Pharmaceutical composition for inhalation

Abstract

Disclosed is a pharmaceutical composition for inhalation, comprising lipid carriers comprising a pharmaceutical agent, the therapeutic uses thereof, and a method of making same.

Description

[0001] PHARMACEUTICAL COMPOSITION FOR INHALATION

[0002] Technical field

[0003] The present invention relates to a lipid composition protecting a pharmaceutical agent and intended to be administered into the respiratory tract to a patient.

[0004] Prior art

[0005] Formulation such as lipid entrapment of pharmaceutical agents is well known.

[0006] In particular, the use for this purpose of lipids and in particular cationic lipids selected according to the nature of the pharmaceutical agent to be trapped and the type of vehicles to be obtained is known. Some of these lipids, however, present clinical toxicity, which limits the options (quantity, nature). In addition, the size of the liposome, including the dispersion of sizes, the stability of the composition in biological media, its capacity to trap the pharmaceutical agent, or to release it at the desired location represent all factors that are difficult to optimize, especially jointly.

[0007] Ji et al., 2023, describe the use of liposomes for the potentiation of STING (Stimulator of Interferon Genes) pathway activation: this approach was designed based on cyclic dinucleotides, which are protected by the lipid structure. Different liposome formulations were made based on DOTAP (1,2,-dioleoyl-3-trimethylamonium-propane), cholesterol and a phosphoethanolamine derivatized by polyethylene glycol. Good encapsulation rates were obtained for DOTAP / agonist molar ratios between 10 and 20, whereas at a ratio of 2.5, the encapsulation efficiency is poor, which is attributed to a too low density of positive charges. Similarly, at these low concentration ratios, the potentiation of the biological effect by the liposome is lost.

[0008] Unfortunately, the low level of encapsulation implies massive administration of liposomes to a patient, which presents risks of toxicity, particularly due to the large amounts of cationic lipids, abundant in the liposomes of this study.

[0009] On the other hand, modifying the content of a compound is not simple because several physicochemical properties of the liposome must correspond to acceptable values ​​for administration to a patient, such as size, size dispersion and electrical charge potential; while ensuring the stability of the formulation. In other words, in a formulation, enrichment in one component is at the expense of the others, which risks strongly affecting the balance: the inventors have noticed that this balance is a function of different concentration ratios, which can be strongly affected even following adaptations of concentrations that might have seemed marginal.

[0010] Thus, the inventors noticed that there is still a need for improved formulations for the benefit of patients.

[0011] Brief summary of the invention

[0012] A first aspect of the present invention relates to a pharmaceutical composition comprising lipid carriers formed from a series of lipids, comprising a pharmaceutical agent selected from the group consisting of an antimicrobial, a nucleic acid, a STING protein agonist or a STING protein antagonist, a Toll-like receptor ligand, an immunomodulant and / or a corticosteroid, a peptide, an antihypertensive, a bronchodilator or a mixture thereof, said composition being for administration by inhalation.

[0013] Advantageously, this pharmaceutical composition can be administered by nebulization and / or by pressurized inhalation and / or by dry powder inhalation and / or by gentle spray inhalation.

[0014] A related aspect of the present invention relates to this pharmaceutical composition for the treatment of metastatic cancer, preferably said metastasis being outside the lung or the central nervous system and the primary tumor being pulmonary or located in the central nervous system, or said metastasis being pulmonary or located in the central nervous system and the primary tumor being outside the lung or the central nervous system.

[0015] Another related aspect of the present invention relates to this pharmaceutical composition for the treatment of an infectious disease at the level of the respiratory and / or systemic tract, or the central nervous system.

[0016] A related aspect of the present invention relates to this pharmaceutical composition comprising a nucleic acid encoding one or more epitope(s), or a peptide comprising one or more epitopes, said pharmaceutical composition being for vaccination.

[0017] A related aspect of the present invention relates to this pharmaceutical composition comprising a nucleic acid being for gene therapy and / or enzyme replacement, said nucleic acid being a DNA or messenger RNA molecule, said gene therapy preferably being somatic.

[0018] A related aspect of the present invention relates to a process for obtaining this pharmaceutical composition and comprising the steps of solubilizing the lipids in an organic solvent, evaporating the solvent so as to form a lipid film, rehydrating with a solution containing the pharmaceutical agent and homogenizing and / or extruding (optional), preferably a second step of contacting a solution containing the pharmaceutical agent with the extruded lipids, preferably, said lipids comprising a neutral lipid at pH 7.0 and positively charged at pH 5, and said rehydration step being carried out at pH below 7.0.

[0019] Alternatively, the pharmaceutical agent is solubilized with the lipids in the organic solvent before evaporation, and is not added again during the rehydration step. Particularly when the pharmaceutical agent is hydrophobic in nature and / or an uncharged molecule at pH 7, such as MSA-2.

[0020] Brief description of the drawings

[0021] Figure 1 compares the stability of different lipid structures.

[0022] Figure 2 compares the effect of functionalization on macrophage activation.

[0023] Detailed description of an embodiment of the invention

[0024] The inventors have succeeded in developing lipid-based formulations that allow significant entrapment of one (or more) pharmaceutical agent(s), while retaining advantageous physicochemical properties compatible with clinical use.

[0025] Thus, a first aspect of the present invention relates to a pharmaceutical composition comprising lipid carriers comprising at least one pharmaceutical agent selected from the group consisting of at least one antimicrobial such as an antibiotic, an antibacterial or an antifungal, a nucleic acid, at least one STING protein agonist or at least one STING protein antagonist, at least one Toll-like receptor ligand, an immunomodulant, a peptide, a bronchodilator, a corticosteroid, an antihypertensive, and mixtures thereof (except the STING agonist with the STING antagonist), this pharmaceutical composition being for inhalation.

[0026] Preferably, this pharmaceutical composition for inhalation is for the activation of macrophages and / or dendritic cells.

[0027] Advantageously, the lipid vehicle forming this pharmaceutical composition for inhalation comprises at least one lipid having functionalization by a ligand of the mannose receptor (CD206).

[0028] The lipid vehicles are advantageously chosen from the group consisting of liposomes, vesicles, micelles, lipoplexes, lipid emulsions, lipid nanocrystals, lipid microspheres, lipid nanoparticles, and mixtures thereof.

[0029] In the context of the present invention, the terminology "by inhalation" relates to both nasal administration (e.g., to reach the nasal cavity, the central nervous system and / or the systemic circulation; e.g., treatment of a tumor, gene therapy, treatment of inflammation or infection) and pulmonary administration (e.g., to treat a disease therein, such as inflammation, infection or tumor), and nasal administration to target both nasal and pulmonary deposition.

[0030] In the context of the present invention, the term "peptide" preferably means any chain of at least 2 amino acids linked by peptide bonds. Advantageous peptides are a protein (including an enzyme), an antigen, an antibody, (or an antibody fragment, a nanobody).

[0031] Advantageously, the pharmaceutical agent comprises at least one nucleic acid selected from the group consisting of single-stranded DNA, double-stranded DNA (including cDNA), single-stranded RNA (including mRNA), double-stranded RNA, siRNA, miRNA, shRNA, oligonucleotide (DNA- and / or RNA-based), and mixtures thereof. The nucleotides, sugars, or phosphodiester bonds may advantageously be modified, for example, uridine may be substituted or partially substituted with pseudouridine, for example, when the nucleic acid is mRNA. The adenosine or cytosine bases may advantageously be methylated, as may the sugars (e.g., in the 2' position). Peptide nucleic acids, morpholino, and / or phosphorothioate bonds may advantageously replace some or all of the phosphodiester bonds. Alternatively, when the nucleic acid is intended to stimulate an immune response and includes CpG motifs, the cytosine is advantageously not methylated.

[0032] Advantageously, the pharmaceutical agent comprises at least one STING protein agonist, preferably selected from the group consisting of cGAMP, 2',3'cGAMP, DMXAA, ADU-S100, MK-1454, MK-21 18, GSK-3745417, BMS-986301, SB-1 1285, IMSA-101, SYN-STING (SYNB1891), Bl-1 387446, TAK-676, E-7766 exoSTING, SNX281, HG-381, DN-015089, PC7A, DiABZI, Di-guanosine monophosphate cyclic (cyclic di-GMP), MSA-2, Ulevostinag, SB 1 1285, IMSA 101, Dazostinag, BMS-986301, Bl 1387446, advantageously the group also consists of cAlMP, cAIMP-di fluor, 3'-3'-cGAMP, c-di-AMP and SR-717, and their mixture, preferably 2'3'cGAMP.

