Inhalation pharmaceutical composition

Abstract

An inhalation pharmaceutical composition containing a lipid carrier containing a drug, its therapeutic use, and a method for producing the same.

Description

Technical Field

[0001] The present invention relates to a lipid composition for protecting a drug, which is intended to be administered to the respiratory system of a patient.

Background Art

[0002] Formulations in which a drug is encapsulated in a lipid are known.

[0003] In particular, it is known to use lipids, especially cationic lipids, selected based on the nature of the drug to be encapsulated and the type of carrier to be obtained for this purpose. However, some of these lipids exhibit clinical toxicity, thus limiting the options (quantity, type). Furthermore, the size of the liposome (including size dispersion), the stability of the composition in a biological medium, the drug encapsulation ability, or the ability to release to a desired site are all factors that are difficult to optimize, and it is particularly difficult to optimize them simultaneously.

[0004] Ji et al. have described using liposomes to enhance the activation of the STING (Stimulator of Interferon Genes) pathway (in 2023). This approach was designed based on cyclic dinucleotides protected by a lipid structure. Various liposome formulations based on DOTAP (1,2-dioleoyl-3-trimethylammonium propane), cholesterol, and polyethylene glycol (PEG)-derived phosphoethanolamine were developed. A good encapsulation rate was obtained when the molar ratio of DOTAP / agonist was 10 - 20, but the encapsulation efficiency was low when the ratio was 2.5, which is attributed to the too low positive charge density.

[0005] Similarly, at such low concentration ratios, the enhancement of the biological effect by liposomes is lost. Unfortunately, due to the low encapsulation level, a large amount of liposomes will be administered to the patient, resulting in a risk of toxicity. This is particularly significant in the case of a large dose of the cationic lipid present in large amounts in the liposomes of this study.

[0006] Furthermore, altering the compound content is not easy. This is because multiple physicochemical properties of the liposomes, such as size, size dispersion, and charge potential, must be adjusted to values ​​acceptable for administration to patients, and the stability of the formulation must be ensured. In other words, increasing the amount of one component in a formulation can sacrifice other components and significantly affect the balance. The inventors found that this balance depends on various concentration ratios and can be greatly affected even after seemingly minor adjustments to the concentration. [Overview of the Initiative] [Problems that the invention aims to solve]

[0007] Therefore, the inventors recognized that there is still a need for improved formulations that will benefit patients. [Means for solving the problem]

[0008] A first aspect of the present invention relates to a pharmaceutical composition comprising a lipid carrier formed from a series of lipids, comprising a drug selected from antimicrobial agents, nucleic acids, STING protein agonists or antagonists, Toll-like receptor ligands, immunomodulators and / or corticosteroids, peptides, antihypertensive agents, bronchodilators, or mixtures thereof, and intended for inhalation administration.

[0009] Preferably, the pharmaceutical composition can be administered by nebulizer administration and / or pressurized inhalation and / or dry powder inhalation and / or soft mist inhalation.

[0010] Related aspects of the present invention relate to pharmaceutical compositions for the treatment of metastatic cancer, preferably when the metastases are outside the lungs or central nervous system and the primary tumor is pulmonary or located in the central nervous system, or when the metastases are pulmonary or located in the central nervous system and the primary tumor is outside the lungs or central nervous system.

[0011] Another related aspect of the present invention relates to pharmaceutical compositions for the treatment of infections of the respiratory and / or systemic routes or the central nervous system.

[0012] A related aspect of the present invention relates to a pharmaceutical composition for vaccination comprising a nucleic acid encoding one or more epitopes, or a peptide containing one or more epitopes.

[0013] Related aspects of the present invention relate to pharmaceutical compositions comprising nucleic acids for gene therapy and / or enzyme supplementation, wherein the nucleic acid is a DNA or messenger RNA molecule, and the gene therapy is preferably somatic cell gene therapy.

[0014] Related aspects of the present invention relate to a method for obtaining a pharmaceutical composition. A step of dissolving lipids in an organic solvent, A step of evaporating the solvent to form a lipid film, A step of rehydrating with a solution containing the drug, and Includes a homogenization and / or extrusion process (optional), Preferably, the process includes a second step of contacting the extruded lipid with a solution containing the drug, Preferably, the lipids include lipids that are neutral at pH 7.0 and positively charged at pH 5, and the rehydration step is carried out at a pH of less than 7.0.

[0015] In one variation, the drug is dissolved with lipids in an organic solvent before evaporation and is not added during the rehydration step. This is especially true if the drug is hydrophobic and / or an uncharged molecule at pH 7, such as MSA-2. [Brief explanation of the drawing]

[0016] [Figure 1] We will compare the stability of different lipid structures. [Figure 2] This study compares the effects of functionalization on macrophage activation. [Modes for carrying out the invention]

[0017] The inventors have successfully developed a lipid-based formulation that enables high encapsulation of one (or more) agents while maintaining advantageous physicochemical properties suitable for clinical use.

[0018] Thus, a first aspect of the present invention relates to a pharmaceutical composition comprising a lipid carrier, comprising at least one antibacterial agent (such as an antibiotic, antibacterial agent or antifungal agent), nucleic acid, at least one STING protein agonist or at least one STING protein antagonist, at least one toll-like receptor ligand, immunomodulator, peptide, bronchodilator, corticosteroid, antihypertensive agent, and mixtures thereof (excluding combinations of STING agonists and STING antagonists), and being for inhalation.

[0019] Preferably, the inhalation pharmaceutical composition is intended for the activation of macrophages and / or dendritic cells.

[0020] Preferably, the lipid carrier forming the inhalation pharmaceutical composition comprises at least one lipid functionalized with a ligand for the mannose receptor (CD206).

[0021] The lipid carrier is preferably selected from the group consisting of liposomes, vesicles, micelles, lipoplexes, lipid emulsions, lipid nanocrystals, lipid microspheres, lipid nanoparticles and mixtures thereof.

[0022] In the present invention, "by inhalation" includes nasal administration (e.g., to reach the nasal cavity, central nervous system and / or systemic circulation; e.g., for tumor treatment, gene therapy, treatment of inflammation or infection) as well as pulmonary administration (e.g., for treating diseases such as inflammation, infection or tumors), and also includes nasal administration aimed at deposition in both the nose and the lungs.

[0023] In the present invention, "peptide" preferably refers to any chain composed of at least two amino acids linked by peptide bonds. Suitable peptides are proteins (including enzymes), antigens, antibodies (or antibody fragments, nanobodies).

[0024] Preferably, the 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, oligonucleotides (based on DNA and / or RNA), and mixtures thereof. Preferably, the nucleotide, sugar, or phosphodiester bond can be modified. For example, when the nucleic acid is mRNA, uridine can be replaced or partially replaced with, for example, pseudouridine. Adenosine or cytosine bases, like the sugar (e.g., at the 2' position), can preferably be methylated. Peptide nucleic acids, morpholinos, and / or phosphorothioate bonds may replace some or all of the phosphodiester bonds. Alternatively, when the nucleic acid contains a CpG motif intended to stimulate an immune response, cytosine is preferably not methylated.

[0025] Preferably, the 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-2118, GSK-3745417, BMS-986301, SB-11285, IMSA-101, SYN-STING (SYNB1891), BI-1387446, TAK-676, E-7766 exoSTING, SNX281, HG-381, DN-015089, PC7A, DiABZl, cyclic diguanosine monophosphate (cyclic diguanylic acid monophosphate), MSA-2, Ulevostinag, SB 11285, IMSA 101, Dazostinag, BMS-986301, BI 1387446. Preferably, this group further consists of cAIMP, cAIMP-difluor, 3'-3'-cGAMP, c-di-AMP, and SR-717, and mixtures thereof, and more preferably 2'3'-cGAMP.

[0026] Alternatively, the drug comprises at least one STING protein antagonist selected from the group consisting of C-176, H-151, Inh-54, RU.521, H-8, A151, SA-1, GSK'932, SN-011, and mixtures thereof.

