Inhalable formulations for treatment of pulmonary bacterial infections

Abstract

The present invention relates to nanocarrier formulations for the treatment of bacterial infections of the lower airway by inhalation therapy.

Description

Technical Field

[0001] The present invention relates to nanocarrier formulations for use in treating bacterial infections of the lower airways by administration by inhalation. Background Art

[0002] A variety of antibiotics have been developed to treat bacterial infections in the lungs, such as tuberculosis. Some of these antibiotics show high tissue accumulation, which is not only due to the lipophilicity of the drug, but also due to its cationic amphiphilicity. It is known that so-called cationic amphiphilic drugs (CAD) bind to phospholipids, resulting in intracellular accumulation of phospholipids in cells and tissues, producing phospholipid inclusion bodies, also known as drug-induced phospholipidosis (DIPL) (Chatman et al., Toxicologic Pathology, 37 (7), 997–1005, 2009). Organs affected by phospholipidosis will experience inflammatory reactions and histopathological changes. Phospholipidosis induced by drugs with cationic amphiphilic structures is a common condition in humans and animals, characterized by intracellular accumulation of phospholipids and the appearance of concentric lamellar bodies. The main mechanism of phospholipidosis is that the drug inhibits the activity of lysosomal phospholipases. Although the mechanism is not fully understood, systemic CAD exposure and DIPL are associated with inhibition of potassium channels encoded by the human ether-à-go-go related gene (hERG). Inhibition of hERG channels can lead to QT interval prolongation, which may lead to life-threatening ventricular tachyarrhythmias (Sun et al., Bioorg. Med. Chem. Lett. 2013; 23(16): 4587-4590). Clinical studies of oral CAD have shown a mild but significant increase in QT prolongation in the treatment group compared with the placebo group, which is usually reversible after discontinuation of treatment. The combined use of CAD with other drugs that cause QT prolongation (such as fluoroquinolones and clofazimine) will produce an additive effect, so the World Health Organization recommends that the combined use of compounds with known QT prolongation be limited to a control regimen that provides QT interval monitoring (Pontali et al., European Respiratory Journal 2016 47: 394-402). Summary of the invention

[0003] Therefore, the object of the present invention is to provide a pharmaceutical preparation for the treatment of bacterial lung diseases, which preparation can reduce or even inhibit the induction of DIPL and / or the inhibition of the potassium ion channel encoded by hERG.

[0004] This object is achieved by the subject matter according to claims 1 and 9. Preferred embodiments are subject matter of the dependent claims.

[0005] Inhaled (particularly pulmonary) therapy of antimicrobial agents provides the opportunity to achieve higher local drug concentrations in the lower airways at the site of infection while generally reducing the potential for systemic adverse reactions. In the case of CAD, inhaled administration reduces systemic drug concentrations, thereby achieving reduced CYP3A4 metabolism and reduced QT prolongation. Therefore, the scope for combined administration of CAD with other therapeutic agents may be significantly broadened.

[0006] Since many antimicrobial agents, especially bedaquiline, have poor water solubility, conventional dry powder formulations for inhalation, especially in the form of interactive powder mixtures, i.e. micronized drugs dispersed on the surface of lactose carrier particles, may be inappropriate, as there is increasing evidence that inhalation of dry powders of poorly soluble compounds is associated with adverse effects in the lower airways, especially the lungs, including particle accumulation in the lower airways (such as the lungs), increased macrophage numbers, increased foam macrophage prevalence, and particle-induced inflammation (Forbes et al., Adv. Drug Deliv. Rev. 2014; 71, pp. 15-33). To overcome this problem, delivery via cell-targeted nanocarriers is used to encapsulate therapeutically relevant concentrations of antimicrobial agents, which are administered in the form of aerosolized liquid or spray-dried dry powder formulations. However, a significant clinical disadvantage of spray-dried powder formulations (as described in Huck, et al., Adv Healthcare Mater, 2022, 11 (11), 2102117) is that a certain amount of additional excipients are required to make inhalable dry powders, which ultimately limits the amount of antibiotics that can be inhaled into the lungs. The aerosolized nanocarrier formulations described herein are not limited in this regard and can be administered at significantly higher concentrations, which is necessary for effectively eliminating bacteria and preventing the development of drug resistance.

[0007] In general, cell-targeted nanocarriers are suitable carriers that can specifically deliver encapsulated compounds to specific cell types expressing corresponding targeting structures. When used therapeutically, this technology can be highly focused on delivering compounds to selected cell types. For example, it has been shown before that compounds delivered by targeted liposomes can selectively reach selected cell types, such as myeloid dendritic cells (mDC), and deliver their contents to such cells (Gieseler RK et al., Scand J Immunol. 2004; 59: 415-24; WO 2005 / 092288A1).

[0008] Therefore, the present invention relates to an inhalable formulation comprising an active agent complex comprising one or more antibacterial agents encapsulated in a DMPG-based or DMPC-based nanocarrier and at least one cell-specific targeting ligand on the surface of the nanocarrier, wherein the targeting ligand specifically binds to a receptor on the surface of a carbohydrate recognition domain (CRD)-positive cell of the lower airways, for use in a method for treating lower airway bacterial infections. BRIEF DESCRIPTION OF THE DRAWINGS

[0009] Figure 1 : A macrophage-like cell line (J774 cells) was used to screen whether three different nanocarrier formulations (1: DOPC / Chol, 2: DMPC / DMPG or 3: PEGylated DOPC / Chol)i) that differed in their lipid composition were cytotoxic to this cell type and whether they could protect cells from BDQ-induced DIPL. Heat maps are used to depict the number of abnormal cells induced by DIPL (the first two columns on the left) and cytotoxicity markers (columns on the right). Black areas indicate that <10% of the cells in the treatment group were abnormal, while gray to white areas indicate an increase in the proportion of cells with abnormalities. The top heat map depicts the DIPL positive control group, which consists of four known CADs (amiodarone, amytryptiline, azithromycin and BDQ) administered to the cells in the form of solutions (non-formulated). The white area in the left column shows that almost 100% of the cells showed the expected abnormal phospholipid accumulation after treatment with CAD. The middle heatmap shows the negative control group, which consists of three different lipid nanocarrier systems (without drug) with and without fucose targeting moieties (+CLR or -CLR) on the surface. The predominantly black area indicates that the empty nanocarrier does not induce DIPL and has low cytotoxicity. The bottom heatmap shows the same nanocarrier loaded with BDQ. The nanocarrier does not completely inhibit the occurrence of DIPL, but in the case of the DMPG / DMPC formulation, the number of cells that develop DIPL is reduced by the formulation. The numbers on the left side of each heatmap indicate the CAD dose (μM) and total lipid concentration (μg / mL) added to each well. The values ​​represent the average percentage of abnormal cells for six replicate formulations tested at four different cell passage numbers.