[0033] Alternatively, the pharmaceutical agent comprises at least one STING protein antagonist selected from the group consisting of Cl 76, H-151, lnh-54, RU.521, H-8, Al 51, SA-1, GSK'932, SN-01 1 and mixtures thereof.

[0034] Advantageously, the antimicrobial (antibiotic) is chosen from the group of aminoglycosides (e.g. amikacin, gentamicin, kanamycin, neomycin, netilmicin, tobramycin, paromycin, streptomycin), carbapenems (e.g. ertapenem, doripenem, cilastatin, meropenem), cephalosporins (e.g. cefadroxil, cefazolin, cephradine, cephapirin, cephalothin, cefalexin, cefaclor, cefoxitin, cefotetan, cefamandole, cefmetazole, cefonicide, loracarbef, cefprozil, cefuroxime, cefixime, cefdinir, cefditoren, cefoperazone, cefotaxime, cefpodoxime, ceftazidime, ceftibuten, ceftizoxime, moxalactam, ceftriaxone, cefepime, ceftaroline fosamil, ceftobiprole), glycopeptides (e.g. teicoplanin, vancomycin, telavancin, dalbavancin, oritavancin), lincosamides (e.g. clindamycin, lincomycin), lipopeptides (e.g. daptomycin), macrolides (e.g. azithromycin, clarithromycin, erythromycin, roxithromycin, telithromycin, spiramycin, fidaxomycin), monobactams (e.g.aztreonam), pénicillines (amoxicline, ampiciline, azlocilline, dicloxacilline, flucloxacilline, mezlocilline, méthicilline, nafcilline, oxacilline, pénicilline G ou V, piperacilline, temocilline, ticarcilline), polypeptides (ex. bacitracine, colistine, polymyxine B), quinolones et fluoroquinolones (ex. ciprofloxacine, enoxacine, gatifloxacine, gemifloxacine, levofloxacine, lomefloxacine, moxifloxacine, nadifloxacine, l’acide nalidixique, norfloxacine, ofloxacine, trovafloxacine, grepafloxacine, sparfloxacine, temafloxacine), sulfamides (ex. mafenide, sulfacetamide, sulfadiazine, sulfadiazine d’argent, sulfadimethoxine, sulfamethizole, sulfamethoxazol, sulfanilimide, sulfasalazine, sulfisoxazo, trimethoprim-sulfamethoxazole (Co- trimoxazole), sulfonamidochrysoidine), tétracyclines (ex. demeclocycline, doxycycline, metacycline, minocycline, oxytetracycline), ou d’autres classes (ex.active against mycobacteria: capreomycin, cycloserine, ethambutol, ethionamide, isoniazid, pyrazinamide, rifampicin, rifabutin, rifapentine, streptomycin).

[0035] Preferably, the series of lipids forming the lipid vehicles has a phase transition temperature (Tm) of between -30°C and 100°C, preferably between 1°C and 90°C, advantageously between 10°C and 80°C, 20°C and 70°C, for example between 20°C and 60°C.

[0036] Preferably (alternatively, or in addition), the series of lipids forming the lipid vehicles has a melting point of between -30°C and 180°C, preferably between 1°C and 160°C, advantageously between 10°C and 155°C, preferably between 20°C and 150°C, for example between 20°C and 80°C.

[0037] These physicochemical properties of the lipid series improve performance in pulmonary administration, in particular by nebulization or inhalation, and increase the release of the pharmaceutical agent in the targeted tissues and / or the cytoplasmic release of the pharmaceutical agent and / or its half-life and / or reduce the release of the pharmaceutical agent in non-targeted tissues. In particular, the inventors noted that a high phase transition temperature (Tm) of the lipid series, for example more than 10°C, more than 20°C, is associated with increased rigidity, which is advantageous, at least for certain formulations or modes of administration to the patient.

[0038] Advantageously, the at least one lipid has a functionalization by a ligand of the mannose receptor (CD206), preferably a phospholipid having a functionalization by a ligand of the mannose receptor (CD206),

[0039] Advantageously (furthermore) at least one lipid, preferably a phospholipid, is derivatized by polyethylene glycol (PEG). A PEG of size between 500 Da and 20 kDa is preferred, for example between 1 and 10 kDa, or even between 2 and 5 kDa.

[0040] Preferably, a lipid, preferably a phospholipid, derivatized by PEG, advantageously a DSPE-PEG, has functionalization by a ligand of the mannose receptor (CD206).

[0041] Advantageously, between 10% and 100% (mole equivalent), preferably between 20 and 90%, preferably between 30 and 80%, or even between 40 and 50% of the PEG-derivatized lipids is further functionalized with the mannose receptor ligand (CD206). Thus, the PEG-derivatized lipids, or a portion of the PEG-derivatized lipids, is not functionalized. The inventors have in fact noted that the double optimization, the PEG content and that of the CD206 receptor ligand, was advantageously carried out in this way.When only a portion of the PEGs is functionalized by the CD206 receptor ligand, this object of the present invention can be achieved either on the basis of a single lipid, preferably a single phospholipid, derivatized by PEG, of which only a proportion would be functionalized, or on the basis of several lipids, preferably comprising at least one phospholipid, which would be derivatized with PEG, with some of these PEG-derivatized molecules which would not be functionalized.

[0042] This increases the ability of the ligand to bind to its receptor. In addition, PEG stabilizes lipid vehicles.

[0043] Advantageously, the ligand of the mannose receptor (CD206) is selected from the group comprising a mannose, a fucose, an N-acetylglucosamine, an N-acetylgalactosamine, a glycoprotein, a galactocomannan, an aD-mannopyranoside (e.g. a-D-Mannopyranose), a-L-fucopyranose-(1-3)-2-acetamido-2-deoxy-[3-D-glucopyranose, Methyl aD-mannopyranoside, a-L-fucopyranose -(1-2)-[3-D-galactopyranose-(1-4)-|3-D- glucopyranose, Methyl 2-acetamido-2-deoxy-aD-glucopyranoside, (3-D- galactopyranose-(l-3)-(aL-Fucopyranose-(l-4)) 2-acetamido-d-deoxy-[3-D-glyco pyranose, a-D-mannopyranose-(l-2)- a -D-mannopyranose, a polymannose (between 4 and 50, preferably between 5 and 30, or even between 6 and 20 mannose residues) an anti-CD206 antibody, and mixtures thereof.

[0044] In the context of the present invention, these ligands may be modified, for example, N-acetylglucosamine may be deacetylated. For example, N-acetylglucosamine is incorporated as chitin or chitosan, preferably chitin or chitosan oligomers (e.g., less than 50 monomers, less than 40 monomers, less than 30 monomers).

[0045] Preferably, the lipid vehicles are lipid nanoparticles (LNPs), preferably liposomes or solid lipid nanoparticles (SLNs) or nanostructured lipid carriers (NLCs).

[0046] In the context of the present invention, "SLN" preferably means colloidal particles (generally 50 to 500 nm in diameter) prepared from solid lipids (solid at room temperature and body temperature), surfactants and water by high speed or high pressure homogenization methods.

[0047] In the context of the present invention, "NLC" preferably means SLNs containing a liquid lipid (oil) fraction that causes structural imperfections of solid lipids leading to a less ordered crystalline arrangement in order to improve the encapsulation of pharmaceutical agent. They are prepared by methods similar to SLNs.

[0048] In the context of the present invention, "liposomes" preferably means spherical lipid vesicles (generally 50 to 500 nm in diameter) composed of one or more lipid bilayers. They result from the emulsification of natural or synthetic lipids in an aqueous medium by methods such as lipid film hydration, the ethanol injection method or by microfluidic methods.

[0049] Preferably, the pharmaceutical agent has an overall negative charge. Indeed, the inventors have succeeded in incorporating large amounts of this type of agent, even in molar proportion to the cationic (or protonable, see below) lipid potentially present.

[0050] Thus, preferably, a cationic or protonatable lipid is incorporated when the pharmaceutical agent has an overall negative charge.

[0051] Conversely, preferably, when the pharmaceutical agent is uncharged, no cationic lipid is added or, then in a content of less than 10% by mole (mole cationic lipid: total moles of incorporated lipids and cholesterol), preferably less than 5% by mole, preferably less than 4.3, 2 or 1% by mole.

[0052] Alternatively, the molar ratio between the cationic lipid possibly present and the uncharged pharmaceutical agent is less than 50% (mole cationic lipid(s): moles uncharged pharmaceutical agent), less than 40, 30, 20, 10, or even less than 5%.

