Liposomes comprising phosphatidylserine, phosphatidic acid and optionally cholesterol, compositions and medical uses

Abstract

The present invention relates to a liposome consisting of an outer leaflet comprising phosphatidylserine and an inner leaflet comprising phosphatidylserine and phosphatidic acid, characterized in that the zeta potential of the liposome is-25 mV to-100 mV and the molar ratio between total phosphatidylserine and phosphatidic acid is 5: 1 to 35: 1, more preferably 16: 1 to 18: 1, even more preferably 17: 1. The invention also relates to the use of said liposomes and / or the composition comprising said liposomes in the treatment of bacterial infections, preferably in the treatment of infections caused by Mycobacterium, in particular by non-tuberculous Mycobacterium.

Description

Technical Field

[0001] The present invention relates to a liposome consisting of an outer leaflet comprising phosphatidylserine and an inner leaflet comprising phosphatidylserine and phosphatidic acid, characterized in that the zeta potential of the liposome is between -25 mV and -100 mV and the molar ratio between total phosphatidylserine and phosphatidic acid is between 5:1 and 35:1, more preferably between 16:1 and 18:1, even more preferably 17:1.

[0002] The present invention also relates to the use of the liposomes and / or compositions comprising the liposomes in the treatment of bacterial infections, preferably in the treatment of infections caused by mycobacteria, in particular by nontuberculous mycobacteria. Background Art

[0003] Mycobacterium is a genus of Gram-variable bacilli, the only genus in the family Mycobacteriaceae that is capable of causing a variety of diseases in humans.

[0004] The cell wall of mycobacteria is characterized by a thin layer of peptidoglycan combined with arabinogalactan, mycolic acids and phenolic glycolipids. Such a complex cell wall gives mycobacteria the advantage of being completely impermeable to some of the most commonly used substances in medical treatment, which partly explains the known resistance of mycobacteria to commonly used antibiotics.

[0005] In medicine, mycobacteria are traditionally divided into 3 groups.

[0006] The first group includes tuberculous mycobacteria, i.e., those capable of causing tuberculosis in animal hosts. This group consists of bacteria of the so-called Mycobacterium tuberculosis complex: Mycobacterium tuberculosis (causes tuberculosis in humans), Mycobacterium africanum (infects organisms similar to those of M. tuberculosis but is less likely to cause disease), and Mycobacterium bovis (cause bovine tuberculosis, a zoonosis that can be transmitted to humans through food).

[0007] The second group consists of nontuberculous mycobacteria, ie mycobacteria that cause a range of pathological conditions other than tuberculosis in the human host but only in the presence of specific conditions that coincide with the reduction of the immune defenses of the colonizing organism (they are therefore opportunistic pathogens).

[0008] The third group is represented by Mycobacterium leprae, the causative agent of leprosy, which, although it can be classified into the group of nontuberculous mycobacteria, has unique clinical and biological features.

[0009] Nontuberculous mycobacteria are a heterogeneous group of bacteria consisting of more than 150 different species, some of which are able to infect humans and cause diseases in humans other than tuberculosis.

[0010] It is known that most subjects infected with nontuberculous mycobacteria will not develop clinical symptoms of infection.

[0011] It has been observed that the subjects most susceptible to infection with nontuberculous mycobacteria are those characterized by a weakened immune system, such as AIDS patients or patients undergoing transplantation, or persons with lung lesions due to smoking or previous tuberculosis, or persons affected by lung diseases such as emphysema, COPD or cystic fibrosis.

[0012] Infections caused by nontuberculous mycobacteria can be difficult to treat and take a long time to eradicate due to these organisms being resistant to commonly prescribed antibiotics used to treat bacterial infections.

[0013] Among nontuberculous mycobacterial species, infections caused by Mycobacterium abscessus are more difficult to treat due to its resistance to commonly used antimicrobial drugs.

[0014] Antibiotic resistance in M. abscessus has been attributed to a variety of mechanisms, among which are intrinsic drug resistance, low permeability of the cell wall, induction of drug efflux pumps, the presence of mutations in mycobacterial enzymes that do not convert prodrugs to active metabolites, and / or the expression of numerous enzymes that can neutralize drugs or alter their specific targets.

[0015] M. abscessus can cause lung disease, skin infections, central nervous system infections, bacteremia, eye infections, and other less common illnesses that can become very serious in immunocompromised patients.

[0016] Furthermore, M. abscessus in particular can cause chronic lung disease in susceptible hosts with underlying lung disease (eg, cystic fibrosis, bronchiectasis, and / or previous tuberculosis).

[0017] M. abscessus can also cause skin infections in immunocompromised patients and those who have had recent surgical intervention, tattoos, or acupuncture.

[0018] The incidence of M. abscessus infections appears to be increasing over time (Lee et al., Mycobacterium abscessus Complex Infections in Humans, Emerg Infect Dis. 2015, Sep 21(9):1638–46), and of particular concern are outbreaks of M. abscessus nosocomial infections reported in immunocompromised patients.

[0019] In view of the above, there is a clear need to provide new products having antimicrobial efficacy against antibiotic resistant bacteria, in particular against mycobacteria, preferably nontuberculous mycobacteria.

[0020] A useful product for the treatment of lung infections caused by bacteria and / or viruses, in particular by mycobacteria, are the liposomes described in WO 2009 / 011007.

[0021] The liposomes described in WO2009 / 011007 are asymmetric liposomes comprising phosphatidylserine molecules in the outer lipid layer and at least one biologically active lipid in the inner lipid layer. The biologically active lipid present in the inner lipid layer of the liposome is selected from phosphatidic acid, lysophosphatidic acid, arachidonic acid, sphingomyelin, sphingosine, sphingosine 1-phosphate, ceramide, leukotrienes, prostanoids, cyclopentenone prostaglandins or derivatives thereof.

[0022] However, such liposomes, characterized by absolute zeta potential values ​​of less than 25 mV, appear to be highly unstable.

[0023] The measurement of the zeta potential is known to predict the stability of electrostatic dispersion or interaction. In particular, the zeta potential is the potential arising from the formation of a double electrical layer.

[0024] In fact, the particles move with the ionic double layer when moving in solution (Brownian motion).

[0025] High absolute zeta potential values ​​(ie, less than -30 mV and greater than +30 mV) cause the nanoparticles to remain apart from one another, rebounding sufficiently to eliminate the possibility of agglomeration, aggregation, and / or flocculation.

[0026] Low zeta potential values, such as those of the liposomes described in WO 2009 / 011007, lead to particle aggregation and flocculation.

[0027] Indeed, liposomes characterized by absolute zeta potential values ​​less than 25 mV are extremely unstable and tend to form aggregates when in solution.

[0028] Besides this, the industrial process for obtaining liposomes characterized by such high instability described in WO2009 / 011007 presents considerable difficulties and is not easily scalable.

[0029] Furthermore, the asymmetric liposomes described in WO2009 / 011007 have a high polydispersity index and there are two or more populations of particles of different sizes. It is therefore evident that such asymmetric liposomes are characterized by a high size heterogeneity.

[0030] Therefore, there is a need to overcome the disadvantages of the known technology by providing products, in particular liposomes, having antimicrobial activity against antibiotic-resistant bacteria, which have higher stability, higher size uniformity and can be obtained by scalable processes. Summary of the invention

[0031] The applicant proposes to solve the technical problem of low stability of liposomes with antimicrobial activity in the prior art by providing liposomes characterized by a zeta potential of -25 mV to -100 mV.

[0032] Compared with asymmetric liposomes in the prior art having an absolute zeta potential value of less than 25 mV, the liposomes according to the present invention are more stable, do not undergo flocculation and aggregation, and are characterized by a higher size uniformity.

[0033] Besides being particularly stable, the liposomes according to the invention are also characterized by high antimicrobial activity, since they are able to stimulate the response of the immune system to one or more pathogens. In fact, as shown in the experimental section, the liposomes of the invention enhance the microbicidal response of macrophages by transporting lipid intermediates (fatty acids, phospholipids, etc.) known to be involved in antibacterial activity (via phagolysosomal biogenesis) or antiviral responses directly into macrophages.

[0034] According to the present invention, these lipid intermediates are transported into macrophages via liposomes characterized by the presence of phosphatidylserine on the outer leaflet.

[0035] Indeed, the presence of phosphatidylserine in the outer leaflet makes the liposomes according to the invention similar to apoptotic bodies. In particular, apoptotic bodies can be phagocytosed by macrophages and target cells of viral and bacterial lung infections, namely fibroblasts, epithelial cells and endothelial cells, which recognize and phagocytose apoptotic bodies.

[0036] Once engulfed by the cells, the liposomes according to the invention release bioactive lipids therein, which are known to be involved in antibacterial activity. In detail, the bioactive lipids participate in all steps of the engulfment process starting from the internalization of the pathogen to the maturation of the phagolysosome, and participate in the activation of the bactericidal response of the cells of the innate immune system by recruiting, retaining and regulating the activity of the specific proteins involved.

[0037] Furthermore, advantageously, phagocytosis of liposomes by macrophages is associated with the production of anti-inflammatory cytokines (Hoffmann PR et al. J. Immunol. 2005; 174: 1393-1404), which reduces the inflammatory response of damaged tissues.