[0010] Figure 2 :J774A.1 cells were cultured with anionic, neutral or PEGylated nanocarriers (TargoSpheres TM Uptake of fluorescent markers Texas Red and BDQ after 48 h of incubation with or without targeting ligands (TLRs). TMBatches were evaluated at four replicate cell passage numbers. Values ​​are expressed as mean ± SD (n = 4). **p < 0.01; ***p < 0.001.

[0011] Figure 3 : A) Schematic diagram of the in vivo anti-tuberculosis activity evaluation of liposome formulations loaded with fucosylated and non-fucosylated BDQ compared to non-formulated BDQ in C3HeB / FeJ mice infected with Mycobacterium tuberculosis (Mtb), which received seven intranasal (in) instillations every two days for 14 days. The BDQ formulation contained 160 μg BDQ (5 mg / kg) per administration, and the non-loaded liposome formulation (fucosylated) was used as a vehicle control. Texas red-PE was incorporated into the liposome formulation to evaluate the changes in liposome content in the lung at day 44 (B), allowing for semi-quantitative evaluation of fluorescence intensity in n=5 lungs (C). CFU / mL in the lung (D) and spleen (E) was determined from organ homogenates. Values ​​are expressed as mean ± standard deviation of n=5 animals per treatment group. UT=untreated Mtb infected control group. *p<0.05; ****p<0.0001.

[0012] Figure 4 : BDQ-loaded DMPG / DPMC nanocarriers (+CLR; labeled TargoSpheres TM ) Pharmacokinetic profiles of BDQ. A) Schematic diagram of the pharmacokinetic study comparing intravenous (iv) and in administration of BDQ-loaded DMPG / DMPC nanocarriers with oral administration of pure BDQ. Plasma concentrations of (B) BDQ and (C) desmethyl-bedaquiline (M2) were determined over 96 hours and compared with BDQ concentrations in (D) lung tissue and (E) lung epithelial lining fluid (ELF). Values ​​are expressed as the mean of n=6 animals per time point. Minimum bactericidal concentrations (MBC) and minimum inhibitory concentrations (MIC) of BDQ and M2, plotted as dashed lines, are from Rouan et al, 2012. In summary, the pharmacokinetic data indicate that intrapulmonary administration of the nanocarrier formulation results in lower systemic exposure and higher lung BDQ concentrations compared with oral administration.

[0013] Figure 5 : A) Absolute bioavailability of BDQ after oral and in administration compared with iv administration. (B) AUC in plasmaM2 :AUC BDQ ratio; (C) BDQ AUC 肺 :AUC 血浆 ratio. DETAILED DESCRIPTION

[0014] Therefore, the present invention relates to an inhalable formulation comprising an active agent complex comprising at least one antibacterial agent encapsulated in a DMPG and / or DMPC-based nanocarrier having at least one cell-specific targeting ligand on the surface of the nanocarrier, wherein the targeting ligand specifically binds to a receptor on the surface of a CRD-positive cell or a CLR-positive cell of the lower airway, for use in a method for treating bacterial infection of the lower airway.

[0015] In a preferred embodiment, the present invention relates to a lipid-drug complex comprising an antibacterial agent encapsulated in a DMPG and / or DMPC-based liposome and at least one carbohydrate or its derivative as a targeting ligand on the outer surface of the liposome, wherein the targeting ligand specifically binds to a receptor expressing a C-type lectin-like carbohydrate recognition domain (CRD or CLR) or other receptors having a CRD or CLR on the surface of lower airway cells.

[0016] In a preferred embodiment, the active agent complex is part of a pharmaceutical composition or combination in combination with a pharmaceutically acceptable carrier.

[0017] The present invention can preferentially deliver antimicrobial agents as active agents to CLR-positive or CRD-positive cells in mammalian subjects, including humans.

[0018] In this application, the terms "nanocarrier", "active agent complex", "active agent formulation" and "active agent system" are used interchangeably and synonymously with each other.

[0019] A "complex" or "preparation" is a mixture or adduct produced by chemical bonding or bonding between and / or among its components or ingredients (including lipids, active agents and other optional components). The nature of the chemical bonding or bonding can be covalent bonds, ionic bonds, hydrogen bonds, van der Waals bonds or hydrophobic bonds, or any combination of these bonding types, connecting the components of the complex at any part or group thereof, wherein one component can have one or more different types of groups. Not every component in the complex or preparation needs to be bound to every other component, but each component in the complex has at least one chemical bond or bond to at least one other component. According to the present invention, examples of lipid-active agent complexes include liposomes (lipid vesicles) or lipid-active agent sheet-disc complexes. In addition, a lipid-conjugated active agent can be part of a lipid-active agent complex.

[0020] Techniques that can be used to prepare lipid-active agent formulations (such as liposomes) are known in the art (e.g., Sullivan SM et al., Antisense Res Dev 1992; 2: 187-97; Laverman P et al., Crit Rev Ther Drug Carrier Syst 2001; 18: 551-66; Oussoren C, Storm G, Adv Drug Deliv Rev 2001; 50: 143-56; U.S. Pat. No. 5,773,027; U.S. Pat. No. 5,223,263; WO 96 / 10399A1; Bailey-Hytholt, CM, et al. Formulating and Characterizing Lipid Nanoparticles using a Microfluidic Mixing Platform., J. Vis. Exp. (168), e62226, doi: 10.3791 / 62226 (2021)).

[0021] For cell targeting, according to the present invention, liposomes or lipid-based nanocarriers can be prepared by any of the above methods. According to the present invention, the lipid is DMPG [1,2-dimyristoyl-sn-glycero-3-phosphoglycerol (preferably sodium salt)] and / or DMPC [1,2-dimyristoyl-sn-glycero-3-phosphocholine]. In a particularly preferred embodiment, the two are used in combination, for example in a 50:50 ratio or DPMG accounts for the majority (e.g. 75%-80% DPMG: 20%-25% DPMC). Optionally, other lipids such as steroids, cholesterol, fatty amines (such as long-chain fatty amines or carboxylic acids), long-chain sulfates and phosphates, diacetyl phosphates, butylated hydroxytoluene, tocopherol, retinol and isoprenoid compounds can be mixed with the phospholipid component to impart certain desired known properties to the formed vesicles. In addition, synthetic phospholipids containing altered aliphatic moieties (such as hydroxyl groups, branched carbon chains, cyclic derivatives, aromatic derivatives, ethers, amides, polyunsaturated derivatives, halogenated derivatives) or modified hydrophilic moieties (containing carbohydrates, diols, phosphates, phosphonates, quaternary amines, sulfates, sulfonates, carboxyl groups, amine groups, thiol groups or imidazole groups) and combinations of these groups can also be mixed with the above-mentioned phospholipids and used according to the present invention.