[0053] Thus, the lipid series preferably comprises a first lipid selected from the group consisting of a cationic lipid, a neutral lipid, an anionic lipid, a phospholipid, a triglyceride, a diglyceride, a glycerophospholipid, a sphingomyelin, a fatty acid, a fatty acid salt, and / or a second lipid selected from the group consisting of a cationic lipid, a neutral lipid, an anionic lipid, a phospholipid, a triglyceride, a diglyceride, a glycerophospholipid, a sphingomyelin, a fatty acid, a fatty acid salt, and / or a third lipid selected from the group consisting of a cationic lipid, a neutral lipid, an anionic lipid, a phospholipid, a triglyceride, a diglyceride, a glycerophospholipid, a sphingomyelin, a fatty acid, a fatty acid salt.

[0054] Preferably, particularly when the pharmaceutical agent has an overall negative charge, the pharmaceutical composition comprises a first lipid being a cationic lipid having at least one quaternary ammonium group, and / or a lipid having at least one protonatable (secondary or tertiary) amine group (and free of negative charge at pH 7.0). Preferably, the lipid having at least one quaternary ammonium group (and / or first lipid) is selected from the group consisting of DOTAP (18:1 TAP) as well as other TAP derivatives (such as 14:0 TAP, 16:0 TAP, 18:0 TAP), DOTMA, DDAB, DC-Chol, DODAP, MVL5, DOSPA, GL67, DOBAQ, EPC derivatives (12:0 EPC, 14:0 EPC, 16:0 EPC, 18:0 EPC, 18:1 EPC, 14:1 EPC, 16:0-18:1 EPC), DORI, DC-6-14, and mixtures thereof. DOTAP is preferred.

[0055] By protonable (secondary or tertiary) amine, it is preferably understood in the context of the present invention, an amine which is predominantly in neutral form at pH 7.0, (e.g. more than 50%, preferably more than 75%, or even more than 90 or 99% in neutral form at pH 7.0) and predominantly in protonated form at pH 5.0 (e.g. more than 50%, preferably more than 75%, or even more than 90 or 99% in protonated form at pH 5.0).

[0056] An example of a lipid comprising a protonatable amine is 1,2-dioleyloxy-3-dimethylaminopropane (DODMA). It is understood, in the context of the present invention, that other lipid chains than oleic acid can be substituted therefor, for example palmitic acid or stearic acid while retaining the Dimethylaminopropane (DMA) derivatives; such as 18:0 DMA, 11:6:0 DMA, 14:0 DMA, 18:1 DMA). Conversely (or additionally) substitutions at the aminopropane group are possible, for example at the methyl groups of DMA.

[0057] Preferably, the second lipid is a sterol and is advantageously selected from the group consisting of cholesterol and its esters (cholesteryl esters), and its glycosylated derivatives (BD-glucosyl cholesterol, Galactosyl Cholesterol, BbGL-1), ox-18:2 cholesterol, desmosterol, stigmasterol, lanosterol, 7-dehydrocholesterol, dihydrolanosterol, zymosterol, lathosterol, and mixtures thereof. Cholesterol is highly preferred.

[0058] Thus, a composition comprising DOTAP and cholesterol is highly preferred, particularly when the pharmaceutical agent has an overall negative charge. Preferably, the phospholipid potentially present in this pharmaceutical composition (second lipid in the absence of sterol, third and / or fourth lipid) is chosen from natural, purified or synthetic phospholipids, such as phospholipids extracted from egg or soy, phosphatidic acids, phosphatidylethanolamines (PE), lysophosphatidylethanolamines (LPE) or phosphatidylcholines (PC) or lysophosphatidylcholine (LPC) or phosphatidylserines (PS), or lysophosphatidylserines (LPS) or phosphatidylglycerol (PG) or lysophosphatidylglycerol (LPG) and mixtures thereof, preferably chosen from the group comprising DSPE, DSPE-PEG, DMPE, DPPC, DSPC, DMPC, DLPC, and their mixtures.

[0059] In the context of the present invention, preferably, the fatty acid chains constituting the phospholipids, but also the cationic or protonable lipids can be saturated, unsaturated or polyunsaturated, provided that the parameters below of melting point and / or phase transition temperature (measured on all the lipids used) are within the ranges described. Thus the composition according to the invention can advantageously contain unsaturated fatty acids and saturated fatty acids.

[0060] The fatty acid chains constituting the above phospholipids have a size advantageously between 14 and 20 carbon atoms, such as 16 or 18 carbon atoms. Shorter or longer sizes are possible (in low contents, e.g. less than 20% by mole of the total lipids) provided that the parameters below of melting point and / or phase transition temperature (measured on all the lipids used) remain within the ranges described.

[0061] Advantageously, the anionic lipid, when present, is chosen from phosphatidylinositol (Pis) and their phosphates (PIPs), phosphatidylserines (PS; e.g. DPPS), phosphatidic acid (PA; e.g. DPPA) and phosphatidylglycerols (e.g. DPPG or DSPG) and mixtures thereof.

[0062] Preferably, the composition comprises a phosphatidylcholine, such as DPPC (1,2-dipalmitoylphosphatidylcholine). The incorporation of phospholipids having long saturated chains (e.g., between 14 and 22 carbons, preferably 16 or 18 carbons, i.e., palmitic acid or stearic acid) allows the rigidity of the particles to be increased, which improves their properties in the event of pulmonary administration, in particular by nebulization or by inhalation, including in the form of DPI.

[0063] The inventors noticed that this lipid having a polar head and two unsaturated hydrocarbon chains (di-palmtoyl and / or di-stearyl) increased the rigidity of lipid structures and / or allowed a reduction of the (relative) DOTAP content. An incorporation of DPPC (and / or DOPC) in a molar ratio of between 0.1 and 0.95, preferably between 0.2 and 0.90, preferably between 0.5 and 0.8, or between 0.6 and 0.7 (mole phosphatidylcholine, e.g. DPPC and / or DOPC: total moles of lipids) relative to all lipids is preferred.

[0064] Preferably, the lipid carriers of the pharmaceutical composition have a molar ratio of the first lipid to the second lipid (or to the third lipid if the second lipid is absent) of between 0.3 and 20, preferably between 0.5 and 10, such as between 1 and 5.

[0065] Preferably, when the latter is incorporated, the lipid vehicles of the pharmaceutical composition have a molar ratio of said at least one lipid having functionalization by a ligand of the mannose receptor (CD206) to total lipids of between 0.01 (or even 0.005 or 0.007 or 0.008 or 0.009) to 0.2, preferably between 0.02 and 0.1, preferably between 0.03 and 0.05.

[0066] Advantageously, when the latter is incorporated, the molar proportion of said at least one pharmaceutical agent relative to said at least one lipid having functionalization by a ligand of the mannose receptor (CD206) is between 5 (or 10) and 150, preferably between 20 and 100, or even between 30 and 80.

[0067] Advantageously, the lipid vehicles have a molar proportion of said cationic and / or ionizable lipid relative to the pharmaceutical agent of between 30 and 0.1, preferably between 10 and 0.2, advantageously between 3 and 0.5, preferably between 2.5 and 0.8, advantageously between 2.4 and 1, in particular when the pharmaceutical agent has an overall negative charge.

[0068] Preferably, the pharmaceutical composition has a loading rate of the pharmaceutical agent of between 0.1 and 80% relative to the mass of the lipid vehicles, preferably between 1 and 70%, preferably between 10 and 60%, preferably between 15 and 50%, such as between 20 and 40% or between 25 and 30%.

[0069] Advantageously, the average size of the lipid vehicles is between 50 and 200 nm, preferably between 60 and 190 nm, advantageously between 70 and 180 nm, preferentially between 80 and 180 nm, preferably between 90 and 180 nm, more preferably between 100 and 180 nm, preferably between 110 and 180 nm, advantageously between 120 and 180 nm, preferentially between 120 and 170 nm, preferably between 120 and 160 nm.

[0070] Alternatively, the average size of the lipid vehicles is between 350 and 1500 nm (or even 2000 nm), preferably between 350 and 1400 nm, advantageously between 350 and 1300 nm, preferentially between 350 and 1200 nm, advantageously between 350 and 1100 nm, preferentially between 350 and 1000 nm, preferably between 350 and 900 nm, preferably between 350 and 800 nm, more particularly between 350 and 700 nm, advantageously between 350 and 600 nm, for example between 400 and 600 nm.

[0071] Alternatively, the average size of lipid vehicles is between 200 and 350 nm, preferably between 225 and 300 nm.