[0027] Preferably, the antibacterial agent (antibiotic) is an aminoglycoside (e.g., amikacin, gentamicin, kanamycin, neomycin, netylmycin, tobramycin, paromomycin, streptomycin), a carbapenem (e.g., ertapenem, doripenem, cilastatin, meropenem), or a cephalosporin (e.g., cefadroxil, cefazolin, cefradin, cefapillin, cephalothin, cephalexin, cefaclor, cefoxitin, cefotetan, cephamandol, cefmetazole, cefonisid, loracalbef, cefprodil, cefuro). (Xime, cefixime, cefdinir, cefditoren, cefoperazone, cefotaxime, cefpodoxam, ceftazidime, ceftibuten, ceftizoxime, moxalactam, ceftriaxone, cefepime, cephthaloline fosamil, ceftobiprole), glycopeptides (e.g., teicoplanin, vancomycin, teravancin, dalbavancin, oritabancin), lincosamides (e.g., clindamycin, lincomycin), lipopeptides (e.g., daptomycin), macrolides (e.g., azithromycin, clarithromycin, erythromycin) Romycin, roxithromycin, telithromycin, spiramycin, fidaxomycin), monobactams (e.g., aztreonam), penicillins (amoxicillin, ampicillin, azurocillin, dicloxacillin, flucloxacillin, mezlocillin, methicillin, nafcillin, oxacillin, penicillin G or V, piperacillin, temocillin, ticalcillin), polypeptides (e.g., bacitracin, colistin, polymyxin B), quinolones and fluoroquinolones (e.g., ciprofloxacin, enoxacin, gatifloxacin, getifloxacin, gynoquinolone) Mifloxacin, levofloxacin, lomefloxacin, moxifloxacin, nadifloxacin, nalidixic acid, norfloxacin, ofloxacin, trovafloxacin, glepafloxacin, sparfloxacin, temafloxacin), sulfonamides (e.g., mafenide, sulfacetamide, sulfadiazine, silver sulfadiazine, sulfadimethoxine, sulfamethizol, sulfamethoxazole, sulfaniramide, sulfasalazine, sulfisoxazole, trimethoprim-sulfamethoxazole (cotrimoxazole),The drug is selected from the group consisting of sulfanamide chrysoidine, tetracyclines (e.g., demeclocycline, doxycycline, metacycline, minocycline, oxytetracycline), or other classifications (e.g., anti-mycobacterial activity: capreomycin, cycloserine, ethambutol, ethionamide, isoniazid, pyrazinamide, rifampicin, rifabutin, rifapentin, streptomycin).

[0028] Preferably, the phase transition temperature (Tm) of the lipid series forming the lipid support is -30 to 100°C, preferably 1 to 90°C, more preferably 10 to 80°C, 20 to 70°C, for example 20 to 60°C.

[0029] Preferably (alternatively or additionally), the melting points of the series of lipids forming the lipid carrier are -30 to 180°C, preferably 1 to 160°C, more preferably 10 to 155°C, and even more preferably 20 to 150°C, for example, 20 to 80°C.

[0030] These physicochemical properties of the lipid series improve performance, particularly during pulmonary administration by nebulizer or inhalation, increasing drug release to target tissues and / or cytoplasmic release and / or half-life, and / or decreasing drug release to non-target tissues. In particular, the inventors have found that a high phase transition temperature (Tm) of the lipid series, for example, above 10°C or 20°C, is associated with increased rigidity and is advantageous, at least in certain formulations or methods of administration to patients.

[0031] Preferably, at least one lipid is functionalized with a ligand for the mannose receptor (CD206), and preferably it is a phospholipid functionalized with a ligand for the mannose receptor (CD206).

[0032] More preferably, at least one lipid, preferably a phospholipid, is derivatized with polyethylene glycol (PEG).

[0033] The molecular weight of PEG is preferably 500 to 20 kDa, for example, 1 to 10 kDa or 2 to 5 kDa.

[0034] Preferably, the PEG-derived lipid, preferably a phospholipid, and more preferably DSPE-PEG, is functionalized with a ligand for the mannose receptor (CD206).

[0035] Preferably, 10-100% (molar equivalent), preferably 20-90%, more preferably 30-80%, and even more preferably 40-50% of the PEG-derived lipid is further functionalized with a mannose receptor (CD206) ligand. Therefore, all or part of the PEG-derived lipid remains unfunctionalized. The inventors have found that dual optimization of PEG content and CD206 receptor ligand content is advantageously achieved by this method. When only a portion of the PEG is functionalized with the CD206 receptor ligand, the object of the present invention can be achieved by functionalizing only a portion of a single PEG-derived lipid (preferably a single phospholipid) or by using multiple lipids containing at least one PEG-derived phospholipid. Some of these PEG-derived molecules remain unfunctionalized.

[0036] This improves the ligand's ability to bind to its receptor. Furthermore, PEG stabilizes the lipid carrier.

[0037] Preferably, the ligand for the mannose receptor (CD206) is mannose, fucose, N-acetylglucosamine, N-acetylgalactosamine, glycoprotein, galactomannan, α-D-mannopyranoside (e.g., α-D-mannopyranose), α-L-fucopyranose-(1-3)-2-acetamide-2-deoxy-β-D-glucopyranose, methyl α-D-mannopyranoside, α-L-fucopyranose-(1-2)-β-D-galactopyranose-(1-4)-β-D -Selected from the group consisting of glucopyranose, methyl 2-acetamide-2-deoxy-α-D-glucopyranose, β-D-galactopyranose-(1-3)-(α-L-fucopyranose-(1-4))2-acetamide-d-deoxy-β-D-glycicopyranose, α-D-mannopyranose-(1-2)-α-D-mannopyranose, polymannose (4-50, preferably 5-30, and even more preferably 6-20 mannose residues), anti-CD206 antibody, and mixtures thereof.

[0038] In the present invention, these ligands may be modified; for example, N-acetylglucosamine may be deacetylated. For instance, N-acetylglucosamine may be incorporated in the form of chitin or chitosan, preferably as chitin or chitosan oligomers (e.g., less than 50 monomers, less than 40 monomers, less than 30 monomers).

[0039] Preferably, the lipid carrier is lipid nanoparticles (LNP), and more preferably liposomes, solid lipid nanoparticles (SLN), or nanostructured lipid carriers (NLC).

[0040] In the present invention, "SLN" preferably refers to colloidal particles (generally with a diameter of 50 to 500 nm) prepared from solid lipids, surfactants, and water that are solid at room temperature and body temperature using a high-speed or high-pressure homogenization method.

[0041] In the present invention, "NLC" preferably refers to SLN containing a liquid lipid (oil) fraction within a solid lipid, thereby creating structural defects in the solid lipid to make the crystal arrangement more disordered and improve the encapsulation of drugs. NLC is prepared by the same method as SLN.

[0042] In the present invention, "liposome" preferably refers to a spherical lipid vesicle (generally 50-500 nm in diameter) consisting of one or more lipid bilayers. Liposomes are obtained by emulsifying natural or synthetic lipids in an aqueous medium using methods such as lipid membrane hydration, ethanol injection, or microfluidics.

[0043] Preferably, the drug as a whole has a negative charge.

[0044] In fact, the inventors succeeded in incorporating large quantities of this type of drug, even in the molar proportion of cationic (or protonable, see below) lipids that may be present.

[0045] Therefore, when the drug as a whole has a negative charge, cationic lipids or protonable lipids are preferably incorporated.

[0046] In contrast, when the drug is uncharged, cationic lipids are preferably not added, or if added, their content is less than 10 mol% (number of moles of cationic lipids: total number of moles of introduced lipids and cholesterol), preferably less than 5 mol%, and more preferably less than 4, 3, 2, or 1 mol%.

[0047] Alternatively, the molar ratio of arbitrarily present cationic lipids to uncharged agents is less than 50% (moles of cationic lipids : moles of uncharged agents), 40%, 30%, 20%, less than 10%, and even less than 5%.