[0038] Therefore, a first aspect of the present invention is a liposome consisting of an outer leaflet comprising phosphatidylserine and an inner leaflet comprising phosphatidylserine and phosphatidic acid, characterized in that the zeta potential of the liposome is between -25 mV and -100 mV and the molar ratio between total phosphatidylserine and phosphatidic acid is between 5:1 and 35:1.

[0039] Preferably, in the liposomes according to the invention, the molar ratio between total phosphatidylserine and phosphatidic acid is between 16:1 and 18:1, even more preferably 17:1.

[0040] According to a preferred aspect, as shown in the experimental part, when the molar ratio of phosphatidylserine, in particular L-α-phosphatidylserine, to phosphatidic acid, in particular L-α-phosphatidic acid, is 17:1 (intended as the molar ratio between total phosphatidylserine and phosphatidic acid), the efficacy of the antimicrobial response against Mycobacterium abscessus is improved. Therefore, in a particularly preferred embodiment, the liposomes of the present invention comprise phosphatidylserine and phosphatidic acid in a molar ratio of 17:1.

[0041] Optionally, the liposomes according to the invention may further comprise cholesterol, which may be arranged on the outer leaflet, on the inner leaflet or on both.

[0042] In one embodiment, the liposomes according to the first aspect of the invention incorporate one or more antibiotics, for example selected from first-line anti-tuberculosis antibiotics; second-line anti-tuberculosis antibiotics; aminoglycosides; glycylcyclines; penicillins; carbapenems; monobactams; quinolones; oxazolidinones; and macrolides.

[0043] The liposomes according to the invention can be produced by lipid membrane hydration (Example 1), or preferably, by microfluidic hydrodynamic focusing technology (Example 2). Therefore, the present invention relates to liposomes produced by lipid membrane hydration or by microfluidic hydrodynamic focusing technology as described above.

[0044] The second aspect of the present invention relates to a pharmaceutical composition comprising at least one liposome according to the first aspect of the present invention, and one or more pharmaceutically acceptable excipients and / or adjuvants.

[0045] A third aspect of the present invention relates to the use of liposomes as defined above and / or pharmaceutical compositions comprising said liposomes for the treatment of bacterial infections, in particular infections caused by mycobacteria, preferably by nontuberculous mycobacteria.

[0046] As shown in the experimental part, the liposomes according to the invention have been shown to be particularly effective against Mycobacterium abscessus, an opportunistic pathogen characterized by extensive and intrinsic antibiotic resistance.

[0047] Therefore, in a particularly preferred embodiment, the liposomes and / or compositions comprising the liposomes are used to treat infections caused by Mycobacterium abscessus.

[0048] A final aspect of the invention is a kit comprising a pharmaceutical composition as defined above and one or more containers. DETAILED DESCRIPTION OF THE INVENTION

[0049] An object of the present invention is a liposome composed of an outer leaflet comprising phosphatidylserine and an inner leaflet comprising phosphatidylserine and phosphatidic acid, characterized in that the zeta potential of the liposome is between -25 mV and -100 mV and the molar ratio between total phosphatidylserine and phosphatidic acid is between 5:1 and 35:1.

[0050] Liposomes are closed vesicles composed of a bilayer membrane composed of phospholipids.

[0051] Phospholipids are amphipathic molecules, specifically a class of lipids with a hydrophilic polar head based on phosphate and a hydrophobic nonpolar tail.

[0052] At the biological level, phospholipids are involved in the structure of cell membranes. Liposomes were discovered quite accidentally in the early 1960s by the British hematologist Alec Bangham during electron microscopy experiments. Since then, liposomes have been widely used as a delivery system for poorly absorbed or poorly soluble active substances, including pharmaceutical substances.

[0053] In fact, liposomes "encapsulate" one or more active substances, owing to the surfactant properties of the phospholipids constituting them, in order to facilitate their release and promote their absorption at the mucosal level.

[0054] Secondly, the components of the phospholipid layer of liposomes are biocompatible, and furthermore, liposomes do not cause side effects.

[0055] In addition, the fact that the substances carried by the liposomes are protected from the action of enzymes (proteases, nucleases) or from denaturing environments (pH) brings further advantages. This also allows to preserve the integrity of the active substances encapsulated in the liposomes.

[0056] Finally, liposomes are biodegradable and can currently be prepared on a large scale according to known techniques.

[0057] According to a preferred aspect, the liposomes according to the invention comprise phosphatidylserine, in particular L-α-phosphatidylserine, and phosphatidic acid, in particular L-α-phosphatidic acid, in a molar ratio of 16: 1 to 18: 1, more preferably 17: 1. In particular, the molar ratio is the molar ratio between total phosphatidylserine and phosphatidic acid.

[0058] In the present invention, the expression "total phosphatidylserine" is intended to mean the amount of phosphatidylserine resulting from the sum of the phosphatidylserine present on the outer leaflet of the liposome and the phosphatidylserine present on the inner leaflet of said liposome.

[0059] Phosphatidylserine (also abbreviated as "PS" in this specification) is a phospholipid component of the plasma membrane of cells, which plays a key role in the signal transduction of apoptosis. Phosphatidylserine in healthy cells is usually exposed on the cytoplasmic side, and when phosphatidylserine is exposed to the outer leaflet of the plasma membrane by flippase ("eat me" signal), it leads to the recognition of the cell by macrophages and subsequent elimination.

[0060] The presence of phosphatidylserine in the outer leaflet of the liposomes according to the invention makes such liposomes similar to apoptotic bodies.

[0061] In biology, the term "apoptotic body" is intended to mean the vesicles generated by the fragmentation of the nucleus and cytoplasm of a cell during the apoptotic process (a form of programmed cell death), which are subsequently engulfed by macrophages. In this way, the liposomes according to the invention will be recognized as apoptotic bodies by macrophages and target cells that may be infected by bacteria (e.g., fibroblasts, epithelial cells, and endothelial cells) and engulfed.

[0062] Advantageously, phagocytosis of the liposomes according to the invention allows the delivery of bioactive lipids involved in antibacterial responses. In particular, these lipids determine the formation and fate of the phagosome by influencing the curvature of the cell membrane into which they are incorporated, contribute to the membrane surface charge, and coordinate protein recruitment mechanisms and association with key proteins in phagocytosis.

[0063] Furthermore, phagocytosis of liposomes is associated with the production of anti-inflammatory cytokines and is therefore responsible for reducing the inflammatory response of damaged tissues.

[0064] According to the present invention, phosphatidylserine is contained in both the outer and inner leaflet of the liposome.

[0065] In the present invention, the terms "comprising", "including" and "containing" are to be understood as open terms, which do not exclude the presence of compounds other than the ones mentioned.

[0066] In particular, the presence of these terms in relation to the outer and inner leaflet of the liposomes indicates that, in addition to phosphatidylserine and phosphatidic acid, other molecules may be present in both the outer and inner leaflet of the liposomes of the invention.

[0067] In one embodiment, the outer leaflet of the liposome according to the invention comprises phosphatidylserine and one or more other phospholipids.

[0068] Preferably, said other phospholipid that may be present in the outer leaflet of the liposome is phosphatidic acid.

[0069] Phosphatidic acid (abbreviated as "PA" in the present invention) is a phosphoglyceride which is formally produced by esterifying glycerol with fatty acids at the 1- and 2-positions and with orthophosphoric acid at the 3-position.

[0070] According to the present invention, the molar ratio between total phosphatidylserine and phosphatidic acid in the liposomes is 5:1 to 35:1.

[0071] In a particularly preferred embodiment, the molar ratio between total phosphatidylserine and phosphatidic acid present in the liposomes is from 16:1 to 18:1, more preferably 17:1.

[0072] Indeed, as shown in the experimental section, liposomes comprising a 17:1 molar ratio between total phosphatidylserine and phosphatidic acid gave the best results in terms of intracellular inhibition of M. abscessus compared to liposomes with different phosphatidylserine to phosphatidic acid ratios.

[0073] Furthermore, the liposomes comprising a molar ratio of total phosphatidylserine to phosphatidic acid of 17:1 significantly enhanced intracellular killing of Mycobacterium abscessus (see Figure 1 A and B).

[0074] Optionally, the liposomes according to the invention may further comprise cholesterol, which may be arranged on the outer leaflet, on the inner leaflet or on both.

[0075] Cholesterol (abbreviated herein by the acronym "CHO") is an organic molecule belonging to the class of lipids and more specifically the class of sterols.

[0076] Cholesterol is a polycyclic aliphatic alcohol with a brute formula of C 27 H 46 O, consists of a perhydro-1,2-cyclopentane-phenanthrene nucleus (a typical tetracyclic nucleus of steroids) with a double bond at C5 and an isooctyl side chain at C17. The suffix "-ol" comes from the presence of a hydroxyl group -OH at C3 of the first carbon atom ring (ring A).

[0077] From a biological perspective, cholesterol has a structural function in cell membranes: it increases the flexibility and stability of the bilayer and maintains its fluidity, even at low temperatures. Cholesterol inserts vertically into the cell membrane, with its hydroxyl group facing outwards and the octyl chain inserted deeply into the membrane.

[0078] In the liposomes according to the invention, cholesterol may optionally be present.

[0079] In particular, cholesterol may optionally be present in both the outer and inner leaflets of the liposome.

[0080] In a particularly preferred embodiment, the molar ratio between total phosphatidylserine, phosphatidic acid and cholesterol in the liposomes according to the invention is 16.5:1:7.5.