[0072] Preferably the (mean) size and mean diameter (Dh) of the lipid vehicles is (are) measured by dynamic light scattering (DLS).

[0073] Advantageously, the lipid vehicles have a polydispersity index of between 0.05 and 0.9, preferably between 0.1 and 0.8, advantageously between 0.2 and 0.75, preferably between 0.3 and 0.7, preferably between 0.4 and 0.6; preferably the polydispersity index being measured by dynamic light scattering (DLS). A preferred apparatus, both for the measurement of particle size, Pdi (see below) or zeta potential (see below; even if other apparatus are also used) is the Malvern Zetasizer nano ZS (Malvern Instruments SA, Worcestershire, UK).

[0074] Advantageously, in the context of the present invention, the polydispersity index is defined by the formula: where = is the average particle diameter, the quantity of particles with size Xi and xi being the diameter of the spherical particle.

[0075] Alternatively, the polydispersity index is between 0.05 and 0.9, preferably between 0.05 and 0.8, advantageously between 0.05 and 0.7, more advantageously between 0.05 and 0.6, even more advantageously between 0.05 and 0.5, preferably between 0.05 and 0.4, advantageously between 0.05 and 0.3, more particularly between 0.05 and 0.25, preferably between 0.05 and 0.2, for example between 0.06 and 0.2, preferably between 0.07 and 0.2, advantageously between 0.08 and 0.2, measured by dynamic light scattering (DLS).

[0076] Indeed, depending on the applications, lipid vehicle particles of the same sizes are preferred, while for other applications, a bimodal or even multimodal distribution is preferred.

[0077] Preferably, the lipid vehicles have a zeta potential of between -60 mV and 100 mV, preferably between -40 mV and 80 mV, advantageously between -20 mV and 60 mV, preferably between -5 mV and 55 mV, preferably between 0 mV and 50 mV, preferably between 10 mV and 40 mV, advantageously between 10 mV and 30 mV, advantageously measured by "laser doppler electrophoresis". Preferably, this measurement is carried out in an aqueous solution of NaCl at 0.009% (mass:volume).

[0078] Advantageously, said pharmaceutical agent is present in a mass proportion of between 0.1 and 80%, preferably between 1 and 70%, preferably between 5 and 60%, preferably between 10 and 50% or between 20 and 40%, relative to the mass of the lipid vehicles.

[0079] Advantageously, the composition contains at least one metal ion such as Mn 2+ , the Co 2+ or Zn 2+in order to potentiate the activity of the pharmaceutical agent. Preferably, this or these metal ions are incorporated in a content greater than 10 PPM (parts per million; weight content; mass of the metal ion(s): total mass (dry) of the lipid composition), preferably at a content of between 100 PPM and 50% by mass (mass of the metal ion(s): total mass (dry) of the lipid composition), preferably between 300 PPM and 10%, preferably between 800 PPM and 1% (by mass).

[0080] A related aspect of the present invention relates to a pharmaceutical composition for inhalation in liquid form such as a solution, dispersion or suspension, or in the form of a powder to be redissolved and redispersed before administration, or in the form of a dry powder for inhalation.

[0081] Advantageously, this pharmaceutical composition for inhalation in liquid form can be frozen to ensure good stability during storage or distribution of the drug. This composition will then be thawed before administration and will allow the lipid vehicles to be reconstituted in an advantageous manner. This also ensures that the formulation retains maximum activity.

[0082] Advantageously, this pharmaceutical composition for inhalation comprises at least one excipient chosen from the group consisting of a buffer chosen from the group comprising phosphate, sulfonate (MES, TES, HERES, MOPS, PIPES, TAPS, TAPSO), acetate, bicine buffers, and / or at least one salt chosen from the group comprising inorganic salts and organic salts, and / or at least one surfactant chosen from the group comprising cholic acids and their salts, phospholipids (e.g. phosphatidylcholines or lecithin, phosphatidylglycerols), lipids or triglycerides, sorbitan esters, polyethoxylated sorbitans, fatty acids, preferably lauric, palmitic, stearic, erucic or behenic acid, esters of these fatty acids, or derivatives of these fatty acids, such as salts, preferably chosen from stearate magnesium, sodium stearyl fumarate and sodium stearyl lactylate, sodium lauryl sulfate, magnesium lauryl sulfate,natural components of pulmonary surfactant such as phospholipids or cholesterol; and sucroesters (sugar esters, for example esters between sucrose or glucose and fatty acids) and / or at least one amino acid selected from the group consisting of histidine, leucine, isoleucine, threonine, lysine, valine, methionine, phenylalanine, mixtures thereof and derivatives thereof, such as acesulfame K or aspartame and / or a sugar, preferably selected from the group of monosaccharides (such as glucose or arabinose), disaccharides (such as lactose, maltose, sucrose, dextrose, trehalose, maltitol and mixtures thereof or a bulking agent selected from polyols such as sorbitol, mannitol and xylitol), polysaccharides (such as dextran, chitosan, starch, cellulose, and its derivatives), oligosaccharides (such as cyclodextrin and dextrins).,

[0083] An advantageous option for the pharmaceutical composition for inhalation is drying in the form of a powder to ensure good stability during storage, to be dissolved or redispersed for example just before administration to a patient (e.g. less than 1 hour). This composition will then be redissolved or redispersed before administration in an aqueous solvent such as water or a physiological solution or buffer such as a 0.9% (mass:volume) NaCl solution or PBS, and will allow the lipid vehicles to be reconstituted in a beneficial manner. This also ensures that the formulation retains maximum activity.

[0084] Alternatively or additionally, the pharmaceutical composition for inhalation according to the invention is in the form of a powder to ensure good stability during storage or distribution of the drug, to be dissolved or redispersed and comprises at least one bulking agent such as a polyol and / or a sugar alcohol such as sorbitol, mannitol, maltitol (sometimes considered a disaccharide) and xylitol, (crystalline) sugars including monosaccharides (glucose, arabinose), disaccharides (lactose, maltose, sucrose, dextrose, trehalose), and polysaccharides (dextran, chitosan, starch, cellulose and its derivatives), or oligosaccharides (dextrins, cyclodextrins) and / or fatty acid salts (e.g. magnesium stearate) or fatty acids or their derivatives (e.g. esters), such as lauric acid, palmitic acid, stearic acid, erucic acid, behenic acid, or a phospholipid (eg.lecithin, phosphatidylcholine phosphatidylglycerol; preferably not incorporated in the lipid vehicle), triglycerides (preferably not incorporated in the lipid vehicle), sugar esters, and / or an amino acid (see above for the liquid composition) and mixtures thereof. Dextran, trehalose, mannitol and lactose are preferred.

[0085] Advantageously, an excipient (bulking agent) will be used, such as lactose, trehalose, sucrose or mannitol. In this case, the composition, even in powder form, will advantageously comprise a buffer and in addition one or more of the compounds listed above, for example a surfactant.

[0086] Preferably, the liquid form to be administered comprising the pharmaceutical composition according to the invention comprises from 50 to 99%, or even 99.9% (or 99.8%), of water (mass of water: total mass of the composition), preferably from 70 to 98% of water, or even from 80 to 95 (or 90%)% of water; in other words, the solution comprises from 1 to 50% of the pharmaceutical composition (sometimes even from 0.1 or 0.2 to 50%), preferably from 2 to 30%, or even from 5 or 10 to 20%.

[0087] The liquid form to be administered comprising the pharmaceutical composition for inhalation further comprises at least one buffer selected from the group consisting of phosphate, sulfonate (MES, TES, HERES, MOPS, PIPES, TAPS, TAPSO), acetate, bicine buffers, and / or at least one salt selected from the group consisting of inorganic salts (sodium chloride, calcium carbonate, sodium or potassium phosphate), organic salts (e.g. sodium lactate, potassium citrate), and / or at least one surfactant selected from the group consisting of cholic acids and their salts (taurocholate, glycocholate), phospholipids (not incorporated into the lipid vehicle), lipids (not incorporated into the lipid vehicle), sorbitan esters (e.g. SPAN 85), polyethoxylated sorbitan (e.g.Tween®80) and / or at least one amino acid selected from the group of histidine, leucine, isoleucine, threonine, lysine, valine, methionine, phenylalanine, their mixtures and their derivatives, such as acesulfame K or aspartame and their mixtures.

