阅读说明：本技术 抑制生物膜形成的脂质体 (Liposomes for inhibiting biofilm formation ) 是由 S·阿泽雷多·达·西尔维拉·拉雅尤尼亚斯 F·拉雅尤尼亚斯 于 2018-03-01 设计创作，主要内容包括：本发明涉及一种组合物,其包含以下并优选由以下组成：(i)单一的空脂质体,其中所述单一的空脂质体选自(a)包含胆固醇的空脂质体,其中胆固醇的量为至少30％(重量/重量)；其中优选地,所述空脂质体包含胆固醇和鞘磷脂,并且进一步优选地由胆固醇和鞘磷脂组成；或(b)由鞘磷脂组成的空脂质体；或(ii)空脂质体的混合物；其中所述空脂质体的混合物包含选自以下的至少一种空脂质体且优选由选自以下的至少一种空脂质体组成：(a)包含胆固醇的空脂质体,其中胆固醇的量为至少30％(重量/重量),其中优选地,所述空脂质体包含胆固醇和鞘磷脂,并且进一步优选地由胆固醇和鞘磷脂组成；(b)由鞘磷脂组成的空脂质体；以及(c)空脂质体,其包含磷脂酰胆碱和鞘磷脂,优选地由磷脂酰胆碱和鞘磷脂组成；以及彼此独立地选自包含脂质或磷脂并优选由脂质或磷脂组成的空脂质体的至少一种空脂质体,所述脂质或磷脂选自胆固醇、鞘磷脂、神经酰胺、磷脂酰胆碱、磷脂酰乙醇胺、磷脂酰丝氨酸、二酰基甘油和含有一个或两个或多个长于4个碳原子并最多达28个碳原子的饱和或不饱和脂肪酸；该组合物用于预防或减少生物膜形成或消除或减少已有生物膜的方法。(The present invention relates to a composition comprising and preferably consisting of: (i) a single empty liposome, wherein the single empty liposome is selected from the group consisting of (a) empty liposomes comprising cholesterol, wherein the amount of cholesterol is at least 30% (weight/weight); wherein preferably the empty liposomes comprise cholesterol and sphingomyelin, and further preferably consist of cholesterol and sphingomyelin; or (b) empty liposomes composed of sphingomyelin; or (ii) a mixture of empty liposomes; wherein the mixture of empty liposomes comprises and preferably consists of at least one empty liposome selected from the group consisting of: (a) empty liposomes comprising cholesterol, wherein the amount of cholesterol is at least 30% (weight/weight), wherein preferably the empty liposomes comprise cholesterol and sphingomyelin, and further preferably consist of cholesterol and sphingomyelin; (b) empty liposomes composed of sphingomyelin; and (c) empty liposomes comprising, preferably consisting of, phosphatidylcholine and sphingomyelin; and at least one empty liposome selected independently of each other from empty liposomes comprising and preferably consisting of lipids or phospholipids selected from the group consisting of cholesterol, sphingomyelin, ceramide, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, diacylglycerol and saturated or unsaturated fatty acids containing one or two or more longer than 4 carbon atoms and up to 28 carbon atoms; the composition is used in a method for preventing or reducing biofilm formation or eliminating or reducing existing biofilms.)

1. A composition comprising and preferably consisting of:

(i) a single empty liposome, wherein said single empty liposome is selected from the group consisting of:

(a) empty liposomes comprising cholesterol, wherein the amount of cholesterol is at least 30% (w/w), wherein preferably said empty liposomes comprise, further preferably consist of, cholesterol and sphingomyelin; or

(b) Empty liposomes composed of sphingomyelin; or

(ii) A mixture of empty liposomes, wherein said mixture of empty liposomes comprises, preferably consists of, at least one empty liposome selected from the group consisting of:

(a) empty liposomes comprising cholesterol, wherein the amount of cholesterol is at least 30% (weight/weight), wherein preferably said empty liposomes comprise cholesterol and sphingomyelin, and further preferably consist of cholesterol and sphingomyelin;

(b) empty liposomes composed of sphingomyelin; and

(c) empty liposomes comprising, preferably consisting of, phosphatidylcholine and sphingomyelin;

and at least one empty liposome, independently of each other, selected from empty liposomes comprising and preferably consisting of a lipid or a phospholipid selected from the group consisting of cholesterol, sphingomyelin, ceramide, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, diacylglycerol and saturated or unsaturated fatty acids containing one or two or more longer than 4 carbon atoms and up to 28 carbon atoms;

the composition is for use in a method of preventing or reducing biofilm formation or eliminating or reducing existing biofilm.

2. The composition for use according to claim 1, wherein the (i) single empty liposomes are selected from (a) empty liposomes consisting of sphingomyelin and cholesterol, wherein the amount of cholesterol is at least 30% (w/w); or (b) empty liposomes composed of sphingomyelin; and wherein the mixture of empty liposomes comprises (a) first empty liposomes comprised of sphingomyelin and cholesterol, wherein the amount of cholesterol is at least 30% (w/w); and (b) a second empty liposome consisting of sphingomyelin.

3. The composition for use according to claim 1, wherein the composition comprises, preferably consists of, a single empty liposome, wherein the single empty liposome is (a) an empty liposome consisting of sphingomyelin and cholesterol, wherein the amount of cholesterol is at least 30% (weight/weight), or (b) an empty liposome consisting of sphingomyelin.

4. Composition for use according to claim 1, wherein the composition comprises, preferably consists of, a mixture of empty liposomes, wherein the mixture of empty liposomes comprises and preferably consists of: (a) a first empty liposome consisting of sphingomyelin and cholesterol, wherein the cholesterol content is at least 30% (w/w); and (b) a second empty liposome consisting of sphingomyelin.

5. The composition for use according to any one of claims 1 to 4, wherein the amount of cholesterol of the empty liposomes (a) is 30-70% (w/w), and wherein preferably the amount of cholesterol of the empty liposomes (a) is 35-60% (w/w).

6. The composition for use according to any one of claims 1 to 5, wherein the amount of cholesterol of the empty liposomes (a) is between 45% and 55% (w/w), and wherein preferably the amount of cholesterol of the empty liposomes (a) is about 50% (w/w).

7. The composition for use according to any one of claims 2, 4 to 6, wherein said mixture of empty liposomes comprises at least 20% (w/w) of said first and said second empty liposomes, and wherein preferably said mixture of empty liposomes comprises at least 30% (w/w) of said first and said second empty liposomes.

8. The composition for use according to any one of claims 2, 4 to 7, wherein said mixture of empty liposomes comprises at least 40% (w/w) of said first and said second empty liposomes.

9. The composition for use according to any one of claims 1 to 8, wherein the use is in a method of preventing or reducing biofilm formation on a surface or in a method of eliminating or reducing existing biofilm on a surface.

10. The composition for use according to any one of claims 1 to 9, wherein the use is for the prevention or treatment of a condition or disease, and wherein preferably the condition or disease is caused by bacteria present in the biofilm.

11. The composition for use according to claim 10, wherein the condition or disease is selected from an infection, and wherein preferably the infection is an airway infection, a sexually transmitted disease, meningitis, urinary system infection, gastrointestinal disease, native valve endocarditis, colitis, vaginitis, urethritis, conjunctivitis, otitis, preferably otitis media, cystic fibrosis, ventilator-associated pneumonia, bacteremia or a wound infection.

12. The composition for use according to claim 10 or claim 11, wherein said condition or disease, preferably said infection, is caused by at least one ESKAPE pathogen.

13. The composition for use according to any one of claims 10 to 11, wherein the condition or disease is selected from the group consisting of streptococcus pneumoniae, bacillus, listeria monocytogenes, staphylococcus, lactic acid bacteria, preferably lactobacillus plantarum and lactococcus lactis, streptococcus sobrinus, streptococcus mutans, escherichia coli, pseudomonas aeruginosa, enterobacteriaceae, salmonella species, preferably salmonella enteritidis, salmonella enteritidis or salmonella typhi, actinobacillus pleuropneumoniae, proteus, shigella dysenteriae, moraxella, preferably moraxella catarrhalis, shigella, preferably helicobacter pylori, stenotrophomonas, bdellovibrio, acetobacter, legionella, preferably legionella pneumophila, cyanobacteria, spirochetes, sulfur-producing bacteria, neisseria, preferably neisseria gonorrhoeae or neisseria meningitidis, Haemophilus influenzae, enterococcus faecalis, staphylococcus aureus, klebsiella pneumoniae, acinetobacter baumannii, serratia marcescens, enterobacter cloacae, and gram-negative or gram-positive bacteria of the genus enterobacter.