[0022] The use of organic solvents can promote the production of lipid-active agent preparations. After the active agent and lipid are mixed, as long as the lipid and active agent are stable at the use temperature, the organic solvent can be removed by, for example, vacuum evaporation, applying heat or hot ethanol injection (such as U.S. Patent No. 4,515,736). Dialysis and / or chromatography (such as affinity chromatography) can also be used to remove the organic solvent. The hydration of the active agent can be carried out with water or any biocompatible aqueous buffer (such as phosphate buffered saline, HEPES or TRIS) to maintain physiological osmotic pressure. The rehydration of liposomes can be completed simultaneously with the removal of organic solvents, or alternatively, it can be postponed to the time when liposomes are more convenient to use (such as U.S. Patent No. 4,235,871). The shelf life of hydratable ("dry") liposomes, which is usually 8-12 months, can be extended by lyophilization.

[0023] In one embodiment of the invention, the lipid-active agent complex is a unilamellar liposome. Such liposomes can accommodate higher levels of active agent, which can interact with the inner and outer surfaces of the liposome. However, multilamellar liposomes and / or pegylated liposomes can also be used according to the invention.

[0024] The diameter of the lipid-active agent complex is preferably, but not necessarily, about 30-200 nm.

[0025] According to the present invention, the lipid-active agent complex can be preserved by any known method, such as lyophilization (eg, Crowe et al., U.S. Pat. No. 4,857,319). Typically, lyophilization or other useful cryopreservation techniques involve the addition of a cryopreservative, such as a disaccharide.

[0026] The targeting ligand can be a small compound, an antibody, a sugar, a peptide or an aptamer. As used herein, the term "antibody" (Ab) refers to an antibody fragment (e.g., Fab and F(ab')2 fragment) that includes a complete immunoglobulin molecule and can specifically bind to a surface receptor. Fab and F(ab')2 fragments lack the Fc fragment of a complete antibody. In addition, antibodies useful for the purposes of the present invention include chimeric antibodies, single-chain antibodies, multifunctional (e.g., bispecific) antibodies, and humanized antibodies or human antibodies.

[0027] The present invention uses nanocarriers, particularly liposomes, whose inner and / or outer surfaces have targeting ligands. These targeting ligands provide surface labels for nanocarriers. For the purposes of the present invention, the targeting ligands are connected to lipid components (such as liposomes) by techniques known in the art (WO 05 / 092288A1), preferably by covalent binding. Preferred examples of targeting ligands are those that specifically bind to receptors expressing C-type lectin-like carbohydrate recognition domains (CRD or CLR). Examples are monofucose, polyfucose or derivatives thereof. A particularly preferred targeting ligand is cholesten-5-yloxy-N-(4-((1-imino-2-D-thiofucosylethyl)amino)alkyl)carboxamide (Fuc-C4-chol), cholestenyl-(4-(3-((2R,3S,4R,5S,6S)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)thiourea)butyl)carbamate (Thiourea-Galac-C4-chol). In a preferred embodiment, fucosylated (Fuc) liposomes are prepared by attaching a fucosyl targeting ligand, which can be attached to a spacer of a suitable lipophilic membrane anchor on the liposome, or can be attached to the distal end of the anchor on the liposome or directly to the lipid component of the liposome. Suitable compounds are polyethylene glycol (PEG) or hydroxyethyl starch (HES).

[0028] CLRs act as "pathogen entry ports" into cells expressing such receptors. As for their biological functions, the endocytosis members of the CLRs expressed by APCs are responsible for the ingestion of pathogens and their intracellular breakdown, followed by controlled processing of pathogen-derived protein antigens into 10-mer or 12-mer peptides, and binding of these antigens to class I or class II proteins of the major histocompatibility complex (MHC) of the APC. These combined molecules allow antigens to be presented on the surface of the APC in an MHC-restricted manner (i.e., strictly controlled), thereby instructing other immune cells to stimulate protective cellular and / or humoral immune responses against the initially internalized pathogens in a delicately balanced and harmonious manner with other surface molecules and soluble signals. Importantly, various pathogens have evolved to resist intracellular degradation by APCs.

[0029] Since the nanocarriers of the present invention have a diameter range of about 50-200 nm, they belong to the class of nanocarriers, and their size is within the range of most bacteria and viruses. Like pathogens, the nanocarriers of the present invention will also be internalized in a clathrin-dependent manner, whereby both the carrier and the payload (=antibacterial agent; such as bedaquiline) will be delivered to the endosome. In addition, like pathogens that form a reservoir, a given therapeutic payload can also escape from the endosome (lysosome) compartment and enter the cytoplasm.

[0030] Targeted CRD-positive cells or CLR-positive cells (the two terms are used interchangeably) include, but are not limited to, airway epithelial cells (such as lung vascular cells) and immune cells such as macrophages (such as alveolar macrophages).

[0031] The present invention is intended to target CRD-positive cells or CLR-positive cells of the lower airways, which line the respiratory mucosa or respiratory epithelium (also known as airway epithelial cells). The lower airways (also known as "lower respiratory tract") include the portion of the larynx below the vocal cords, the trachea, bronchi, and bronchioles. The lungs are also part of the lower respiratory tract, including respiratory bronchioles, alveolar ducts, alveolar sacs, and alveoli.

[0032] According to the present disclosure, the terms "compound", "drug", "medicament" or "drug" include their tautomers, stereoisomers and mixtures and salts thereof, in particular pharmaceutically acceptable salts thereof, as well as solvates and hydrates of such compounds, including solvates and hydrates of such tautomers, stereoisomers and salts.

[0033] The terms "treat," "treatment," and "treating" include therapeutic, i.e., curative and / or palliative treatments. Thus, the terms "treat," "treatment," and "treating" include therapeutic treatment of a patient who already has the condition (especially in a manifested form). Therapeutic treatment may be symptomatic treatment to relieve the symptoms of a particular indication; it may also be causal treatment to reverse or partially reverse the condition of the indication, or to arrest or slow the progression of the disease. Thus, the compositions and methods of the invention may be used for both therapeutic treatment, e.g., for a period of time, as well as chronic therapy.