[0088] Another advantageous option for the pharmaceutical composition for inhalation is to be in the form of a dry powder for inhalation and / or intended to be administered using a dry powder inhaler. This composition will have advantageous properties of dispersion and aerosolization of the powder in the air, allowing the delivery of adapted doses of powder into the patient's respiratory tract (inhalable powder). These properties include an aerodynamic size adapted to the route of administration. This composition will allow the lipid vehicles to be advantageously reconstituted in physiological fluids once the powder has been deposited in the respiratory tract. This further ensures that the formulation retains maximum activity.

[0089] Preferably the geometric diameter and / or aerodynamic size for pulmonary administration is less than 5 μm, preferably between 0.5 μm and 5 μm, preferably between 1 μm and 3 μm.

[0090] Preferably the geometric diameter and / or the aerodynamic size for nasal administration is greater than 5 pm, preferably between 5 pm and 120 pm, preferably between 10 pm and 60 pm, preferably between 20 and 40 pm, preferably between 20 and 30 pm.

[0091] Thus, a bimodal distribution of particle size and / or aerodynamic size of the pharmaceutical composition in the form of powder for inhalation is advantageous and allows nasal deposition and pulmonary deposition by nasal administration.

[0092] In the context of the present invention, the term "aerodynamic size" preferably means a parameter describing the aerodynamic behavior of the particles; thus, this parameter takes into account the geometric diameter (therefore the particle size), but also the density and shape of the particles.

[0093] The particle size (and geometric diameter) is determined by laser diffraction. The aerodynamic size (and aerodynamic diameter) is determined using an impaction test (using cascade impactors) described in pharmacopoeias (such as the European Pharmacopoeia for example).

[0094] Alternatively or additionally, the pharmaceutical composition for inhalation according to the invention is administered in the form of a dry powder and comprises at least one bulking agent such as a polyol and / or a sugar alcohol such as sorbitol, mannitol, maltitol (sometimes considered a disaccharide) and xylitol, (crystalline) sugars including monosaccharides (glucose, arabinose), disaccharides (lactose, maltose, sucrose, dextrose, trehalose), and polysaccharides (dextran, chitosan, starch, cellulose and its derivatives), or oligosaccharides (dextrins, cyclodextrins) and / or fatty acid salts (e.g. magnesium stearate) or fatty acids or their derivatives (e.g. esters), such as lauric acid, palmitic acid, stearic acid, erucic acid, behenic acid, or a phospholipid (eg.lecithin, phosphatidylcholine phosphatidylglycerol; preferably not incorporated in the lipid vehicle), triglycerides (preferably not incorporated in the lipid vehicle), sugar esters, and / or an amino acid (see above for the liquid composition) and mixtures thereof. Dextran, trehalose, mannitol and lactose are preferred.

[0095] Advantageously, an excipient (bulking agent) will be used, such as lactose, trehalose, sucrose or mannitol.

[0096] Advantageously, when the pharmaceutical composition is intended for administration to the patient in the form of a dry powder for inhalation, the above excipients comprise at least one lipid or fatty acid-based substance (e.g., magnesium stearate, phospholipid, sucroester). This promotes good mixing homogeneity and / or advantageous aerosolization of the solid particles. Another excipient is also advantageous for good aerodynamic properties, such as leucine.

[0097] It should be noted that some surfactants include fatty acids, or even phospholipids or triglycerides, which are also potential components of the lipid vehicles of the present invention. However, these surfactants are not incorporated within the structure of the lipid vehicles and are advantageously incorporated into a dry formulation of these already formed lipid vehicles. In this case, the composition, even in powder form, will advantageously comprise a buffer and in addition one or more of the compounds listed above, for example a surfactant.

[0098] Examples of drying methods are freeze-drying, spray-drying, spray-congealing or spray-chilling, spray-freeze-drying, supercritical fluid drying.

[0099] In the case of lyophilization with the lipid vehicles according to the invention, the inventors have obtained good results when sucrose is added as a protective agent, preferably in a content higher than that of the lipids, preferably in a mass ratio of at least 2:1, 3:1, 5:1, or even about 10:1 (weight of sucrose: weight of all the lipids).

[0100] Advantageously, the protective agent (sucrose) is added during the lipid film hydration stage.

[0101] Advantageously, this pharmaceutical composition can be administered into the respiratory tract, preferably by nebulization and / or by pressurized inhalation and / or by dry powder inhalation and / or by gentle spray inhalation (for example carried out using the Respimat® SofMist system or an Aerogen nebulizer).

[0102] A related aspect of the present invention is a pharmaceutical composition for vaccination at the level of the respiratory tract (as described above), or of the central nervous system, this composition being administered by inhalation.

[0103] Advantageously, this pharmaceutical composition for vaccination is for the activation of macrophages and / or dendritic cells.

[0104] This advantageously allows the treatment of tumors, infectious diseases or inflammatory diseases in the lung or central nervous system.Furthermore, according to a further associated aspect of the present invention, this pharmaceutical composition (for activating macrophages and / or dendritic cells) administered by inhalation is particularly advantageous in the case of metastatic cancer: either the primary tumor is located in the lung or the central nervous system and the composition according to the present invention stimulates the activity of the immune system at the level of metastases outside the lung or the central nervous system (abscopal effect), or the primary tumor is located outside the lung or the central nervous system and a metastasis is found at the level of the lung or the central nervous system: the macrophages are activated there, and the immune system is then stimulated to attack the primary tumor and / or metastases outside the lung or the central nervous system.

[0105] Furthermore, according to another related aspect of the present invention, this pharmaceutical composition (for activating macrophages and / or dendritic cells) administered by inhalation is particularly advantageous in combination with other therapies such as radiotherapy, chemotherapy, targeted therapies and immunotherapies, preferably immune brake inhibitors, preferably anti-PDl, anti-PD-Ll, anti-CTLA-4 and combinations thereof, in cancer.

[0106] Another aspect of the present invention relates to this pharmaceutical composition for use in (somatic) gene therapy, in which the active ingredient is a nucleic acid (DNA or messenger RNA, antisense or interfering RNA; siRNA), according to one alternative, encoding a peptide to be synthesized by the patient (including via genetic editing) and, according to the other alternative, intended to reduce the expression of a deleterious factor in the patient. In the context of the present invention, according to the first alternative, "gene therapy" preferably means the administration to the patient of any genetic construct which corrects a gene expression defect, whether qualitative (e.g. replacement therapy, in the case of a deleterious mutation) or quantitative (e.g. correction of a deleterious over-expression of a gene). In the context of the present invention,Gene therapy is advantageously applied for the treatment of a disease chosen from cystic fibrosis (active principle DNA, or messenger RNA, encoding CFTR), severe immunodeficiency (SCID; active principle, DNA, or messenger RNA, encoding adenosine deaminase; ADA), lysosomal diseases chosen from Gaucher disease (active principle DNA, or messenger RNA, encoding glucocerebrosidase), Niemann-Pick disease (active principle DNA, or messenger RNA, encoding SMPD1 or NPC1 or NPC2, depending on the subtypes of the disease), Tay-Sachs disease (active principle DNA, or messenger RNA, encoding hexosamindase A), metachromatic leukodystrophy (active principle DNA, or messenger RNA, encoding arylsulfatase A), Krabbe disease (active principle DNA, or messenger RNA, encoding Galactosylceramidase), Canavan disease (active principle DNA, or messenger RNA, encoding aspartoacylase), X-linked adrenoleukodystrophy (active principle DNA,or messenger RNA, encoding the peroxisome ABC transporter), Alexander disease (active principle DNA, or messenger RNA, encoding GFAP), Hunter syndrome (active principle DNA, or messenger RNA, encoding MPS II), Hurler syndrome (active principle DNA, or messenger RNA, encoding MPS-IH), Pompe disease (active principle DNA, or messenger RNA, encoding acid alpha glucosidase), Danon disease (active principle DNA, or messenger RNA, encoding LAMP2), Fabry disease (active principle DNA, or messenger RNA, encoding alpha-galactosidase A), Schindler disease (active principle DNA, or messenger RNA, encoding alpha-galactosidase B), hemophilia (Coagulation Factor VIII or IX), and spinal muscular atrophy (SMA; active principle DNA, or messenger RNA, encoding SMN1, for other SMA, active principle DNA, or messenger RNA, encoding VAPB, DYNC1 H1, BICD2, UBA1). Preferably, gene therapy is somatic. According to the other advantageous alternative,the active ingredient of the composition according to the present invention is a nucleic acid intended to reduce the expression of a gene (antisense oligonucleotide, siRNA), which is particularly advantageous for pulmonary infectious diseases (e.g. respiratory viruses, including SARS-type viruses such as Covid19) or respiratory syncytial virus; RSV), for infections of the central nervous system (e.g. viral meningitis) or for certain degenerative diseases of the nervous system or affecting the cognitive performance of the patient (Huntington's disease; target, the expression of the mutated HTT gene in heterozygous patients; Down syndrome; targets, cystathionine beta-synthase (CBS) and / or DYRK1 A).,

[0107] Other features and advantages of the present invention will be drawn from the following non-limiting description, and with reference to the drawings and examples.

