectionsTechnical field

[0001] The present invention relates to gallium-apramycin complexes, methods of preparation thereof, and their use in the treatment of bacterial infections.Background

[0002] In the decades following the discovery of penicillin in 1929 numerous antibiotics for treating bacterial infections have been developed. However, the development of new antibiotics has considerably slowed down in the last decades, mainly due to the pharmaceutical industries shifting attention towards other, more profitable fields, such as chronic diseases. This decline is particularly problematic as at the same time the emergence of new bacterial strains and an increased resistance of bacteria to conventional antibiotics has been observed.

[0003] In addition, when an efficient antibiotic is available for a certain bacterial strain, it cannot always effectively be delivered to the site of infection and may suffer from poor bioavailability. This is for example the case with pulmonary infections, whereby difficulties arise to deliver the antibiotic to all areas of the lungs.

[0004] As an alternative to classic modes of administration, such as oral or intravenous administration, pulmonary drug delivery is a promising strategy to treat lung infectious diseases as it can allow for high local drug concentrations and low systemic side effects.

[0005] Overall, an urgent need for new efficient antibacterial agents increasingly exists, in particular agents that show adequate antibacterial effects and can efficiently be delivered to the patient.Summary

[0006] The present disclosure provides a novel compound which shows excellent properties as an antibacterial agent.

[0007] A first object of the present invention is a complex of gallium and apramycin.

[0008] In another aspect of the invention, there are proposed methods for producing a complex according to the invention.

[0009] Thus, a second object of the present invention relates to a method for preparing a complex of gallium and apramycin, wherein the method comprises a step a-1 ) of solubilizing a gallium salt and apramycin, or a salt thereof, to obtain an aqueous solution, and a step b-1 ) of spray-drying said aqueous solution, to obtain the complex of gallium and apramycin in the form of spray-dried particles.

[0010] Thus, a third object of the present invention relates to a method for preparing a complex of gallium and apramycin, wherein the method comprises a step a-2) of providing a solution comprisinga gallium salt and apramycin, or a salt thereof, in a solvent, followed by a step b-2) of stirring of the solution of step a-2) to obtain a suspension comprising the complex in the form of a precipitate.

[0011] A fourth object of the present invention is a complex of gallium and apramycin, obtainable by the methods as described herein.

[0012] A fifth object of the present invention relates to a complex of gallium and apramycin for use in a method of treatment of the human or animal body.

[0013] A sixth object of the present invention relates to a complex of gallium and apramycin for use in the treatment of a bacterial infection.

[0014] The inventors have identified a new compound useful in the treatment of bacterial infections. It has been found that a complex of gallium and apramycin, surprisingly shows advantageous antibacterial properties when compared to either gallium alone, apramycin alone, or a simple admixture of gallium and apramycin. The observed enhanced antimicrobial properties are particularly surprising, in view of the observation that several other aminoglycosides (such a tobramycin or amikacin), structurally related to apramycin, do not show enhanced efficiency when provided in the form of a gallium complex.Brief description of the FiguresFigure 1

[0015] Figure 1 shows standard electron microscope (SEM) images of the powder isolated by precipitation according to example 1 .Figure 2

[0016] Figure 2 shows standard electron microscope (SEM) images of six spray-dried particles prepared using six different formulations, according to example 2.Figure 3

[0017] Figure 3 represents a graph showing the aerodynamic properties of six spray-dried particles prepared using six different formulations, according to example 2. The x-axis represents the effective cut-off diameter (ECD), expressed in pm. The y-axis represents the cumulative fraction of particles, expressed in %.Figure 4

[0018] Figure 4 represents a graph showing the minimum inhibition concentration (MIC) of apramycin, gallium nitrate and a gallium-apramycin complex prepared according to example 2, against 24 clinical isolates of Acinetobacter baumannii, as measured according to example 4. Figure 4A shows number of strains (y-axis) for which a certain MIC, expressed in equivalent apramycin mass concentration (x-axis), was measured both for apramycin alone and the gallium-apramycin complexes prepared according to example 2. Figure 4B shows number of strains (y-axis) for whicha certain MIC, expressed in equivalent gallium mass concentration (x-axis), was measured both for gallium nitrate alone and the gallium-apramycin complexes prepared according to example 2.Figure 5

[0019] Figure 5 represents a graph showing the antibacterial activity of spray dried particles according to the invention against 3 clinical isolates of Acinetobacter baumannii. Figure 5A shows number of strains (y-axis) for which a certain MIC, expressed in equivalent apramycin mass concentration (x-axis) was measured for apramycin alone, a gallium-apramycin complex before spray-drying and a gallium-apramycin complex after spray-drying. Figure 5B represents a graph showing the optical density change at 600 nm, expressed in % (y-axis), as a function of the apramycin concentration expressed in pg / ml of equivalent apramycin concentration (x-axis), for apramycin alone, a gallium-apramycin complex before spray-drying and a gallium-apramycin complex after spray-drying.Figure 6

[0020] Figure 6 represents a graph showing the antibacterial activity of spray dried particles comprising a gallium-apramycin complex against 3 clinical isolates of Acinetobacter baumannii. The Figure shows number of strains (y-axis) for which a certain MIC (x-axis) was measured for apramycin alone, a gallium-apramycin complex before spray-drying and a gallium-apramycin complex after spray-drying.Figure 7

[0021] Figure 7 represents time-kill curves showing the bactericidal effect of apramycin, and a gallium-apramycin complex prepared according to example 2, against 3 clinical isolates of Acinetobacter baumannii having various susceptibility to apramycin. The Figures show the bacterial concentration, expressed in Log10 of the CFU / mL (y-axis) as a function of time (x-axis) expressed in hours. Concentrations of apramycin used (mg / L), alone or complexed with gallium, are indicated in the legends.Figure 8

[0022] Figure 8 represents comparative data for the antibacterial activity against Acinetobacter baumannii of aminoglycosides other than apramycin. Figure 8A represents a graph showing the number of strains (y-axis) for which a certain MIC (x-axis) was measured for tobramycin alone, and for a 1 / 1 gallium-tobramycin complex. Figure 8B represents a graph showing the number of strains (y-axis) for which a certain MIC (x-axis) was measured for amikacin alone, and a 1 / 1 gallium-amikacin complex.Figure 9Figure 9 represents NMR spectra showing the formation of a complex according to the present invention. The top-spectrum represents the spectrum of a solution comprising a mere admixture of a gallium salt and apramycin. The bottom-spectrum represents the spectrum of a solution comprising the complex.Figure 10

[0023] Figure 10 represents an experimental set up and flow chart for the determination of the apparent permeability of apramycin through Calu-3 and Cacao-2 monolayers according to example 4.Figure 11

[0024] Figure 11 represents graphs showing the results of the apparent permeability determination according to examples 4A and 4B. Figure 11A represents the P_app determination of apramycin with respect to Calu-3 cells, and Figure 11B represents the P_app determination of gallium(lll) with respect to Calu-3 cells. Figure 11C represents a comparison of the P_app determination of Apramycin alone and the gallium-apramycin complex. Figure 11 D represents a comparison of the P_app determination of Gallium alone and the gallium-apramycin complex.Figure 12

[0025] Figure 12 represents graphs showing the results of the TEER measurements according to example 4B. Figure 12A represents Apramycin alone, Figure 12B represents Gallium alone, and Figure 12C represents the gallium-apramycin complex.Figure 13

[0026] Figure 13 represents the PD evaluation according to example 5C, comparing the gallium- apramycin complex of the invention with apramycin alone, non-complexed.Description of the embodiments

[0027] A first object of the present invention is a complex of gallium and apramycin.