14. The composition for use according to any one of claims 1 to 13, wherein the use is in combination with an antimicrobial agent, wherein preferably the antimicrobial agent is an antibiotic, an antifungal agent, an antitoxic agent, an antiseptic agent or a combination thereof, and wherein further preferably the antimicrobial agent is an antibiotic.

15. The composition for use according to any one of claims 1 to 14, wherein the method is an ex vivo method, wherein preferably the method comprises contacting a surface, preferably a surface of a medical device, with the composition according to any one of claims 1 to 14.

Technical Field

The present invention relates to empty liposomes or mixtures of empty liposomes having a defined lipid composition for preventing or reducing biofilm formation or for eliminating or reducing existing biofilms. The invention also relates to the treatment or prevention of such biofilm formation by the use of empty liposomes or mixtures of empty liposomes of the invention alone or in combination with standard antimicrobial therapy.

Background

Reference documents: davies, nat. Rev. drug Discov. (2003)2: 114-; bjarnsholt et al, nat. Rev. drug Discov. (2013)12: 791-; wu et al, International Journal of Oralccience 7 (2015); rasamiravaka et al, BioMed Research International Volume (2015); bhattacharya et al, Expert Rev Anti infection Ther. (2015)13 (12); penessan et al, Molecules (2015) 20; rabin et al, Future med. chem. (2015)7 (4).

The microorganisms may be present in the form of plankton (especially free-floating bacteria or fungi) or biofilm (especially biofilm bacteria or fungi). Typically, these biofilms consist of tightly packed and highly organized microbial communities encased in a secretory polymer matrix consisting of proteins, extracellular DNA, amyloid fibrils and polysaccharides, which forms a highly hydrated polar mixture that contributes to the overall structural scaffolding and structure of the biofilm.

In contrast to planktonic microorganisms, biofilm microorganisms dominate in almost all ecosystems under well-nourished conditions. Thus, biofilms can be established in a variety of host environments because they can adhere to non-biological surfaces, for example, to implant surfaces such as catheters, vascular prostheses, heart valves, cardiac pacemakers, cerebrospinal fluid shunts, urinary catheters, peritoneal dialysis catheters, joint prostheses and orthopedic fixation devices, intrauterine devices, biliary stents, breast implants, contact lenses, dentures, to caries and periodontitis in the dental area, and to biological surfaces such as lung, skin, bone, teeth, leading to chronic airway infections, chronic obstructive pulmonary disease, natural valve endocarditis, chronic otitis media, chronic sinusitis, or chronic wound infections in patients with, for example, cystic fibrosis. The biofilm also forms plaque, which contributes to the development of dental disease, as both bacteria and fungi adhere to teeth and are embedded in salivary polymers and microbial extracellular products. Biofilm is also a problem in the food industry due to its ability to form on the surfaces of food equipment and in industrial processes in food plants.

This disclosure is not important if the biofilm microorganisms are simply plankton adhering to the surface, but the biofilm microorganisms are very different from plankton. The biofilm polymer matrix can form a barrier against host immune defenses and protect submerged bacteria and fungi from antibacterial and antifungal agents, respectively. In addition, biofilms can act as infectious niches (niches) contributing to chronic infections and recurrences. Biofilm-associated infections are chronic, persistent and difficult to cure. Even in the case of biofilm formation on implants such as heart valves, the sloughed biofilm cells can migrate with the blood stream and cause infection of other organs.

Biofilms are involved in a variety of infections and gram-positive and gram-negative bacteria can form as well as mycoplasma, spirochetes and fungi. They are also involved in infections caused by ESKAPE pathogens. The number of diseases associated with biofilms is considered to be very large, with colitis, vaginitis, urethritis, conjunctivitis and otitis media being only a small subset of the common examples. As noted, biofilms also serve as colonization of important medical devices, including urinary ducts, venous and arterial catheters, shunts, and the like. Biofilm-associated infections are often chronic, for example, infection of patients with Cystic Fibrosis (CF) with p.aeruginosa in bronchopneumonia, due to antimicrobial resistance and immune defense mechanisms of bacteria and fungi present in biofilms.

In fact, biofilm bacteria are 1000 times more resistant to antibiotics than planktonic bacteria. Traditionally, antibiotics have been developed to target cellular mechanisms involved in the growth and survival of free-floating bacteria (i.e., planktonic bacteria). However, biofilm bacteria are quite different from planktonic bacteria. Notably, the extracellular proteins, virulence factors and surfactants expressed by biofilm bacteria are different from those expressed by free-floating bacteria. Moreover, the protective biofilm matrix is able to evade host immune responses, promote persistence and bacterial spread. In addition, antimicrobial resistance is an inherent property of biofilms. Thus, despite the intact host immune defenses and frequent antibiotic treatment, bacteria persist. Furthermore, antibiotic therapy using planktonic bacteria alone to target infections is generally ineffective against biofilm phenotypes. Due to the toxicity and side effects of high doses of antibiotics, as well as limitations in renal and hepatic function, it is difficult for conventional antibiotic administration to achieve an effective antibiotic dose for eradication of biofilms. In addition, exposure of bacteria in biofilms to antibiotics further increases the selection pressure associated with the development of antibiotic resistance. Similarly, fungal infections associated with biofilms are often refractory to conventional therapies due to resistance to antibacterial agents, due in part to surface-induced up-regulation of drug efflux pumps.

Current methods may include physical removal of the source of infection (removal of orthopedic hardware, non-absorbable sutures, necrotic tissue by catheter or surgery) or treatment with antibiotics. However, although antibiotics and antifungals directly target biofilm-forming organisms, they have been very difficult to succeed in treating biofilm-associated diseases. Indeed, current methods are not always successful in eliminating infections, infections involving biofilm resident bacteria or fungi often prove incurable and eventually progress to a chronic state. Despite the fact that the ability to form biofilms is a common attribute of bacteria, and that many medically important fungi produce biofilms, the exact mechanism of biofilm formation is not well understood, which makes biofilm-associated infections particularly difficult to treat and eradicate. The main goals of the non-bactericidal anti-biofilm agents under development are (i) to avoid the attachment of microorganisms on surfaces, (ii) to influence the maturation of biofilms and/or to induce their dispersion and degradation, e.g. by targeting bacterial systems and messengers, especially bacterial Quorum Sensing (QS), nucleotides, especially c-di-GMP or small non-coding rnas (srna), which have an important role in the regulation of biofilm formation, or (iii) to avoid the attachment of microorganisms on surfaces, e.g. by disrupting amyloid structures involved in biofilm formation. Treatment of biofilm-related disorders has proven to be a considerable unmet clinical need.

Customized empty liposomes (e.g. those consisting of Cholesterol (CHOL) and/or Sphingomyelin (SM)) and their use in the treatment of bacterial infections have been described as traps for bacterial toxins (WO 2013/186286; Henry BD et al, Nat Biotechnol 2015; 33(1): 81-88; Azeredo da Silvereira, S and Perez, A, Expertrev. anti infection. The. 2015; 13(5): 531-533).

It is therefore an object of the present invention to provide compositions having anti-biofilm activity and compositions for the treatment of biofilm-related disorders and diseases.

Disclosure of Invention

We have surprisingly found that a mixture of empty liposomes of defined lipid composition and empty liposomes of defined lipid composition according to the invention prevents or reduces biofilm formation and eliminates or reduces existing biofilms. In particular, it has been surprisingly found that the empty liposomes of the invention and mixtures thereof demonstrate reduced biofilm formation by pseudomonas aeruginosa. Thus, empty liposomes and mixtures of empty liposomes of defined lipid composition according to the invention can prevent or reduce biofilm formation or eliminate or reduce existing biofilms and thus can prevent and treat conditions and diseases such as acute and chronic bacterial infections or cystic fibrosis.

Furthermore, the disruption or weakening of the biofilm or the avoidance or reduction of biofilm formation by the single empty liposomes or mixtures of empty liposomes of the invention will result in an increased efficacy of the antimicrobial agent, in particular the biocide (antibacterial agent), especially the antibiotic or antifungal agent, applied simultaneously or subsequently. This is a highly synergistic effect, as the released planktonic bacteria or fungi are more sensitive (up to 1000 times) to antibacterial and antibiotic agents. By simultaneous or subsequent antibiotic or antifungal treatment, plankton removal and infection spread to other parts of the body can be avoided. In addition, the released organisms are more susceptible to host immune defense mechanisms and thus may be more effectively cleared by the host's own immune system.