[0034] When the present disclosure refers to a patient in need of treatment, it is primarily concerned with the treatment of mammals, especially humans.

[0035] "Therapeutically effective amount" or "pharmaceutically effective amount" refers to one or more compounds disclosed in the present invention that have a therapeutic effect. The dosage of the compound disclosed in the present invention for treatment is a therapeutically effective amount. Therefore, as used herein, a therapeutically effective amount refers to the amount of the compound that can produce the expected therapeutic effect based on the results of clinical trials and / or model animal infection studies. In a specific embodiment, the compound is administered in a predetermined dose, so the therapeutically effective amount is the amount of the administered dose. This amount and the amount of the compound can be routinely determined by those skilled in the art and will vary due to various factors (such as the specific microbial strains involved). The amount can also depend on the patient's height, weight, gender, age and medical history.

[0036] "Therapeutic effect" relieves one or more symptoms of infection to a certain extent, and includes curing infection to a certain extent. "Cure" refers to the elimination of symptoms of active infection, including complete or substantial elimination of excess members of live microorganisms involved in infection, to a point at or below the detection threshold of traditional measurements. However, even after obtaining a cure, some long-term or permanent effects of acute or chronic infection may also exist (such as extensive tissue damage). As used herein, "therapeutic effect" is defined as a statistically significant reduction in bacterial load in the host, an improvement in lung function, or an improvement in infection symptoms or functional status.

[0037] The phrase "pharmaceutically acceptable" as used herein refers to compounds, substances, compositions and / or dosage forms that are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problems or complications, and are commensurate with a reasonable benefit / risk ratio.

[0038] The term "pharmaceutically acceptable salt" refers to a salt that retains the biological effectiveness and properties of the compound and has no adverse biological or other effects. In many cases, due to the presence of amino and / or carboxyl groups or similar groups, the compound is able to form acid and / or base salts.

[0039] The term "administering" or "administering" refers to a method of administering a dose of a pharmaceutical composition to a mammal, such as by inhalation. The method of administration may vary depending on factors such as the components of the pharmaceutical composition, the site of potential or actual bacterial infection (e.g., the lungs), the microorganisms involved, and the severity of the actual microbial infection.

[0040] The term "microbial infection" or "bacterial infection" refers to the unwanted proliferation or presence of pathogenic microorganisms in a host organism. This includes the overgrowth of microorganisms that normally exist in or on a mammal or other organism (e.g., in the lungs). More generally, a microbial infection can be any situation in which the presence of a microbial population causes damage to the host mammal. Thus, a microbial infection exists when there is an excessive microbial population in or on a mammal, or when the presence of a microbial population causes damage to the cells or other tissues of a mammal.

[0041] The term "sequential" refers to the administration of more than one therapeutic agent at different times. The therapeutic agents can be administered in any order. Unless the drugs are formulated together, they are considered to be administered sequentially. In one embodiment, if two or more therapeutic agents are administered within 24 hours of each other, they are considered to be administered sequentially. In another embodiment, two or more therapeutic agents may be administered in a cycle of less than 24 hours. In another embodiment, two or more therapeutic agents may be administered in a cycle of less than 12 hours. In another embodiment, two or more therapeutic agents may be administered in a cycle of less than 6 hours. In another embodiment, two or more therapeutic agents may be administered in a cycle of less than 3 hours. In one embodiment, the therapeutic agents are administered immediately one after another, or are no more than 60 minutes apart from each other.

[0042] “One or more” and “at least one” include one to ten (specifically one, two, three, four, five, six, seven, eight, nine or ten) samples.

[0043] Non-limiting examples of antibacterial agents active against lower airway bacterial infections, particularly for the treatment of tuberculosis (TB), include bedaquiline (BDQ); aminoglycoside antibiotics (e.g., kanamycin A, amikacin, tobramycin, dibekacin, gentamicin, sisomicin, netilmicin, neomycin B, neomycin C, paromomycin and streptomycin); fluoroquinolones (e.g., moxifloxacin, levofloxacin, sparfloxacin, nalidixic acid, ciprofloxacin, cinoxacin, oxolinic acid, piromidic acid, pipemidic acid, rosoxacin, enoxacin, fleroxacin, lome ... In one embodiment, the antibacterial agent is bedaquiline. In one embodiment, the aminoglycoside antibiotic is amikacin. In one embodiment, the fluoroquinolone is selected from levofloxacin and moxifloxacin. In one embodiment, the nitroimidazole antibiotic is metronidazole.

[0044] In one embodiment, the drug treatment comprises administering an active agent complex comprising a lipid-drug complex comprising bedaquiline encapsulated in a DMPG and / or DMPC-based nanocarrier, alone or sequentially or in combination with one or more encapsulated or non-encapsulated antibacterial agents such as amikacin or linezolid.

[0045] The present invention contemplates that drug therapy may be administered by inhalation.

[0046] For pulmonary administration, the upper airways are avoided and the middle and lower airways are preferentially acted on. Pulmonary drug delivery can be achieved by inhaling an aerosolized suspension of the active agent complex through the nose, mouth and throat. Particles with a mass median aerodynamic diameter (MMAD) greater than about 5 microns generally do not reach the lungs; instead, they tend to hit the back of the throat and be swallowed and may be absorbed orally. Particles with a diameter of about 2 to about 5 microns are small enough to reach the middle and upper regions of the lungs (conducting airways), but are too large to reach the alveoli. Smaller particles, i.e., about 0.5 microns to about 2 microns, can reach the alveolar region. Particles with a diameter less than about 0.5 microns can also be deposited in the alveolar region by sedimentation, but very small particles may also be exhaled.

[0047] In one embodiment, the basis for selecting the nebulizer is to allow the formation of an active agent complex aerosol disclosed herein, whose MMAD is mainly between about 0.5 microns and about 5 microns. In one embodiment, the delivery amount of the active agent complex can provide a therapeutic effect for respiratory infections, particularly tuberculosis, and most preferably multidrug-resistant tuberculosis. The nebulizer can deliver an aerosol with a mass median aerodynamic diameter from about 0.5 microns to about 5 microns, a mass median aerodynamic diameter from about 1.0 microns to about 3.0 microns, or a mass median aerodynamic diameter from about 1.5 microns to about 2.5 microns. In some embodiments, MMAD can be about 0.5 microns, about 1.0 microns, about 1.5 microns, about 2.0 microns, about 2.5 microns, about 3.0 microns, about 3.5 microns, about 4.0 microns, about 4.5 microns or about 5.0 microns. In one embodiment, the range of MMAD is from about 2.5 microns to about 5.0 microns. In another embodiment, the range of MMAD is from about 3.0 to about 4.5 microns. In some embodiments, the nebulizer can be a breath-actuated nebulizer (BAN). In some embodiments, a vibrating mesh nebulizer can be used to generate the aerosol. Examples of vibrating mesh nebulizers include PARI Nebulizer or nebulizer using PARI eFlow technology. Further commercial examples of nebulizers that can be used with the formulations described herein include Respirgard Aeroneb Aeroneb AERx Freeway PARI and PARI In one embodiment, the nebulizer is a breath-actuated nebulizer.