[0048] Therefore, the lipid series preferably includes a first lipid selected from the group consisting of cationic lipids, neutral lipids, anionic lipids, phospholipids, triglycerides, diglycerides, glycerophospholipids, sphingomyelin, fatty acids, and fatty acid salts, and / or a second lipid selected from the group consisting of cationic lipids, neutral lipids, anionic lipids, phospholipids, triglycerides, diglycerides, glycerophospholipids, sphingomyelin, fatty acids, and fatty acid salts, and / or a third lipid selected from the group consisting of cationic lipids, neutral lipids, anionic lipids, phospholipids, triglycerides, diglycerides, glycerophospholipids, sphingomyelin, fatty acids, and fatty acid salts.

[0049] Preferably, especially when the drug as a whole has a negative charge, the pharmaceutical composition comprises a first lipid which is a cationic lipid having at least one quaternary ammonium group, and / or a lipid having at least one protonable (secondary or tertiary) amine group (which does not have a negative charge at pH 7.0).

[0050] Preferably, the lipid having at least one quaternary ammonium group (and / or the first lipid) is selected from the group consisting of DOTAP (18:1 TAP) and other TAP derivatives (14:0 TAP, 16:0 TAP, 18:0 TAP, etc.), 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.

[0051] In the present invention, "protonable (secondary or tertiary) amine" preferably means an amine that exists mainly in a neutral form at pH 7.0 (for example, more than 50%, preferably more than 75%, and even more than 90% or 99%, in a neutral form at pH 7.0) and that exists mainly in a protonated form at pH 5.0 (for example, more than 50%, preferably more than 75%, and even more than 90% or 99%, in a protonated form at pH 5.0).

[0052] An example of a lipid containing a protonable amine is 1,2-dioleyloxy-3-dimethylaminopropane (DODMA). In the present invention, it is understood that fatty acids other than oleic acid, such as palmitic acid or stearic acid, may be used as the lipid chain, and that the dimethylaminopropane (DMA) derivative may be retained (e.g., 18:0 DMA, 16:0 DMA, 14:0 DMA, 18:1 DMA). Alternatively (or in addition), substitution at the aminopropane group, such as substitution at the methyl group of DMA, is also possible.

[0053] Preferably, the second lipid is a sterol, and is preferably selected from the group consisting of cholesterol and its esters (cholesteryl esters), and their glycosylated derivatives (β-D-glucosylcholesterol, galactosylcholesterol, BbGL-1), ox-18:2 cholesterol, desmosterol, stigmasterol, lanosterol, 7-dehydrocholesterol, dihydrolanosterol, zymosterol, lathosterol, and mixtures thereof. Cholesterol is particularly preferred.

[0054] Therefore, compositions containing DOTAP and cholesterol are particularly preferred, especially when the drugs as a whole have a negative charge.

[0055] Preferably, the phospholipids optionally present in the pharmaceutical composition (second, third, and / or fourth lipids in the absence of sterols) consist of natural, purified, or synthetic phospholipids such as phospholipids extracted from eggs or soybeans, phosphatidic acid, phosphatidylethanolamine (PE), lysophosphatidylethanolamine (LPE), phosphatidylcholine (PC), lysophosphatidylcholine (LPC), phosphatidylserine (PS), lysophosphatidylserine (LPS), phosphatidylglycerol (PG), or lysophosphatidylglycerol (LPG), and mixtures thereof, and are preferably selected from the group consisting of DSPE, DSPE-PEG, DMPE, DPPC, DSPC, DMPC, DLPC, and mixtures thereof.

[0056] In the context of the present invention, the fatty acid chains constituting phospholipids, and the fatty acid chains constituting cationic or protonable lipids, are preferably saturated, unsaturated, or polyunsaturated, provided that the melting point and / or phase transition temperature parameters measured for the total lipids used are within the range described below. Accordingly, the compositions of the present invention may advantageously contain unsaturated and saturated fatty acids.

[0057] The length of the fatty acid chain constituting the above phospholipids is advantageously 14 to 20 carbon atoms, for example, 16 or 18 carbon atoms. Shorter or longer chain lengths are possible (for example, at low content such as less than 20 mol% of the total lipid). However, this is conditional on the melting point and / or phase transition temperature parameters measured for the total lipid used remaining within the specified range.

[0058] Advantageously, in the presence of anionic lipids, phosphatidylinositol (PI) and its phosphorylated derivatives (PIP), phosphatidylserine (PS; e.g., DPPS), phosphatidic acid (PA; e.g., DPPA), phosphatidylglycerol (e.g., DPPG or DSPG), and mixtures thereof are selected.

[0059] Preferably, the composition contains phosphatidylcholine such as DPPC (1,2-dipalmitoylphosphatidylcholine).

[0060] Introducing phospholipids having long-chain saturated chains (e.g., 14-22 carbon atoms, preferably 16 or 18, e.g., palmitic acid or stearic acid) increases particle rigidity and improves properties during pulmonary administration, particularly with nebulizers or inhalation (including DPI form).

[0061] The inventors found that this lipid, having a polar head and two unsaturated hydrocarbon chains (dipalmitoyl and / or distearyl), increases the rigidity of the lipid structure and / or allows for a reduction in the (relative) DOTAP content. It is preferable to incorporate DPPC (and / or DOPC) with total lipids in a molar ratio of 0.1 to 0.95, preferably 0.2 to 0.90, more preferably 0.5 to 0.8, or 0.6 to 0.7 (number of moles of phosphatidylcholine, e.g., DPPC and / or DOPC:total number of moles of lipids).

[0062] Preferably, the molar ratio of the first lipid to the second lipid (or third lipid if there is no second lipid) in the lipid carrier of the pharmaceutical composition is 0.3 to 20, preferably 0.5 to 10, for example, 1 to 5.

[0063] Preferably, when the latter is introduced, the molar ratio of at least one lipid functionalized with a mannose receptor (CD206) ligand to the total lipid in the lipid carrier of the pharmaceutical composition is 0.01 (and may be further 0.005, 0.007, 0.008, or 0.009) to 0.2, preferably 0.02 to 0.1, and more preferably 0.03 to 0.05.

[0064] Advantageously, when the latter is introduced, the molar ratio of at least one agent to at least one lipid functionalized with a mannose receptor (CD206) ligand is 5 (or 10) to 150, preferably 20 to 100, and even more preferably 30 to 80.

[0065] Advantageously, the molar ratio of the cationic and / or ionizable lipid in the lipid carrier to the drug is 30 to 0.1, preferably 10 to 0.2, more preferably 3 to 0.5, preferably 2.5 to 0.8, and even more preferably 2.4 to 1, and is particularly suitable when the drug as a whole has a negative charge.

[0066] Preferably, the drug content (ratio to the lipid carrier mass) in the pharmaceutical composition is 0.1 to 80%, more preferably 1 to 70%, more preferably 10 to 60%, and even more preferably 15 to 50%, for example, 20 to 40% or 25 to 30%.

[0067] Advantageously, the average size of the lipid carrier is 50-200 nm, preferably 60-190 nm, more preferably 70-180 nm, preferredly 80-180 nm, preferably 90-180 nm, more preferably 100-180 nm, preferably 110-180 nm, even more preferably 120-180 nm, preferredly 120-170 nm, and preferably 120-160 nm.

[0068] Alternatively, the average size of the lipid carrier is 350-1500 nm (and may be 2000 nm), preferably 350-1400 nm, more preferably 350-1300 nm, preferredly 350-1200 nm, more preferably 350-1100 nm, preferredly 350-1000 nm, preferably 350-900 nm, preferably 350-800 nm, more specifically 350-700 nm, and even more preferably 350-600 nm, for example, 400-600 nm.

[0069] Alternatively, the average size of the lipid carrier is 200-350 nm, preferably 225-300 nm.

[0070] Preferably, the (average) particle size and average diameter (Dh) of the lipid carrier are measured by dynamic light scattering (DLS).

[0071] Advantageously, the polydispersity index (PDI) of the lipid carrier is 0.05 to 0.9, preferably 0.1 to 0.8, more preferably 0.2 to 0.75, preferably 0.3 to 0.7, and most preferably 0.4 to 0.6. The polydispersity index is preferably measured by dynamic light scattering (DLS). A suitable instrument for measuring particle size, PDI (see below), or zeta potential (see below; other instruments may also be used) is the Malvern Zetasizer nano ZS (Malvern Instruments SA, Worcestershire, UK).