[0028] Apramycin, also known under the name “Nebramycin II” is a compound belonging to the class of aminoglycosides.Chemical structure of apramycin

[0029] Apramycin is commercially available as a sulfate salt and may be converted to the free base for the purpose of the present invention.

[0030] The expression “complex of gallium and apramycin" refers to a coordination compound of gallium cation and apramycin. Without wishing to be bound to theory, the amine and / or hydroxyl functional groups in the molecular structure of apramycin may form a coordination bond with gallium cation species. The ability of apramycin to form a complex with cations had previously been shownfor the case of a copper complex, in which a complex was formed comprising a Cu2+cation and two apramycin molecules (Balenci et al. Dalton Trans., 2009, 1 123-1 130).

[0031] The complex according to the invention may be provided in various forms. For instance, the complex may be in powder form, i.e. in the form of particles. As an example, when the complex is prepared using spray-drying as described below.

[0032] In an embodiment, the complex of gallium and apramycin according to the invention may be in the form of a particles or may be comprised in particles, in particular spray-dried particles.

[0033] Within the context of the invention, a particle denotes an individual nanoscopic unit, i.e. a nanoparticle or an individual microscopic unit such as a microparticle, a microsphere, or a microcapsule of the complex of gallium and apramycin with or without excipients. The particles according to the invention may be of various shapes, such as of spherical shape or oval shape, and in various sizes.

[0034] In an embodiment, the particles as defined above have a mean average size from 300 nm to 400 nm, in particular of from 340 nm to 380 nm.

[0035] In particular, powders which are obtained by precipitation as described hereafter may have a mean average particle size within this range.

[0036] In the context if the invention, the particle size refers to the largest dimension of the particle. When the particles are in the form of a round sphere, the particle size refers to the diameter of the sphere. When the particles are of elliptical shape, such as an oval shape, the particle size is measured as the largest dimension of this shape, i.e. in the length direction.

[0037] Thus, in particular, the particles according to an embodiment the invention may be substantially round, having a diameter of from 300 nm to 400 nm, in particular of from 340 nm to 380 nm.

[0038] In another embodiment, the particles may have an elongated shape, such as an elliptical or oval shape. In this embodiment, the particles may in particular have a length of from 300 nm to 400 nm, in particular of from 340 nm to 380 nm, and may have a width of in particular from 150 nm to 250 nm, in particular of from 170 nm to 210 nm. In this embodiment, the particle size corresponds to the length of the particles.

[0039] Particle sizes may be measured using Scanning Electron Microscopy (SEM).

[0040] In an embodiment, the particles as defined above have a median mass aerodynamic diameter MMAD comprised from 1 to 5 pm, in particular from 1 to 3 pm.

[0041] In particular, particles having an MMAD comprised in these ranges are spray-dried particles.

[0042] Advantageously, particles having a median mass aerodynamic diameter within the above range may be optimal for inhalation purposes. In particular, particles having a median mass aerodynamic diameter within this range may efficiently reach deep into the lungs, when inhaled, where they can be absorbed.

[0043] The median mass aerodynamic diameter (MMAD) is a statistical value defining a particle sample in terms of aerodynamic diameters. A given median mass aerodynamic diameter denotes that 50% of the particles in the sample have an aerodynamic diameter below said value, and 50% of the particles of the sample have an aerodynamic diameter greater than said value. For instance, a median mass aerodynamic diameter of 5 pm means that 50 % of the total sample mass are present as particles having aerodynamic diameters less than 5 pm, and that 50 % of the total sample mass is present in particles having an aerodynamic diameter larger than 5 pm. Within this context, the aerodynamic diameter is the diameter of a spherical particle with a density of 1 g / cm3 that behaves the same way as the actual particle in the air, in terms of sedimentation and diffusion.

[0044] The MMAD may be measured an NG I (Next Generation Impactor) cascade impactor.

[0045] In an embodiment, the particles as defined above have a fine particle fraction FPF of more than 30%, in particular between 30% and 65%.

[0046] In particular, may be more than 50%, in particular between 50% and 65%.

[0047] Particles having and FPF within the above ranges correspond to a powder which may show reproducible results when administered by inhalation.

[0048] The fine particle fraction FPF is defined as the mass of particles with aerodynamic diameters smaller than 5.0 pm expressed as a percentage of the mass recovered on the NG I emitted from a gelatine capsule loaded in a dry powder inhaler. As an example of a dry particle inhaler that can be used for the FPF determination, a Handihaler® can be cited.

[0049] In an embodiment, the particles as defined above, have a geometric standard deviation GSD of 1 .0 to 5, in particular of 1 .1 to 2.5, or of 1 to 2.

[0050] Particles having and GSD within the above ranges correspond to a powder which may show reproducible results when administered by inhalation.

[0051] In an embodiment, the complex of gallium and apramycin as defined above has a molar ratio gallium I apramycin in the range of 4:1 to 1 :4, in particular in the range of 1 :1 to 1 :4.

[0052] In particular, the molar ratio gallium I apramycin is in the range if 1 :1 to 1 :3, more in particular, about 1 :1 , about 1 :2 or about 1 :3.

[0053] Preferably, the molar ratio gallium I apramycin is about 1 :1 .

[0054] It is to be understood that the above-described molar ratios for the complex of gallium and apramycin also apply to the particles as described above.

[0055] The complex of gallium and apramycin as defined above may be formulated with a pharmaceutically acceptable excipient.

[0056] In particular, the particles comprising the complex of gallium and apramycin as defined above may further comprise pharmaceutically acceptable excipients.

[0057] For the purposes of the present invention, the expression “pharmaceutically acceptable" is understood to mean what is useful in the preparation of a pharmaceutical composition which is of sufficient purity and quality for human or veterinary use.

[0058] "Pharmaceutically acceptable excipients" are understood to mean substances which are nontoxic, biologically tolerable and biologically suitable for administration to a subject, such as an inert substance, added to a pharmacological composition or used as a vehicle, carrier or diluent to facilitate the administration of an agent and which is compatible therewith.

[0059] Examples of suitable pharmaceutically acceptable excipients include anti-adherents.

[0060] Thus, in an embodiment, the complex of gallium and apramycin as defined above may be formulated with an anti-adherent.

[0061] In particular, the particles as described above, comprising the complex of gallium and apramycin according to the invention, may further comprise an anti-adherent as defined herein.

[0062] An anti-adherent refers to a compound which may tend to decrease the cohesion between the particles and between particles and any other particles present in a composition. As such, the anti-adherent may give better flow of the particles, for example in a dry powder inhaler which will lead to a better dose reproducibility.

[0063] Examples of anti-adherent materials which may be used in the present invention include lipophilic amino acids or di or t / 7-peptides derived from lipophilic amino acids, phospholipids, or fatty acids or salts thereof.

[0064] With “lipophilic amino acids” is meant amino acids with non-polar chain. Examples of lipophilic amino acids include Alanine, Valine, Isoleucine, leucine.

[0065] With “di- or tri-peptides derived from lipophilic amino acids" is meant a compound in which two or three lipophilic amino acids as defined above are attached to each other through a peptide bond. In other words, a di or t / 7-peptide derived from lipophilic amino acids is di- or trimer of lipophilic amino acids. Examples of di- or tri-peptides derived from lipophilic amino acids include di-Alanine, tri-Alanine, di-Valine, tri-Valine, di-lsoleucine, tri-lsoleucine, di-leucine, tri-leucine

[0066] Phospholipids useful as anti-adherents include different glycerophospholipids, especially phosphatidylcholines such as for example dipalmitoylphosphatidylcholine, but also phosphatidylglycerol

[0067] Fatty acids useful in the present invention are acids having a linear or branched carbon chain of 6 to 22 carbon atoms, such as for example stearic acid. Suitable salts of fatty acids include magnesium salts or sodium salts. An example of a salt of fatty acids is magnesium stearate.