[0048] The preparation may include conventional pharmaceutical carriers, diluents, excipients, buffers, surfactants, etc., which have been approved for inclusion in inhalation products by the US National Formulary and the approved excipient database maintained by the USFDA and other regulatory agencies. Non-limiting examples of carriers and excipients include, for example, water, ethanol, glycerol, propylene glycol, PEG1000, sorbitol trioleate, soybean lecithin, lecithin, oleic acid, magnesium stearate, sodium lauryl sulfate, lactose, mannitol, glucose, methylparaben, propylparaben, chlorobutanol, benzalkonium chloride, cetylpyridinium chloride, thymol, ascorbic acid, sodium bisulfate, sodium pyrosulfite, sodium bisulfate, EDTA, NaOH, tromethamine, ammonia, HCl, H2SO4, HNO2, citric acid, CaCl2 and CaCO3.

[0049] Liquid pharmaceutical compositions can be prepared, for example, by dissolving, dispersing, etc., the active agent complex defined above and optional pharmaceutical adjuvants in a carrier (such as water, saline, aqueous glucose solution, glycerol, ethylene glycol, ethanol, etc.) to form a solution or suspension. The solution to be aerosolized can be prepared in a conventional form, which can be a liquid solution or suspension, an emulsion, or a solid form suitable for dissolving or suspending in a liquid before aerosol production and inhalation. The percentage of active compound contained in such aerosol compositions depends largely on its specific properties, as well as the activity of the compound and the needs of the subject. However, the percentage of active ingredient in the solution is feasible from about 0.01% to about 90%.

[0050] The formulation can be administered in a therapeutically effective dose, e.g., a dose sufficient to provide treatment for the disease state described above. Of course, the amount of active compound administered depends on the subject and disease state being treated, the severity of the affliction, the mode and schedule of administration, and the judgment of the prescribing physician.

[0051] In one embodiment, the formulation and one or more therapeutic agents can be administered to the lung in less than about 60 minutes, about 55 minutes, about 50 minutes, about 45 minutes, about 40 minutes, about 35 minutes, about 30 minutes, about 25 minutes, about 20 minutes, about 15 minutes, about 10 minutes, about 5 minutes, about 4 minutes, about 3 minutes, about 2 minutes, and about 1 minute.

[0052] The methods and compositions described herein can be used to treat lung infections and lower airway disorders. Other examples of such disorders may include tuberculosis, cystic fibrosis, pneumonia and chronic obstructive pulmonary disease, including chronic bronchitis and some asthma, with tuberculosis being preferred, particularly multidrug-resistant tuberculosis. Some embodiments include treating an infection comprising one or more bacteria selected from the group consisting of: Pseudomonas aeruginosa, Pseudomonas fluorescens, Pseudomonas acidovorans, Pseudomonas alcaligenes, Pseudomonas putida, Bordetella pertussis, Bordetella parapertussis, Bordetella bronchiseptica, Haemophilus influenzae, Haemophilus parainfluenzae, Legionella pneumophila, Burkholderia cepacia, Streptococcus pneumoniae, pneumoniae) and Mycobacterium tuberculosis (Mtb).

[0053] The present invention shows that DMPC or DMPC-based nanocarrier formulations, especially when they have Fuc-C4-Chol as a CLR targeting ligand on their surface, are superior to neutral and PEGylated formulations in delivering antimicrobial agents, especially BDQ, to the intracellular environment of macrophage-like cells. Despite higher levels of cell stress markers, which may be due to stronger interactions of anionic DMPC and / or DMPC-based nanocarriers with cells, they also appear to reduce the number of cells with significant DIPL in the cell population compared to neutral and PEGylated nanocarriers. Based on the results of these examples, it is reasonable to speculate that higher concentrations of antimicrobial agents (such as BDQ) in the lungs will lead to steeper concentration gradients locally. Such gradients will drive the diffusion of drugs throughout the lungs and are believed to increase the levels of active drugs in difficult to reach compartments (such as fibrogranuloma cysts). This will lead to improved treatment efficiency and reduced development of drug resistance.

[0054] Another advantage of inhaled aerosolized BDQ nanocarriers is that they bypass the first pass through the liver (the so-called "first-pass effect"), thereby delaying the metabolism of BDQ to N-desmethyl-BDQ (M2). Encapsulation of BDQ may further prevent or delay distribution processes such as protein and albumin binding and diffusion processes in other tissues, leading to altered biodistribution and pharmacokinetics in vivo. The BDQ metabolite N-desmethyl-BDQ (M2) is mainly formed in the liver through cytochrome P450 34A biotransformation and is 3 to 5 times less active against Mycobacterium tuberculosis strains compared to BDQ. This metabolite is also responsible for most of the systemic side effects such as phospholipidosis and "QT prolongation".

[0055] Inhaled administration of antimicrobial agents such as BDQ encapsulated in nanocarriers offers advantages in terms of both drug bioavailability and local lung tolerability, as inhalation of high doses of powder formulations of poorly soluble compounds is known to be associated with particle accumulation and side effects such as cough. In a murine TB infection model, intranasal administration of a liposomal BDQ formulation (5 mg / kg BDQ administered every two days for 2 weeks) reduced the burden of infectious pathogens such as Mtb in the lungs by approximately half-log, a result comparable to the efficacy data of Irwin et al. (2016) using the same Mtb infection model (C3HeB / FeJ mice) but with daily oral administration of 10 mg / kg BDQ. The present results demonstrate that inhaled administration significantly reduces systemic exposure to the M2 metabolite, a compound associated with an increased risk of QT prolongation in some patients, thus supporting the proposition that inhaled liposomal CADs such as BDQ may exhibit clinically relevant activity while reducing the side effect profile.

[0056] The following examples should be understood as illustrative rather than limiting the scope of the present invention. The use of the antibacterial agent bedaquiline (BDQ) in the examples should be understood as an exemplary compound of the various suitable antibacterial compounds described above.