[0072] Advantageously, in the context of the present invention, the polyvariance index is defined by the following formula:

number

[0073] Alternatively, the polydispersion index is 0.05 to 0.9, preferably 0.05 to 0.8, more preferably 0.05 to 0.7, more preferably 0.05 to 0.6, even more preferably 0.05 to 0.5, preferably 0.05 to 0.4, even more preferably 0.05 to 0.3, more specifically 0.05 to 0.25, preferably 0.05 to 0.2, for example 0.06 to 0.2, preferably 0.07 to 0.2, and even more preferably 0.08 to 0.2. The polydispersion index is measured by dynamic light scattering (DLS).

[0074] In fact, depending on the application, lipid carrier particles of the same size may be preferable, while in other applications, a bimodal or multimodal distribution may be preferable.

[0075] Preferably, the zeta potential of the lipid carrier is -60 to 100 mV, preferably -40 to 80 mV, more preferably -20 to 60 mV, preferably -5 to 55 mV, preferably 0 to 50 mV, and preferably 10 to 40 mV. Preferably, it is 10 to 30 mV and is preferably measured by laser Doppler electrophoresis. Preferably, this measurement is performed in a 0.009% (mass:volume) NaCl aqueous solution.

[0076] Advantageously, the agent is present in a mass ratio of 0.1 to 80%, preferably 1 to 70%, preferably 5 to 60%, preferably 10 to 50%, or 20 to 40%, relative to the mass of the lipid carrier.

[0077] Advantageously, this composition contains Mn to enhance the activity of the drug. 2+ Co 2+ or Zn 2+ It contains at least one metal ion, such as the following. Preferably, this metal ion is present in a content of more than 10 PPM (parts per million) (by weight), and the (mass of metal ions: total mass (dry) of the lipid composition) is preferably in the range of 100 PPM to 50% (by mass), more preferably 300 PPM to 10%, and more preferably 800 PPM to 1% (by mass).

[0078] Related aspects of the present invention relate to pharmaceutical compositions for inhalation in liquid form such as solutions, dispersions, or suspensions, or to pharmaceutical compositions in powder form intended to be redissolved and redispersed before administration, or in the form of dry powder for inhalation.

[0079] Advantageously, inhalation pharmaceutical compositions in liquid form can be frozen to ensure the stability of the drug during storage or distribution. This composition is thawed before administration, allowing for advantageous reconstitution of the lipid carrier. This ensures that the formulation is maximally active.

[0080] Advantageously, this inhalation pharmaceutical composition comprises at least one excipient selected from the following: A buffer selected from phosphates, sulfonates (MES, TES, HEPES, MOPS, PIPES, TAPS, TAPSO), acetates, 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 the following: Cholic acid and its salts, Phospholipids (e.g., phosphatidylcholine or lecithin, phosphatidylglycerol), Lipids or triglycerides, Sorbitan ester, polyoxyethylene sorbitan, Fatty acids, preferably lauric acid, palmitic acid, stearic acid, erucic acid, or behenic acid, esters or derivatives of these fatty acids, such as salts, preferably selected from magnesium stearate, sodium stearyl fumarate, sodium stearyl lactylate, sodium lauryl sulfate, and magnesium lauryl sulfate. Natural components of lung surfactants such as phospholipids or cholesterol; and Sugar esters (e.g., 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 (e.g., acesulfame K or aspartame), and / or The bulking agent is selected from the group consisting of sugars (preferably monosaccharides (e.g., glucose or arabinose), disaccharides (e.g., lactose, maltose, saccharose, dextrose, trehalose, maltitol, and mixtures thereof), or polyols such as sorbitol, mannitol, and xylitol), polysaccharides (e.g., dextran, chitosan, starch, cellulose, and derivatives thereof), and oligosaccharides (e.g., cyclodextrin and dextrin).

[0081] An advantageous option for inhalation pharmaceutical compositions is to ensure good storage stability by drying them into a powder form so that they can be dissolved or redispersed immediately before administration to the patient (e.g., within one hour). This composition is redissolved or redispersed in an aqueous solvent such as water, physiological saline, 0.9% NaCl solution (mass:vol), or a buffer such as PBS before administration, which advantageously reconstitutes the lipid carrier. This ensures that the formulation is maximally active.

[0082] Alternatively or additionally, the inhalation pharmaceutical composition according to the present invention is in a powder form that is dissolved or redispersed to ensure the stability of the drug during storage or distribution, and comprises at least one extender such as polyols and / or sugar alcohols such as sorbitol, mannitol, maltitol (sometimes considered a disaccharide) and xylitol, monosaccharides (glucose, arabinose), (crystalline) sugars, disaccharides (lactose, maltose, sucrose, dextrose, trehalose), polysaccharides (dextran, chitosan, starch, cellulose and its derivatives) The composition includes (for liquid compositions), or oligosaccharides (dextrin, cyclodextrin) and / or fatty acid salts (e.g., magnesium stearate), or fatty acids or their derivatives (e.g., esters) (e.g., lauric acid, palmitic acid, stearic acid, erucic acid, behenic acid), or phospholipids (e.g., lecithin, phosphatidylcholine, phosphatidylglycerol; preferably not incorporated into a lipid carrier), triglycerides (preferably not incorporated into a lipid carrier), sugar esters, and / or amino acids (see above for liquid compositions), and mixtures thereof. Dextran, trehalose, mannitol, and lactose are preferred.

[0083] Advantageously, lactose, trehalose, sucrose, or mannitol may be used as excipients (bulking agents). In this case, even in powder form, the composition may advantageously contain a buffer and further contain one or more of the compounds listed above, such as surfactants.

[0084] Preferably, the administered liquid form containing the pharmaceutical composition according to the present invention contains 50-99%, more precisely 99.9% (or 99.8%) water (mass of water: total mass of composition), preferably 70-98%, more precisely 80-95% (or 90%) water; in other words, the solution contains 1-50% (optionally 0.1 or 0.2-50%), preferably 2-30%, more precisely 5 or 10-20% of the pharmaceutical composition.

[0085] The administered liquid form comprising the inhalation pharmaceutical composition further comprises at least one buffer selected from the group consisting of phosphates, sulfonic acids (MES, TES, HEPES, MOPS, PIPES, TAPS, TAPSO), acetates, and bicine buffers, and / or at least one salt selected from the group consisting of inorganic salts (sodium chloride, calcium carbonate, sodium phosphate, or potassium phosphate), organic salts (e.g., sodium lactate, potassium citrate), and / or cholic acid and its salts (taurocholate, glycocholate), phospholipids (not incorporated into a lipid carrier), lipids (not incorporated into a lipid carrier), sorbitan esters (e.g., SPAN 85), polyoxyethylene sorbitan (e.g., Tween® 80), 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 (e.g., acesulfame K or aspartame, and mixtures thereof).

[0086] Another advantageous option for inhalation pharmaceutical compositions is in the form of a dry powder for inhalation and / or a form intended to be administered using a dry powder inhaler. This composition exhibits excellent powder dispersibility and aerosolization properties in air, thereby enabling the delivery of an appropriate dose of powder to the patient's respiratory system (inhalable powder). These properties include an aerodynamic size suitable for the administration route. This composition allows the lipid carrier to be favorably reconstituted in physiological fluids after the powder has deposited in the respiratory system, thereby ensuring that the formulation is maximally active.

[0087] Preferably, the geometric diameter and / or aerodynamic size for lung administration is less than 5 μm, preferably 0.5 to 5 μm, and more preferably 1 to 3 μm.

[0088] Preferably, the geometric diameter and / or aerodynamic size for nasal administration is greater than 5 μm, preferably 5 to 120 μm, more preferably 10 to 60 μm, even more preferably 20 to 40 μm, and preferably 20 to 30 μm.

[0089] Therefore, it is advantageous for the particle size and / or aerodynamic size of the pharmaceutical composition in powder form for inhalation to have a bimodal distribution, which enables nasal and pulmonary deposition through nasal administration.