[0068] In an embodiment, the anti-adherent is chosen from L-Leucine, tri-Leucine and magnesium stearate, said anti-adherent being in particular L-leucine.

[0069] The anti-adherent may be present in an amount of 5% to 15%, in particular of 10% to 15%, by weight with respect to the total dry weight of the formulation comprising the complex of gallium and apramycin.

[0070] In particular, the particles as described above, comprising the complex of gallium and apramycin according to the invention, may comprise the anti-adherent in an amount of 5% to 15%, in particular of 10% to 15%, by weight with respect to the total weight of the particles.

[0071] Below 5%, the amount of anti-adherent may not be sufficient to adequately decrease the cohesion between the materials, in particular between particles. In an amount above 15%, too low amounts of gallium apramycin complex may be present in the powder, i.e. in the particles, requiring a high dose of powder, i.e. in the particles to be administered to the patient to achieve an adequate antibacterial effect.

[0072] In another embodiment, the complex of gallium and apramycin according to the present invention may be protonated.

[0073] When the gallium apramycin complex is protonated, its aqueous solubility may be increased. Protonation of the complex may be achieved by adding an acid, such as an organic acid or a mineral acid to the complex, preferably during preparation of the complex.

[0074] In particular, the organic acid is chosen from citric acid, acetic acid, formic acid, succinic acid, oxalic acid, lactic acid, malic acid, tartaric acid and butyric acid.

[0075] In particular, the mineral acid is chosen from hydrochloric acid, nitric acid, sulfuric acid and phosphoric acid.Preparation of the complex

[0076] The inventors have found that the complex of gallium and apramycin according to the invention, and particles consisting of or comprising said complex of gallium and apramycin can be readily prepared by different techniques.

[0077] For instance, particles consisting of the complex of gallium and apramycin according to the invention or comprising the complex of gallium and apramycin according to the invention may be obtained by spray-drying.

[0078] Thus, a second object of the invention relates to a method for preparing a complex of gallium and apramycin, wherein the method comprises a step a-1 ) of solubilizing a gallium salt and apramycin, or a salt thereof, to obtain an aqueous solution, and a step b-1 ) of spray-drying said aqueous solution, to obtain the complex of gallium and apramycin in the form of spray-dried particles.

[0079] In particular, the apramycin used in the method as described herein is in the form of a free base. For this purpose, commercial apramycin sulfate may be converted into the free base using standard techniques. Alternatively, an apramycin salt may be used, such as commercial apramycin sulfate.

[0080] The gallium salt useful in step a-1 ) of the method may be any gallium salt, such as gallium nitrate, gallium citrate, gallium chloride, gallium sulfate, gallium acetate, gallium formiate, gallium tartrate, gallium succinate, gallium acetate, gallium butyrate or gallium propionate, in particular gallium nitrate.

[0081] These salts may be mixed with apramycin or a salt thereof, in an aqueous solvent, to obtain the aqueous solution to be spray-dried in step b-1 ).

[0082] Alternatively, the gallium salt may be formed in situ by providing a gallium compound which is able to be converted to a gallium salt. Examples of such gallium compounds include gallium metal or gallium oxides. Gallium metal or gallium oxide may be converted to gallium salts by addition of acids. Thus, in an embodiment, the aqueous solution of step a-1 ) is prepared by mixing a gallium compound, which is a precursor of a gallium salt, such as gallium metal or gallium oxide with apramycin in the presence of acid and stirring the resulting mixture.

[0083] In an embodiment, the molar ratio gallium salt I apramycin in the range of 4:1 to 1 :4, in particular in the range of 1 :1 to 1 :4. In particular, the molar ratio gallium salt I apramycin is in the range if 1 :1 to 1 :3. More in particular, the molar ratio is about 1 :1 , about 1 :2 or about 1 :3.

[0084] In an embodiment, the aqueous solution to be spray-dried in step b-1 ) further comprises an acid, in particular an organic acid or a mineral acid.

[0085] In particular, said organic acid is chosen from citric acid, acetic acid, formic acid, succinic acid, oxalic acid, lactic acid, malic acid, tartaric acid and butyric acid, more in particular the organic acid is acetic acid and formic acid.

[0086] In particular, said mineral acid is chosen from hydrochloric acid, nitric acid, sulfuric acid and phosphoric acid.

[0087] In particular, the pH of the aqueous solution is in the range of 2 to 7 to be spray-dried in step b-1 ), in particular of 2 to 4, more in particular of 2.5 to 3.5, even more in particular about 3.5.

[0088] The amount of organic acid comprised in the aqueous solution to be spray-dried may be such as to obtain the above disclosed pH values.

[0089] The inventors have surprisingly found that the presence of organic acid may allow to obtain particles having advantageous aerodynamic properties, such as a smaller median mass aerodynamic diameter MMAD, combined with a higher fine particle fraction FPF. Such particles may be especially useful administration by inhalation.

[0090] In addition, when the aqueous solution is formed from a gallium salt precursor, such as gallium metal or gallium oxide, the addition of acid allows to transform said gallium salt precursor into a gallium salt.

[0091] In another embodiment, the aqueous solution obtained in step a-1 ) further comprises a pharmaceutical excipient, in particular an anti-adherent, which may be added during step a-1 ).

[0092] Examples of anti-adherent materials which may be used in the processes according to the present invention include lipophilic amino acids or di- or tri peptides derived from lipophilic amino acids, phospholipids, or fatty acids or salts thereof.

[0093] In an embodiment, the anti-adherent is chosen from L-Leucine, tri-Leucine or magnesium stearate, said anti-adherent being in particular L-leucine.

[0094] In particular, the anti-adherent is present in the aqueous solution to be spray-dried an amount of 5% to 15% by dry weight of the aqueous solution.

[0095] In an embodiment, apramycin, or a salt thereof, is included in the aqueous solution of step a- 1 at a concentration of 0.5 to 20 mM, in particular about 5 mM.

[0096] Spray drying in step b-1 ) may be performed using standard spray drying equipment, such as a Buchi B-290 mini-dryer.

[0097] For instance, spray-drying may be performed using a Buchi B-290 mini-dryer (Buchi Labortechnik, Flawil, Schweiz) mounted with a nozzle having a 0.5 mm nozzle tip and / or a 1 .5 mm nozzle gap. The air flow rate used for spraying may be in the range of 200-1000 L / hr, in particular about 900 L / hr. the air flow rate for drying may in particular be in the range of 30 000 to 35 000 L / hr, in particular about 35 000 L / hr. The inlet temperature may in particular be comprised from 1 10 to 180 °C, in particular about 120 °C.

[0098] According to an alternative method to spray-drying, the complex of gallium and apramycin may be formed in standard reactors by simply mixing a gallium salt, or a precursor of a gallium salt, with apramycin, or a salt thereof in a solvent.

[0099] It is to be understood that a gallium apramycin complex obtained by a method other than spray-drying may subsequently be spray-dried so as to obtain spray-dried particles.

[0100] It has been found for instance that the solubility of the complex of gallium and apramycin was different from that the starting gallium salt and the apramycin. Formation of the complex from solution of a gallium compound and apramycin may thus lead to precipitation of the complex, allowing for its isolation by filtration.

[0101] Thus, a third object of the present invention relates to a method for preparing a complex as described above, wherein the method comprises a step a-2) of providing a solution comprising a gallium salt and apramycin, or a salt thereof in a solvent, followed by a step b-2) of stirring the solution of step a-2) to obtain a suspension comprising the complex of gallium and apramycin in the form of a precipitate.