[0057] Example

[0058] Example 1: Preparation of nanocarriers encapsulating bedaquiline (BDQ)

[0059] BDQ Liposomes The formulations were prepared by thin film hydration followed by extrusion. First, lipids, BDQ, and targeting ligand (Fuc-C4-Chol) were dissolved in chloroform, ethanol, or a mixture of water and ethanol, respectively. The stock solutions were then combined in a round-bottom flask at the molar fractions shown in Table 1, and the solvent was slowly removed using a rotary evaporator to obtain a uniform film. To remove any residual solvent, the film was dried in a vacuum for two days. For the production of lipid-based nanocarriers, the dried film was hydrated with PBS to a film concentration of 40 mg / mL. The crude sample was then extruded through a polycarbonate membrane ( Nuclepore TM Track-Etched Membrane) and then extruded through a 50 nm membrane.

[0060] Table 1: Different Composition of liposome formulations. DMPG [1,2-dimyristoyl-sn-glycero-3-phosphoglycerol (preferably sodium salt)], DMPC [1,2-dimyristoyl-sn-glycero-3-phosphocholine], DOPC [1,2-dioleoyl-sn-glycero-3-phosphocholine], Chol [cholesterol], m-PEG-2000-DSPE [N-(carbonyl-methoxypolyethylene glycol-2000)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine, sodium salt]

[0061] Size, PDI and zeta potential: BDQ-Lipo fuc The hydrodynamic diameter and ζ-potential of the BDQ-Lipo formulations were determined by dynamic light scattering (DLS) and laser Doppler anemometer, using a Zetasizer ZS series (Malvern Instruments Limited, Malvern, UK). fuc The systems are small (50-100 nm), predominantly unilamellar, with a small fraction of multilamellar vesicles. Fucosylation does not significantly alter the hydrodynamic diameter or zeta potential.

[0062] Example 2: In vitro evaluation of respirable bedaquiline-loaded nanocarriers for inhaled therapy of multidrug-resistant tuberculosis

[0063] High content analysis was initially used as a rapid screening tool to evaluate lipid-based nanocarriers applied in liquid dispersion form. Interaction between formulations and macrophages. As described in Example 1, cells were treated with five different formulations (n=6 batches) containing BDQ encapsulated in liposome formulations of different lipid compositions (DOPC / Chol, PEG-DOPC / Chol and DMPG / DMPC). Two of the formulations (DOPC / Chol and DMPG / DMPC) contained Fuc-C4-Chol as a C-type lectin receptor targeting ligand (+CLR). Four concentrations of 15-700 μg / mL (total solid content) were tested. After an incubation time of 48 hours, three assays were performed with different fluorescent markers. To assess the health and morphology of the cells, the cells were incubated with 100 μl of a 5% PBS containing Hoechst 33342 (10 mg / ml, Thermo Fischer, Dreieich, Germany), (300 nM, Thermo Fischer, Dreieich, Germany) and Image-IT TM DEAD Green TM The cells were stained with a mixture of dyes (35 nM, Thermo Fischer, Dreieich, Germany) at 37°C for 30 min. After a washing step with 100 μl PBS, the cells were fixed with 100 μl 4% paraformaldehyde for 15 min at room temperature in the dark. After the second washing step, the fixed cells were stained with HSC CellMask TM Deep Red (10 mg / ml, Thermo Fischer, Dreieich, Germany) was stained overnight. To determine the content of phospholipids, cells were treated with HCS LipidTOX TM Red (Thermo Fischer, Dreieich, Germany) was incubated and the cells were treated for 48 hours. After a washing step with 100 μl PBS, the cells were fixed with a dye mixture containing Hoechst 33342 (10 mg / ml, Thermo Fischer, Dreieich, Germany) and 4% paraformaldehyde at room temperature in the dark for 30 min. The cells were washed again and HCS LipidTOX was diluted 1:1000 (according to the manufacturer's protocol). TM Green (Thermo Fischer, Dreieich, Germany) was incubated for another 30 min at room temperature in the dark to detect neutral lipids. Before imaging, cells were washed once with PBS. Both assays were stored at 4°C.

[0064] For high-content analysis, use Cytation TMImages were recorded using a 40x objective lens in standard 2D imaging mode using a 5-cell imaging multimode reader (BioTek, Bad Friedrichshall, Germany). Six images were analyzed per well using Gen5 software (BioTek, Bad Friedrichshall, Germany). Hoechst 33342 nuclear staining was used to identify nucleated cells for cell health and morphology analysis. Accumulation in active mitochondria by detecting changes in mitochondrial membrane potential. Image-IT TM DEAD Green TM HSC CellMask is an impermeable dye that is important for healthy cells and becomes permanent when the cell plasma membrane is damaged. Fluorescence intensity values ​​for both dyes were measured. TM Deep Red dye was used to highlight the cytoplasmic region within the cell. The area and size of the vacuole were determined and measured based on the negative staining. For lipid determination, Hoechst 33342 was used again to identify nucleated cells. TM Green has a high affinity and is used to detect neutral lipids in cellular areas. To detect and quantify the accumulation of intercellular phospholipids (also known as phospholipidosis), HCS LipidTOX TM Red. Nine basic parameters were collected and characterized: cell health (mitochondrial activity, increased or decreased membrane permeability), cell morphology (cell area, nuclear area, multinucleated cells) and vacuolar properties (vacuolar area, phospholipidosis, neutral lipid content). Each experiment was repeated at least 6 times with different passage numbers. For any plate, there were 6 wells containing untreated cells, 3 wells containing amiodarone (phospholipidosis control) and 3 wells with FCCP (mitochondrial control). If the number of cells per well was lower than the number of untreated cells, the well was excluded. Table 2 shows the inclusion criteria for each cell parameter. The limit was set at twice the standard deviation of the mean compared to untreated cells.

[0065] Table 2: Basic parameters and inclusion criteria for high-content analysis.

[0066] When cells were exposed to bedaquiline (BDQ)-loaded formulations, the anionic DMPG / DMPC nanocarrier formulations reduced the number of cells in the population with significant DIPL to some extent compared to the neutral and PEGylated formulations. This is an interesting observation considering that the anionic nanocarrier formulations showed higher intracellular uptake of BDQ (and Texas Red as an encapsulated fluorescent marker) in subsequent uptake experiments performed under the same conditions ( Figure 1). Therefore, it can be concluded that DMPC / DMPC nanocarrier formulations are superior to neutral and PEGylated formulations in delivering BDQ to the intracellular environment of macrophage-like cells, and although the levels of cell stress markers were higher (which may be due to stronger interactions of anionic DMPC / DMPC nanocarriers with cells), they also appear to reduce the number of cells with significant DIPL in the cell population compared to neutral and PEGylated nanocarriers.