Furthermore, it is believed that the non-toxicity of the compositions of the invention and the empty liposomes of the invention represent another beneficial and advantageous feature of the invention.

Accordingly, in a first aspect, the present invention provides a composition for use in a method of preventing or reducing biofilm formation or eliminating or reducing existing biofilm, wherein the composition comprises and preferably consists of: (i) a single empty liposome, wherein said single empty liposome is selected from the group consisting of: (a) empty liposomes comprising cholesterol, wherein the amount of cholesterol is at least 30% (weight/weight), wherein preferably said empty liposomes comprise cholesterol and sphingomyelin, and further preferably consist of sphingomyelin and sphingomyelin; (b) empty liposomes composed of sphingomyelin; or (ii) a mixture of empty liposomes; wherein the mixture of empty liposomes comprises, preferably consists of, at least one empty liposome selected from the group consisting of: (a) empty liposomes comprising cholesterol, wherein the amount of cholesterol is at least 30% (weight/weight), wherein preferably said empty liposomes comprise cholesterol and sphingomyelin, and further preferably consist of cholesterol and sphingomyelin; (b) empty liposomes composed of sphingomyelin; (c) empty liposomes comprising and preferably consisting of phosphatidylcholine and sphingomyelin; and at least one empty liposome, independently of each other, selected from the group consisting of empty liposomes comprising and preferably consisting of a lipid or phospholipid selected from the group consisting of cholesterol, sphingomyelin, ceramide, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, diacylglycerol and saturated or unsaturated fatty acids containing one or two or more longer than 4 carbon atoms and up to 28 carbon atoms.

In another aspect, the present invention provides an empty liposome consisting of sphingomyelin and cholesterol, wherein the amount of cholesterol is at least 30% (weight/weight), for use in a method for preventing or reducing biofilm formation or for eliminating or reducing existing biofilm, preferably in a mammal, further preferably in a human.

In another aspect, the present invention provides empty liposomes composed of sphingomyelin for use in a method for preventing or reducing biofilm formation or for eliminating or reducing existing biofilms, preferably in a mammal, further preferably in a human.

In another aspect, the present invention provides a mixture of empty liposomes comprising: (a) a first empty liposome consisting of sphingomyelin and cholesterol, wherein the amount of cholesterol is at least 30% (w/w); and (b) a second empty liposome consisting of sphingomyelin; it is used in a method for preventing or reducing biofilm formation or for eliminating or reducing existing biofilms, preferably in a mammal, more preferably in a human.

In another aspect, the invention provides a method for reducing biofilm formation or for eliminating or reducing existing biofilm, the method comprising administering to a mammal, preferably a human, in need of treatment a therapeutically effective amount of a composition of the invention, a single empty liposome of the invention, or a mixture of empty liposomes of the invention. In another aspect, the invention provides a method of preventing biofilm formation in a subject at risk comprising administering to a mammal, preferably a human, at risk an effective amount of a composition of the invention, a single empty liposome of the invention or a mixture of empty liposomes of the invention. In another aspect, the present invention provides a method for preventing or reducing biofilm formation or for eliminating or reducing existing biofilm, the method comprising administering to a mammal, preferably a human, in need of treatment a therapeutically effective amount of a composition of the present invention, a single empty liposome of the present invention, or a mixture of empty liposomes of the present invention without co-administration of standard antimicrobial agents. Furthermore, the present invention relates to the prevention or reduction of biofilm formation or for the elimination or reduction of existing biofilms comprising administering a therapeutically effective amount of a composition of the invention, a single empty liposome of the invention or a mixture of the invention to a mammal, preferably a human, in need of treatment before, after, together with or concurrently with standard antibacterial treatment of infections.

Other aspects and embodiments of the invention will become apparent as the description proceeds.

Drawings

FIG. 1: using 1X 10⁷Bacterial density per cell/well, inhibition of the biofilm by the empty liposome mixture of the present invention against the multidrug resistant strain of pseudomonas aeruginosa 6077. The graph shows the percentage of biofilm inhibition compared to the biofilm formed in the absence of empty liposomes.

Detailed Description

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

The term "about" as used herein shall have the meaning of +/-10%. For example, about 50% means 45% to 55%. Preferably, the term "about" as used herein shall have the meaning of +/-5%. For example, about 50% means 47.5% to 52.5%. When the terms "a" and "an" are used herein, they mean "at least one" unless otherwise specified. In particular, the terms "a" or "an" are used in conjunction with the single empty liposome, the first empty liposome and the second empty liposome to describe the mixture of empty liposomes and empty liposomes according to the invention. Generally and preferably refers to a mixture of single empty liposomes and empty liposomes comprising said first empty liposomes and said second empty liposomes.

Accordingly, in a first aspect, the present invention provides a composition for use in a method for preventing or reducing biofilm formation or eliminating or reducing existing biofilm, wherein the composition comprises and preferably consists of: (i) a single empty liposome, wherein said single empty liposome is selected from the group consisting of: (a) empty liposomes comprising cholesterol, wherein the amount of cholesterol is at least 30% (weight/weight), wherein preferably said empty liposomes comprise cholesterol and sphingomyelin, and further preferably consist of sphingomyelin and sphingomyelin; (b) empty liposomes composed of sphingomyelin; or (ii) a mixture of empty liposomes; wherein the mixture of empty liposomes comprises, preferably consists of, at least one empty liposome selected from the group consisting of: (a) empty liposomes comprising cholesterol, wherein the amount of cholesterol is at least 30% (weight/weight), wherein preferably said empty liposomes comprise cholesterol and sphingomyelin, and further preferably consist of cholesterol and sphingomyelin; (b) empty liposomes composed of sphingomyelin; (c) empty liposomes comprising and preferably consisting of phosphatidylcholine and sphingomyelin; and at least one empty liposome, independently of each other, selected from the group consisting of empty liposomes comprising and preferably consisting of a lipid or phospholipid selected from the group consisting of cholesterol, sphingomyelin, ceramide, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, diacylglycerol and saturated or unsaturated fatty acids containing one or two or more longer than 4 carbon atoms and up to 28 carbon atoms.

As used herein, the term "empty liposomes" refers to liposomes, preferably artificial liposomes, having an average diameter of from 20nm to 10 μm, preferably from 20 to 500nm, and further preferably having an average diameter of from 20nm to 400nm, even further preferably from 40nm to 400nm or from 20nm to 200nm, and consisting of one or more phospholipid bilayers, and typically and preferably unilamellar vesicles and multilamellar vesicles, more preferably Small Unilamellar Vesicles (SUVs). In a preferred embodiment, the term "empty liposomes" as used herein generally and preferably refers to liposomes that do not incorporate any drug, generally and preferably refers to liposomes that do not incorporate any drug. As used herein, "incorporated/incorporated" when referring to empty liposomes of the invention generally and preferably means encapsulated/encapsulated in the lumen of the liposome, within a potential bilayer of the liposome or as part of the liposome membrane layer. In another preferred embodiment, the term "empty liposomes" as used herein generally and preferably refers to liposomes consisting of sphingomyelin and cholesterol or consisting of sphingomyelin according to the invention and further comprising only water-soluble inorganic compounds and/or water-soluble organic molecules, wherein generally and preferably said water-soluble inorganic compounds and/or water-soluble organic molecules are derived from the synthesis of said inventive empty liposomes, and wherein generally and preferably said water-soluble inorganic compounds are inorganic salts, preferably selected from NaCl, KCl, MgCl₂Wherein said water soluble organic molecule is a buffer, wherein preferably said water soluble organic molecule is selected from the group consisting of glucose and HEPES. Typically and preferably, the water-soluble inorganic compounds and/or water-soluble organic molecules are incorporated into the empty liposomes of the invention due to their presence during the production process of the empty liposomes of the invention. In another preferred embodiment, the term "empty liposomes" as used herein generally and preferably refers to liposomes consisting of sphingomyelin and cholesterol or consisting of sphingomyelin according to the invention, wherein said empty liposomes do not comprise an antioxidant. In another preferred embodiment, the term "empty liposomes" as used herein generally and preferably refers to a composition consisting of sphingomyelin and bile according to the inventionLiposomes consisting of sterols or consisting of sphingomyelin and further comprising only water-soluble inorganic compounds and/or water-soluble organic molecules, wherein typically and preferably said water-soluble inorganic compounds and/or water-soluble organic molecules are derived from the synthesis of empty liposomes of said invention, and wherein typically and preferably said water-soluble inorganic compounds are preferably selected inorganic salts selected from the group consisting of NaCl, KCl, MgCl, and wherein₂Wherein said water soluble organic molecule is a buffer, wherein preferably said water soluble organic molecule is selected from glucose and HEPES, and wherein said empty liposomes consist of sphingomyelin and cholesterol or consist of sphingomyelin the compounds of the invention do not comprise an antioxidant.