[0102] In particular, the precipitate obtained after step b-2) is in the form of a powder, in particular, said powder consisting of, or comprising particles consisting of the complex of gallium and apramycin according to the invention or comprising the complex of gallium and apramycin according to the invention.

[0103] In an embodiment, the solvent in step a-2) is water.

[0104] In another embodiment, the solvent in step a-2) is an organic solvent, in particular chosen from alcohols, in particular methanol or ethanol, or a mixture of said alcohols and water.

[0105] The gallium salt useful in step a-2) of the method as described above may be any gallium salt. Examples of such salts include gallium nitrate, gallium citrate, gallium chloride, gallium sulfate, gallium acetate, gallium formiate, gallium tartrate, gallium succinate, gallium acetate, gallium butyrate and gallium propionate, in particular gallium nitrate.

[0106] Alternatively, the gallium salt may be formed by providing a gallium compound, i.e. a gallium salt precursor, which is able to be converted to a gallium salt. Examples of such gallium compounds include gallium metal and gallium oxides. Gallium metal or gallium oxide may be converted to gallium salts by addition of acids. Thus, in this embodiment, the aqueous solution of step a-2) is prepared by mixing a gallium compound, which is a precursor of a gallium salt, such as gallium metal or gallium oxide with apramycin in the presence of acid and stirring the resulting mixture.

[0107]

[0108] In an embodiment, the molar ratio gallium compound I apramycin in the range of 4:1 to 1 :4, in particular in the range of 1 :1 to 1 :4. In particular, the molar ratio gallium salt I apramycin is in the range if 1 :1 to 1 :3. More in particular, the molar ratio is about 1 :1 , about 1 :2 or about 1 :3.

[0109] In an embodiment, the mixture in step a-2) further comprises an acid, such as an organic acid or a mineral acid.

[0110] In particular, said organic acid is chosen from citric acid, acetic acid, formic acid, succinic acid, oxalic acid, lactic acid, malic acid, tartaric acid and butyric acid, more in particular the organic acid is acetic acid and formic acid.

[0111] In particular, said mineral acid is chosen from hydrochloric acid, nitric acid, sulfuric acid and phosphoric acid.

[0112] In particular, the pH of the mixture is in the range of 2 to 8, in particular about 7.

[0113] In another embodiment, the solution in step a-2) further comprises an anti-adherent.

[0114] In an embodiment, the anti-adherent is chosen from L-Leucine, tri-Leucine or magnesium stearate, said anti-adherent being in particular L-leucine.

[0115] In particular, the anti-adherent is present in the solution in an amount of 5% to 15% by dry weight of the solution.

[0116] In an embodiment, the solution provided in step a-2) comprises apramycin or a salt thereof in a concentration of 0.1 to 500 mM, in particular between 1 to 50 mM, and more particular about 10 mM.

[0117] The mixture provided in step a-2) may be stirred during step b-2) at room temperature, i.e. at a temperature comprised from 15 °C to 25 °C, or may be cooled. In particular the solution may be cooled to a temperature comprised from 0 °C to 5 °C.

[0118] Cooling of the solution may in particular be implemented when the pH of the solution is acidic, in particular when the pH of the solution is comprised from 2 to 4. It has been found that at such acidic pH, the complex has higher solubility in the solvent, in particular in the aqueous solvent. By cooling the aqueous solution, precipitation of the complex may be achieved.

[0119] In an embodiment, the process further comprises a step c-2) of isolation of the complex, for instance by filtration.

[0120] In an embodiment, the process further comprises a step d-2) of drying of the complex, for instance by using a vacuum pump.

[0121] The complex of gallium and apramycin as disclosed herein, and the particles consisting of or comprising said complex, may be characterized by different analytical techniques, other than the particle size determination and the MMAD determination disclosed above.

[0122] For instance, the apramycin content may be analyzed using LC-MS / MS.

[0123] For instance, the gallium content may be analyzed using ICP-MS.

[0124] For instance, the elemental composition may be analyzed using elemental analysis or Energy-dispersive X-ray spectroscopy (EDX).

[0125] A fourth object of the present invention pertains to a complex of gallium and apramycin obtainable by the methods as described above.Medical use

[0126] A fifth object of the present invention relates to a complex of gallium and apramycin as defined above, or a complex of gallium and apramycin obtainable by the methods as described above, for use in a method of treatment of the human or animal body.

[0127] A sixth object of the present invention relates to a complex of gallium and apramycin as defined above, or a complex of gallium and apramycin obtainable by the methods as described above, for use in the treatment of a bacterial infection.

[0128] With " Treatment is meant the action of treating a declared pathology, e.g. a bacterial infection, the symptoms of which are visible.

[0129] In embodiment, the bacterial infection is caused by Acinetobacter baumannii, Klebsiella pneumoniae, Enterobacteriaceae or Pseudomonas aeruginosa, Mycobacterium abscessus, Mycobacterium tuberculosis, Enterococcus faecium, Staphylococcus aureus, Helicobacter pylori, Campylobacter spp., Salmonellae, Neisseria gonorrhoeae, Streptococcus pneumoniae, Haemophilus influenzae or Shigella spp.

[0130] Notably, the bacterial infection is caused by Acinetobacter baumannii.

[0131] In particular, the bacterial infection is a pulmonary infection.

[0132] The complex or particles according to the present invention may be administered by usual means, for instance by oral administration, topical administration or inhalation.

[0133] In particular, the complex of gallium and apramycin as described herein is administered by inhalation, the bacterial infection being in particular a pulmonary infection.

[0134] In an embodiment, for administration by inhalation, the complex is administered in powder form, in solution or as a suspension, such as an aqueous solution or suspension.

[0135] It has been found the gallium apramycin complex in the form of spray dried particles is particularly suitable to be administered by inhalation.

[0136] In an alternative, the suspension obtained in step b-2) of the method according to the third object of the invention may directly be used for administration by inhalation.

[0137] In another alternative, when the method according to the third object of the invention is implemented at acidic pH as described above, such as at pH=3, cooling may be needed to precipitate the complex, as described above. In this case, the solution obtained before cooling may be used for administration by inhalation.

[0138] Inhalation is preferably performed by using a nebulizer. Suitable inhalers include for instance a dry powder inhaler (DPI) with various air resistance, such as the Handihaler®, Boehringer Ingelheim.