[0081] In the liposomes according to the invention, the phospholipids of the liposomes, mainly phosphatidylserine and phosphatidic acid, have the structure and biological activity functions as defined above, while cholesterol mainly has a structural function.

[0082] The liposomes described so far can advantageously be used as a delivery system for one or more drugs (such as drugs commonly used to treat bacterial infections).

[0083] Thus, in one embodiment, the liposomes may encapsulate one or more antibiotics inside them, such as first-line anti-tuberculosis antibiotics, second-line anti-tuberculosis antibiotics, aminoglycosides, glycylcyclines, tetracyclines, cephalosporins, penicillins, carbapenems, monobactams, quinolones, oxazolidinones and macrolides.

[0084] In one embodiment, the liposomes embed in their interior: first-line anti-tuberculosis antibiotics, preferably selected from isoniazid, rifampin, rifabutin, ethambutol, pyrazinamide and streptomycin; second-line anti-tuberculosis antibiotics, preferably selected from cycloserine, ethionamide, levofloxacin, moxifloxacin, ciprofloxacin, gatifloxacin acid, paraaminosalicylic acid, acid, kanamycin and capreomycin; aminoglycosides, preferably selected from gentamicin and amikacin; glycylcyclines, preferably tigecycline; tetracyclines, preferably minocycline; cephalosporins, preferably selected from cefoxitin and ceftobiprole medocaryl sodium; penicillins, preferably amoxicillin; carbapenems, preferably imipenem; monobactams, preferably aztreonam lysine salt; quinolones, preferably moxifloxacin; Oxazolidinones, preferably linezolid; macrolides, preferably selected from azithromycin, clarithromycin, erythromycin and fidaxomycin.

[0085] The size of the liposomes according to the present invention may be between 30 nm and 350 nm, preferably between 50 nm and 300 nm, even more preferably between 100 nm and 250 nm.

[0086] The polydispersity index of the liposomes of the present invention is from 0.10 to 0.55, preferably from 0.20 to 0.40, even more preferably from 0.25 to 0.35.

[0087] The zeta potential of the liposomes of the present invention is lower than -25 mV, preferably lower than -30 mV, more preferably lower than -35 mV, and higher than -100 mV, preferably higher than -70 mV, more preferably higher than -50 mV.

[0088] Preferably, the zeta potential of the liposome according to the present invention is -25mV to -85mV, more preferably -30mV to -75mV, even more preferably -35mV to -50mV. The above zeta potential values ​​indicate that the liposome has good stability and will not tend to form aggregates in solution because the nanoparticles are kept far enough apart from each other and the repulsive force is sufficient to eliminate the possibility of agglomeration, aggregation and / or flocculation. The liposome objects of the present invention are produced by lipid film hydration (see Example 1 and the results of Example 1), or more preferably by microfluidic hydrodynamic focusing technology (see Example 2 and the results of Example 2).

[0089] In particular, microfluidic hydrodynamic focusing (MHF) is based on the use of a device (chip) with a cross-flow geometry. Typically, a lipid flow in an alcohol solution is forced to flow in the central (or internal) channel of the device. The lipid flow is crossed and covered by two lateral (or coaxial) flows of an aqueous phase (usually distilled water or an aqueous buffer). In this way, the lipid-containing flow is hydrodynamically focused into a thin sheet with a rectangular cross-section. In particular, the size of the focused flow can be adjusted by adjusting the flow ratio (Flow Rate Ratio-FRR) between the flows of the aqueous phase and the lipid phase and the total flow rate (Total Flow Rate-TFR). The formation of liposomes in the MHF chip is regulated by the diffusion of a variety of molecular substances (mainly alcohol and water, but also lipids) at the liquid interface between the solvent (alcohol) and non-solvent (water) phases. The alcohol that initially dissolves the lipid diffuses into the water (while the water also diffuses into the alcohol) until the alcohol concentration drops below the lipid solubility limit. Therefore, the diffusion of alcohol triggers the formation of liposomes through a mechanism known as "self-assembly". In particular, the interdiffusion of alcohol and water at the focusing interface is thought to lead to lipid precipitation followed by the formation of intermediate structures that subsequently close on themselves to form liposomal vesicles.

[0090] Microfluidic hydrodynamic focusing technology is particularly advantageous for producing liposomes according to the present invention, because the size of the liposomes can be controlled by adjusting the FRR flow ratio. As is evident from Table 3a and Table 3b, the liposomes produced by microfluidic hydrodynamic focusing technology have a higher uniformity. A second aspect of the present invention relates to a pharmaceutical composition comprising at least one liposome as defined above and at least one pharmaceutically acceptable excipient.

[0091] The composition of the present invention may comprise at least one antibiotic, which is ultimately embedded in the liposomes and / or outside the liposomes.

[0092] In particular, in one embodiment of the composition of the present invention, at least one antibiotic is present within the liposome, encapsulated therein.

[0093] In another embodiment, at least one antibiotic is present in the pharmaceutical composition of the invention outside of the liposomes.

[0094] In yet another embodiment, at least one antibiotic is present in the composition both inside and outside the liposomes.

[0095] When at least one antibiotic is present in the composition both inside and outside the liposomes, the antibiotic may be the same, or a combination of two or more antibiotics.

[0096] For example, the antibiotics that may be present in the composition according to the invention are: first-line anti-tuberculosis antibiotics, preferably selected from isoniazid, rifampicin, rifabutin, ethambutol, pyrazinamide and streptomycin; second-line anti-tuberculosis antibiotics, preferably selected from cycloserine ethionamide, levofloxacin, moxifloxacin, ciprofloxacin, gatifloxacin acid, para-aminosalicylic acid, kanamycin and capreomycin; aminoglycosides, preferably selected from gentamicin and amikacin; glycylcyclines, preferably tigecycline; tetracyclines, preferably minocycline; cephalosporins, preferably selected from cefoxitin and cefoperazone sodium; penicillins, preferably amoxicillin; carbapenems, preferably imipenem; monobactams, preferably aztreonam lysine salt; quinolones, preferably moxifloxacin; Oxazolidinones, preferably linezolid; macrolides, preferably selected from azithromycin, clarithromycin, erythromycin and fidaxomicin.

[0097] The at least one pharmaceutically acceptable excipient present in the composition according to the invention may be an excipient and / or adjuvant usually present in formulations suitable for administration via the air, the skin or a mucosa (eg the intestinal mucosa).

[0098] In a preferred embodiment, the composition according to the invention comprises one or more pharmaceutically acceptable excipients and / or adjuvants for administration by inhalation.

[0099] The liposomes according to the invention have proven to be particularly useful for the treatment of bacterial infections, in particular infections caused by nontuberculous mycobacteria.

[0100] Furthermore, a third aspect of the present invention relates to the use of a liposome as defined above and / or a pharmaceutical composition comprising at least one such liposome for the treatment of a bacterial infection.

[0101] In one embodiment, the liposomes and compositions comprising the liposomes according to the present invention can be used to treat bacterial infections caused by Pseudomonas aeruginosa, Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Enterobacter spp., Propionibacterium acnes, Mycobacterium tuberculosis complex, Mycobacterium tuberculosis, Mycobacterium africanum, Mycobacterium bovis, Mycobacterium ulcerans, Mycobacterium leprae, Mycobacterium abscessus, Mycobacterium avium complex (MAC), Mycobacterium chimaera, Mycobacterium kansasii, kansasii), Mycobacterium bolletii, Mycobacterium massiliense and Mycobacterium abscessus, preferably Mycobacterium abscessus.

[0102] The release of bioactive lipids by the liposomes of the invention in the macrophages into which they have been incorporated generally allows an enhanced antimicrobial response. In fact, the lipids released by the liposomes of the invention, which are recognized as apoptotic bodies by macrophages and incorporated into them, participate in the internalization of pathogens until phagolysosomal maturation.

[0103] As is known, phagolysosomes are vesicles formed by the fusion of phagosomes (endocytic vesicles containing foreign substances (e.g., pathogens)) and lysosomes (vesicles containing lysosomal enzymes capable of degrading foreign substances (e.g., pathogens) contained in phagosomes) within phagocytes (cells capable of engulfing and killing pathogens intracellularly, including macrophages).

[0104] It is therefore evident that the enhancement of the antimicrobial response does not depend on the type of microorganism causing the infection but rather on the specific mechanism by which the liposomes according to the invention act.

[0105] In a preferred embodiment, the liposomes and compositions comprising the liposomes of the present invention are used to treat infections caused by mycobacteria, preferably by nontuberculous mycobacteria.

[0106] Some examples of mycobacteria for which the liposomes according to the first aspect of the invention and the compositions according to the second aspect of the invention can be used are Mycobacterium tuberculosis complex, Mycobacterium avium complex (MAC), Mycobacterium chimaera, Mycobacterium kansasii, Mycobacterium bolei, Mycobacterium massiliense and Mycobacterium abscessus.

[0107] Nontuberculous mycobacteria can be divided into two groups: slow-growing and fast-growing. Organisms belonging to the first group include, for example, the Mycobacterium tuberculosis complex, the Mycobacterium avium complex (MAC), M. chimaera, and M. kansasii, while those belonging to the second group include the M. abscessus complex, which includes M. abscessus ssp. abscessus, M. abscessus ssp. bolletii, and M. abscessus ssp. massiliense.

[0108] In one embodiment, the liposomes according to the first aspect of the invention and the composition according to the second aspect of the invention are used to treat bacterial infections caused by Mycobacterium abscessus.