[0090] In the context of this invention, "aerodynamic size" is understood to preferably mean a parameter that describes the aerodynamic behavior of a particle. Therefore, this parameter takes into account not only the geometric diameter (i.e., particle size) but also the density and shape of the particle.

[0091] Particle size (and geometric diameter) is determined by laser diffraction. Aerodynamic size (and aerodynamic diameter) is determined by impact tests (using cascade impactors) as described in pharmacopoeias (e.g., the European Pharmacopoeia).

[0092] Alternatively or additionally, the inhalation pharmaceutical composition according to the present invention is administered in dry powder form and comprises at least one extender containing polyols and / or sugar alcohols such as sorbitol, mannitol, maltitol (which may be considered a disaccharide), xylitol, monosaccharides (glucose, arabinose), disaccharides (lactose, maltose, sucrose, dextrose, trehalose), polysaccharides (dextran, chitosan, starch, cellulose and its derivatives), or oligosaccharides (dextrin, cyclodextrin) (crystalline). The composition comprises sugars 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 phospholipids (e.g., lecithin, phosphatidylcholine, phosphatidylglycerol; preferably not incorporated into a lipid carrier), triglycerides (preferably not incorporated into a lipid carrier), sugar esters, and / or amino acids (see above for liquid compositions) and at least one filler selected from the group consisting of mixtures thereof. Dextran, trehalose, mannitol, and lactose are preferred.

[0093] Lactose, trehalose, sucrose, or mannitol are used as excipients (bulking agents) to enhance the product.

[0094] Advantageously, when the pharmaceutical composition is intended to be administered to a patient in the form of a dry powder for inhalation, the excipients include at least one lipid or fatty acid-based substance (e.g., magnesium stearate, phospholipids, sucrose esters). This ensures good uniformity of the mixture and / or promotes favorable aerosolization of solid particles. Other excipients, such as leucine, are also advantageous due to their good aerodynamic properties.

[0095] It should be noted that some surfactants may contain fatty acids, and furthermore, phospholipids or triglycerides, which are also potential components of the lipid carrier of the present invention. However, these surfactants are not incorporated into the structure of the lipid carrier but are advantageously incorporated into the already formed dry formulation of the lipid carrier. In this case, even in powder form, the composition may advantageously contain a buffer and further contain one or more of the compounds listed above (e.g., surfactants).

[0096] Examples of drying methods include freeze-drying, spray drying, spray congealing or spray chilling, spray freeze-drying, and supercritical fluid drying.

[0097] The inventors have found that when the lipid carrier according to the present invention is freeze-dried, good results can be obtained by adding sucrose as a protective agent, preferably in a higher content than lipids, and preferably in a mass ratio of at least 2:1, 3:1, 5:1, and even about 10:1 (weight of sucrose:weight of total lipids).

[0098] Advantageously, the protective agent (sucrose) is added during the lipid membrane hydration process.

[0099] Advantageously, this pharmaceutical composition can be administered to the respiratory system, preferably by nebulizer administration and / or pressurized inhalation and / or dry powder inhalation and / or soft mist inhalation (e.g., using a Respimat® SofMist system or an Aerogen nebulizer).

[0100] Aspects related to the present invention relate to a pharmaceutical composition for vaccination in the respiratory or central nervous system, which is administered by inhalation.

[0101] Advantageously, this vaccine-containing pharmaceutical composition aims to activate macrophages and / or dendritic cells.

[0102] This will allow for advantageous treatment of tumors, infections, or inflammatory diseases of the lungs or central nervous system.

[0103] Furthermore, according to another relevant aspect of the present invention, this pharmaceutical composition administered by inhalation (for activating macrophages and / or dendritic cells) is particularly advantageous in the case of metastatic cancer: If the primary tumor is located in the lung or central nervous system, and the composition of the present invention stimulates immune system activity at metastatic sites outside the lung or central nervous system (abscopal effect), Alternatively, if the primary tumor is located outside the lungs or central nervous system, and metastases are found in the lungs or central nervous system, macrophages are activated there, stimulating the immune system to attack the primary tumor and / or the metastases outside the lungs or central nervous system.

[0104] Furthermore, according to another relevant aspect of the present invention, this pharmaceutical composition administered by inhalation (for activating macrophages and / or dendritic cells) is particularly advantageous in cancer when used in combination with other treatments such as radiotherapy, chemotherapy, targeted therapy and immunotherapy (preferably immune checkpoint inhibitors, preferably anti-PD1, anti-PD-L1, anti-CTLA-4, and combinations thereof).

[0105] Another aspect of the present invention relates to the pharmaceutical composition for use in (somatic cell) gene therapy, wherein the active ingredient is nucleic acid (DNA or messenger RNA, antisense or interfering RNA; siRNA) and is intended to encode a peptide synthesized by the patient (including by gene editing) and / or to reduce the expression of a harmful factor in the patient.

[0106] In the context of the present invention, according to the first alternative, “gene therapy” means administering to a patient any gene construct that corrects a gene expression defect, whether qualitatively (e.g., replacement therapy in the case of harmful mutations) or quantitatively (e.g., correction of harmful overexpression of a gene). In the present invention, gene therapy is advantageously a lysosomal storage disorder selected from cystic fibrosis (active ingredient: DNA or messenger RNA encoding CFTR), severe immunodeficiency (SCID; active ingredient: DNA or messenger RNA encoding adenosine deaminase (ADA)), Gaucher disease (active ingredient: DNA or messenger RNA encoding glucocerebrosidase), Niemann-Pick disease (active ingredient: DNA or messenger RNA encoding SMPD1, NPC1, or NPC2 depending on the disease subtype), Tay-Sachs disease (active ingredient: DNA or messenger RNA encoding hexosaminidase A), or (Messenger RNA), Metachromatic leukodystrophy (active ingredient: DNA or messenger RNA encoding arylsulfatase A), Krabbe disease (active ingredient: DNA or messenger RNA encoding galactosylceramidase), Canavan disease (active ingredient: DNA or messenger RNA encoding aspartoacylase), X-linked adrenoleukodystrophy (active ingredient: DNA or messenger RNA encoding peroxisome ABC transporter), Alexander disease (active ingredient: DNA or messenger RNA encoding GFAP), Hunter syndrome (active ingredient: MPS) DNA or messenger RNA encoding II), Hurler syndrome (active ingredient: DNA or messenger RNA encoding MPS-IH), Pompe disease (active ingredient: DNA or messenger RNA encoding acid α-glucosidase), Danon disease (active ingredient: DNA or messenger RNA encoding LAMP2), Fabry disease (active ingredient: DNA or messenger RNA encoding α-galactosidase A), Schindler's disease (active ingredient: DNA or messenger RNA encoding α-galactosidase B), hemophilia (coagulation factor VIII or IX), and spinal muscular atrophy (SMA; active ingredient: DNA or messenger RNA encoding SMN1).For other SMAs, the treatment is applied to diseases selected from the group consisting of active ingredients (DNA or messenger RNA encoding VAPB, DYNC1H1, BICD2, or UBA1). Preferably, the gene therapy is somatic cell gene therapy.

[0107] According to other favorable alternatives, the active ingredient of the composition according to the present invention is a nucleic acid (antisense oligonucleotide, siRNA) intended to reduce gene expression, which is particularly favorable against lung infections (e.g., respiratory viruses (including SARS-like viruses such as COVID-19)) or respiratory syncytial virus (RSV), central nervous system infections (e.g., viral meningitis), or certain degenerative neurological disorders or diseases affecting a patient's cognitive function (Huntington's disease; target: expression of mutated HTT gene in heterozygous patients; Down syndrome; target: cystathionine β-synthase (CBS) and / or DYRK1A).

[0108] Other features and advantages of the present invention will be understood from the following non-limiting description and reference to the drawings and embodiments. [Examples]

[0109] The present invention is not limited in any way to the embodiments described above, and it should be understood that many modifications are possible without departing from the scope of the appended claims.