[0139] In an embodiment, for administration by inhalation, the powder inhaled is contained in hard gelatin or Hydroxypropyl methylcellulose (HPMC) capsules loaded into a dry powder inhaler (DPI), or into another powder reservoir of a DPI.The following examples serve to illustrate the invention without restricting its scope.ExamplesGeneral experimental conditionsApramycin base (APR) was obtained from apramycin sulfate and Gallium nitrate (Ga(NOs)3) was purchased from Sigma Aldrich. To adjust the pH, acetic acid, formic acid, butyric acid, hydrochloric acid, purchased from Merck, and citric acid purchased, from Carl ROTH was used.■ Nuclear Magnetic Resonance (NMR) spectrometryNMR was used to characterize the interaction of gallium and apramycin within the complex. Apramycin was dissolved in D2O at 10 mM and supplemented with different amount of gallium (gallium nitrate) to obtain different APR / Ga molar ratio (0.2, 0.4, 0.6, 1 ). Then pH of the solutions was adjusted to 3 with hydrochloric acid. NMR measurements were made with a controlled temperature of 20°C using a Bruker Avance 500-MHz spectrometer (Bruker Biospin, Rheinstetten, Germany) at 1 1.74T. For1H NMR spectrum, 64 scans were acquired using a 30° flip angle (zg30 in Bruker language), an acquisition time of 2.62 s. D2O signal was used for the calibration. For13C NMR spectrum, 1024 scans were acquired using a 30° flip angle (zg30 in Bruker language), an acquisitiontime of 1 .09 s Spectra analysis were made using Top Spin version 3.6.5 software. Proton assignments were made using 2D COSY and detection and assignment of carbon using1H13C HSQC.■ Assay ofApramycin by LC-MS / MSThe concentration of APR was determined using liquid chromatography coupled with tandem mass spectrometry (LC-MS / MS). A Nexera -20AD system (Shimadzu, Marne-la-Valle, France) equipped with a Kinetex® XB-C18 100-A column (5pm, 150 mm, 2.1 mm ; Phenomenex, Le Pecq, France) was used for chromatographic separation. The mobile phase consisted of 0.1 % formic acid in water (A) and 0.1 % formic acid in acetonitrile (B) in gradient mode. Detection and quantification were performed using a Triple Quad 3500 mass spectrometer (SCIEX Headquarters, Framingham, Massachusetts, United States). A calibration curve was prepared in the matrix of the sample, with APR base concentration ranging from 0.1 to 250 pg / mL.■ Assay of Gallium by ICP-MSGallium concentration was determined using inductively coupled plasma mass spectrometry (ICP- MS). Analyses were performed on a quadrupole Agilent 7800 ICP-MS (Agilent Technologies, Marcy I'Etoile, France) operating in helium (He) mode. The instrument utilized a Micromist nebulizer with a Scott spray chamber (Agilent Technologies) for sample introduction. Calibration was achieved using a range of gallium standards from 0.1 to 4.1 pg / mL.■ Determination of the particle morphology by Scanning Electron Microscopy (SEM)Spray-dried powders were coated with a 10-nanometer-thick tungsten film. SEM micrographs were then acquired using a Teneo Volume Scope microscope (FEI, Hillsboro, OR, USA) at an accelerating voltage of 5 kV. The micrographs were formed by collecting secondary electrons emitted from the sample. Mean particle size dimensions were measured from pictures of samples taken at randomly selected locations.■ Elemental Composition of the complexesEnergy-dispersive X-ray spectroscopy (EDX) was employed to quantify the mass percentages and spatial distribution of gallium within the uncoated, spray-dried powders. The analysis employed an Oxford X-MAX probe (Oxford Instruments, Abingdon-on-Thames, England) coupled to a Teneo Volume Scope scanning electron microscope (FEI, Hillsboro, OR, USA).Aerodynamic particle size analysisThe aerodynamic size distribution of particles emitted from a dry powder inhaler (DPI) was measured using a Next Generation Impactor (NGI, Copley Scientific Limited, Nottingham, UK) following the 2009 pharmacopoeial guidelines). Two size 3 hard gelatin capsules, each loaded with 20 ± 3 mg of powder, were used in a high-resistance DPI (Handihaler®, Boehringer Ingelheim). An airflow rate of 50 L / min was applied to achieve a pressure drop of 4 kPa across the DPI, mimicking patient inhalation. The aspiration time was adjusted to simulate a typical inhalation volume of 4 L. Before and after powder release from the DPI, filters on the NGI stages were weighed to determine the mass deposited at each stage. To ensure accuracy, the test was repeated three times. The total amount of particles with aerodynamic diameters smaller than 5.0 pm was calculated by interpolation from the inverse of the standard normal cumulative mass distribution less than stated size cut-off against the natural logarithm of the cut-off diameter of the respective stages. This amount was considered as the fine particle fraction (FPF) and expressed as a percentage of the emitted recovered dose (ED). The mass median aerodynamic diameter (MMAD) of the particles was defined from the same plot as the particle size at which the line crosses the 50% mark.The total emitted dose (ED) was measured using a Gallium assay (described below) after dissolving the emitted powder in a dosage unit sampling apparatus (DUSA, Copley Scientific Limited, Nottingham, UK).Example 1 Synthesis of a Gallium Apramycin complex - precipitation1a) From aqueous solutionOne volume of 5 mM aqueous APR base solution was mixed with the same volume of 5 mM Ga(NOs)3 solution. After one hour's mixing at room temperature, the APR-Ga complex formed had a lower aqueous solubility than free APR and precipitated. The precipitate was recovered by centrifugation, then washed 4 times with pure water and dried.■ SEM analysis - particle sizeThe obtained powders were analysed using SEM microscopy using the general procedure described above. The results are shown in Figure 1. From the SEM image of Figure 1 , it was measured that the particles had an average length comprised from 340 to 380 nm, and an average particle width of 185 to 195 nm.■ EDX analysis - composition of the complexEnergy dispersive X-ray spectroscopy was used to determine the composition of the complex, using the general procedure as described above.It was determined that the particles had the following elemental composition, expressed in weight percent: C 50.8%, O 28.1 %, N 16.5%, Ga 4.5%. It follows that the precipitated complex had an apramycin-gallium molar ratio in the order of 3:1 .NMR spectrometry before precipitation of the particlesSamples were analysed by NMR according to the general procedure.A 1 :1 mixture of Gallium salt and Apramycin was mixed in D2O, at pH 3. Figure 9 shows the NMR spectrum of the admixture of the 2 starting materials (top). Formation of the complex is evidenced by deshielding of the NH-CH and NH-CH3 signals (bottom). lb) From aqueous solution under acidic conditionsThe complex was prepared in the same way as in example 1 a), but acid was added to the solution. Acidification of the solution at pH 3 prevented the precipitation of the complex at room temperature (20 °C), but the precipitation was obtained after cooling the solution at 4 °C for 2 days. The thus formed precipitate was isolated by filtration, as in example 1 a). lc) From organic solutionAn excess of apramycin base was reacted with Ga(NOs)3 hydrate in methanol, increasing the solubility of apramycin. The mixture was stirred for one day at room temperature (RT) and then centrifuged. After filtration through a 0.45 micrometer filter, the filtrate volume was reduced in vacuo to about 10 mL. The solution was then stored at 4 °C for 24 hours, leading to precipitation. The solid precipitate was collected by filtration and washed with small portions of ethanol.Example 2 Synthesis a Gallium Apramycin complex - spray-dryingAqueous solutions containing a mixture of apramycin (dissolution of the base form) and gallium (dissolution of the gallium nitrate salt), each at an equivalent molar concentration (5mM) to give a molar ratio of 1 , were spray-dried using a Buchi B-290 mini-dryer (Buchi Labortechnik, Flawil, Schweiz) to obtain particles suitable for pulmonary inhalation. Different pH values were tested (2.5, 3 or 3.5), controlled with different acids (acetic acid, formic acid, butyric acid and citric acid). L- Leucine (Sigma-Aldrich) and tri-Leucine (Sigma-Aldrich) at 5 wt.% or 15 wt.% of the total solid contents present in the solutions were tested as particle dispersants. These molecules concentrate on the surface of particles during spray drying, reducing their surface energy and facilitating their dispersion during the inhalation process. The spray dryer was operated in open suction mode with the following parameters: a standard two-fluid nozzle was used, coupled with a 0.5 mm nozzle tip and a 1 .5 mm diameter nozzle cap. The solution feed pump had a flow rate of 8 ml / min, the air flow rate used for spraying was 15 l / min and the air flow rate for drying was 630 l / h. Inlet temperatures were set at 120°C, giving outlet temperatures of 48°C + / - 3°C.Example 2a Synthesis of Gallium / Apramycin complexesUsing the above procedure, particles were prepared according to the compositions as shown in Table 1. Thus, particles have been prepared without addition of acids (entries 1 -4), or with theaddition of citric acid (entries 5-6). In addition, complexes formulated with L-Leucine were prepared (entries 2, 4, 6), as well as complexes without L-Leucine (entries 1 , 3, 5).Table 1 analysis of the spray dried Apramycin-Gallium complexes■ Shape of the spray-dried particlesThe obtained spray-dried powders were analysed using SEM microscopy using the general procedure described above. The results are shown in Figure 2. According to Figure 2, the spray- dried particles are spherical in shape, irrespective of the composition.