[0109] As shown in the experimental section, the replication of M. abscessus was reduced in PSPA-L liposomes having a molar ratio between total phosphatidylserine and phosphatidic acid of 5:1 to 35:1.

[0110] from Figure 1 In B), it is evident that liposomes with a molar ratio between total phosphatidylserine and phosphatidic acid of 8.5:1 and 34:1 showed antimicrobial activity against M. abscessus and reduced the replication of M. abscessus in macrophages compared to macrophages not treated with the liposomes according to the invention.

[0111] Even more advantageously, liposomes with a molar ratio of 17:1 between total phosphatidylserine (the sum of PS present in the outer and inner leaflets of the liposomes) and phosphatidic acid enhanced the antimicrobial response of macrophages to M. abscessus.

[0112] Therefore, preferably, the liposomes and compositions comprising the liposomes of the present invention are used to treat infections caused by M. abscessus.

[0113] In particular, the composition and / or the liposomes contained therein may be used to treat nontuberculous mycobacterial lung disease, a relatively rare but chronic and debilitating pathological condition that results in decreased lung function.

[0114] It is advantageously observed that combination therapy based on antibiotics and bioactive liposomes can be an effective strategy to simultaneously target extracellular and intracellular pathogens, since, on the one hand, the liposomes of the present invention act by promoting the activation of bactericidal responses of cells of the innate immune system by recruiting, retaining and modulating the activity of specific proteins involved, and, on the other hand, the antibiotics act directly on the pathogens.

[0115] As reported in the experimental section, treatment with liposomes comprising phosphatidylserine and phosphatidic acid in a molar ratio of 17:1 or phosphatidylserine, phosphatidic acid and cholesterol in a ratio of 16.5:1:7.5 in combination with amikacin additively reduced the intracellular growth of Mabs in human macrophages. In particular, the combined treatment with the liposomes induced a significantly greater reduction in the Mab replication index relative to the single treatments (see Figure 3 ).

[0116] Another object of the present invention is a kit comprising a pharmaceutical composition according to the second aspect of the invention and one or more containers.

[0117] In one embodiment, the kit comprises a container for a pharmaceutical composition comprising one or more liposomes having an antibiotic incorporated therein.

[0118] In another embodiment, the kit comprises: a container for a pharmaceutical composition comprising one or more liposomes without an antibiotic incorporated therein; and one or more containers for an antibiotic associated with treatment with the liposomes according to the invention.

[0119] In yet another embodiment, the kit comprises: a container for a pharmaceutical composition comprising one or more liposomes having an antibiotic incorporated therein; and one or more containers for an antibiotic associated with treatment with the liposomes according to the invention.

[0120] One aspect of the present description relates to a method for treating one or more bacterial infections, preferably infections caused by mycobacteria, in particular by nontuberculous mycobacteria, comprising administering to a subject suffering from one or more bacterial infections one or more of the liposomes according to the invention and / or the pharmaceutical compositions according to the invention.

[0121] The effective dosage and administration regimen of the liposomes and / or compositions comprising the liposomes according to the present invention depends on many factors such as, for example, the mode of administration or the degree of disease of the individual being treated.

[0122] Furthermore, both the effective dosage and administration regimen will be determined by the physician treating the subject being treated.

[0123] In a preferred embodiment, the method of treating one or more bacterial infections, preferably mycobacteria, comprises administering by inhalation, more preferably by aerosol, one or more liposomes as defined above and / or compositions comprising them. BRIEF DESCRIPTION OF THE DRAWINGS

[0124] The following abbreviations are shown in the drawings accompanying this patent application and in the following examples: PS: phosphatidylserine; PS-NBD: phosphatidylserine conjugated with NBD fluorescent dye; PA: phosphatidic acid; CHO: cholesterol; Mab: Mycobacterium abscessus; Ctrl: control; PSPA-L: liposomes obtained by lipid membrane hydration method, which contain PS on the outer leaflet and PS and PA on the inner leaflet; PSPACHO-L: liposomes obtained by lipid membrane hydration method, which contain PS on the outer leaflet and PS, PA and CHO on the inner leaflet; PS / PA: prior art asymmetric liposomes obtained by the Weitz method as described in WO 2009 / 011007 A2, comprising PS only on the outer leaflet and PA only on the inner leaflet; PSPA-M: liposomes obtained by microfluidic hydrodynamic focusing technology, which contain PS on the outer leaflet and PS and PA on the inner leaflet; AMK: amikacin; dTHP-1: THP-1 cells were stimulated with phorbol-12-myristate-13-acetate (PMA) to induce differentiation and then used as a human macrophage model.

[0125] Figure 1 In vitro comparison of the efficacy of various formulations of PSPA-L liposomes in intracellular killing of Mabs.

[0126] 5×10 5 dTHP-1 cells / well were seeded in 24-well plates and infected with the Mab reference strain (ATCC 19977) for 3 h, followed by incubation with PSPA-L ( Figure 1 A) and PSPA-L ( Figure 1B) Treat for 18 hours. The growth of Mab bacteria was assessed by CFU assay. The replication index was calculated as the ratio between the CFU obtained 18 hours after infection in the absence of (control) or the presence of liposome preparations and the CFU obtained before adding liposomes. The results are shown as the mean ± standard deviation of the values ​​obtained from triplicates of each condition of two independent experiments. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001; ·p<0.00001; ·p<0.00001, Student t test from two-tailed distribution.

[0127] Figure 2 Evaluation of the efficacy of PSPA-L 17:1 liposomes in intracellular killing of Mabs following addition of cholesterol and dose variation.

[0128] 5×10 5 dTHP-1 cells / well were seeded in 24-well plates and infected with the Mab reference strain (ATCC 19977) for 3 hours and then treated with PSPA-L at a molar ratio of 17:1, double dose of PSPA-L at a molar ratio of 17:1 (2×PSPA-L 17:1), or PSPACHO-L for 18 hours. Bacterial growth was assessed by CFU assay. The replication index was calculated as the ratio between the CFU obtained 18 hours after infection in the absence (control) or presence of liposome formulations and the CFU obtained before the addition of liposomes. Results are shown as mean ± standard deviation of values ​​obtained from triplicates for each condition. nsp = not significant; ***p<0.001; ****p<0.0001, Student's t-test from two-tailed distribution.

[0129] Figure 3 Evaluation of the efficacy of a combination strategy consisting of PSPA-L 17:1 or PSPACHO-L liposomes and amikacin in terms of extracellular and intracellular killing of Mabs.

[0130] 5×10 5dTHP-1 cells / well were inoculated in 24-well plates and infected with Mab reference strain (ATCC 19977) for 3 hours, then treated with PSPA-L or PSPACHO-L at a molar ratio of 17:1 for 18 hours, and / or treated with 4 μg / ml of amikacin (AMK) for another 18 hours. Finally, supernatants were collected, cells were lysed, and both intracellular (A and B) and extracellular (C and D) bacterial growth were analyzed. Replication index was calculated as the ratio between the CFU obtained after infection 18 hours in the absence (control) or presence of liposome preparations and / or AMK and the CFU obtained before adding liposomes. Results are shown as the mean ± standard deviation of the values ​​obtained from triplicates of each condition. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001; ·p<0.00001; ··p<0.00001, Student's t-test from two-tailed distribution.

[0131] Figure 4 Evaluation of cell viability after treatment with PSPA-L 17:1 and PSPACHO-L liposomes.

[0132] 2×10 5 dTHP-1 cells / well were seeded in 96-well plates and incubated in complete medium (control, Ctrl), or treated with PSPA-L, PSPACHO-L or saponin (Sap, used as a negative control for cell viability) at a molar ratio of 17:1 for 18 hours, and then the viability of dTHP1 cells was monitored using the MTT assay. The results are shown as the mean ± standard deviation of the percentage of cell viability of triplicates for each condition.

[0133] Figure 5 Comparison of the arrangement of PS in asymmetric PS / PA liposomes and PSPA-L liposomes.

[0134] The fluorescence intensity of PS-NBD / PA (A), PS-NBDPA-L 17:1 (B), PS-NBDPACHO-L (C), PS-NBDPA-L 8.5:1 (D), PS-NBDPA-L 34:1 (E) liposomes was kinetically monitored by 12 measurements every 30 seconds (for a total of 5 minutes) in the presence or absence of quencher solution. The results are expressed as a fluorescence intensity index, which is calculated as the ratio between the 12 measurements over time (0, 30, 60, 90, 120, 150, 180, 210, 240, 270, 300, 330 seconds) and the measurement at 0 seconds.

[0135] Figure 6Evaluation of the size distribution of PSPA-L, PSPACHO-L and PS / PA liposome formulations.

[0136] The size distribution of PS / PA (A), PSPA-L 17: 1 (B), PSPACHO-L (C), PSPA-L 8.5: 1 (D) and PSPA-L 34: 1 (E) formulation samples was analyzed using Zeta-Sizer Advance Ultra-Malvern Panalytical. The figure shows a single batch acquisition and represents triplicate acquisitions of two independent batches of liposome formulations. Batch 1 and batch 2 relative to the same liposome formulation were produced at different times. The size distribution is expressed as the intensity percentage (% intensity) associated with the particle diameter in nanometers (d.nm).

[0137] Figure 7 Assessment of cell viability after treatment with PSPA-M 17:1 liposomes.