As used herein, the term "biofilm" shall refer to a population of bacterial and/or fungal cells that adhere to each other, to surfaces, to interfaces including biological and non-biological surfaces, or to interfaces associated with tissue or mucus. This definition also includes small and large microorganism aggregates and flocs, as well as attached populations within the pore spaces of porous media. As used herein, the term "anti-biofilm activity" or "anti-biofilm only" refers to the prevention or reduction of biofilm formation or the elimination or reduction of existing biofilm, the at least partial release of microorganisms, preferably bacteria, or the at least partial lysis of biofilm. As used herein, the term "biofilm bacteria" refers to non-planktonic bacteria present in a biofilm.

The term "ESKAPE pathogen" as used herein shall mean a pathogen selected from the group consisting of enterococcus faecalis, staphylococcus aureus, klebsiella pneumoniae, acinetobacter baumannii, pseudomonas aeruginosa and enterobacter.

The terms "inhibit", "disrupt", "reduce" and "eliminate" as used herein and in relation to a biofilm or biofilm formation refer to a complete or partial inhibition (preferably more than 20%, further preferably more than 30%, further preferably more than 50%, further preferably more than 90%, still more preferably more than 95% or even more than 99%) of biofilm formation, typically and preferably in terms of residual cell number and/or development, also including the reversal of processes associated with biofilm development or biofilm formation and/or development. With respect to the latter, it preferably means that existing biofilms are eliminated at a greater rate than untreated biofilms or biofilms treated with compounds known to have no effect on the stability of biofilms. In addition, inhibition may be permanent or temporary. For temporary inhibition, biofilm formation and/or development may be inhibited for a time sufficient to produce a desired effect (e.g., at least 5 days, preferably at least 10 days). Preferably, inhibition of the biofilm is complete and/or permanent (preferably without retentive bacteria) ("eliminated").

As used herein, the term "medical device intended to be introduced into a subject" refers to a surgically invasive device or an implantable device as defined herein, such as, but not limited to, european commission committee DG Health and Consumer council (european commission DG Health and Consumer director) B, B2 unit "cosmetics and medical equipment" relates to guidelines for the application of council instructions 93/42/EEC to medical equipment.

As used herein, the terms "treat," "treating," or "therapy" refer to a means of obtaining a desired physiological effect. The effect may be therapeutic in terms of a partial or complete cure for the disease or condition and/or symptoms attributable to the disease or condition. The term refers to inhibiting a disease or condition, i.e., arresting its development, or ameliorating a disease or condition, i.e., causing regression of the disease or condition. The term "treatment" or "treating" as used herein and in the context of an ex vivo method according to the invention or in the context of treating a surface (e.g. a surface of a mammalian cell, tissue or structure, or a surface of a plant cell, tissue or structure, or a surface of a food device, or selected from a surface of a medical device or a surface intended to be contacted with water or an aqueous solution) with a composition of the invention shall refer to and include (but is not limited to) coating and applying to said surface in said ex vivo method according to the invention or contacting said composition of the invention.

As used herein, the term "preventing" refers to preventing or delaying the onset of a disease or disorder and/or symptoms attributed to the disease or disorder.

As used herein, the term "effective amount" refers to an amount of active ingredient (typically and preferably a composition according to the present invention) that is sufficient to produce a beneficial or desired result when administered or applied to a subject or patient or surface. In the context of prophylaxis or therapy, the term "surface of a medical device" is generally preferably used for intervention in a patient. More specifically, the term "therapeutically effective amount" as used herein refers to an amount of active ingredient (e.g., a composition according to the present invention) that is sufficient to produce a beneficial or desired result when administered to a subject or patient. A therapeutically effective amount may be applied in one or more administrations, dosages. A therapeutically effective amount of a composition according to the invention can be readily determined by one of ordinary skill in the art. In the context of the present invention, a "therapeutically effective amount" is an amount that produces an objectively measured change in one or more parameters associated with the prevention or reduction of biofilm formation or the treatment for the elimination or reduction of an existing biofilm or a condition or disease associated with said existing biofilm, wherein preferably said condition or disease is caused by bacteria present in said biofilm, and wherein preferably said condition or disease is selected from an infection, and wherein further preferably said infection is an airway infection-transmitting disease, meningitis, urinary tract infection, gastrointestinal disease, native valve endocarditis, colitis, vaginitis, urethritis, conjunctivitis, otitis, (preferably otitis media), cystic fibrosis, ventilator-associated pneumonia, bacteremia or wound infection. Of course, a therapeutically effective amount will depend upon the particular subject and condition being treated, the weight and age of the subject, the severity of the disease condition, the particular composition selected, the dosing regimen to be followed, the time of administration, the mode of administration, and the like, all of which can be readily determined by one of ordinary skill in the art.

As used herein, the term "subject" or "animal" or "patient" or "mammal" refers to any subject, particularly a mammalian subject, in need of diagnosis, prognosis, prevention or treatment, e.g., a human or a domesticated mammal (e.g., dog, cat, or horse) or a food animal (e.g., cow, sheep, or pig), preferably a human.

In a preferred embodiment, the at least one empty liposome is selected independently of each other from empty liposomes comprising and preferably consisting of a lipid or phospholipid selected from the group consisting of cholesterol, sphingomyelin, ceramide, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, diacylglycerol and phosphatidic acid comprising one or two or more saturated or unsaturated fatty acids longer than 4 carbon atoms and up to 28 carbon atoms, from (a) empty liposomes comprising cholesterol, wherein the amount of cholesterol is at least 30% (weight/weight), wherein preferably the empty liposomes comprise cholesterol and sphingomyelin, and further preferably consist of cholesterol and sphingomyelin; (b) empty liposomes composed of sphingomyelin; (c) empty liposomes comprising and preferably consisting of phosphatidylcholine and sphingomyelin.

In a preferred embodiment, the (i) single empty liposomes are selected from (a) empty liposomes comprising cholesterol and sphingomyelin, wherein the amount of cholesterol is at least 30% (weight/weight); (b) empty liposomes composed of sphingomyelin; and wherein the empty liposome mixture comprises: (a) a first empty liposome comprising cholesterol and sphingomyelin, wherein the amount of cholesterol is at least 30% (weight/weight); (b) a second empty liposome consisting of sphingomyelin.

In a preferred embodiment, the (i) single empty liposomes are selected from (a) empty liposomes composed of sphingomyelin and cholesterol, wherein the amount of cholesterol is at least 30% (w/w); (b) empty liposomes composed of sphingomyelin; and wherein the empty liposome mixture comprises (a) first empty liposomes comprised of sphingomyelin and cholesterol, wherein the amount of cholesterol is at least 30% (weight/weight); (b) a second empty liposome consisting of sphingomyelin.

In a preferred embodiment, the composition comprises, preferably consists of, a single empty liposome selected from the group consisting of: (a) an empty liposome comprising cholesterol and sphingomyelin, wherein the amount of cholesterol is at least 30% (weight/weight).

In a preferred embodiment, said composition for use in the method according to the invention comprises, preferably consists of, a single empty liposome, wherein said single empty liposome is selected from the group consisting of (a) empty liposomes consisting of sphingomyelin and cholesterol, wherein the cholesterol content is at least 30% (weight/weight); or (b) empty liposomes composed of sphingomyelin.

In another preferred embodiment, said composition for use in the method according to the invention comprises, preferably consists of, a single empty liposome, wherein said single empty liposome is an empty liposome consisting of sphingomyelin and cholesterol, wherein the amount of cholesterol is at least 30% (w/w). Preferably, the amount of cholesterol of the empty liposomes is 30% to 70% (weight/weight), further preferably, the amount of cholesterol of the empty liposomes is 35% to 60% (weight/weight). In another preferred embodiment, the amount of cholesterol of the empty liposomes is from 45% to 55% (w/w), and even more preferably, the amount of cholesterol of the empty liposomes is about 50% (w/w). Thus, in a very preferred embodiment of the invention, the single empty liposome comprises and preferably consists of 50% (w/w) sphingomyelin and 50% (w/w) cholesterol, for use in a method for preventing or reducing biofilm formation or for eliminating or reducing existing biofilms, preferably in a mammal, more preferably in a human.