[0110] <Example 1: Soy lecithin-based lipid carrier> The inventors first tested a lipid mixture of soy lecithin, cholesterol, and DSPE-PEG, setting the molar ratio of soy lecithin lipid:cholesterol:DSPE-PEG to 8:1:0.05. In short, the lipids were first weighed into a container and dissolved in a dichloromethane:methanol mixed solvent (50:50; V:V), and stirred for 30 minutes. Once dissolved, the solvent was evaporated at 30°C and 16,000 Pa for 1 hour (using a rotary evaporator) to form a lipid film. Next, the film was subjected to an airflow to remove any traces of residual solvent. Then, an aqueous solution (water or buffer solution) was added to the film, and the mixture was stirred for about 30 minutes at a temperature higher than the highest phase transition temperature characterizing the lipids in the mixture. The suspension was then extruded using a Liposofast LP-50 (four times through three types of filters - 1, 0.4, and 0.1 μm - at 600 psi; approximately 4.1 × 10⁻⁶). 6 Pa) and finally obtained a lipid carrier.

[0111] The obtained lipid supports had an average diameter (Dh) of approximately 110–150 nm and a dispersion index (PDI) of approximately 0.1–0.2; the zeta potential was neutral to slightly negative in the absence of DSPE-PEG and strongly negative (-20–-35 mV) in its presence. To measure the zeta potential, the samples were diluted with a 0.009% NaCl solution (mass / volume) to obtain suitable attenuator and conductivity values ​​for successful measurement.

[0112] This formulation was used to encapsulate a drug, in this case 2'3'-cGAMP (cgAMP), with an encapsulation efficiency (EE%) of 30-45%. The best results were obtained with compositions having a higher cGAMP / lipid ratio (0.08 mol per mol of lipid). In addition to showing low EE% values, these formulations did not show improvement in STING pathway activation in THP1-dual monocytes (THP1-Dual® Cells, invivogen #thpd-nfis) in vitro.

[0113] <Example 2: DOTAP / cholesterol-based lipid carrier> The inventors used DOTAP cationic lipids (Lipid 1 according to the present invention) and cholesterol (Lipid 2) at concentrations of 3.9 mg / mL and 1.4 mg / mL, respectively, and incorporated 100 μg / mL of 2'3'-cGAMP (cGAMP). These carriers were prepared in the absence of DSPE-PEG (Composition 1) or in the presence of 0.05 molar equivalents of DSPE-PEG relative to cholesterol. The physical properties are shown in Table 1 below.

[0114] [Table 1]

[0115] Furthermore, the inventors achieved near 100% energy efficiency (EE%). In in vitro testing of STING pathway activation in THP1-dual monocytes (THP1-Dual® Cells, in vivogen #thpd-nfis), both compositions showed a four-fold increase in efficacy compared to free cGAMP at the same concentration. Similarly, the inventors confirmed in vitro that DOTAP / cholesterol-based formulations better protect cGAMP from enzymatic degradation by better preservation of STING pathway activation in THP1-dual monocytes after incubation of recombinant human ENPP1 enzyme (#6136-EN-010, biotechne, USA) and a lipid carrier (vs. free cGAMP) in vitro.

[0116] Although the addition of DSPE-PEG negatively affected the variance index (PDI), the inventors noted that this addition was not excessively problematic.

[0117] <Example 3: Effects of DSPE-PEG addition> The inventors then tested different DSPE-PEG ratios (Figure 1). Composition 2 was adopted. Other compositions (3-5) were developed with DSPE-PEG in amounts of 0.1, 0.5, and 1 molar equivalent. The cGAMP content decreased from 1.88% (composition 2) to 1.55%, 0.94%, and finally to 0.60%.

[0118] Composition 3 exhibits properties (diameter, PDI, encapsulation rate) very similar to Composition 2. Particle diameter decreases to 115 nm in Compositions 4-5, and then to 55 nm, while PDI increases to approximately 0.26. Zeta potential decreases to almost 0 in Composition 5. EE% decreases to approximately 89% in Compositions 4-5, and then to 62%. Composition 3 still enables very good activation of THP1-dual macrophages in vitro, but this is reduced in Composition 4, and the effect is almost completely lost in Composition 5.

[0119] On the other hand, compositions 3 and 4 provide better protection against enzymatic degradation of cGAMP by better maintaining STING pathway activation in THP1-dual monocytes after in vitro incubation of recombinant human ENPP1 enzyme and lipid carrier (vs. free cGAMP).

[0120] <Example 4: Functionalization> Based on compositions 2 and 4 (0.05 and 0.5 molar equivalents of DSPE-PEG), compositions 6 and 7 were prepared by substituting all of the DSPE-PEG with DSPE-PEG-mannose, or by substituting half of it with DSPE-PEG-mannose (compositions 8 and 9).

[0121] Composition 7 showed a significant increase in diameter and PDI index. Composition 9 also showed an increase in PDI index. The EE% remained nearly 100% each time.

[0122] The inventors then performed activation tests on immunosuppressive macrophages (type M2) differentiated with IL-10 and IL-4 or tumor-associated macrophages (TAM-like) LLC1 (Lewis lung cancer, ATCC CRL1642), obtained from bone marrow-derived macrophages (BMDM) using a protocol similar to that described by Kwart et al. (Cell Reports 41 (2022) 111769). Both compositions induced strong macrophage activation, but composition 6 showed significantly higher activation than composition 2 in both types of macrophages (M2 and TAM-like), as shown in Figure 2 for M2.

[0123] Furthermore, it was observed that when composition 6 was administered to healthy mice via the intratracheal route, alveolar macrophages and dendritic cells were activated.

[0124] <Example 5: Increase in content> To increase the cGAMP content in the lipid carrier, the amount of DOTAP was reduced to 1.5 molar equivalents relative to cholesterol, and in compositions 10-16, the amounts of both lipids were further reduced to 1 / 10 of that in composition 1. Various cGAMP concentrations from 100 (composition 10) to 150, 200, 250, 300, 360, and 720 μg / mL were tested. The content 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. Particle diameter increases slightly up to composition 12 (loading rate 28.86%), and then increases sharply with increasing cGAMP concentration. PDI remains constant up to composition 12, but then increases sharply. Conversely, zeta potential decreases beyond composition 12. EE% remains nearly 100% up to this composition. STING pathway activation in Dual THP1 monocytes remains significantly higher than with free cGAMP.

[0125] Composition 12 allows for the addition of DPPC (in a 2:1 molar ratio here) to DOTAP, but the cGAMP content decreases. STING pathway activation in Dual THP1 monocytes remains significantly higher than with free cGAMP, but is halved at low cGAMP concentrations. However, this type of formulation is interesting because it is associated with increased stiffness (also reflected in increased Tm of the lipid series) and is useful in specific administration methods.

[0126] <Example 6: Improvement of the rigidity properties of the formulation> The inventors observed that when these formulations were diluted with large amounts of PBS, the cGAMP encapsulated in the compositions was immediately released. To address this limitation, the inventors prepared new formulations by adding excipients (DPPCs) with a higher Tm. From composition 12, 3, 5, 8, and 10 molar equivalents of DPPC were added to cholesterol as compositions 13, 14, 15, and 16, respectively. The results showed that increasing the amount of DPPC reduced the percentage of cGAMP immediately released after 200-fold dilution with PBS. Compositions 12, 13, 14, 15, 16: 100%; 59.48%; 69.57%; 47.83%; 32.18%.

[0127] <Example 7: Use of ionizable lipids> From composition 14, the inventors substituted DODMA with DOTAP in the same molar ratio. Applying the lipid carrier preparation protocol described above, the lipid membrane rehydration buffer was set to 0.01 M acetate buffer at pH 5.0, and DODMA was ionized to promote cGAMP encapsulation. The EE% was similar to that obtained under condition 14.

[0128] <Example 8: Combination with standard treatment> The composition of the present invention was administered intratracheally to mice with lung tumors in combination with an anti-PD1 monoclonal antibody (clone RPM1-14 #BP0146, BioXcell). This combination therapy yielded higher activity compared to the corresponding monotherapy.

[0129] <Example 9: Formulations using other STING agonists and other active ingredients> The inventors developed several formulations using the compositions (molar ratios) listed in Table 2 below: DPPC / DOTAP / AgoSting / Cholesterol / DSPE-PEG-Mannose.