[0138] 2×10 5 dTHP-1 cells / well were seeded in 96-well plates and incubated in complete medium (control, Ctrl) or treated with 2.54 μM, 1.27 μM, or 0.635 μM of PSPA-M or saponin (Sap, used as a negative control for cell viability) at a molar ratio of 17:1 for 18 h with or without dialyzation ( Figure 7 A) or 5 days ( Figure 7 B) Cell viability was then monitored using the MTT assay. Results are shown as the mean ± standard deviation of the percentage of cell viability of triplicates for each condition.

[0139] Figure 8 Evaluation of the efficacy of dialyzed or non-dialyzed PSPA-M 17:1 liposomes in intracellular killing of Mabs.

[0140] 5×10 5 dTHP-1 cells / well were seeded in 24-well plates and infected with a Mab reference strain (ATCC 19977) for 3 hours, followed by treatment with 1.27 μM PSPA-M at a molar ratio of 17:1, dialyzed or undialyzed, for 18 hours. Bacterial growth was assessed by CFU assay. The replication index was calculated as the ratio between the CFU obtained 18 hours after infection in the absence (control) or presence of a liposome formulation and the CFU obtained before the addition of liposomes. The results are shown as the mean ± standard deviation of the values ​​obtained from triplicates for each condition. ns is not significant, and ***p<0.001, Student's t-test from a two-tailed distribution.

[0141] Fig. 9Comparison of the arrangement of PS in dialyzed PSPA-M 17:1, PSPA-M 17:1, and PSPA-L liposomes.

[0142] Fluorescence intensity of dialyzed PS-NBDPA-M 17:1 (A), PS-NBDPA-M 17:1 (B) and PS-NBD-PA-L 17:1 (C) liposomes was kinetically monitored by 12 measurements every 30 seconds (for a total of 5 minutes) in the presence or absence of quencher solution. The results are expressed as a fluorescence intensity index calculated as the ratio between the 12 measurements over time (0, 30, 60, 90, 120, 150, 180, 210, 240, 270, 300, 330 seconds) and the measurement at 0 seconds.

[0143] Fig.10 Evaluation of cell viability of primary M1 or M2 macrophages after treatment with PSPA-M 17:1 liposomes.

[0144] 2×10 5 M1 cells / well or M2 / well were seeded in 96-well plates and incubated in complete medium (control, Ctrl), or treated with PSPA-M or saponin (Sap, used as a negative control for cell viability) at a molar ratio of 17:1 at a concentration of 2.54 μM, 1.27 μM or 0.635 μM, dialyzed or not, for 18 hours (A and C) or 5 days (B and D), and then cell viability was monitored using the MTT assay. Results are shown as the mean ± standard deviation of the percentage of cell viability of triplicates for each condition.

[0145] Fig.11 Evaluation of the efficacy of dialyzed or non-dialyzed PSPA-M 17:1 liposomes in intracellular killing of Mabs.

[0146] 5×10 5 M1 cells / well or M2 cells / well were inoculated in 24-well plates and infected with Mab reference strain (ATCC19977) for 3 hours, followed by 1.27 μM dialyzed PSPA-M at a molar ratio of 17:1 for 18 hours. Bacterial growth was assessed by CFU assay. The replication index was calculated as the ratio between the CFU obtained 18 hours after infection in the absence (control) or presence of a liposome formulation and the CFU obtained before the addition of liposomes. The results are shown as the mean ± standard deviation of the values ​​obtained from triplicates for each condition. ****p<0.0001, Student t test from a two-tailed distribution. Example Example 1 - Lipid membrane hydration method: Preparation of liposome formulations with different molar ratios between lipids and their efficacy against Mycobacterium abscessus infection Research on force Materials and Methods Preparation of liposome formulations

[0147] Using the lipid film hydration method, a liposomal preparation (PSPA-L) composed of L-α-phosphatidylserine (PS; Avanti Polar Lipids) and L-α-phosphatidic acid (PA; Avanti Polar Lipids) was produced, and finally cholesterol (CHO; Avanti Polar Lipids) was added (PSPACHO-L). Specifically, 7 liposomal preparations with different molar ratios between lipids were produced, as shown in Table 1. Table 1

[0148] For each preparation, PS and PA phospholipids with the mutual ratios shown in Table 1, with a total amount equal to 42.4 nanomoles, were placed in a glass tube, and finally 12.7 nanomoles of CHO was added thereto to produce PSPA-L and PSPACHO-L liposomes, respectively (Table 1). Then the lipids pre-dissolved in chloroform were dehydrated using a Rotavapor R-100 (Buchi). After evaporation of the chloroform, the lipid film was rehydrated with 1 ml of saline solution (0.9% NaCl). Then the preparation was shaken for 10 minutes and subsequently sonicated in a sonicator bath for 30 minutes to promote the formation of unilamellar vesicles. Finally, the liposomes were extruded using a mini-extruder (Avanti Polar Lipids) with a membrane having a pore size equal to 200 nm (Avanti Polar Lipids).

[0149] PS / PA liposome preparations were produced using the Weitz method (Proc Natl Acad Sci USA. 2003. 100(19): 10718-21) as described in WO2009 / 011007A2. Briefly, the lipid L-α-phosphatidic acid (PA; Avanti Polar Lipids), which constitutes the inner leaflet, was suspended in anhydrous dodecane (Sigma) at a concentration of 0.05 mg / ml. To produce the outer leaflet, L-α-phosphatidylserine (PS; Avanti Polar Lipids) was used and added to a solution of dodecane: silicone 99:1 to obtain a final concentration of 0.05 mg / ml. Liposomes were prepared by adding 2 ml of the outer lipid leaflet suspension to 3 ml of saline solution (0.9% NaCl). Finally, 100 μl of the inner lipid leaflet suspension was added to 2 ml of the outer lipid leaflet suspension, and the sample was centrifuged at 120 g for 10 minutes. After centrifugation, the aqueous phase containing the vesicles was collected using a 5 ml syringe with a 16-gauge stainless steel needle to yield PS-outer / PA-inner liposomes (PS / PA). Liposomes were then quantified using a FACSCalibur cytofluorimeter (Becton Dickinson), allowing quantification of monodisperse vesicles with a diameter >0.2 μm. Cell culture

[0150] The monocyte / macrophage cell line THP-1 was provided by the European Collection of Authenticated Cell Culture (ECACC 88081201) and cultured in RPMI 1640 containing fetal bovine serum (10%), gentamicin (5 μg / ml), L-glutamine (2 mM), non-essential acidic amino acids (1 mM), sodium pyruvate (1 mM) in 75 cm2 polystyrene bottles. Prior to the experiment, cells (5×105) were seeded in 24 or 96-well plates and induced to differentiate by stimulating with phorbol-12-myristate 13-acetate (PMA) (20 ng / ml) for 72 hours and used as a human macrophage model (dTHP-1). Bacterial culture

[0151] By Mycobacterium abscessus (Mab) ( 19977) were inoculated in Middlebrook 7H10 (7H10-BD Difco TM)A single colony of the mycobacterium was obtained on solid medium and then resuspended in 15 ml of Middlebrook 7H9 broth (7H9-BD Difco) supplemented with albumin, dextran, and catalase (ADC), and cultured with agitation in Erlenmeyer flasks at 37 °C for 48 h. The growth of the bacterial culture was monitored by measuring the optical density at a wavelength of 600 nm using a spectrophotometer (Varioskan LUX multimode microplate reader, ThermoFisher Scientific). After suspending the bacilli in Microorganism Preservation System-Protect (Technical Service Consultants Ltd), they were stored at -80 °C until use. Infection and assessment of intracellular bacterial growth TM )and, and cultured with agitation in Erlenmeyer flasks at 37 °C for 48 h. The growth of the bacterial culture was monitored by measuring the optical density at a wavelength of 600 nm using a spectrophotometer (Varioskan LUX multimode microplate reader, ThermoFisher Scientific). After suspending the bacilli in Microorganism Preservation System-Protect (Technical Service Consultants Ltd), they were stored at -80 °C until use. Infection and assessment of intracellular bacterial growth (Technical Service Consultants Ltd), they were stored at -80 °C until use. Infection and assessment of intracellular bacterial growth

[0152] To assess intracellular bacterial growth, monocyte / macrophage dTHP-1 cells were seeded at a concentration of 5 × 10 5 cells / ml in 24-well plates. The cells were infected with Mab at an MOI of 10 at 37 °C for 3 h, and after infection, extracellular bacilli were killed by incubating with 250 μg / ml amikacin for 1 h. The cells were then washed and incubated for an additional 18 h with PSPA-L at a molar ratio of 1:1, PSPA-L at a molar ratio of 2:1, PSPA-L at a molar ratio of 1:2, PSPA-L at a molar ratio of 17:1, PSPA-L at a molar ratio of 8.5:1, PSPA-L at a molar ratio of 34:1, or PSPACHO-L at a molar ratio of 16.5:1:7.5, where the phospholipid concentration was equal to 1.27 μM. Finally, the cells were lysed with 1% deoxycholate (Sigma), the samples were diluted in PBS-tween 80, and CFUs were quantified by plating the bacilli in triplicate on 7H10.