In another preferred embodiment, said composition for use in said method according to the invention comprises, preferably consists of, a single empty liposome, wherein said single empty liposome is an empty liposome consisting of sphingomyelin.

In one embodiment, the composition comprises, preferably consists of, a mixture of empty liposomes, wherein said mixture of empty liposomes comprises and preferably consists of: (a) a first empty liposome comprising cholesterol, wherein the amount of cholesterol is at least 30% (weight/weight); (b) a second empty liposome consisting of sphingomyelin; (c) a third empty liposome comprising and preferably consisting of phosphatidylcholine and sphingomyelin.

In another embodiment, the composition for use comprises, preferably consists of, a mixture of empty liposomes, wherein said mixture of empty liposomes comprises and preferably consists of: (a) a first empty liposome comprising cholesterol, wherein the amount of cholesterol is at least 30% (w/w); (b) a second empty liposome consisting of sphingomyelin; (c) empty liposomes comprising and preferably consisting of phosphatidylcholine and sphingomyelin; and further optionally (d) at least one empty liposome selected independently of each other from the group consisting of empty liposomes comprising and preferably consisting of a lipid or phospholipid selected from the group consisting of cholesterol, sphingomyelin, ceramide, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, diacylglycerol and saturated or unsaturated fatty acids containing one or two or more carbon atoms longer than 4 and up to 28.

In one embodiment, the composition comprises, and preferably consists of, a mixture of empty liposomes, wherein the mixture of empty liposomes comprises (a) first empty liposomes consisting of sphingomyelin and cholesterol, wherein the amount of cholesterol is at least 30% (weight/weight); (b) a second empty liposome consisting of sphingomyelin; (c) a third empty liposome comprising and preferably consisting of phosphatidylcholine and sphingomyelin.

In one embodiment, the composition comprises, preferably consists of, a mixture of empty liposomes, wherein said mixture of empty liposomes comprises and preferably consists of: (a) a first empty liposome consisting of sphingomyelin and cholesterol, wherein the amount of cholesterol is at least 30% (per weight/weight); (b) a second empty liposome consisting of sphingomyelin; (c) a third empty liposome comprising and preferably consisting of a lipid or phospholipid selected from the group consisting of cholesterol, sphingomyelin, ceramides, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, diacylglycerol and saturated or unsaturated fatty acids containing one or two or more carbon atoms longer than 4 and up to 28.

In a preferred embodiment, the composition comprises, preferably consists of, a mixture of empty liposomes, wherein said mixture of empty liposomes comprises and preferably consists of: (a) a first empty liposome comprising cholesterol and sphingomyelin, wherein the amount of cholesterol is at least 30% (weight/weight); (b) a second empty liposome consisting of sphingomyelin.

In another preferred embodiment, the composition for use in the method according to the invention comprises and preferably consists of a mixture of empty liposomes, wherein said mixture of empty liposomes comprises (a) first empty liposomes consisting of sphingomyelin and cholesterol, wherein the cholesterol content is at least 30% (w/w); (b) a second empty liposome consisting of sphingomyelin. Preferably, the amount of cholesterol of the first empty liposome is 30% to 70% (weight/weight), further preferably, the amount of cholesterol of the first empty liposome is 35% to 60% (weight/weight). In another preferred embodiment, the first empty liposome has an amount of cholesterol of 45% to 55% (w/w), and still further preferably, the first empty liposome has an amount of cholesterol of about 50% (w/w). Thus, in a very preferred embodiment of the invention, the first empty liposome comprises, preferably consists of, 50% (w/w) sphingomyelin and 50% (w/w) cholesterol, for use in a method for preventing or reducing biofilm formation or for eliminating or reducing existing biofilms, preferably in a mammal, more preferably in a human.

In another preferred embodiment, the composition for use in the method according to the invention comprises a mixture of empty liposomes, wherein said mixture of empty liposomes consists of: (a) a first empty liposome consisting of sphingomyelin and cholesterol, wherein the amount of cholesterol is at least 30% (w/w), preferably 30-70% (w/w); (b) a second empty liposome consisting of sphingomyelin. Preferably, the cholesterol content of the first empty liposomes is between 35% and 60% (w/w), further preferably the cholesterol content of the first empty liposomes is between 45% and 55% (w/w), again preferably the cholesterol amount of the first empty liposomes is about 50% (w/w). Thus, in a very preferred embodiment of the invention, said first empty liposomes of said empty liposome mixture consist of 50% (w/w) sphingomyelin and 50% (w/w) cholesterol, for use in a method for preventing or reducing biofilm formation or for eliminating or reducing existing biofilms, preferably in a mammal, more preferably in a human.

In another preferred embodiment, the composition for use in the method according to the invention consists of a mixture of empty liposomes, wherein said mixture of empty liposomes consists of: (a) a first empty liposome consisting of sphingomyelin and cholesterol, wherein the cholesterol is at least 30% (w/w), preferably 30-70% (w/w); (b) a second empty liposome consisting of sphingomyelin. Preferably, the cholesterol content of said first empty liposomes is between 35% and 60% (w/w), further preferably, the cholesterol content of said first empty liposomes is between 45% and 55% (w/w), and yet further preferably, said amount of cholesterol of said first empty liposomes is about 50% (w/w). Thus, in a very preferred embodiment of the invention, said first empty liposomes of said empty liposome mixture consist of 50% (w/w) sphingomyelin and 50% (w/w) cholesterol, for use in a method for preventing or reducing biofilm formation or for eliminating or reducing existing biofilms, preferably in a mammal, more preferably in a human.

In another preferred embodiment, said mixture of empty liposomes comprises at least 1%, preferably at least 5%, further preferably at least 10%, still further preferably at least 20%, still further preferably at least 30% (w/w) of said first empty liposomes and at most 99%, preferably at most 95%, further preferably at most 90%, still further preferably at most 80%, still further preferably at most 70% (w/w) of second empty liposomes, wherein again preferably said mixture of empty liposomes comprises at least 40% (w/w) of said first empty liposomes and at most 60% (w/w) of said second empty liposomes.

In another preferred embodiment, said mixture of empty liposomes comprises at least 1%, preferably at least 5%, further preferably at least 10%, still further preferably at least 20%, still further preferably at least 30% (w/w) of said second empty liposomes and at most 99%, preferably at most 95%, further preferably at most 90%, still further preferably at most 80%, still further preferably at most 70% (w/w) of said first empty liposomes, and wherein, further preferably, said mixture of empty liposomes comprises at least 40% (w/w) of said second empty liposomes and at most 60% (w/w) of said first empty liposomes.

In another preferred embodiment, said mixture of empty liposomes comprises 1-99%, preferably 5-95%, further preferably 10-90%, still further preferably 20-80%, still further preferably 30-70% (w/w) of said first and said second empty liposomes, and wherein still preferably said mixture of empty liposomes further comprises 40-60% (w/w) of said first and said second empty liposomes.

In another preferred embodiment, said mixture of empty liposomes comprises at least 30% (w/w) of said first empty liposomes and at most 70% (w/w) of said second empty liposomes, and wherein preferably said mixture of empty liposomes comprises at least 40% (w/w) of said first empty liposomes and at most 60% (w/w) of said second empty liposomes. In another preferred embodiment, said empty liposome mixture comprises at least 45% (w/w) of said first empty liposome and at most 55% (w/w) of said second empty liposome, and wherein preferably said empty liposome mixture comprises about 50% (w/w) of said first empty liposome and about 50% (w/w) of said second empty liposome.

In another preferred embodiment, said mixture of empty liposomes comprises at least 30% (w/w) of said second empty liposomes and at most 70% (w/w) of said first empty liposomes, and wherein preferably said mixture of empty liposomes comprises at least 40% (w/w) of said second empty liposomes and at most 60% (w/w) of said first empty liposomes. In another preferred embodiment, said mixture of empty liposomes comprises at least 45% (w/w) of said second empty liposomes and at most 55% (w/w) of said first empty liposomes, and wherein preferably said mixture of empty liposomes comprises about 50% (w/w) of said second empty liposomes and about 50% (w/w) of said first empty liposomes.

In another preferred embodiment, said mixture of empty liposomes comprises at least 20% (w/w) of said first and said second empty liposomes, and wherein preferably said mixture of empty liposomes comprises at least 30% (w/w) of said first and said second empty liposomes. In another preferred embodiment, said mixture of empty liposomes comprises at least 40% (w/w) of said first and said second empty liposomes.