[0067] Example 3: Uptake of Bedaquiline

[0068] The encapsulated lipid-based nanocarriers (DMPG / DMPC-TargoSphere) described in Example 1 were tested in phosphate buffered saline (0.1 M, pH 7.4) supplemented with 10.8 mg / mL albumin at 37°C. TM ) system (Patel, 2020, J Control Release. 2020 Dec 10; 328: 339-349). The receiving fluid was supplemented with albumin to ensure sedimentation conditions and simulate the protein concentration in the lung lining fluid [Bicer, 2011, Drug Deliv. to Lungs 22, Edinburgh (2011)]. The nanocarrier (TargoSphere TM ) preparation (10 mg / mL, 1 mL) was placed in a dialysis tube (Fischer, MWCO 10 kDa), and the dialysis membrane was then immersed in a beaker containing 10 mL of receiving solution. The beaker was incubated in a 37°C water bath (Grant Instruments, Cambridge, UK) with gentle stirring (100 times / min). The receiving solution sample was extracted from the beaker and fresh culture medium was replaced regularly to maintain sedimentation conditions. BDQ was extracted from the receiving solution sample by mixing BDQ with dichloromethane (DCM; 10 mL), ultrasonically treating for 20 minutes, and then standing for 60 minutes for phase separation. The denser DCM layer was carefully separated from the buffer aqueous phase by dripping and evaporated overnight at room temperature. The extraction process was repeated three times. The dried sample containing BDQ was dissolved in 1 mL DMSO and analyzed by UV spectroscopy (n=3 repetitions).

[0069] from Figure 2 As can be seen from the figure, the cellular uptake can be significantly improved by using DMPG / DMPC nanocarriers.

[0070] Example 4: In vivo anti-tuberculosis activity after intranasal administration

[0071] Female C3HeB / FeJ (Jackson Laboratories, USA) mice aged six to eight weeks were housed in a specific pathogen-free BSL3 laboratory. C3HeB / FeJ mice were selected because they can form caseous necrotic granulomas (unlike other mouse strains such as Balb / c), which better simulate the human lung's response to Mycobacterium tuberculosis infection in terms of granuloma formation and higher resistance to drug therapy. On day 0, animals were infected with a virulent (H37Rv) strain of Mycobacterium tuberculosis by aerosol route. Thirty days after infection, mice were divided into five treatment groups (Table 3) and given BDQ-loaded liposomes (fucosylated / non-fucosylated) or controls of Example 1, for a total of seven applications. Dosing was performed every two days for two weeks. 20 μL of sample was instilled intranasally (in) in each nostril as a minimally invasive and material-saving method to achieve upper and lower respiratory tract delivery. In the drug containing samples, the volume contained 160 μg BDQ / 40 μL dose (5 mg / kg) and approximately 2.5 mg total lipid.Texas Red labeled phosphatidylethanolamine was used to confirm the presence of the formulation in the lungs following intranasal administration.

[0072] Table 3: Treatment groups for in vivo efficacy studies

[0073] After the animals were sacrificed, the bacterial load of the lungs and spleens was assessed. The whole organs were harvested, weighed, and mechanically ground in 1 mL of WTA (water: Tween-80 (0.01%): albumin (0.05%)) buffer inside a Whirpak plastic bag using a 50 mL Falcon tube and a petri dish. The organ homogenate was serially diluted 10-fold in WTA buffer, and 100 μL was inoculated onto a Middlebrook 7H11 agar plate using a glass rod and incubated at 37°C. After 21-28 days, mycobacterial colonies were counted. Figure 4 As shown, BDQ-loaded liposomes achieved a half-log reduction in Mtb.CFU / mL.

[0074] Example 5: In vivo pharmacokinetic evaluation of bedaquiline-loaded nanocarriers designed for inhaled therapy of multidrug-resistant tuberculosis

[0075] Compared with intravenous and oral administration, intrapulmonary administration of BDQ-loaded lipid-based nanocarriers (TargoSpheres TM ) can fully increase the lung concentration of antibiotics.

[0076] Methods: BDQ-loaded nanocarriers (DPMG / DMPC TargoSpheres TM) was administered intravenously (IV) and intranasally (IN, Southam et al., Am. J. Physiol. Lung Cell Mol. Physiol., 282 2002, L833-L839, a recognized form of intrapulmonary administration) at a dose of 2.5 mg / kg. Unformulated BDQ crystal suspension dispersed in a glucose solution containing carmellose was administered orally (po). After 0.5, 3, 24, 48, 72, and 96 hours, animals were sacrificed and samples were taken from plasma, lung tissue, lung epithelial cell fluid, and the cell fraction of lung epithelial cell fluid. BDQ concentrations in the four tissue compartments were determined using LC-MS / MS.

[0077] Two comparative studies were used to validate this approach. The first study by Rouan et al. (Antimicrob Agents Chemother. 2012 Mar; 56 (3): 1444-51) showed plasma concentrations over a period of 168 hours in male Swiss mice after oral administration of BDQ (called TMC207) (30 mg / kg). Irwin et al. (ACS Infect Dis. 2016 2: 251-267) showed plasma and lung concentrations over 168 hours in male Balb / c mice after oral administration of 25 mg / kg BDQ. Both studies were conducted by dissolving the drug in an aqueous solution containing 20% ​​2-hydroxypropyl-β-cyclodextrin before administration, so the dissolution rate of the very lipophilic drug should not have any effect on pharmacokinetics. In this study, the active substance was administered in the form of a suspension (25 mg / kg) because it was considered that the dissolution rate of unprocessed BDQ particles ("pure BDQ") in the stomach better reflects the conditions of the standard therapeutic dosage form (tablets).

[0078] After both intravenous and oral administration, BDQ is distributed to various peripheral compartments including lung tissue. Literature shows that BDQ is distributed in the lung with an AUC of 20 肺 :AUC 血浆 Ratio accumulation (Rouan et al., 2012). In this example, oral administration resulted in a lung accumulation ratio of 11, while the lung accumulation of intravenous administration was 15. In contrast, intranasal administration achieved an AUC of 62 肺 :AUC 血浆 ratio, thus increasing lung exposure 3-fold after intrapulmonary administration.

[0079] Table 4. BDQ PK data in plasma and lung tissue, comparing the results of this study with previously published data.

[0080] The higher concentration of BDQ in the lung appears to create a steeper concentration gradient locally. This gradient drives diffusion of the drug throughout the lung and is believed to increase active drug levels in hard-to-reach compartments such as fibrogranuloma cysts. This would increase the efficacy of treatment and reduce the development of drug resistance.