[0153] To assess the in vitro efficacy of combination therapy on the viability of extracellular and intracellular mycobacteria, dTHP-1 cells were infected with Mab at an MOI of 10 at 37 °C for 3 h. The cells were then stimulated with PSPA-L at a molar ratio of 17:1 or PSPACHO-L at a molar ratio of 16.5:1:7.5 and / or 4 μg / ml amikacin (AMK) for 18 h. CFU assays were performed by plating the bacilli in triplicate on 7H10 to assess both extracellular and intracellular bacterial growth. MTT assay for assessing cell viability

[0154] The viability of dTHP-1 cells stimulated for 24 h with either PSPA-L at a molar ratio of 17:1 or PSPACHO-L at a molar ratio of 16.5:1:7.5 at a phospholipid concentration of 1.27 μM was assessed by using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The MTT (Molecular Probe) assay is based on the intracellular reduction of tetrazolium salts to formazan by the mitochondrial enzyme succinate dehydrogenase (SDH). The optical density of crystals of a blue product, which can only be produced in metabolically active cells, was measured by using a spectrophotometer at a wavelength of 540 nm. The assay was performed according to the guidelines indicated in the user manual. Distribution of phosphatidylserine on the liposome surface

[0155] Assessment of PS organization was analyzed by fluorometry as described previously (Proc Natl Acad Sci USA. 2003. 100(19): 10718-21).

[0156] Briefly, as previously described in the section “Preparation of liposome formulations”, a fluorescent analog of PS (1-palmitoyl-2-{12-[(7-nitro-2-1,3-benzo PSPA-L, PSPACHO-L, and PS / PA liposomes were generated with molar ratios of 17:1, 8.5:1, and 34:1 to evaluate their presence on the external surface of liposomes. Specifically, after adding a quenching solution (1 M sodium dithionite (Na 2 S 2 O 4 ) in 5mM TES of pH 9, prepared daily) before and after monitoring the fluorescence of PS-NBD. Adding quencher to the vesicles of the suspension only reduces the fluorescence of PS-NBD located on the outer leaflet of the liposome, but cannot pass through the lipid bilayer. Using Varioskan LUX multi-mode microplate reader fluorometer (Thermo Fisher Scientific), by setting the excitation wavelength and emission wavelength to 470nm and 550nm, respectively, the fluorescence measurement is carried out kinetically every 30 seconds for 5 minutes (12 measurements). DLS characterization of liposomes

[0157] PSPA-L, PSPACHO-L and PS / PA samples with a molar ratio of 8.5:1, 17:1 and 34:1 stored in a refrigerator at 4°C were analyzed using a Zeta-Sizer Advance Ultra-Malvern Panalytical provided with a He-Ne laser (λ=633nm) with a maximum power of 10mW. The measurements were performed at a constant temperature value of 25°C, with a scattering angle of 12.78° or 90°, a conductivity of 0.006 to 0.187mS / cm, and an attenuation factor value of 0.12 to 1.

[0158] Two different production batches were analyzed in triplicate, for each of which 1 mL of sample was transferred to a suitable cuvette. No dilution was required as the count rate values ​​for the events were within the acceptable limits of the instrument. Results of Example 1 PSPA-L 17:1 liposomes enhance antimicrobial responses against Mab

[0159] Liposomes composed of PS and PA (PSPA-L) were generated using the classical lipid membrane hydration method and their efficacy in enhancing intracellular killing of Mabs was evaluated. Specifically, human macrophages were infected with Mabs in vitro and then inoculated with different PS and PA molar ratios [PS:PA molar ratio: 0.5:1, 1:1, 2:1 and 17:1 ( Figure 1 A); 8.5:1, and 17:1 and 34:1 ( Figure 1 The results showed that all the tested preparations could significantly improve the intracellular killing of Mab, and the PSPA-L preparation with a molar ratio of 17:1 was particularly effective in the intracellular killing of Mab (see Figure 1 A and B). Addition of cholesterol to the PSPA-L 17:1 liposomal formulation or its dose variation did not interfere with the efficacy in intracellular killing of Mab

[0160] PSPA-L 17:1 liposome formulations with or without the addition of 30% CHO (PSPACHO-L, molar ratio of 16.5:1:7.5) were generated to analyze the efficacy of both formulations in determining intracellular killing of Mab on Mab-infected macrophages. In addition, it was evaluated whether treatment consisting of a double dose of liposomes (2×PSPA-L 17:1) could result in increased killing of intracellular mycobacteria. Figure 2 The results in showed no significant differences in intracellular killing of Mab, emphasizing that cholesterol does not affect the bioactivity of the liposomal formulation and that the bioactivity of the liposomal formulation is independent of the dose of the liposomes. PSPA-L 17:1 or PSPACHO-L-amikacin combination treatment additively reduces the intracellular growth of Mabs in human macrophages

[0161] To determine the effect of combined antibiotic and bioactive liposome-based treatment, in vitro treatment with PSPA-L 17:1 or PSPACHO-L and amikacin was tested on dTHP1 cells infected with Mab. The results showed that PSPA-L17:1 or PSPACHO-L reduced the intracellular replication of Mab in dTHP-1 cells ( Figure 3 A and B), but not any direct effect on extracellular pathogens ( Figure 3 C and D). It should be noted that the combination treatments with PSPA-L 17:1 or PSPACHO-L and AMK ( Figure 3 A and B) induced a significantly greater decrease in the Mab replication index. Treatment with PSPA-L 17:1 and PSPACHO liposomes does not affect cell viability of human macrophages

[0162] To evaluate whether PSPA-L 17:1 and PSPACHO-L liposomal formulations produced using the classical lipid membrane hydration method do not induce any toxic effects on human macrophages, an MTT cell viability assay was performed. Figure 4 The results shown indicate that there was no change in cell viability after treatment with PSPA-L (17:1) or PSPACHO-L, confirming the non-toxicity of the liposomal formulation in vitro. Asymmetric PS / PA liposomes and PSPA-L 17:1 liposomes have different PS distributions on the outer leaflet of the liposome

[0163] The distribution of phosphatidylserine of PSPA-L 17:1 and PSPACHO-L (liposomes according to the invention) as well as PS / PA liposomes was evaluated.

[0164] In particular, fluorescent analogs of PS (PS-NBD) and PA were used, and liposome preparations were generated using the classical lipid membrane hydration method (PS-NBDPA-L and PS-NBDPACHO-L) or using the Weitz method (PS-NBD / PA). To assess the presence of PS-NBD on the internal surface of the liposomes, the liposomes were exposed to a quenching solution that can only reduce the fluorescence of the PS-NBD located on the external leaflet of the liposomes, but cannot penetrate its lipid bilayer. Figure 5The results in A show that after adding the quencher to the PS-NBD / PA liposomes, the fluorescence gradually decreased and almost reached complete extinction, which verifies the presence of phosphatidylserine in the outer leaflet (Proc Natl Acad Sci US A. 2012; 109(21): E1360-E1368). Figure 5 In B and C, it can be noted that the addition of the quencher to the liposomes resulted in a partial decrease in fluorescence, but the decrease was greater in the case of PS-NBDPACHO-L liposomes, indicating the presence of PS in both leaflets of the liposomes and subsequently confirming the structural differences of the two liposome preparations. Asymmetric PS / PA liposomes have different PS distribution on the outer leaflet compared with PSPA-L 8.5:1 and PSPA-L 34:1 liposomes

[0165] The distribution of phosphatidylserine of PSPA-L 8.5:1 and 34:1 (liposomes according to the invention) and PS / PA liposomes was evaluated using the same method as described above.

[0166] In particular, fluorescent analogs of PS (PS-NBD) and PA were used, and liposome preparations (PS-NBDPA-L 8.5:1 and PS-NBDPA-L 34:1) were generated by classical lipid membrane hydration and compared with PS / PA liposomes (PS-NBD / PA) generated by the Weitz method.

[0167] To assess the presence of PS-NBD on the internal surface of liposomes, liposomes were exposed to a quenching solution that can only reduce the fluorescence of PS-NBD located on the outer leaflet of the liposomes but cannot penetrate its lipid bilayer.

[0168] Applicants noticed that the addition of quenchers to liposomes resulted in a partial decrease in fluorescence with a trend similar to Figure 5 The trend is similar to that shown in B, and the fluorescence is stable at about 0.7 ( Figure 5 D and 5E), which demonstrated the presence of phosphatidylserine on both leaflets of the liposomes and subsequently determined the structural differences between the two liposome preparations. Compared with asymmetric PS / PA liposomes, PSPA-L 8.5:1, PSPA-L 17:1, PSPA-L 34:1, and PSPACHO-L liposomes have different chemical / physical characteristics.

[0169] Table 2 below shows the zeta potential values, polydispersity index and size values ​​of PSPA-L 8.5:1, PSPA-L 17:1, PSPA-L 34:1, PSPACHO-L and PS / PA detected in samples of two batches of liposomes. For each batch, triplicate measurements were performed. Batch 1 and batch 2 relative to the same liposome formulation were produced at different times, and the different results for each batch are due to the variability of the laboratory method. Table 2a - Batch 1 Table 2b - Batch 2 Polydispersity Index

[0170] The results obtained showed that the PSPA-L 8.5:1, PSPA-L 17:1, PSPA-L 34:1 and PSPACHO-L formulations had a single peak corresponding to a single particle population, resulting in a more uniform ( Figure 6 B to E and Table 2).