In another preferred embodiment, said empty liposome mixture comprises at least 20% (w/w) each of said first and said second empty liposomes, and wherein preferably said empty liposome mixture comprises at least 30% (w/w) each of said first and said second liposomes. In another preferred embodiment, said mixture of empty liposomes comprises at least 40% (w/w) of each of said first and said second empty liposomes.

In another preferred embodiment, the mixture of empty liposomes consists of the first empty liposome and the second empty liposome.

In another preferred embodiment, said mixture of empty liposomes consists of said first empty liposome and said second empty liposome, and wherein said mixture of empty liposomes consists of at least 40% (w/w) of said first empty liposome and at most 60% (w/w) of said second empty liposome, and preferably wherein said mixture of empty liposomes consists of about 50% (w/w) of said first empty liposome and about 50% (w/w) of said second empty liposome.

In another preferred embodiment, said mixture of empty liposomes consists of said first empty liposome and said second empty liposome, and wherein said mixture of empty liposomes consists of at least 40% (w/w) of said second empty liposome and at most 60% (w/w) of said first empty liposome, and preferably wherein said mixture of empty liposomes consists of preferably about 50% (w/w) of said second empty liposome and about 50% (w/w) of said first empty liposome.

In another preferred embodiment, said mixture of empty liposomes consists of 1-99%, preferably 5-95%, further preferably 10-90%, still further preferably 20-80%, still further preferably 30-70% (weight/weight) of said first and second empty liposomes, and wherein yet preferably said mixture of empty liposomes consists of 40-60% (weight/weight) of said first and second empty liposomes.

In another preferred embodiment, said mixture of empty liposomes consists of said first empty liposome and said second empty liposome, and wherein said mixture of empty liposomes consists of at least 40% (w/w) of said first empty liposome and at most 60% (w/w) of said second empty liposome, and wherein preferably said mixture of empty liposomes consists of about 50% (w/w) of said first empty liposome and about 50% (w/w) of said second empty liposome, and wherein said first empty liposome has a cholesterol content of 45% -55% (w/w), and wherein preferably said first empty liposome has a cholesterol content of about 50% (w/w).

In another preferred embodiment, said mixture of empty liposomes consists of said first empty liposome and said second empty liposome, and wherein said mixture of empty liposomes consists of at least 40% (w/w) of said second empty liposome and at most 60% (w/w) of said first empty liposome, and wherein preferably said mixture of empty liposomes consists of about 50% (w/w) of said second empty liposome and about 50% of the weight (w/w) of said first empty liposome, and wherein the amount of cholesterol of said first empty liposome is between 45% and 55% (w/w), and wherein preferably the amount of cholesterol of said first empty liposome is about 50% (w/w).

In another preferred embodiment, the empty liposomes for use in the present invention are liposomes, preferably artificial liposomes, having an average diameter of from 20nm to 10 μm, preferably from 20 to 500nm, and further preferably having an average diameter of from 20nm to 200 nm. Empty liposomes for use in the present invention are liposomes that do not encapsulate any drug. The empty liposomes for use in the present invention do not contain any drug. Empty liposomes for use in the present invention do not incorporate other drugs. As used herein, "incorporated" generally and preferably refers to encapsulation in the lumen of a liposome, within a potential bilayer of a liposome, or as part of the membrane layer of a liposome. Liposomes for use in the present invention are composed of one or more phospholipid bilayers, typically and preferably unilamellar and multilamellar vesicles. Most preferred are Small Unilamellar Vesicles (SUVs).

In another aspect, the present invention provides an empty liposome consisting of sphingomyelin and cholesterol, wherein the amount of cholesterol is at least 30% (w/w), for use in a method for preventing or reducing biofilm formation or for eliminating or reducing existing biofilm, preferably in a mammal, more preferably in a human. In another preferred embodiment, said empty liposomes for use in the method according to the invention comprise, preferably consist of, sphingomyelin and cholesterol, wherein the amount of cholesterol is between 30% and 70% (w/w), further preferred said empty liposomes have cholesterol between 35% and 60% (w/w). In another preferred embodiment, the amount of cholesterol of the empty liposomes is from 45% to 55% (w/w), and even more preferably, the amount of cholesterol of the empty liposomes is about 50% (w/w). Thus, in a very preferred embodiment of the invention, the empty liposomes comprise and preferably consist of 50% (w/w) sphingomyelin and 50% (w/w) cholesterol, a method for preventing or reducing biofilm formation or a method for eliminating or reducing existing biofilms, preferably in mammals, more preferably in humans.

In another aspect, the present invention provides empty liposomes comprised of sphingomyelin for use in a method for preventing or reducing biofilm formation or for eliminating or reducing existing biofilms, preferably in a mammal, further preferably in a human.

In another aspect, the present invention provides a mixture of empty liposomes comprising: (a) a first empty liposome consisting of sphingomyelin and cholesterol, wherein the amount of cholesterol is at least 30% (w/w); (b) a second empty liposome consisting of sphingomyelin; in a method for preventing or reducing biofilm formation or for eliminating or reducing existing biofilm, preferably in a mammal, more preferably in a human. Preferably, the amount of cholesterol of the first empty liposome is 30% to 70% (weight/weight), further preferably, the amount of cholesterol of the first empty liposome is 35% to 60% (weight/weight). In another preferred embodiment, the first empty liposome has an amount of cholesterol of 45% to 55% (w/w), and even more preferably, the first empty liposome has an amount of cholesterol of about 50% (w/w). Thus, in a very preferred embodiment of the invention, said first empty liposome comprises, preferably consists of, 50% (w/w) sphingomyelin and 50% (w/w) cholesterol, for use in a method for preventing or reducing biofilm formation or for use in a method for eliminating or reducing existing biofilms, preferably in a mammal, more preferably in a human. In another preferred embodiment, said mixture of empty liposomes comprises at least 30% (w/w) of said first empty liposomes and at most 70% (w/w) of said second empty liposomes, and wherein preferably said empty lipid mixture liposomes comprise at least 40% (w/w) of said first empty liposomes and at most 60% (w/w) of said second empty liposomes. In another preferred embodiment, said empty liposome mixture comprises at least 45% (w/w) of said first empty liposome and at most 55% (w/w) of said second empty liposome, and wherein preferably said empty liposome mixture comprises about 50% (w/w) of said first empty liposome and about 50% (w/w) of said second empty liposome.

In another aspect, the present invention provides a mixture of empty liposomes comprising: (a) a first empty liposome consisting of sphingomyelin and cholesterol, wherein the amount of cholesterol is at least 30% (w/w); and (b) a second empty liposome consisting of sphingomyelin; in a method for preventing or reducing biofilm formation or for eliminating or reducing existing biofilm, preferably in a mammal, more preferably in a human. Preferably, the amount of cholesterol of the first empty liposome is 30% to 70% (weight/weight), further preferably, the amount of cholesterol of the first empty liposome is 35% to 60% (weight/weight). In another preferred embodiment, the first empty liposome has an amount of cholesterol of 45% -55% (w/w), and yet further preferably, the first empty liposome has an amount of cholesterol of about 50% (w/w). Thus, in a very preferred embodiment of the invention, said first empty liposome comprises, preferably consists of, 50% (w/w) sphingomyelin and 50% (w/w) cholesterol, for use in a method for preventing or reducing biofilm formation or for use in a method for eliminating or reducing existing biofilms, preferably in a mammal, more preferably in a human. In another preferred embodiment, said empty liposome mixture comprises at least 30% (w/w) of said second empty liposomes and at most 70% (w/w) of said first empty liposomes, and wherein preferably said empty lipid mixture liposomes comprise at least 40% (w/w) of said second empty liposomes and at most 60% (w/w) of said first empty liposomes. In another preferred embodiment, said mixture of empty liposomes comprises at least 45% (w/w) of said second empty liposomes and at most 55% (w/w) of said first empty liposomes, and wherein preferably said mixture of empty liposomes comprises about 50% (w/w) of said second empty liposomes and about 50% (w/w) of said first empty liposomes.