[0108] Exe m pies. -

[0109] It is understood that the present invention is in no way limited to the embodiments described above and that many modifications may be made thereto without departing from the scope of the appended claims.

[0110] Example 1 - Lipid vehicles based on soy lecithin

[0111] The inventors first tested a lipid mixture of soy lecithin, cholesterol and DSPE-PEG, in a soy lecithin lipid: cholesterol: DSPE-PEG molar ratio of 8:1:0.05. Briefly, the lipids were first weighed in a container, then dissolved, here in a dichloromethane:methanol mixture (50:50; V:V) with stirring for 30 minutes. When solubilization is complete, the solvent is evaporated (rotovapor) at 30°C under a pressure of 16000 Pa for 1 hour, leading to the formation of the lipid film. The film is then subjected to an air flow, to remove traces of residual solvent. An aqueous solution (water or buffer) is then added to the film and the mixture is stirred for approximately 30 minutes at a temperature above the highest phase transition temperature characterizing the lipids in the mixture.The suspension is then extruded, here using Liposofast LP-50 (4 extrusions through 3 filters - 1, 0.4 and 0.1 μm, at 600 psi; so approximately 4.1 10. 6 Pa) to finally obtain the lipid vehicles.

[0112] The obtained lipid vehicles had a mean diameter (Dh) of the order of 110 to 150 nm and polydispersity indices (Pdl) of approximately 0.1 to 0.2; the zeta potential was neutral to slightly negative in the absence of DSPE-PEG and strongly negative in its presence (-20 to -35 mV). For zeta potential measurement, the sample was diluted in a 0.009% NaCl (mass / volume) solution so as to obtain attenuator and conductivity values ​​suitable for a good measurement.

[0113] This formulation was used for the entrapment of a pharmaceutical agent, here, 2'3'-cGAMP (cgAMP) with entrapment efficiencies (EE%) of 30 to 45% obtained. The best results were obtained for compositions involving a higher cGAMP / lipid ratio (0.08 mole / mole of lipid). These formulations, in addition to presenting low EE% values, did not show any improvement in STING pathway activation in THPl-dual monocytes (THPl-Dual™ Cells, invivogen #thpd-nfis) in vitro.

[0114] Example 2 - DOTAP / cholesterol-based lipid vehicles

[0115] The inventors used a cationic lipid DOTAP (lipid 1 according to the present invention) with cholesterol (lipid 2) at a concentration of 3.9 mg / mL and 1.4 mg / mL respectively and incorporating 100 g / mL of 2'3'-cGAMP (cGAMP). These vehicles were prepared in the absence (composition 1) or in the presence of DSPE-PEG at 0.05 mole equivalents (relative to cholesterol).

[0116] The physical characteristics are shown in the table below: Furthermore, the inventors obtained EE% close to 100%. In vitro tests of STING pathway activation in THPl-dual monocytes (THF 1 - Dual™ Cells, invivogen #thpd-nfis) showed a 4-fold increased efficacy for both compositions, compared to the same concentration of free cGAMP. Similarly, the inventors verified in vitro that DOTAP / cholesterol-based formulations better protect cGAMP from enzymatic degradation by observing a better preservation of STING pathway activation in THPl-dual monocytes after in vitro incubation of lipid vehicles (vs free cGAMP) with the recombinant human ENPP1 enzyme (#6136-EN-010, biotechne, USA).

[0117] Although the addition of DSPE-PEG negatively affected the polydispersity index pdi, the inventors note that this addition is not too problematic.

[0118] Example 3 - effect of adding DSPE-PEG

[0119] The inventors then tested different proportions of DSPE-PEG (Figure 1). Composition 2 above is retained. Other compositions (3-5) are developed with 0.1, 0.5 and 1 mole equivalent of DSPE-PEG. The cGAMP loading rate is then reduced from 1.88% (composition 2), then 1.55%, then 0.94% and finally 0.60%.

[0120] Composition 3 shows properties (diameter, pdi, % encapsulation) very close to composition 2. The particle diameter is reduced to 115 then 55 nm at compositions 4-5, while the pdi increases to about 0.26. The zeta potential drops to almost 0 for composition 5. EE% drops to about 89% then 62% at compositions 4-5. Composition 3 still allows very good activation of THP1-dual macrophages in vitro, reduced for 4, and the effect is almost completely lost for 5.

[0121] On the other hand, compositions 3 and 4 offer better protection of cGAMP against enzymatic degradation by observing a better preservation of the activation of the STING pathway in THP1-dual monocytes after in vitro incubation of lipid vehicles (vs free cGAMP) with the recombinant human ENPP1 enzyme. Example 4 - functionalization

[0122] Compositions 2 and 4 (0.05 and 0.5 mole equivalents of DSPE-PEG) were used as a basis, with replacement of either all of the DSPE-PEG by DSPE-PEG-mannose (compositions 6 and 7), or half (compositions 8 and 9).

[0123] The diameter and the pdi index increase sharply for composition 7. The pdi index also increases for composition 9. The EE% remains close to 100% each time.

[0124] The inventors then performed an activation test of immunosuppressive macrophages (M2 type), differentiated with IL-10 and IL-4) or tumor-associated macrophages (TAMs-like) LLC1 (Lewis lung carcinoma, ATCC CRL1642) obtained according to a protocol similar to that described in Kwart et al. (Cell Reports 41 (2022) 1 1 1769), from bone marrow-derived macrophages (BMDMs). Although both compositions induce strong macrophage activation, composition 6 shows much greater activation than composition 2 on both types of macrophages (M2 and TAMs-like), as illustrated in Figure 2 for M2.

[0125] The inventors also observed that composition 6 administered endotracheally to healthy mice induced activation of alveolar macrophages and dendritic cells.

[0126] Example 5 - increasing the load rate

[0127] In order to increase the cGAMP loading rate in lipid vehicles, the amount of DOTAP was reduced to 1.5 mole equivalents relative to cholesterol and the amount of both lipids was reduced to 1 / 10 relative to composition 1 for compositions 10-16. Different concentrations of cGAMP were tested from 100 (composition 10) to 150, 200, 250, 300, 360, and 720 g / mL. The loading rate increased from 16.86 (composition 10) to 23.33; 28.86; 33.65; 37.83; 42.20 and 59.35%. The DOTAP / cGAMP ratio decreased from 3.5 / 1 (composition 10) to 2.35 / 1; 1.76 / 1; 1.4 / 1; 1.2 / 1, 1 / 1 and 0.5 / 1. The particle diameter increases slightly up to composition 12 (loading rate 28.86%), then strongly when the cGAMP concentration is increased. The pdi is constant up to composition 12, then increases strongly. Conversely, the zeta potential drops beyond composition 12. The EE% remains close to 100% up to this composition.STING pathway activation in dual THP1 monocytes remains significantly higher than for free cGAMP.

[0128] Starting from composition 12, the addition of DPPC to DOTAP (here, in a 2:1 molar ratio) is possible, but the cGAMP loading rate is reduced. The activation of the STING pathway in dual THP1 monocytes remains significantly higher than for free cGAMP, but is reduced by half at low cGAMP concentrations. However, this kind of formulation is interesting because it is associated with increased stiffness (also reflected by an increased Tm of the lipid series), which is useful in certain modes of administration.

[0129] Example 6 - Improving the stiffness properties of formulations

[0130] The inventors observed an instantaneous release of cGAMP trapped in compositions when these formulations were diluted in a large volume of PBS. In order to overcome this limitation, the inventors prepared new formulations by adding an excipient with a higher Tm (DPPC). Starting from composition 12, DPPC was added at 3, 5, 8 and 10 mole equivalents relative to cholesterol for compositions 13, 14, 15 and 16, respectively. The results demonstrated that increasing the amount of DPPC decreases the fraction of cGAMP released instantly after a 200X dilution in PBS. Compositions 12, 13, 14, 15 and 16: 100%; 59.48; 69.57; 47.83; 32.18%.