[0130] [Table 2]

[0131] For each composition, particle diameter was kept within acceptable limits, although larger particles were obtained in some compositions. EE% values ​​remained high in each case (at least 87%), and in most cases exceeded 95% (and even 100%).

[0132] Furthermore, the inventors tested the encapsulation of rifampicin in the DPPC / DOTAP / Chol / rifampicin / DSPE-PEG-mannose (3 / 1.5 / 1 / 0.48 / 0.05) formulation. The amount of encapsulated rifampicin was measured by absorbance at 479 nm, and the EE% was 79%. The diameter, PDI, and zeta potential values ​​were of a similar order to those mentioned above.

[0133] Similarly, when siRNA was encapsulated in similar types of liposomes, the EE% was 85% (measured at 260 nm), and the size, PDI, and zeta potential values ​​were of a similar order to those mentioned above.

[0134] <Example 10: STING agonist activity> The inventors observed that in macrophages prepared in vitro from mouse bone marrow, formulations applied to immunosuppressive M2 macrophages induced polarization into pro-inflammatory M1 macrophages. This was true for both 2'3'cGAMP and the other STING agonists in Example 9, and the polarization was dose-dependent, more pronounced with c-AIMP, particularly with more potent agonists such as c-AIMP difluor and ADU-S100. This polarization from immunosuppressive M2 macrophages to pro-inflammatory M1 macrophages was also observed in the lungs of M109 lung tumor mice after pulmonary administration of nanoparticle formulations containing 2'3'cGAMP.

[0135] The inventors then tested the activity of these encapsulated STING agonists in activating the STING pathway in THP1-dual monocytes.

[0136] Encapsulated 3'3' cGAMP, cAIMP, cAIMP-difluor, and di-AMP resulted in strong, even very strong, activation. Only c-di-GMP showed very weak activation, only slightly higher than the unencapsulated 2'3' cGAMP control.

[0137] <Example 11: T cell proliferation> T cells obtained from freshly isolated splenocytes of Balb / c mice were stimulated with anti-CD3 / anti-CD28 antibody and IL-2-modified beads, with or without 2'3'cGAMP. cGAMP strongly suppressed CD3 / CD28 and IL-2-induced T cell proliferation, and this effect was dose-dependent (mild at 1.38 μM, pronounced at 22 μM).

[0138] <Example 12: Indications for the Formulation> To counteract the harmful effects on T cell proliferation, the inventors came up with an idea and tested its development: a larger size, here about 250 nm (by adjusting the pore size of the extrusion filter to 0.4 μm).

[0139] The potential effects were examined in terms of CD4+ and CD8+ lymphocyte proliferation.

[0140] Larger formulations demonstrated the best performance, and at the tested cGAMP concentrations (0.34, 1.38, 5.5, 22 μM), the survival rates were nearly equivalent to those under untreated conditions.

[0141] <Example 13: Spray Test> The inventors compared two compositions: one containing DOTAP / DPPC / Chol / cGAMP / DSPE-PEG-Man, and the same composition without DPPC, in spray tests using Aerogen® Solo and Ultra vibrating mesh nebulizers. The first formulation achieved high content and bioactivity, while the second formulation did not. Specifically, in the presence of DPPC, the particle diameter was unaffected, remaining at approximately 140 nm, and the PDI index did not substantially increase. The second formulation was more affected, with the diameter increasing from 130 to 170 nm and the PDI index increasing from 0.12 to 0.42, clearly indicating a lack of stability in this formulation during spraying.

[0142] These spray tests demonstrated that, under these conditions where the structure is maintained, liposomes can be effectively delivered deep into the lungs, droplet size is within the desired range (average diameter D4.3 of 5–6 μm), and exhibit an acceptable deposition profile with Next Generation Impactor-type cascade impactors (NGI).

[0143] <Example 14: Other Formulations> The inventors used the above formulation consisting of DPPC / cholesterol / AgoSTING / DSPE-PEG-mannose, excluding DOTAP.

[0144] Two nucleotide-free, neutral-charged STING agonists, SR-717 and MSA-2, were encapsulated.

[0145] Empty structures without an agonist had 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, with experimentally measured PDIs of 0.2–0.3 and experimentally measured zeta potentials of -1–-2 mV. Structures incorporating SR-717 showed greater size variation, with diameters of 100 nm or 160 nm, a maximum PDI of 0.38, and experimentally measured zeta potentials of -3.5–-2.7 mV. The EE% ranged from 70% to almost 90% for MSA-2 and 40–55% for SR-717.

Claims

1. It comprises a lipid carrier selected from the group consisting of a series of lipids, including liposomes, vesicles, micelles, lipoplexes, lipid emulsions, lipid nanocrystals, lipid microspheres, lipid nanoparticles, and mixtures thereof. The aforementioned lipid carrier Antimicrobial agents (preferably antibiotics), nucleic acid, STING protein agonist or STING protein antagonist, Toll-like receptor ligand, Immunomodulators and / or corticosteroids, peptide, Antihypertensive drugs, Bronchodilators, Or containing a drug selected from the group including mixtures thereof, A pharmaceutical composition characterized by being administered by inhalation.

2. The pharmaceutical composition according to claim 1, characterized in that the series of lipids forming the lipid carrier have a phase transition temperature (Tm) of 10 to 80°C, preferably 20 to 60°C.

3. The pharmaceutical composition according to claim 1 or 2, wherein at least one lipid is functionalized with a ligand for the mannose receptor (CD206), and is preferably intended for the activation of macrophages and / or dendritic cells.

4. The pharmaceutical composition according to claim 3, characterized in that at least one lipid functionalized with the ligand of the mannose receptor (CD206) is a phospholipid functionalized with the ligand of the mannose receptor (CD206), preferably a PEG-phospholipid functionalized with the ligand of the mannose receptor (CD206), and more advantageously a DSPE-PEG functionalized with the ligand of the mannose receptor (CD206).

5. The pharmaceutical composition according to claim 3 or 4, characterized in that the ligand for the mannose receptor (CD206) is selected from the group comprising mannose, fucose, N-acetylglucosamine, N-acetylgalactosamine, glycoprotein, galactomannan, α-D-mannopyranoside, polymannose, anti-CD206 antibody, and mixtures thereof.

6. The pharmaceutical composition according to any one of claims 3 to 5, characterized in that the lipid carrier has a molar ratio of at least one lipid functionalized with the ligand of the mannose receptor (CD206) to the total lipids of 0.01 to 0.2, preferably 0.02 to 0.1, and preferably 0.03 to 0.

05.

7. The pharmaceutical composition according to any one of claims 3 to 6, characterized in that the lipid carrier has a molar ratio of at least one drug to at least one lipid functionalized with the ligand of the mannose receptor (CD206) in the range of 5 to 150.

8. The pharmaceutical composition according to any one of claims 1 to 7, characterized in that the lipid carrier is lipid nanoparticles (LNP), preferably liposomes, solid lipid nanoparticles (SLN), or nanostructured lipid carriers (NLC).

9. A pharmaceutical composition according to any one of claims 1 to 8, characterized in that it has a negative charge as a whole.

10. The pharmaceutical composition according to any one of claims 1 to 9, characterized in that the series of lipids includes a cationic and / or ionizable first lipid.

11. The pharmaceutical composition according to claim 10, characterized in that the lipid carrier has a molar ratio of cationic and / or ionizable lipid to the drug of 30 to 0.1, preferably 10 to 0.2, advantageously 3 to 0.5, preferably 2.5 to 0.8, and particularly preferably 2.4 to 1.

12. The pharmaceutical composition according to claim 10 or 11, characterized in that the series of lipids comprises a second lipid selected from the group consisting of sterols, phospholipids, triglycerides, diglycerides, glycerophospholipids, and sphingomyelin, preferably a sterol or a phospholipid, and particularly preferably a sterol.

13. The pharmaceutical composition according to claim 12, wherein the lipid carrier has a molar ratio of the first lipid to the second lipid in the range of 0.3 to 20, preferably in the range of 0.5 to 10, and more preferably in the range of 1 to 5 (moles of the first lipid:moles of the second lipid), and the second lipid is preferably a sterol such as cholesterol and / or a phospholipid such as DPPC and / or DSPC.