In another aspect, the present invention provides a mixture of empty liposomes comprising: (a) a first empty liposome consisting of sphingomyelin and cholesterol, wherein the amount of cholesterol is at least 30% (w/w); and (b) a second empty liposome consisting of sphingomyelin; a method for preventing or reducing biofilm formation or for eliminating or reducing existing biofilm, preferably in a mammal, more preferably in a human. Preferably, the amount of cholesterol of the first empty liposome is 30% to 70% (weight/weight), further preferably, the amount of cholesterol of the first empty liposome is 35% to 60% (weight/weight). In another preferred embodiment, the first empty liposome has an amount of cholesterol of 45% to 55% (w/w), and even more preferably, the first empty liposome has an amount of cholesterol of about 50% (w/w). Thus, in a very preferred embodiment of the invention, said first empty liposome comprises, preferably consists of, 50% (w/w) sphingomyelin and 50% (w/w) cholesterol, for use in a method for preventing or reducing biofilm formation or for use in a method for eliminating or reducing existing biofilms, preferably in a mammal, more preferably in a human. In another preferred embodiment, said mixture of empty liposomes comprises 1-99%, preferably 5-95%, further preferably 10-90%, still further preferably 20-80%, still further preferably 30-70% (w/w) of said first and second empty liposomes, wherein still further preferably said mixture of empty liposomes comprises 40-60% (w/w) of said first and second empty liposomes.

Thus, in a further aspect, the present invention provides a composition comprising, preferably consisting of, a mixture of empty liposomes, wherein said mixture of empty liposomes comprises, preferably consists of: (a) a first empty liposome consisting of sphingomyelin and cholesterol, wherein the cholesterol content is at least 30% (w/w); (b) a second empty liposome consisting of sphingomyelin; in a method for preventing or reducing biofilm formation or for eliminating or reducing existing biofilm, preferably in a mammal, more preferably in a human. Preferably, the amount of cholesterol of the first empty liposome is 30% to 70% (weight/weight), further preferably, the amount of cholesterol of the first empty liposome is 35% to 60% (weight/weight). In another preferred embodiment, the first empty liposome has an amount of cholesterol of 45% to 55% (w/w), and even more preferably, the first empty liposome has an amount of cholesterol of about 50% (w/w). Thus, in a very preferred embodiment of the invention, the first empty liposome comprises, preferably consists of, 50% (w/w) sphingomyelin and 50% (w/w) cholesterol, for use in a method for preventing or reducing biofilm formation or for eliminating or reducing existing biofilms, preferably in a mammal, more preferably in a human. In another preferred embodiment, the mixture of empty liposomes comprises 1-99%, preferably 5-95%, further preferably 10-90%, still further preferably 20-80%, still further preferably 30-70% (w/w) of said first and second empty liposomes, and wherein again preferably the weight percentage of said first and second empty liposomes is preferably 40-60% (w/w).

Thus, in a very preferred embodiment of the invention, the mixture of empty liposomes comprises and preferably consists of: (i) a first empty liposome consisting of about 50% (w/w) sphingomyelin and about 50% (w/w) cholesterol, and (ii) a second empty liposome consisting of (100%) sphingomyelin; wherein said mixture of empty liposomes comprises at least 45% (w/w) of said first empty liposomes and at most 55% (w/w) of said second empty liposomes, and wherein preferably said mixture of empty liposomes comprises about 50% (w/w) of said first empty liposomes and about 50% (w/w) of said second empty liposomes, a method for preventing or reducing biofilm formation or a method for eliminating or reducing existing biofilms, preferably in a mammal, more preferably in a human.

Thus, in another aspect, the present invention provides a composition comprising, preferably consisting of, a mixture of empty liposomes, wherein said mixture of empty liposomes comprises and preferably consists of: (i) a first empty liposome consisting of about 50% (w/w) sphingomyelin and about 50% (w/w) cholesterol, and (ii) a second empty liposome consisting of (100%) sphingomyelin; wherein said mixture of empty liposomes comprises at least 45% (w/w) of said first empty liposomes and at most 55% (w/w) of said second empty liposomes, and wherein preferably said mixture of empty liposomes comprises about 50% (w/w) of said first empty liposomes and about 50% (w/w) of said second empty liposomes, for use in a method of preventing or reducing biofilm formation or eliminating or reducing existing biofilm, preferably for use in a mammal. Preferably in humans.

In another preferred embodiment, the first empty liposomes consist of about 50% (w/w) sphingomyelin and about 50% (w/w) cholesterol, and the second empty liposomes consist of (100%) sphingomyelin, with an average diameter of 20 to 500nm, preferably 20nm to 400nm, and even more preferably 40nm to 400 nm. In another preferred embodiment, said first empty liposome consisting of about 50% (w/w) sphingomyelin and about 50% (w/w) cholesterol, and the second empty liposome consisting of (100%) sphingomyelin, comprise a pH of 6.6-8.0. In another preferred embodiment, said first empty liposomes, consisting of about 50% (w/w) sphingomyelin and about 50% (w/w) cholesterol, and second empty liposomes, consisting of (100%) sphingomyelin, comprise a polydispersity index < 0.45. In another preferred embodiment, said first empty liposomes, consisting of about 50% (w/w) sphingomyelin and about 50% (w/w) cholesterol, and second empty liposomes, consisting of (100%) sphingomyelin, comprise a zeta potential of-25 to +2 mV.

Liposomes are manufactured according to extrusion or sonication or microfluidization (e.g., high pressure homogenization) methods known in the art. For example, the lipids are mixed in an organic solvent such as chloroform. The chloroform is evaporated and the dried lipid film is hydrated in an aqueous solution such as normal saline (0.9% NaCl), Krebs solution, or Tyrode solution and further sonicated to produce liposomes. If desired, the size of the liposomes can be controlled by extruding the liposomes through a membrane filter of fixed pore size. Separately produced liposomes having different lipid compositions are typically and preferably mixed in the desired proportions immediately prior to administration. As another example, liposomes are prepared using standard liposome hydration, extrusion, and diafiltration processes, in which the lipids are dissolved in a solvent (e.g., ethanol) while mixing, and then the lipid solution is added to the PBS buffer solution while mixing different sized liposomes to spontaneously mix to form and fully hydrate. The resulting vesicles were repeatedly extruded through a series of polycarbonate track-etched membranes until the desired particle size was obtained, as measured by dynamic light scattering. The extruded process fluid was diafiltered with PBS to remove the solvent from the process fluid, and finally concentrated and/or diluted with PBS buffer to the target concentration of total lipids.

The lipid surface (bilayer) of liposomes forms spontaneously in water-based solvents, thus entrapping water and other water-soluble inorganic and organic molecules that may be present during liposome production within the liposomes. The empty liposomes used in this study were in the form of liposomes containing water and simple organic or inorganic molecules (e.g., NaCl, KCl, MgCl)₂Glucose, HEPES and/or CaCl₂) The buffer of (3) to produce liposomes.

In a very preferred embodiment of the invention, the composition comprises, preferably consists of, a mixture of empty liposomes comprising (a) first empty liposomes consisting of sphingomyelin and cholesterol, wherein the amount of cholesterol is about 50% (w/w); (b) a second empty liposome consisting of sphingomyelin; a method for preventing or reducing biofilm formation or a method for eliminating or reducing existing biofilm, preferably in a mammal, further preferably in a human, yet further preferably in a method for preventing or reducing biofilm formation in a human. Preferably, the mixture of empty liposomes comprises at least 40% (w/w) of the first empty liposomes and at most 60% (w/w) of the second empty liposomes. More preferably, the mixture of empty liposomes comprises about 50% (w/w) of the first empty liposome and about 50% (w/w) of the second empty liposome.

In another preferred embodiment, the use is a method for preventing or reducing biofilm formation, wherein preferably the use is a method for preventing or reducing biofilm formation on a surface. Preferably, the surface is a surface of a cell, tissue or structure of a mammal, preferably a human, and wherein further preferably the cell or tissue is a lung, muscle, bone or skin cell or tissue and the structure is a tooth.

In another preferred embodiment, said use is a method for preventing or reducing biofilm formation in a mammal, preferably in a human, wherein further preferably said use is a method for preventing or reducing biofilm formation on a surface in a mammal, preferably in a human.

In another preferred embodiment, the use is a method for eliminating or reducing existing biofilm, wherein preferably the use is a method for preventing or reducing biofilm formation on a surface. Preferably, the surface is a surface of a cell, tissue or structure of a mammal, preferably a human, and wherein further preferably the cell or tissue is a lung, muscle, bone or skin cell or tissue and the structure is a tooth.

In another preferred embodiment, the use is a method for preventing or reducing biofilm formation on a surface or a method for eliminating or reducing existing biofilm on a surface.