[0131] Example 7 - Use of an ionizable lipid

[0132] Starting from composition 14, the inventors replaced DOTAP with DODMA in the same molar proportions. The protocol for preparing the lipid vehicles described above was adapted so that the lipid film rehydration buffer was a 0.01 M acetate buffer at pH 5.0 in order to ionize the DODMA and thus promote the trapping of cGAMP. The EE% was similar to that obtained for condition 14. Example 8 - combination of a composition with a standard treatment

[0133] A composition of the present invention was administered endotracheally in combination with an anti-PD1 monoclonal antibody (clone RPM1-14 #BP0146, BioXcell) to mice bearing lung tumors. The treatment combination led to superior activity compared to the corresponding monotherapies.

[0134] Example 9 - formulation with other STING agonists and other active ingredients The inventors have produced several formulations according to the following compositions (molar ratios):

[0135] DPPC / DOTAP / AgoSting / cholesterol / DSPE-PEG-Mannose.

[0136] For each of the compositions, the diameters remain within the acceptable ranges, and some compositions result in larger particles.

[0137] The EE% values ​​remained high each time (at least 87%), almost always above 95% (or even 100%). In addition, the inventors tested the encapsulation of rifampicin in a DPPC / DOTAP / Chol / Rifampicin / DSPE-PEG-Mannose formulation: 3 / 1.5 / 1 / 0.48 / 0.05; The amount of entrapped rifampicin was measured by absorbance at 479 nm, leading to an EE% of 79%. The diameter, Pdl and zeta potential values ​​were of the same order of magnitude as those previously described.

[0138] Similarly, a siRNA was encapsulated in the same type of liposome, with an EE% of 85% (measured at 260 nm) and size, Pdl and zeta potential values ​​of the same order of magnitude as presented above.

[0139] Example 10 - STING agonist activity

[0140] The inventors have noticed on macrophages (generated in vitro from murine bone marrow) that the formulations applied to immunosuppressive macrophages of type M2 induce a polarization into macrophages of type M1, pro-inflammatory. This is true both for 2'3' cGAMP and for the other STING agonists of Example 9, and even that this polarization is dose-dependent, and more marked for the more potent agonists, such as c-AlMP and, especially, c-AIMP difluor and ADU-S100. This polarization of immunosuppressive macrophages of type M2 into pro-inflammatory macrophages of type M1 was also observed in the lungs of mice bearing M109 lung tumors following pulmonary administration of a nanoparticle formulation containing 2'3' cGAMP.

[0141] The inventors then tested the activity of these encapsulated STING agonists in activating the STING pathway in THPl-dual monocytes.

[0142] Encapsulated 3'3' cGAMP, cAlMP, cAIMP-difluor, and di-AMP resulted in strong, even very strong, activations. Only c-di-GMP resulted in very weak activation, barely higher than the unencapsulated 2'3' cGAMP control. Example 1 1 - T cell proliferation

[0143] T lymphocytes from freshly isolated spleen cells of Balb / c mice were stimulated using beads decorated with anti-CD3 / anti-CD28 and IL-2 antibodies, with or without 2'3'cGAMP.

[0144] cGAMP strongly reduces CD3 / CD28- and IL-2-induced T cell proliferation and this effect is dose-dependent (minor at 1.38 pM; major at 22 pM).

[0145] Example 12 - Adaptation of the formulation.

[0146] In an attempt to counter the deleterious effect on lymphocyte proliferation, the inventors had the intuition and then tested a development: a larger size, here around 250 nm (via adaptation of the porosity of the extrusion filter, porosity of 0.4 pm.

[0147] The possible effect was verified at the level of proliferation of CD4+ lymphocytes and CD8+ lymphocytes.

[0148] The larger formulation gave the best results with approximately the same viability as the untreated condition at the cGAMP concentrations tested, 0.34, 1.38, 5.5, and 22 pM.

[0149] Example 13 - Nebulization tests

[0150] The inventors compared two compositions, one DOTAP / DPPC / Chol / cGAMP / DSPE-PEG-Man and the same one without DPPC, in nebulization tests using the Aerogen® solo and Ultra vibrating mesh nebulizers. The first formulation achieved a high loading rate and biological activity, but the second did not. In more detail, in the presence of DPPC, the particle diameter was not affected; here, around 140 nm, the PDI index did not increase, so to speak.The second formulation was more affected with a diameter increasing from 130 to 170 nm and the pdi index from 0.12 to 0.42, clearly indicating a lack of stability of this formulation during nebulization. These nebulization tests showed that, under these conditions, where the structures are preserved, the liposomes can be administered effectively in the deep lungs, with droplet sizes observed in the desired range (average diameter D4.3 of 5-6 µm) and acceptable deposition profiles in the cascade impactor (next generation impactor NGI).

[0151] Example 14 - other formulations

[0152] The inventors have taken up the above formulations, with the exception of DOTAP, therefore composed of DPPC / cholesterol / AgoSTING / DSPE-PEG-Mannose.

[0153] Two non-nucleotide, neutrally charged STING agonists were encapsulated, SR-717 and MSA-2.

[0154] Empty structures (without agonist) have a diameter of approximately 120 nm and a zeta potential of -2.7 mV. Structures incorporating MSA-2 had a slightly larger average diameter of 130 nm and a polydispersity (pdi) of 0.2 to 0.3, depending on the experiment, for a zeta potential of -1 to -2 mV, depending on the experiment. Structures incorporating SR-717 showed greater variability in size, with a diameter of either 100 nm or 160 nm and a polydispersity (pdi) of up to 0.38, for a zeta potential of -3.5 to -2.7, depending on the experiment. The EE% ranged from 70 to almost 90% for MSA-2, and from 40 to 55% for SR-717.

Claims

CLAIMS 1. Pharmaceutical composition comprising lipid vehicles formed from a series of lipids, said lipid vehicles being selected from the group consisting of liposomes, vesicles, micelles, lipoplexes, lipid emulsions, lipid nanocrystals, lipid microspheres, lipid nanoparticles, and mixtures thereof, said lipid vehicles comprising a pharmaceutical agent selected from the group consisting of An antimicrobial, preferably an antibiotic, a nucleic acid, a STING protein agonist or a STING protein antagonist, a Toll-like receptor ligand, an immunomodulant and / or a corticosteroid, a peptide, an antihypertensive, a bronchodilator or a mixture thereof, said composition being for administration by inhalation.

2. Pharmaceutical composition according to claim 1, in which the series of lipids forming the lipid vehicles has a phase transition temperature (Tm) of between 10°C and 80°C, preferably between 20°C and 60°C.

3. Pharmaceutical composition according to claim 1 or 2 in which at least one lipid has functionalization by a ligand of the mannose receptor (CD206), preferably for the activation of macrophages and / or dendritic cells.

4. Pharmaceutical composition according to claim 3, wherein said at least one lipid exhibits functionalization by a mannose receptor ligand (CD206) is a phospholipid having functionalization by a mannose receptor ligand (CD206), preferably a phospholipid-PEG having functionalization by a mannose receptor ligand (CD206), advantageously a DSPE-PEG having functionalization by a mannose receptor ligand (CD206).

5. A pharmaceutical composition according to claim 3 or 4, wherein said mannose receptor ligand (CD206) is selected from the group consisting of mannose, fucose, N-acetylglucosamine, N-acetylgalactosamine, glycoprotein, galactocomannan, aD-mannopyranoside, polymannose, anti-CD206 antibody, and mixtures thereof.

6. Pharmaceutical composition according to any one of claims 3 to 5, in which the lipid vehicles have a molar proportion of said at least one lipid having functionalization by a ligand of the mannose receptor (CD206) to the total lipids of between 0.01 and 0.2, preferably between 0.02 and 0.1, preferably between 0.03 and 0.

05.

7. Pharmaceutical composition according to any one of the preceding claims 3 to 6, in which the lipid vehicles have a molar proportion of said at least one pharmaceutical agent relative to said at least one lipid having functionalization by a ligand of the mannose receptor (CD206) of between 5 and 150.

8. Pharmaceutical composition according to any one of the preceding claims, wherein the lipid vehicles are lipid nanoparticles (LNPs), preferably liposomes or solid lipid nanoparticles (SLN) or nanostructured lipid carriers (NLC).

9. A pharmaceutical composition according to any preceding claim, wherein the pharmaceutical agent has an overall negative charge.

10. Pharmaceutical composition according to any one of the preceding claims, wherein the series of lipids comprises a first lipid being cationic and / or ionizable. 1 1. Pharmaceutical composition according to claim 10, in which the lipid vehicles have a molar proportion of said cationic and / or ionizable lipid relative to the pharmaceutical agent of between 30 and 0.1, preferably between 10 and 0.2, advantageously between 3 and 0.5, preferably between 2.5 and 0.8, preferentially between 2.4 and 1.