[0081] Another advantage of inhaled aerosolized BDQ nanocarriers is that they can bypass the first pass through the liver (the so-called "first-pass effect"), thereby delaying the metabolism of BDQ to N-desmethyl-BDQ. Encapsulation of BDQ may further prevent or delay distribution processes such as protein and albumin binding and diffusion processes in other tissues, leading to changes in in vivo biodistribution and pharmacokinetics. The BDQ metabolite N-desmethyl-BDQ is mainly formed in the liver through cytochrome P45034A biotransformation and is 3 to 5 times less active against Mycobacterium tuberculosis strains compared to BDQ. This metabolite is also responsible for most of the systemic side effects such as phospholipidosis and "QT prolongation". Figure 4 C and 5B show that the amount of M2 in plasma after intranasal administration was lower than that after intravenous and oral administration, which proves that the intrapulmonary administration route can reduce the metabolism of BDQ to M2.

Claims

1. An active agent complex comprising one or more antibacterial agents encapsulated in a nanocarrier based on 1,2-dimyristoyl-sn-glycero-3-phosphoglycerol (DMPG) and / or 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), having at least one cell-specific targeting ligand on the surface of the nanocarrier, wherein the targeting ligand specifically binds to a receptor on the surface of a CRD-positive cell or a CLR-positive cell of the lower airway, for use in a method for treating lower airway bacterial infection by inhalation.

2. The active agent complex according to claim 1, wherein the antibacterial agent is bedaquiline (BDQ), kanamycin A, amikacin, tobramycin, dibekacin, gentamicin, sisomicin, netilmicin, neomycin B, neomycin C, paromomycin, streptomycin; moxifloxacin, levofloxacin, sparfloxacin, nalidixic acid, ciprofloxacin, cinoxacin, oxolinic acid, piromidic acid, pipemidic acid, rosoxacin, enoxacin, fluroxacin, floxacin, lomefloxacin, nadifloxacin, norfloxacin, ofloxacin, pefloxacin, rufloxacin, balofloxacin, grepafloxacin, pazufloxacin, temafloxacin, tosufloxacin, clinafloxacin, gatifloxacin, sitafloxacin, prulifloxacin, delafloxacin, nemofloxacin, danofloxacin, difloxacin, enrofloxacin, ibafoxacin, marbofloxacin, orbifloxacin, sarafloxacin, trovafloxacin, linezolid, metronidazole, tinidazole, and / or nimorazole.

3. The active agent complex of claim 2, wherein the antibacterial agent is bedaquiline (BDQ).

4. The active agent complex according to any one of claims 1 to 3, wherein the targeting ligand is monofucose, polyfucose, cholesteryl-5-yloxy-N-(4-((1-imino-2-D-thiofucosylethyl)amino)alkyl)formamide (Fuc-C4-chol) or cholesteryl-(4-(3-((2R,3S,4R,5S,6S)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)thiourea)butyl)carbamate (Thiourea-Galac-C4-chol).

5. The active agent complex of any one of claims 1-4, wherein the lower airway includes the portion of the larynx below the vocal cords, the trachea, bronchi, bronchioles, and lungs.

6. The active agent complex according to any one of claims 1 to 5, wherein the bacterial infection of the lower airways comprises tuberculosis, cystic fibrosis, pneumonia and chronic obstructive pulmonary disease.

7. The active agent complex according to claim 6, wherein the tuberculosis is multidrug-resistant tuberculosis.

8. The active agent complex according to claim 6 or 7, wherein the infection is caused by one or more bacteria selected from the group consisting of: Pseudomonas aeruginosa, Pseudomonas fluorescens, Pseudomonas acidovorans, Pseudomonas alcaligenes, Pseudomonas putida, Bordetella pertussis, Bordetella parapertussis, Bordetella bronchiseptica, Haemophilus influenzae, Haemophilus parainfluenzae, Legionella pneumophila, Burkholderia cepacia, Streptococcus pneumoniae and Mycobacterium tuberculosis.

9. A pharmaceutical preparation comprising (a) an active agent complex comprising one or more antimicrobial agents encapsulated in a 1,2-dimyristoyl-sn-glycero-3-phosphoglycerol (DMPG)-based and / or 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC)-based nanocarrier having at least one cell-specific targeting ligand on its surface, wherein the targeting ligand specifically binds to a receptor on the surface of a CRD-positive cell or a CLR-positive cell of the lower airway, and (b) a pharmaceutically acceptable carrier, diluent, excipient, buffer and / or surfactant, The pharmaceutical formulation is used in a method of treating lower airway bacterial infections by inhalation.

10. The pharmaceutical formulation for use according to claim 9, wherein the inhalation is achieved by inhaling the aerosolized suspension containing the active agent complex through the mouth and throat.

11. The pharmaceutical preparation for use according to claim 9 or 10, wherein the antibacterial agent is bedaquiline (BDQ), kanamycin A, amikacin, tobramycin, dibekacin, gentamicin, sisomicin, netilmicin, neomycin B, neomycin C, paromomycin, streptomycin; moxifloxacin, levofloxacin, sparfloxacin, nalidixic acid, ciprofloxacin, cinoxacin, oxolinic acid, piromidic acid, pipemidic acid, rosoxacin, enoxacin, Fleroxacin, lomefloxacin, nadifloxacin, norfloxacin, ofloxacin, pefloxacin, rufloxacin, balofloxacin, grepafloxacin, pazufloxacin, temafloxacin, tosufloxacin, clinafloxacin, gatifloxacin, sitafloxacin, prulifloxacin, delafloxacin, nemofloxacin, danofloxacin, difloxacin, enrofloxacin, ibafoxacin, marbofloxacin, orbifloxacin, sarafloxacin, trovafloxacin, linezolid, metronidazole, tinidazole, and / or nimorazole.

12. The pharmaceutical formulation for use according to claim 11, wherein the antibacterial agent is bedaquiline (BDQ).

13. The pharmaceutical formulation for use according to any one of claims 9 to 12, wherein the targeting ligand is monofucose, polyfucose, cholesteryl-5-yloxy-N-(4-((1-imino-2-D-thiofucosylethyl)amino)alkyl)formamide (Fuc-C4-chol) or cholesteryl-(4-(3-((2R,3S,4R,5S,6S)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)thiourea)butyl)carbamate (Thiourea-Galac-C4-chol).

14. A pharmaceutical preparation for use according to any one of claims 9 to 13, wherein the lower airways include the part of the larynx below the vocal cords, the trachea, bronchi, bronchioles and lungs.

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