[0171] The results related to the analysis of the PS / PA samples showed the presence of three peaks corresponding to three different particle populations with different particle sizes, which can be in the micrometer range ( Figure 6 A). In fact, the liposome formulation turned out to be highly heterogeneous, with a polydispersity index of 0.8857±0.187 measured in batch 1 and 0.49±0.111 measured in batch 2. Zeta potential

[0172] Analysis of the zeta potential of PSPA-L and PSPACHO-L liposomes showed values ​​lower than -30 mV or -50 mV in batch 2 and even lower than -60 mV in batch 1, which emphasizes the high stability of the samples due to the presence of electrostatic repulsion suitable for achieving good physical colloidal stability.

[0173] In contrast, the results related to the zeta potential of the PS / PA samples showed values ​​ranging from about -18 mV to about -22 mV, indicating low electrostatic repulsion associated with sample instability, which can lead to particle aggregation and flocculation due to attractive van der Waals forces acting on the particles. size

[0174] The liposome size values ​​shown in Table 2 are related to the mean Z size detected by DLS.

[0175] Given that DLS is not the preferred technique for analyzing samples approaching micrometers in size, measurements on PS / PA samples were performed in specially made cuvettes that allow larger particles (up to 10 μm) to be measured with higher accuracy due to the inherent physical properties of the capillary sizing cell, eliminating errors associated with multiple scattering, and also allowing samples to be measured over a wider dynamic concentration range than is typically possible under side scatter (90°). PSPA-L 8.5:1, PSPA-L 17:1, PSPA-L 34:1, and PSPACHO-L liposomes resulted in liposomes with smaller sizes than asymmetric PS / PA liposomes.

[0176] The data obtained show that there are significant differences between PSPA-L 8.5:1, PSPA-L 17:1, PSPA-L 34:1 and PSPACHO-L liposomes and asymmetric PS / PA liposomes, both in terms of size and zeta potential values, indicating that the preparation method used leads to obtaining two products that differ in terms of chemical-physical characteristics and therefore in terms of stability and homogeneity.

[0177] Furthermore, PSPA-L 8.5:1, PSPA-L 17:1, PSPA-L34:1, and PSPACHO-L liposomes generated by the classical lipid membrane hydration method had very similar distribution characteristics despite not having the same lipid composition. Example 2: Microfluidic hydrodynamic focusing technology Materials and Methods Production of liposome PSPA-M 17:1

[0178] In Example 2, the abbreviation "PSPA-M" indicates liposomes produced by microfluidic hydrodynamic focusing technology, which contain PS on the outer leaflet and PS and PA on the inner leaflet.

[0179] To generate these liposomes, L-α-phosphatidylserine (PS; Avanti Polar Lipids) and L-α-phosphatidic acid (PA; Avanti Polar Lipids) were initially reconstituted in methanol at concentrations of 10 mg / ml and 1 mg / ml, respectively. To obtain 1 ml of a final solution with a total lipid concentration (PS+PA) of 5 mg / ml, in which the molar ratio between PS and PA was maintained at 17:1, 496 μl of PS, 238 μl of PA were taken and 267 μl of methanol were added.

[0180] This solution was used as the alcohol phase in the Dolomite microfluidic system, while deionized water was used as the aqueous phase. The flow ratio (FRR) between the two solutions was set to 7.5 (300 μl / min for the aqueous phase solution and 40 μl / min for the lipid solution). The resulting liposomes (5 ml of the final solution) were collected in a 5 ml tube, and half of the solution was dialyzed in one liter of deionized water using a PUR-A-LYZER MAXI 6000 dialysis membrane (molecular weight cutoff 6 to 8 kDa) for 18 hours. Cell culture dTHP-1

[0181] The monocyte / macrophage cell line THP-1 was provided by the European Collection of Certified Cell Cultures (ECACC88081201) and cultured in 75 cm2 polystyrene bottles in RPMI 1640 containing fetal bovine serum (10%), gentamicin (5 μg / ml), L-glutamine (2 mM), non-essential acidic amino acids (1 mM), and sodium pyruvate (1 mM). Before the experiment, cells (5×10 5 or 2×10 5 ) were seeded in 24- or 96-well plates, and cell differentiation was induced by stimulation with phorbol-12-myristate 13-acetate (PMA) (20 ng / ml) for 72 h, and used as a human macrophage model (dTHP-1). Type 1 (M1) or type 2 (M2) macrophages

[0182] Peripheral blood mononuclear cells (PBMCs) from healthy donors were isolated using a Ficoll density gradient. Monocytes were then positively selected using an anti-CD14 monoclonal antibody conjugated to magnetic microbeads (Miltenyi Biotec) according to the manufacturer's instructions. Monocytes (5 × 10 5 or 2×10 5 ) were seeded in 24- or 96-well plates and induced to differentiate in the presence of granulocyte-macrophage colony-stimulating factor (GM-CSF) (35 ng / ml) or macrophage colony-stimulating factor (M-CSF) (50 ng / ml) (R&D Systems) to obtain type 1 (M1) or type 2 (M2) macrophages, respectively. Bacterial culture

[0183] By Mycobacterium abscessus (Mab) ( 19977) were inoculated in Middlebrook 7H10 (7H10-BD DifcoTM ) solid medium to obtain a single colony of the mycobacterium, which was then resuspended in 15 ml of Middlebrook 7H9 broth (7H9-BD Difco TM ) and cultured in a conical flask at 37°C with stirring for 48 hours. The growth of bacterial culture was monitored by measuring the optical density at a wavelength of 600 nm using a spectrophotometer (Varioskan LUX multi-mode microplate reader, Thermo Fisher Scientific). After the bacilli were suspended in Microorganism Preservation System-Protect (Technical Service Consultants Ltd), they were stored at -80°C until use. MTT assay for assessing dTHP-1 cell viability

[0184] The viability of dTHP-1 cells stimulated for 18 hours or 5 days with PSPA-M 17:1 liposomes generated using microfluidic hydrodynamic focusing technology and containing phospholipids at concentrations of 2.54 μM, 1.27 μM, or 0.635 μM, with or without dialysis, was assessed by using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The MTT (Molecular Probe) assay is based on the intracellular reduction of tetrazolium salts to a phospholipid called formazan by the mitochondrial enzyme succinate dehydrogenase (SDH). The optical density of crystals of a blue product, which can only be produced in metabolically active cells, was measured by using a spectrophotometer at a wavelength of 540 nm. The assay was performed according to the guidelines indicated in the user manual. MTT assay for assessing cell viability of M1 and M2 macrophages

[0185] The cell viability of M1 and M2 macrophages stimulated for 18 h or 5 days with PSPA-M 17:1 liposomes generated using microfluidic hydrodynamic focusing technology and containing phospholipids at concentrations of 2.54 μM, 1.27 μM, or 0.635 μM, with or without dialysis, was assessed by using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The Molecular Probe assay is based on the intracellular reduction of tetrazolium salts to formazan by the mitochondrial enzyme succinate dehydrogenase (SDH). The optical density of crystals of a blue product, which can only be produced in metabolically active cells, was measured by using a spectrophotometer at a wavelength of 540 nm. The assay was performed according to the guidelines indicated in the user manual. Infection and assessment of bacterial intracellular growth in vitro - dTHP-1 cells

[0186] To assess intracellular bacterial growth, dTHP-1 cells were cultured at 5 × 10 5 The cells were distributed in 24-well plates at a concentration of 10 cells / ml. The cells were infected with Mab at an MOI of 10 for 3 hours at 37°C, and after infection, the extracellular bacilli were killed by incubation with 250 μg / ml of amikacin for 1 hour. The cells were then washed and incubated for another 18 hours with PSPA-M 17:1 liposomes produced using microfluidic hydrodynamic focusing technology with or without dialysis, whose phospholipid concentration was equal to 1.27 μM. Finally, the cells were lysed with 1% deoxycholate (Sigma), the samples were diluted in PBS-tween 80, and CFU were quantified by plating the bacilli on solid Middlebrook 7H10 agar growth medium in triplicate. Infection and assessment of bacterial intracellular growth in vitro - M1 and M2 macrophages

[0187] To evaluate intracellular bacterial growth, M1 and M2 were cultured at 5 × 10 5 The cells were distributed in 24-well plates at a concentration of 10 cells / ml. The cells were infected with Mab at an MOI of 10 for 3 hours at 37°C, and after infection, the extracellular bacilli were killed by incubation with 250 μg / ml of amikacin for 1 hour. The cells were then washed and incubated for another 18 hours with dialyzed PSPA-M 17:1 liposomes whose phospholipid concentration was equal to 1.27 μM, produced using microfluidic hydrodynamic focusing technology. Finally, the cells were lysed with 1% deoxycholate (Sigma), the samples were diluted in PBS-tween 80, and CFU were quantified by plating the bacilli on solid Middlebrook 7H10 agar growth medium in triplicate. Distribution of phosphatidylserine on the liposome surface

[0188] The distribution of phosphatidylserine of PSPA-M 17:1, dialyzed PSPA-M 17:1, and PSPA-L 17:1 liposomes was evaluated.

[0189] In particular, fluorescent analogs of PS (PS-NBD) and PA were used and liposome preparations were generated using either classical lipid membrane hydration (PS-NBDPA-L 17:1) or microfluidic hydrodynamic focusing techniques (PS-NBDPA-M 17:1 and dialyzed PS-NBDPA-M17:1). To assess the presence of PS-NBD on the internal surface of the liposomes, the liposomes were exposed to a quenching solution that only reduced the fluorescence of the PS-NBD located on the outer leaflet of the liposomes but could not penetrate its lipid bilayer.