■ Composition of the spray-dried particlesThe apramycin content of the different complexes were analysed using LC-MS / MS, and the gallium content was analysed using IPC-MS and EDX, as described above.The results are shown in Table 1 above. It follows that the Apramycin-Gallium complexes had a molar ratio of 2:1 to 4:1 .■ Aerodynamic properties of the particlesThe aerodynamic properties of the different particles were analyzed by determining the mass median aerodynamic diameter (MMAD) of the particles, using the general procedure above.The results are shown in Figure 3 and in Table 2.Table 2: aerodynamic properties of spray-dried particlesIt was found that the presence of L-Leu makes the mean aerodynamic diameter (MMAD) of the particles prepared by spray-drying less than 5 (pm), and in most cases comprised from 1 to 3 pm, making them ideal candidates for administration by inhalation and predicting deposition in the alveolar space of the lung.In the absence of L-Leu, the MMAD of the particles is above 5 pm, making them less adapted for administration by inhalation.Example 2b Synthesis a Gallium Apramycin complex various pHUsing the above procedure of example 2a, particles were prepared by adding acids into the formulation to be spray dried (Table 3). The addition of acid caused the solution to be spray-dried to have different pH values, ranging from 2.5 to 3.5.Table 3: spray-drying at low pH, by addition of acids.All particles thus prepared showed MMAD values compatible with administration by inhalation.Example 3 antimicrobial activityThe antimicrobial properties of the Apramycin complexes prepared according to the above procedures were determined.Example 3a) minimum inhibition concentration.A complex was prepared using an equimolar aqueous solution of gallium nitrate and apramycin, at pH 7. The minimum inhibition concentration (MIC) of the thus formed complex was determined against 24 clinical isolates of Acinetobacter baumannii. Acinetobacter baumannii clinical isolates were grown overnight on Muller-Hinton Agar plates. A single colony was then transferred to ID-MHB to achieve a density of 1 x 106CFU / mL. APR-Ga complex was prepared by mixing equal volumes of 10 mM Ga(NOs)3 and 10 mM APR solutions. The pH was adjusted with acetic acid if required. Serial dilutions of APR, Ga(NOs)3, and APR-Ga complex were prepared in a 96-well plate to create a range of concentrations in ID-MHB. An equal volume ofthe bacterial suspension was then added to each well to get 5 x 105CFU / mL. The 96-well plates were incubated at 35°C ± 1 °C for 16-20 hours. MICs were determined by measuring the absorbance at 600 nm using an Infinite 200 Pro automatic plate reader (Tecan Trading AG, Mannedorf, Switzerland).In addition, the minimum inhibition concentration (MIC) of apramycin alone and gallium nitrate alone was also determined. The results are shown in Figures 4A and 4B, and where as follows:■ complex - versus apramycin aloneThe complex according to the invention showed apramycin equivalent MIC values ranging from 0.270 to 0.017 pg / ml.In contrast, non-complexed apramycin, i.e. in the absence of gallium, showed MIC values ranging from 64 to 2 pg / ml. It was thus observed that the complex according to the invention showed a minimum inhibition concentration which was substantially lower than the minimum inhibition concentration of apramycin alone.■ complex - versus gallium aloneThe complex according to the invention showed gallium equivalent MIC values ranging from 0,035 to 0,0022 pg / mL.In contrast, non-complexes Gallium showed MIC values from 87 to 22 pg / mLThese results show that the apramycin / gallium complex according to the invention shows much lower minimum inhibition values than either Gallium or Apramycin alone.Example 3b) complex prepared by spray-drying.Spray dried particles of a 1 / 1 complex of gallium and apramycin were prepared according to the procedure of example 2.The minimum inhibition concentration was determined against 3 clinical isolates of Acinetobacter baumannii, according to the procedure described in example 3a. MIC values have thus been determined for apramycin alone, a gallium-apramycin complex before spray-drying and a gallium- apramycin complex after spray-drying and the results are shown in Figures 5 and 6.It can be seen that the complex according to the invention, both before and after spray-drying, showed lower MIC values as compared to apramycin alone.The spray-dried particles showed slightly higher MIC values as compared to the complex before spray-drying, but these spray-dried particles are particularly adapted to administration by inhalation.Example 3c time kill experiments.A a 1 / 1 complex of gallium and apramycin were prepared according to the procedure of example 1 .Microbial experiments were conducted in iron-depleted Mueller-Hinton broth (ID-MHB). To achieve iron depletion, 100 g of Chelex® 100 resin (Bio-Rad, Hercules, CA, USA) was added to 1 liter of MHB (Sigma-Aldrich, St-Quentin-Fallavier, France). The suspension was stirred at 4°C for 1 hour, followed by filtration through a 3 pm filter to remove the resin. The pH was then adjusted to 7.3 using2 M hydrochloric acid. Essential cations were selectively replenished: calcium to 22.5 ± 2.5 pg / mL (CaCl2 from VWR, Radnor, PA, USA), magnesium to 1 1 .25 ± 1 .25 pg / mL (MgCl2 from Carl Roth, Karlsruhe, Germany), and zinc to 0.56 ± 0.06 pg / mL (ZnSCk from Sigma-Aldrich, St-Quentin- Fallavier, France). The medium was then autoclaved and final cation concentrations verified by ICP- MS (see analytical section).Time-dependant killing assays were performed according to the procedure below:Exponentially growing bacteria were diluted to 2.0 x 106CFU / mL in ID-MHB. A 1 :1 v / v mixture of bacterial suspension and ID-MHB containing varying concentrations of APR or APR-Ga complex (0.5x, 1 x, 2x, 4x, 8x MIC) was added to wells of a 24-well plate (final volume: 2 mL). Pure ID-MHB served as a growth control. Plates were incubated at 37°C with shaking.Bacterial viability was assessed at time points of 0, 4, 6, 24, and 30 hours by measuring the CFU / mL. Serial dilutions were plated on MHA plates, and CFUs were counted after a 24-hour incubation at 37°C. The limit of quantification was 2.96 Iog10 CFU / mL.The results of these experiments are shown in Figure 7.The data show that the 1 / 1 (mol / mol) apramycin / gallium complex achieves rapid killing of various Acinetobacter baumannii clinical isolates within 5 hours, reducing the bacterial concentration below the limit of quantification. This occurs at APR-equivalent concentrations that are ineffective when APR is used alone.Example 4A: In-vitro transport studiesGallium and apramycin in solution were used in in vitro transport studies across a monolayer of Calu-3 cells was determined according to the following procedure (see also Figure 10 for the experimental setup of these experiments).The transport of free and gallium-complexed APR across epithelial barrier models was investigated. Caco-2 and Calu-3 epithelial cell lines were used to create these models of barriers. The culture medium consisted of Eagle's Minimum Essential Medium (EMEM, Sigma-Aldrich, St-Quentin- Fallavier, France), supplemented with L-glutamine (1 % v / v), foetal calf serum (10% v / v), MEM Non- essential Amino Acid (1 % v / v). Then, cells were incubated at 37°C under 90-95% of RH and 5% of v / v of CO2 in the air.To assess the apparent permeability (P_app) of free and gallium-complexed APR across intestinal and lung epithelial barriers, transport experiments were conducted using Caco-2 and Calu-3 cell lines, respectively. Cells were seeded at a density of 1 .5 x 105cells / cm2on Transwell® inserts(Corning, Glendale, AZ, USA) in 12-well plates. Caco-2 cultures were maintained under liquid-liquid conditions (LLC) for 21 days. Calu-3 cultures were initially grown under LLC until their transepithelial electrical resistance (TEER) exceeded 600 Q cm2. Subsequently, the cultures were switched to airliquid interface (ALI) conditions until the TEER reached a value above 300 Q cm2.Free and gallium-complexed apramycin solutions in culture medium, with concentration ranging from 200 pM to 500 pM, were added either in the apical or basal side of the cell barriers. The incubation time was set to 6 hours. Apramycin concentrations in acceptor compartment were determined by LC / MSMS (Triple Quad 3500 mass spectrometer (SCIEX Headquarters, Framingham, Massachusetts, United States). Apramycin apparent permeability (P_app) was calculated in apical to basolateral (AB) and apical to basolateral (BA) directions.The concentration of Ga(lll) in the donor medium ranged from 200 to 500 pM, and the incubation time was set at 6 hours. Gallium concentrations in acceptor compartment were determined by inductively coupled plasma mass spectrometry (ICP-MS - Agilent Technologies, Marcy I'Etoile, France). Ga(lll) apparent permeability (P_app) were calculated in apical to basolateral (AB) and apical to basolateral (BA) directions.To assess the integrity of the cell monolayer after the experiment, fluorescein P_app was measured. P_app values were calculated using the following equation:Where Q is the amount of drug that appeared into the acceptor compartment, T is the incubation time (6 h), A is the semi-permeable membrane surface area (0.33 cm2) and Co is the initial drug concentration in the donor compartment.The results of these experiments are shown in Figures 11 A and 11 B.Figure 11 A shows apramycin apparent permeability (cm / s) measured in apical-to-basolateral (AB) and basolateral-to-apical (BA) directions across a Calu-3 monolayer cultured in air-liquid condition (ALI). The pH of the solution was adjusted to 7 (n= 3-6)Figure 11 B shows gallium (Ga(lll) apparent permeability (cm / s) measured in apical-to-basolateral (AB) and basolateral-to-apical (BA) directions across a Calu-3 monolayer cultured in air-liquid condition (ALI). The pH of the solution was adjusted to 7. (n= 6).It can be seen from these data that the apparent permeability of both compounds is low, of the same order as antibiotics (tobramycin, amikacin) whose pulmonary elimination is slow after inhalation due to their low apparent permeability, which predicts a prolonged retention time in the lungs after inhalation and low pulmonary penetration after intravenous administration, justifying the development of an inhaled formulation of these compounds.Example 4B: In-vitro transport studiesCalu-3 cells were cultured in EMEM supplemented with 10% FBS, 1 % NEA solution, 1 % L-glutamine and 1 % penicillin-streptomycin solution at 37°C in a humidified air incubator with 5% CO2. For transport studies, cells were seeded at a density of 4x105 cells / cm2 on Transwell® inserts (Corning® 24 mm Transwell® with 0.4 pm pore polyester membrane) under liquid-liquid interface for one week. After reaching of confluence, cells were maintained in Air-liquid interface (ALI) conditions for 2 weeks until a tight monolayer formed, confirmed by transepithelial electrical resistance (TEER) measurements (> 300 Q-cm2). Medium was renewed every 2 - 3 days. 350 pL of 500 pM Apramycin solution was applied to the apical chamber and samples (900 pL) were collected from the basolateral chamber after 6 h of incubation. TEER measurements and fluorescein transport were performed at the end of the experiment to verify the barrier integrity. Quantification of apramycin transport was performed using LC-MS / MS.The results are shown in Figures 11C and 11 D, and in Figure 12.It can be seen from these data that the complexation between gallium and apramycin did not alter the apparent permeability (P_app) of apramycin in the apical-to-basolateral direction, while it slightly but significantly reduced the gallium P_app by approximately 2-fold. These apparent permeability values, in the range of 1 X 1 0“7cm / s, are very low and suggest a limited absorption rate of the complex across the lung-blood barrier following inhalation. The TEER values measured across the Calu-3 cell monolayers before and after the 6-hour transport study remained unchanged, indicating the absence of cytotoxic effects at this high concentration (500 pM, equivalent to approximately 270 mg / L of apramycin)Example 5 - Studies in lung-infected RatsExample 5A - Neutropenic Rat Pulmonary Infection ModelAll animal experiments complied with the European Directive 2010 / 63 / EU and were approved by the local ethics committee (COMETHEA), registered under French Ministry of Higher Education and Research (approval no. 2024081713224359). Seven-week-old male and female Sprague-Dawley rats (250-300 g) were obtained from Janvier Labs (Saint Berthevin, France). Animals were housed in ventilated racks under controlled temperature and a 12-hour I ig ht / dark cycle, with ad libitum access to food and water, and acclimated for at least five days before experimentation. Thirty minutes prior to infection and throughout the duration of the experiments, animals received analgesia with buprenorphine to minimize pain and distress. Neutropenia was induced by intraperitoneal injections of cyclophosphamide at 100 mg / kg and 75 mg / kg administered 4 and 1 day prior to infection, respectively. Pneumonia was established by intratracheal instillation of 0.1 mL bacterial suspension at 108CFU / mL, delivering approximately 107CFU per lung. Following infection, rats were randomly assigned to three groups: (i) treatment by nebulization with apramycin solution at a total inhaled dose of 1 mg / kg, (ii) treatment by nebulization with an apramycin-gallium nanocomplex suspension at the same equivalent inhaled apramycin dose (1 mg / kg), and (iii) untreated controls.Example 5B - Apramycin Administration via InhalationTwo hours post infection, rats were exposed to apramycin aerosols using a nose only inhalation system (TSE Systems, Germany) for 20 minutes. Apramycin solution and apramycin-gallium nanocomplex suspension was prepared in water at at the same equivalent apramycin concentration (37.5 mg / mL), to get the same equivalent inhaled apramycin dose of 1 mg / kg. Aerosols were generated by the integrated nebulizer connected to a syringe pump delivering solution at 1 mL / min. Aerosol concentration within the inhalation tower was continuously monitored by spectroscopic measurements to ensure stability before and during each exposure. To minimize stress and ensure acclimation to the exposure protocol, all animals underwent two nebulization sessions of 0.9% NaCI four and two days prior to apramycin administration. A Mercer like 7-stage cascade impactor connected to an air-sampling pump (0.5 mL / min) was positioned in place of the animal restrainer and operated simultaneously to characterize aerosol properties, calculate the apramycin concentration in the air of the inhalation system and estimate the total inhaled dose (TID) and the fraction of the TID deposited in the lung. The TID was normalized to individual body weight to express the dose in mg / kg. Particles collected on each impactor stage were weighed with a high precision microbalance (Mettler Toledo, sensitivity 0.01 pg), dissolved in a known volume of water, and apramycin concentrations were quantified by LC-MS / MS. Aerodynamic parameters, including mass median aerodynamic diameter (MMAD) and geometric standard deviation (GSD), were derived from the impactor data.Example 5C - Sampling for Pharmacokinetics and PharmacodynamicsPK / PD analyses were conducted on the same animals. At defined time points after administration (2 and 20 h), rats (n = 2-12 per time point) were anesthetized with isoflurane and placed in a supine position with cervical hyperextension to perform bronchoalveolar lavage (BAL). A polyethylene catheter was inserted 50 mm into the trachea, and 1 mL of sterile 0.9% NaCI at 37 °C was instilled. The BAL fluid (300-800 pL) was retrieved by gentle aspiration, centrifuged at 500 g for 5 min, and the supernatant stored at -20 °C until analysis. Immediately after BAL, blood was collected by intracardiac puncture into heparinized tubes and plasma isolated by centrifugation at 4,000 rpm for 10 min. Apramycin concentrations in BAL fluid and plasma were quantified using liquid chromatography-tandem mass spectrometry (LC-MS / MS). Drug concentrations in the epithelial lining fluid (ELF) were calculated from BAL values using the urea dilution method, based on the BAL- to-plasma urea concentration ratio, as described previously. Given that ELF protein concentrations are approximately 10-fold lower than in plasma, total apramycin concentrations in ELF were considered representative of the unbound (free) fraction.For pharmacodynamic evaluations, animals were euthanized after BAL and blood sampling, and lungs were aseptically harvested, homogenized in 5 mL of sterile saline, serially diluted, and plated on Mueller-Hinton agar. Bacterial counts were determined after overnight incubation at 37 °C andexpressed as log™ CFU per lung. Additional animals were sacrificed at the time of apramycin dosing (i.e., 2 h after infection) to determine baseline lung bacterial burden (time 0) before treatment initiation.Figure 13 shows that in a rat model of acute lung infection caused by Acinetobacter baumannii, inhalation of pure apramycin solution at a total inhaled dose (TID) of 1 mg / kg resulted in a modest reduction of lung bacterial burden, ranging from 1 to 2 log™ at 2 and 20 hours post-infection. In contrast, inhalation of the apramycin-gallium nanocomplex (1 :1 molar ratio) at the same equivalent apramycin dose (1 mg / kg) led to a significantly greater reduction, with approximately 4 log™ at 2 hours and up to 6 log™ at 20 hours post-infection.Comparative Example 1 Gallium complexes of Tobramycin and AmikacinGallium complexes of Tobramycin and Amikacin were prepared using the same procedure as for the Gallium Apramycin complex as described in example 3a.Antibacterial activity of the complexes was measured against clinical isolates of Acinetobacter baumannii, both for free tobramycin, free amikacin and the respective Gallium complexes.The measured MICs are shown in Figure 8A (Tobramycin) and Figure 8B (Amikacin). No decrease, or a slight decrease of MIC was observed using these complexes, as compared to Tobramycin and Amikacin alone.Contrary to Apramycin, these compounds do not show a beneficial effect of the use of a Gallium complex with respect to using free, uncomplexed compounds.ConclusionsIt has been shown that a complex of gallium and apramycin according to the invention shows antibacterial activity against several strains of bacteria, in particular Acinetobacter baumannii, which is on the WHO priority list because of the critical need to develop new antibiotics to treat infections caused by this bacterium.It has been shown that the complex according to the invention has lower MIC as compared to gallium alone, Apramycin alone or an admixture of these compounds. The complexes according to the invention thus show advantageous antibacterial properties and are particularly useful for administration by inhalation.Surprisingly it has been shown that other aminoglycosides, i.e. Tobramycin and Amikacin do not show a synergistic effect when a gallium complex of these compounds is used.