12. Pharmaceutical composition according to claim 10 or 11, wherein the series of lipids comprises a second lipid selected from the group of sterols, a phospholipid, a triglyceride, a diglyceride, a glycerophospholipid and a sphingomyelin, preferably a sterol or a phospholipid, very preferably a sterol.

13. Pharmaceutical composition according to claim 12, in which the lipid vehicles have a molar ratio of the first lipid to the second lipid of between 0.3 and 20, preferably between 0.5 and 10, preferably between 1 and 5 (moles of the first lipid: moles of the second lipid), said second lipid preferably being a sterol, such as cholesterol and / or a phospholipid such as DPPC and / or DSPC.

14. Pharmaceutical composition according to any one of the preceding claims, wherein the lipid vehicles have an average size, measured by dynamic light scattering (DLS), of between 50 and 200 nm, preferably between 60 and 190 nm, advantageously between 70 and 180 nm, preferentially between 80 and 180 nm, preferably between 90 and 180 nm, more preferably between 100 and 180 nm, preferably between 110 and 180 nm, advantageously between 120 and 180 nm, preferentially between 120 and 170 nm, preferably between 120 and 160 nm.

15. Pharmaceutical composition according to any one of the preceding claims 1 to 13, in which the lipid vehicles have an average size, measured by dynamic light scattering (DLS), of between 200 and 350 nm, preferably between 225 and 300 nm.

16. Pharmaceutical composition according to any one of the preceding claims 1 to 13, in which the lipid vehicles have an average size, measured by dynamic light scattering (DLS), between 350 and 1500 nm, preferably between 350 and 1400 nm, advantageously between 350 and 1300 nm, preferentially between 350 and 1200 nm, advantageously between 350 and 1100 nm, preferentially between 350 and 1000 nm, preferably between 350 and 900 nm, preferably between 350 and 800 nm, more particularly between 350 and 700 nm, advantageously between 350 and 600 nm, for example between 400 and 600 nm.

17. Pharmaceutical composition according to any one of the preceding claims, in which the lipid vehicles have a polydispersity index of between 0.05 and 0.9, preferably between 0.1 and 0.8, advantageously between 0.2 and 0.75, preferentially between 0.3 and 0.7, preferably between 0.4 and 0.6 measured by dynamic light scattering (DLS), preferably this polydispersity index being defined by the formula: where = is the average particle diameter, the quantity of particles with size Xi and xi being the diameter of the spherical particle.

18. Pharmaceutical composition according to any one of the preceding claims, in which the lipid vehicles have a zeta potential of between -60 mV and 100 mV, preferably between -40 mV and 80 mV, advantageously between -20 mV and 60 mV, preferably between 0 mV and 50 mV, preferably between 10 mV and 40 mV, advantageously between 10 mV and 30 mV, preferably measured by laser doppler electrophoresis, preferably in an aqueous solution of NaCl at 0.009% (mass:volume).

19. Pharmaceutical composition according to any one of the preceding claims, wherein said pharmaceutical agent is present in a loading rate of between 0.1 and 80% relative to the mass of the lipid vehicles, preferably between 1 and 70%, preferably between 10 and 60%, preferably between 15 and 50%, such as between 20 and 40% or between 25 and 20. Pharmaceutical composition according to any one of the preceding claims being in dry form, preferably intended to be dissolved or redispersed in a physiological aqueous solvent, or to be administered using a dry powder inhaler.

21. Pharmaceutical composition according to any one of the preceding claims 1 to 18 comprising from 50 to 98% of water (mass of water: total mass of the composition), preferably from 70 to 95% of water, or even from 80 to 90% of water.

22. Pharmaceutical composition according to any one of the preceding claims further comprising a buffer selected from the group consisting of phosphate, sulfonate (MES, TES, HERES, MOPS, PIPES, TAPS, TAPSO), acetate, bicine buffers, and / or at least one salt selected from the group consisting of inorganic salts and organic salts, and / or at least one surfactant selected from the group consisting of cholic acids and their salts, phospholipids (eg.phosphatidylcholines or lecithin, phosphatidylglycerols), lipids or triglycerides, sorbitan esters, polyethoxylated sorbitans, fatty acids, preferably lauric, palmitic, stearic, erucic or behenic acid, esters of these fatty acids, or derivatives of these fatty acids, such as salts, preferably chosen from magnesium stearate, sodium stearyl fumarate and sodium stearyl lactylate, sodium lauryl sulfate, magnesium lauryl sulfate, natural pulmonary surfactants; and sucroesters (sugar esters, for example esters between sucrose or glucose and fatty acids) and / or. a sugar, preferably selected from the group consisting of monosaccharides (such as glucose or arabinose), disaccharides (such as lactose, maltose, sucrose, dextrose, trehalose, maltitol and mixtures thereof or a bulking agent selected from polyols such as sorbitol, mannitol and xylitol), polysaccharides (such as dextran, chitosan, starch, cellulose, and its derivatives), oligosaccharides (such as cyclodextrin and dextrins), and / or at least one amino acid selected from the group consisting of histidine, leucine, isoleucine, threonine, lysine, valine, methionine, phenylalanine, mixtures thereof and derivatives thereof, such as acesulfame K or aspartame.

23. Pharmaceutical composition according to any one of the preceding claims further comprising a metal ion, preferably chosen from Mn 2+ , the Co 2+ or Zn 2+, and / or at a content of at least 10 PPM.

24. Pharmaceutical composition according to any one of the preceding claims being administrable by nebulization and / or by pressurized inhalation and / or by dry powder inhalation and / or by gentle spray inhalation.

25. Pharmaceutical composition according to any one of the preceding claims being for the treatment of a cancer, preferably a metastatic cancer, preferably said metastasis being outside the lung or the central nervous system and the primary tumor being pulmonary or located at the level of the central nervous system, or said metastasis being pulmonary or located at the level of the central nervous system and the primary tumor being outside the lung or the central nervous system.

26. Pharmaceutical composition according to any one of the preceding claims 1 to 24 being for the treatment of an infectious disease at the level of the respiratory and / or systemic tract, or of the central nervous system.

27. Pharmaceutical composition according to any one of the preceding claims 1 to 24 comprising a nucleic acid encoding one or more epitope(s), or a peptide comprising one or more epitopes, said pharmaceutical composition being for vaccination.

28. Pharmaceutical composition according to any one of the preceding claims 1 to 24 comprising a nucleic acid being for gene therapy and / or enzyme replacement, said nucleic acid being a DNA or messenger RNA molecule, said gene therapy preferably being somatic.

29. A process for obtaining the pharmaceutical composition according to any one of the preceding claims comprising the steps of solubilizing the lipids in an organic solvent, evaporating the solvent so as to form a lipid film, rehydrating with a solution containing the pharmaceutical agent and extruding, preferably a second step of bringing a solution containing the pharmaceutical agent into contact with the extruded lipids, said lipids comprising a neutral lipid at pH 7.0 and positively charged at pH 5, and said rehydration step being carried out at a pH below 7.

0.

30. The method of claim 29 further comprising the step of inserting a PEG-derivatized lipid and, advantageously, further by a mannose receptor ligand (CD206), into the extruded pharmaceutical composition.

31. Process for obtaining the pharmaceutical composition according to any one of the preceding claims 1 to 28, comprising the steps of solubilizing the lipids and the pharmaceutical agent in an organic solvent or in a mixture of organic solvents, of injecting the organic solvent (or into a mixture of organic solvents) into an aqueous solution which may contain a (mixture of) surfactant(s), according to defined injection and stirring speeds, of removing the organic solvent(s), by evaporation or dialysis for example, preferably of inserting a lipid derivatized with a ligand of the CD206 receptor, 32. Method for obtaining the pharmaceutical composition according to any one of the preceding claims 1 to 28 by microfluidic technologies during which a solvent (or mixture of solvents) containing the lipids and the pharmaceutical agent is mixed, at a controlled speed, with an aqueous solution which may contain one or more surfactants, using a device having microchannels.

33. Process for obtaining the pharmaceutical composition according to any one of the preceding claims 1 to 28 by freezing-thawing method of multilamellar vesicles (MLV) or monolamellar vesicles (SUV).

34. Process for obtaining the pharmaceutical composition according to any one of the preceding claims 1 to 28 by double centrifugation method.

35. Process for obtaining the pharmaceutical composition according to any one of the preceding claims 1 to 28 by high pressure homogenization of multi-lamellar vesicle (MLV) suspension obtained after rehydration of lipid film.

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