[0190] Fluorescence measurements were performed kinetically every 30 seconds for 5 minutes (12 measurements) using a Varioskan LUX multi-mode microplate reader fluorometer (Thermo Fisher Scientific) by setting the excitation and emission wavelengths to 470 nm and 550 nm, respectively. DLS characterization of liposomes

[0191] Using a Zeta-Sizer Advance Ultra-Malvern Panalytical instrument provided with a maximum power of 10 mW He-Ne laser (λ = 633 nm), samples stored in a 4 ° C refrigerator were subjected to experiments for the characterization of PSPA-M 17:1 liposomes with or without dialysis. The measurements were performed at a constant temperature value of 25 ° C, with a scattering angle of 12.78 ° or 90 °, a conductivity of 0.006 to 0.187 mS / cm, and an attenuation factor value of 0.12 to 1. Two different production batches were analyzed in triplicate, and for each of them, 1 mL of sample was transferred to a suitable cuvette. Since the count rate values ​​of the events were within the acceptable limits of the instrument, no dilution was required. Results of Example 2 Treatment with dialyzed PSPA-M 17:1 or with PSPA-M 17:1 liposomes does not affect cell viability of dTHP-1 human macrophages

[0192] Cell viability assays highlighted that liposomes generated using microfluidic hydrodynamic focusing technology, whether dialyzed or not, did not reduce cell viability of dTHP-1 human macrophages after either 18 h or 5 days of stimulation, indicating a lack of toxicity (see Figure 7 A and B). Treatment with dialyzed PSPA-M 17:1 or with PSPA-M 17:1 liposomes did not affect cell viability of primary M1 or M2 macrophages

[0193] Cell viability assays highlighted that liposomes generated using microfluidic hydrodynamic focusing technology, whether dialyzed or not, did not reduce cell viability of primary M1 or M2 macrophages after 18 h or 5 days of stimulation, indicating their lack of toxicity (see Fig.10 ). PSPA-M 17:1 liposomes enhance antimicrobial responses against Mab

[0194] The results obtained on dTHP-1 human macrophages indicate that the liposomes produced using the microfluidic hydrodynamic focusing technique are able to significantly enhance the intracellular killing of mycobacteria. Furthermore, it can be noted that the dialysis process does not interfere with the biological activity of the preparation (see Figure 8 ). Dialyzed PSPA-M 17:1 liposomes enhance antimicrobial responses against Mabs in primary M1 or M2 macrophages

[0195] The results previously obtained on dTHP-1 human macrophages highlight that there are no significant differences in the biological effects between PSPA-M 17:1 liposomes and dialyzed PSPA-M 17:1. Furthermore, dialyzed PSPA-M 17:1 liposomes have not proven toxic after MTT assays on primary M1 or M2 macrophages, and their physico-chemical characterization shows that they are more stable in terms of zeta potential compared to non-dialyzed PSPA-M 17:1 liposomes. For these reasons and also considering that for liposomes produced with the MHF technique, the dialysis process is a key step to remove any remaining traces of organic solvents, only dialyzed PSPA-M 17:1 liposomes were selected for the subsequent bioactivity tests on primary M1 or M2 macrophages.

[0196] Fig.11 The results reported in showed that dialyzed PSPA-M 17:1 liposomes were able to significantly enhance the intracellular killing of mycobacteria. PSPA-M 17:1, dialyzed PSPA-M 17:1, and PSPA-L 17:1 liposomes have identical PS distribution on the outer leaflet of the liposomes

[0197] Fig. 9 The results in show that the addition of a quencher to the liposomes resulted in a partial decrease in fluorescence, indicating that the PS is present in both leaflets of the liposomes and subsequently indicating the structural similarity of these three liposome preparations. Homogeneity, stability and size of PSPA-M 17:1 liposomes

[0198] Tables 3a and 3b below show the average zeta potential, polydispersity index and size values ​​of PSPA-M 17:1 liposomes detected in two batches with or without dialyzation. Table 3a - Batch 1 Not on dialysis Dialysis Average-Z(nm) 239.6±2.745 175±2.124 Polydispersity Index 0.1276±0.061 0.1246±0.023 Zeta potential (mV) -55.26±1.632 -73.31±2.342 Table 3b - Batch 2 Not on dialysis Dialysis Average-Z(nm) 285.9±7.881 161±1.331 Polydispersity Index 0.1765±0.02 0.1789±0.023 Zeta potential (mV) -56.81±1.429 -81.6±0.4547

[0199] The characterization of the liposomes produced using the microfluidic hydrodynamic focusing technique in terms of size, polydispersity index and zeta potential highlighted that they were more homogeneous than those produced using the lipid membrane hydration method. In addition, the applicant also advantageously noted that the dialysis process had an effect on the liposomes: the liposomes became smaller and their zeta potential decreased, but there was no difference in the polydispersity index.

Claims

1. A liposome consisting of an outer leaflet comprising phosphatidylserine and an inner leaflet comprising phosphatidylserine and phosphatidic acid, It is characterized in that The zeta potential of the liposomes is -25 mV to -100 mV, and the molar ratio between total phosphatidylserine and phosphatidic acid is 5:1 to 35:

1.

2. The liposome according to claim 1, wherein the molar ratio between total phosphatidylserine and phosphatidic acid is 16:1 to 18:

1.

3. The liposome according to claim 1 or 2, wherein the molar ratio between total phosphatidylserine and phosphatidic acid is 17:

1.

4. The liposome according to any one of the preceding claims, wherein the zeta potential of the liposome is from -25 mV to -85 mV, preferably from -30 mV to -75 mV, even more preferably from -35 mV to -50 mV.

5. The liposome according to any one of the preceding claims, further comprising cholesterol, said cholesterol being present on the outer leaflet and / or the inner leaflet of the liposome.

6. The liposome according to claim 5, wherein the molar ratio between the total phosphatidylserine, the phosphatidic acid and the cholesterol is 16.5:1:7.

5.

7. The liposome according to any one of the preceding claims, comprising at least one first antibiotic therein.

8. The liposome of claim 7, wherein the at least one first antibiotic is selected from the group consisting of: first-line anti-tuberculosis antibiotics; second-line anti-tuberculosis antibiotics; aminoglycosides; glycylcyclines; penicillins; carbapenems; monobactams; quinolones; oxazolidinones; and macrolides.

9. Pharmaceutical composition comprising at least one liposome according to any one of claims 1 to 8 and at least one pharmaceutically acceptable excipient.

10. The pharmaceutical composition according to claim 9, further comprising at least one second antibiotic outside the liposome, wherein the at least one second antibiotic is selected from: first-line anti-tuberculosis antibiotics, preferably selected from isoniazid, rifampicin, rifabutin, ethambutol, pyrazinamide and streptomycin; second-line anti-tuberculosis antibiotics, preferably selected from cycloserine ethionamide, levofloxacin, moxifloxacin, ciprofloxacin, gatifloxacin, para-aminosalicylic acid, kanamycin and capreomycin; aminoglycosides, preferably selected from gentamicin and amikacin; glycylcyclines, preferably tigecycline; tetracyclines, preferably minocycline; cephalosporins, preferably selected from cefoxitin and cefoperazone sodium; penicillins, preferably amoxicillin; carbapenems, preferably imipenem; monocyclic β-lactams, preferably aztreonam lysine salt; quinolones, preferably moxifloxacin; Oxazolidinones, preferably linezolid; macrolides, preferably selected from azithromycin, clarithromycin, erythromycin and fidaxomicin.

11. The liposome according to any one of claims 1 to 8 or the composition according to any one of claims 9 to 10 for use in treating a bacterial infection.

12. The liposome or composition for use according to claim 11, wherein the infection is caused by Pseudomonas aeruginosa, Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Enterobacter spp., Propionibacterium acnes, Mycobacterium tuberculosis, Mycobacterium africanum, Mycobacterium bovis, Mycobacterium ulcerans, Mycobacterium leprae, Mycobacterium chimaera, Mycobacterium kansasii, kansasii), Mycobacterium tuberculosis complex, Mycobacterium avium complex (MAC), and Mycobacterium abscessus complex (M. abscessus complex).

13. The liposome or composition for use according to claim 11 or 12, wherein the infection is an infection caused by mycobacteria, preferably by nontuberculous mycobacteria.

14. The liposome or composition for use according to claim 13, wherein the infection is an infection caused by nontuberculous mycobacteria, the nontuberculous mycobacteria being selected from the group consisting of Mycobacterium avium complex (MAC), Mycobacterium chimera, Mycobacterium kansasii and Mycobacterium abscessus complex, the Mycobacterium abscessus complex comprising M. abscessus ssp. abscessus, M. abscessus ssp. bolletii and M. abscessus ssp. massiliense, preferably M. abscessus ssp. abscessus.

15. The liposome or composition for use according to claim 14, wherein the bacterial infection is an infection caused by Mycobacterium abscessus.

16. A kit comprising the composition according to claim 9 or 10 and one or more containers.

17. A method for treating one or more bacterial infections, preferably bacterial infections caused by mycobacteria, in particular nontuberculous mycobacteria, comprising administering to a subject suffering from one or more bacterial infections one or more of the liposomes according to any one of claims 1 to 8 and / or one or more of the pharmaceutical compositions according to claim 9 or 10.

18. The method according to claim 17, wherein the one or more liposomes and / or the pharmaceutical composition are administered by inhalation, preferably by aerosol administration.

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