Claims

Claims[Revendication 1] A complex of gallium and apramycin.[Revendication 2] The complex according to claim 1 , wherein the complex is in the form of a particles or is comprised in particles, in particular spray-dried particles.[Revendication 3] The complex according to claim 2, wherein the particles have a median mass aerodynamic diameter MMAD comprised from 1 to 5 pm, in particular from 1 to 3 pm, said particles being in particular spray-dried particles.[Revendication 4] The complex according to any one of claims 1 to 3, wherein the molar ratio gallium I apramycin is in the range of 0.5:1 to 1 :4, in particular of 1 :1 to 1 :4, preferably about of about 1 :1 .[Revendication 5] The complex according to any of claims 1 to 4, wherein the complex is formulated with a pharmaceutically acceptable excipient, in particular wherein the particles according to claims 2 to 4 a pharmaceutically acceptable excipient, said pharmaceutically acceptable excipient being in particular an anti-adherent, in particular chosen from lipophilic amino acids or di or t / 7-peptides derived from lipophilic amino acids, phospholipids, or fatty acids or salts thereof.[Revendication 6] The complex according to claim 5, wherein the anti-adherent is chosen from L- Leucine, tri-Leucine and magnesium stearate, in particular L-leucine.[Revendication 7] The complex according to any of claims 5 or 6, wherein the anti-adherent is present in an amount of 5% to 15% by weight with respect to the total weight of the formulation.[Revendication 8] The complex according to any of claims 1 to 7, wherein the complex is protonated, in particular wherein the complex is protonated with an organic acid or with a mineral acid.[Revendication 9] A method for preparing the complex according to any of claims 1 to 8, wherein the method comprises a step a-1 ) of solubilizing a gallium salt and apramycin, or a salt thereof, to obtain an aqueous solution, and a step b-1 ) of spray-drying said aqueous solution, to obtain the complex of gallium and apramycin in the form of spray-dried particles.[Revendication 10] The method according to claim 9, wherein in step a-1 ) the gallium salt is chosen from gallium nitrate, gallium citrate, gallium chloride, gallium sulfate, gallium acetate, gallium formiate, gallium tartrate, gallium succinate, gallium acetate, gallium butyrate or gallium propionate, in particular gallium nitrate, or wherein the aqueous solution of step a-1 ) is prepared by mixing a gallium compound, which is a precursor of a gallium salt, such as gallium metal or gallium oxide with apramycin in the presence of acid and stirring the resulting mixture.[Revendication 11] The method according to any of claims 9 or 10, wherein the aqueous solution to be spray-dried in step a-1 ) further comprises an organic acid or a mineral, wherein said organic acid is in particular chosen from citric acid, acetic acid, formic acid, succinic acid, oxalic acid, lactic acid, malic acid, tartaric acid and butyric acid, more in particular the organic acid is acetic acid andformic acid, and wherein said mineral acid is in particular chosen from hydrochloric acid, nitric acid, sulfuric acid and phosphoric acid.[Revendication 12] The method according to any of claims 9 to 1 1 , wherein the aqueous solution obtained in step a-1 ) further comprises a pharmaceutical excipient, in particular an anti-adherent, which is in particular added during step a-1 ).[Revendication 13] A method for preparing a method for preparing the complex according to any of claims 1 to 8, wherein the method comprises a step a-2) of providing a solution comprising a gallium salt and apramycin, or a salt thereof in a solvent, followed by a step b-2) of stirring the solution of step a-2) to obtain a suspension comprising the complex of gallium and apramycin in the form of a precipitate.[Revendication 14] The complex according to any of claims 1 to 8, for use in a method of treatment of the human or animal body.[Revendication 15] The complex according to any of claims 1 to 8, for use in the treatment of a bacterial infection.[Revendication 16] The complex for use according to claim 15, wherein the bacterial infection is caused by Acinetobacter baumannii, Klebsiella pneumoniae, Enterobacteriaceae or Pseudomonas aeruginosa, Mycobacterium abscessus, Mycobacterium tuberculosis, Enterococcus faecium, Staphylococcus aureus, Helicobacter pylori, Campylobacter spp., Salmonellae, Neisseria gonorrhoeae, Streptococcus pneumoniae, Haemophilus influenzae or Shigella spp, in particular Acinetobacter baumannii.[Revendication 17] The complex for use according to any of claims 15 or 16, wherein the complex is administered by inhalation, said complex being in particular in the form of spray-dried particles.

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