14. The pharmaceutical composition according to any one of claims 1 to 13, characterized in that the lipid carrier has an average size measured by dynamic light scattering (DLS) of 50 to 200 nm, preferably 60 to 190 nm, advantageously 70 to 180 nm, particularly preferably 80 to 180 nm, preferably 90 to 180 nm, more preferably 100 to 180 nm, preferably 110 to 180 nm, advantageously 120 to 180 nm, particularly preferably 120 to 170 nm, and preferably 120 to 160 nm.

15. The pharmaceutical composition according to any one of claims 1 to 13, characterized in that the lipid carrier has an average size of 200 to 350 nm, preferably 225 to 300 nm, as measured by dynamic light scattering (DLS).

16. The pharmaceutical composition according to any one of claims 1 to 13, characterized in that the lipid carrier has an average size measured by dynamic light scattering (DLS) of 350 to 1500 nm, preferably 350 to 1400 nm, advantageously 350 to 1300 nm, particularly preferably 350 to 1200 nm, advantageously 350 to 1100 nm, particularly preferably 350 to 1000 nm, preferably 350 to 900 nm, preferably 350 to 800 nm, particularly preferably 350 to 700 nm, advantageously 350 to 600 nm, for example, 400 to 600 nm.

17. The pharmaceutical composition according to any one of claims 1 to 16, wherein the lipid carrier has a polydispersity index measured by dynamic light scattering (DLS) of 0.05 to 0.9, preferably 0.1 to 0.8, advantageously 0.2 to 0.75, particularly preferably 0.3 to 0.7, and preferably 0.4 to 0.6, and preferably the polydispersity index is defined by the following formula. [Math 1]

18. The pharmaceutical composition according to any one of claims 1 to 17, characterized in that the lipid carrier has a zeta potential of -60 to 100 mV, preferably -40 to 80 mV, advantageously -20 to 60 mV, preferably 0 to 50 mV, preferably 10 to 40 mV, advantageously 10 to 30 mV, which is preferably measured by laser Doppler electrophoresis, preferably in a 0.009% (mass:volume) aqueous NaCl solution.

19. The pharmaceutical composition according to any one of claims 1 to 18, characterized in that the drug is present in an amount of 0.1 to 80%, preferably 1 to 70%, preferably 10 to 60%, preferably 15 to 50%, for example, 20 to 40% or 25 to 30%, relative to the mass of the lipid carrier.

20. The pharmaceutical composition according to any one of claims 1 to 19, characterized in that it is in a dry form and is preferably intended to be dissolved or redispersed in a physiological aqueous solvent or administered using a dry powder inhaler.

21. The pharmaceutical composition according to any one of claims 1 to 18, characterized by comprising 50 to 98% water (by mass of water relative to the total mass of the composition), preferably 70 to 95% water, or 80 to 90% water.

22. Furthermore, a buffer selected from the group comprising phosphoric acid, sulfonates (MES, TES, HEPES, MOPS, PIPES, TAPS, TAPSO), acetate, and bicine buffer, and / or At least one salt selected from the group comprising inorganic salts and organic salts, and / or At least one surfactant selected from the following group: Cholic acid and its salts, Phospholipids (e.g., phosphatidylcholine or lecithin, phosphatidylglycerol), Lipids or triglycerides, Sorbitan ester, polyoxyethylene sorbitan, Fatty acids (preferably lauric acid, palmitic acid, stearic acid, erucic acid, or behenic acid), esters of these fatty acids, or derivatives of these fatty acids (salts, etc.) (preferably selected from the group consisting of magnesium stearate, sodium stearyl fumarate, sodium stearyl lactylate, sodium lauryl sulfate, and magnesium lauryl sulfate), Natural lung surfactants, and, Sucroesters (sugar esters, e.g., esters between sucrose or glucose and fatty acids), and / or Sugars selected from the group consisting of the following: monosaccharides (glucose or arabinose, etc.), disaccharides (lactose, maltose, saccharose (sucrose), dextrose, trehalose, maltitol and mixtures thereof, etc.), or polyols (sorbitol, mannitol, xylitol, etc.) as fillers, polysaccharides (dextran, chitosan, starch, cellulose and their derivatives, etc.), oligosaccharides (cyclodextrin and dextrin, etc.), and / or A pharmaceutical composition according to any one of claims 1 to 21, characterized by comprising at least one amino acid selected from the group consisting of the following: histidine, leucine, isoleucine, threonine, lysine, valine, methionine, phenylalanine, mixtures thereof, and derivatives thereof (such as acesulfame K or aspartame).

23. Furthermore, metal ions, preferably Mn ２＋ Co ２＋ or Zn ２＋ A pharmaceutical composition according to any one of claims 1 to 22, characterized in that it contains a metal ion selected from the group consisting of and / or in an amount of at least 10 ppm.

24. The pharmaceutical composition according to any one of claims 1 to 23, characterized in that it can be administered by nebulizer spraying and / or pressurized inhalation and / or dry powder inhalation and / or soft mist inhalation.

25. A pharmaceutical composition according to any one of claims 1 to 24, for the treatment of cancer, preferably metastatic cancer, and preferably characterized in that the metastases are outside the lungs or central nervous system and the primary tumor is pulmonary or located in the central nervous system, or the metastases are pulmonary or located in the central nervous system and the primary tumor is outside the lungs or central nervous system.

26. A pharmaceutical composition according to any one of claims 1 to 24, characterized in that it is for the treatment of infections of the respiratory tract and / or systemic pathways or the central nervous system.

27. A pharmaceutical composition according to any one of claims 1 to 24, characterized in that it comprises a nucleic acid encoding one or more epitopes, or a peptide containing one or more epitopes, and is for the purpose of vaccination.

28. A pharmaceutical composition according to any one of claims 1 to 24, comprising nucleic acids for gene therapy and / or enzyme supplementation, wherein the nucleic acids are DNA or messenger RNA molecules, and the gene therapy is preferably somatic cells.

29. A step of solubilizing the aforementioned lipid in an organic solvent, A step of evaporating the solvent to form a lipid film, A step of rehydrating with a solution containing the aforementioned drug, The process includes an extrusion process, Preferably, the process further includes a second step of contacting the extruded lipid with a solution containing the drug, A method for obtaining a pharmaceutical composition according to any one of claims 1 to 28, characterized in that the lipids include lipids that are neutral at pH 7.0 and positively charged at pH 5, and the rehydration step is carried out at a pH of less than 7.

0.

30. A method for obtaining the pharmaceutical composition according to claim 29, further comprising the step of inserting a lipid derivatized with PEG and, advantageously, further derivatized with a ligand for a mannose receptor (CD206) into an extruded pharmaceutical composition.

31. A method for obtaining the pharmaceutical composition described in any one of claims 1 to 28, A step of solubilizing the lipid and the drug in an organic solvent or a mixture of organic solvents, A step of injecting the organic solvent (or mixture of organic solvents) into an aqueous solution which may contain one or more surfactants, using a predetermined injection rate and stirring rate, For example, a step of removing the organic solvent by evaporation or dialysis, Includes, Preferably, a method for obtaining a pharmaceutical composition characterized by further comprising the step of inserting a lipid derivatized with a ligand for the CD206 receptor.

32. A method for obtaining a pharmaceutical composition according to any one of claims 1 to 28, characterized by using microfluidic technology to mix a solvent (or solvent mixture) containing the lipid and the drug with an aqueous solution which may contain one or more surfactants at a controlled rate using a device having microchannels.

33. A method for obtaining a pharmaceutical composition according to any one of claims 1 to 28, characterized in that it is obtained by a freezing and thawing method of multilayer vesicles (MLVs) or monolayer vesicles (SUVs).

34. A method for obtaining the pharmaceutical composition described in any one of claims 1 to 28, characterized by being obtained by a double centrifugation method.

35. A method for obtaining a pharmaceutical composition according to any one of claims 1 to 28, characterized by treating a multilayer vesicle (MLV) suspension obtained after rehydration of a lipid membrane by a high-pressure homogenization method.

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