In another preferred embodiment, the use is a method for preventing or reducing biofilm formation on a surface, preferably the surface is a surface of a mammalian cell, tissue or structure.

In another preferred embodiment, the use is a method for preventing or reducing biofilm formation on a surface, preferably the surface is a surface of a plant cell, tissue or structure. In another preferred embodiment, the plant may be, but is not limited to, a plant that produces carrots, potatoes, cucumbers, onions, tomatoes, lettuce, apples, citrus, or plums. In certain embodiments, the plant cell is from a plant and the tissue or structure is selected from the group consisting of leaf, root, flower, fruit, or other edible structure of the plant. In another preferred embodiment, the surface is a surface of a food plant.

In another preferred embodiment, the use is a method for eliminating or reducing existing biofilm on a surface. Preferably, the surface is a surface of a cell, tissue or structure of a mammal, preferably a human, and wherein further preferably the cell or tissue is a lung, muscle, bone or skin cell or tissue and the structure is a tooth.

In another preferred embodiment, the surface is a surface of a wound dressing.

In another preferred embodiment, the surface is generally and preferably non-biological, selected from the surface of a medical device or a surface intended to be in contact with water or an aqueous solution.

In another preferred embodiment, the surface is a surface of a medical device, typically and preferably non-biological, which medical device is typically and preferably intended for intervention in the body of a subject, wherein the medical device is selected from the group consisting of pacemakers, pacemaker leads, catheters, stents, vascular prostheses, prosthetic heart valves, cardiac pacemakers, cerebrospinal fluid shunts, urinary catheters, peritoneal dialysis catheters, joint prostheses and orthopedic fixation devices, intrauterine devices, biliary stents, breast implants, contact lenses, dental prostheses.

In another preferred embodiment, the surface is a surface intended to be in contact with water or an aqueous solution, wherein the surface intended to be in contact with water or an aqueous solution is a surface of a ship hull, a pipe, a filter, a strain or a pump, wherein preferably the surface is stainless steel or polypropylene.

In certain preferred embodiments, the medical device intended to be introduced into the body of the subject is a surgically invasive device intended for short-term use (>60 minutes, <30 days), such as, but not limited to, clamps, infusion cannulas, skin closure devices, temporary filling materials, tissue stabilizers used in cardiac surgery, cardiovascular catheters, cardiac output probes, temporary pacemaker leads, thoracic catheters intended to drain the heart (including pericardium), carotid shunts, ablation catheters, nervous system catheters, cortical electrodes, or brachytherapy devices.

In certain preferred embodiments, the medical device intended for introduction into the body of a subject is an implantable device or a long-term surgically invasive device (>30 days), such as artificial joint replacements, ligaments, shunts, stents and valves (e.g. pulmonary valves), nails and plates, intraocular lenses, internal closure devices (including vascular closure devices), tissue augmentation implants, peripheral vascular catheters, peripheral vascular grafts and stents, penile implants, non-absorbable sutures, bone cements and maxillofacial implants, elastosurgical devices, specialized for anterior segment surgery, bridges and crowns, dental filling materials and pins, dental alloys, ceramics and polymers, artificial heart valves, aneurysm clips, artificial vessels and stents, central vascular catheters, spinal stents, CNS electrodes, cardiovascular sutures, permanent and retrievable vena cava filters, permanent and retrievable vena cava filters, surgical devices, and the like, A septal occlusion device, an intra-aortic balloon pump, and an external left ventricular assist device.

In the food processing industry, biofilms can cause chronic bacterial contamination in food processing equipment, such as pasteurized tubes and pipes.

In the marine-based industry, marine pollution is often described as including multiple stages, an early step of bacterial adhesion beginning with biofilm formation, followed by colonization by spores (e.g., enteromorpha, filamentaria) and protozoa (e.g., bellybirds, polypipes) of a second batch of macroalgae. Finally, a third batch of colonists, large fouling organisms, including tunicates, mollusks and aphtylenchus. Thus, biofilm formation provides a foundation for biofouling of underwater surfaces such as ship hulls, ship propellers, cages, underwater dock structures, underwater structures on offshore oil platforms, subsea mines, buoys, subsea cables, cooling systems for power plants, pipelines and filter desalination plants, and the like.

In another preferred embodiment, the use is a method for preventing or reducing bacterial biofilm formation on a surface.

The biofilm inhabiting bacteria may be any bacteria, i.e. gram negative or gram positive bacteria or mycoplasma and spirochetes. Within these categories, there are bacteria associated with animal cells, plant cells or artificial surfaces. In certain embodiments, the bacteria produced and/or present in a biofilm in question are gram-negative bacteria or gram-positive bacteria.

In another preferred embodiment, said use is for the prevention or treatment of a condition or disease, and wherein preferably said condition or disease is caused by bacteria present in said biofilm.

In another preferred embodiment, said use is for prophylaxis, typically and preferably for a condition or disease, and wherein further preferably said condition or disease is caused by bacteria present in said biofilm. In another preferred embodiment, said use is for the treatment of a condition or disease, and wherein preferably said condition or disease is caused by bacteria present in said biofilm.

In another preferred embodiment, the use is for the prevention or treatment of a condition or disease, and wherein the disease is an infectious disease. In another preferred embodiment, the use is for the prevention of a condition or disease in general and preferably, and wherein the condition or disease is an infectious disease. In another preferred embodiment, the use is for the treatment of a condition or disease, and wherein the condition or disease is an infectious disease.

In another preferred embodiment, said use is for the prevention or treatment of a condition or disease in a mammal, preferably a human, and wherein preferably said condition or disease is an infectious disease. In another preferred embodiment, said use is for the treatment of a condition or disease in a mammal, preferably a human, and wherein preferably said condition or disease is an infectious disease.

In another preferred embodiment, the condition or disease is selected from an infection, and wherein preferably the infection is an airway infection, a sexually transmitted disease, meningitis, a urinary tract infection, a gastrointestinal tract disease, natural valve endocarditis, colitis, vaginitis, urethritis, conjunctivitis, otitis (preferably otitis media), cystic fibrosis, ventilator-associated pneumonia, bacteremia or a wound infection.

In another preferred embodiment, the condition or disease is an airway infection, a sexually transmitted disease, meningitis, a urinary tract infection, a gastrointestinal tract disease, natural valve endocarditis, colitis, vaginitis, urethritis, conjunctivitis, otitis (preferably otitis media), cystic fibrosis, ventilator-associated pneumonia, bacteremia or a wound infection.

In another preferred embodiment, the condition or disease is an infection.

In another preferred embodiment, the condition or disease is selected from the group consisting of an airway infection, a sexually transmitted disease, meningitis, a urinary tract infection, a gastrointestinal tract disease, natural valve endocarditis, colitis, vaginitis, urethritis, conjunctivitis, otitis (preferably otitis media), cystic fibrosis, ventilator-associated pneumonia, bacteremia or a wound infection.

In another preferred embodiment, the condition or disease is an airway infection. In another preferred embodiment, the condition or disease is a sexually transmitted disease. In another preferred embodiment, the condition or disease is meningitis. In another preferred embodiment, the condition or disease is a urinary tract infection. In another preferred embodiment, the condition or disease is a gastrointestinal disease. In another preferred embodiment, the condition or disease is native valve endocarditis. In another preferred embodiment, the condition or disease is colitis. In another preferred embodiment, the condition or disease is vaginitis. In another preferred embodiment, the condition or disease is urethritis. In another preferred embodiment, the condition or disease is conjunctivitis. In another preferred embodiment, the condition or disease is otitis, preferably otitis media. In another preferred embodiment, the condition or disease is ventilator-associated pneumonia. In another preferred embodiment, the condition or disease is bacteremia. In another preferred embodiment, the condition or disease is a (chronic) wound infection, typically and preferably a chronic wound infection.

In another preferred embodiment, the condition or disease is a chronic airway infection, preferably the chronic airway infection is cystic fibrosis, chronic obstructive pulmonary disease, or chronic sinusitis. In another preferred embodiment, the condition or disease is cystic fibrosis. In another preferred embodiment, the condition or disease is caused by a bacterium, mycoplasma, spirochete or fungus. In another preferred embodiment, the condition or disease is caused by bacteria. In another preferred embodiment, the condition or disease is caused by mycoplasma. In another preferred embodiment, the disorder or disease is caused by spirochetes. In another preferred embodiment, the condition or disease is caused by a fungus, wherein preferably the fungus is selected from the group consisting of candida, aspergillus, cryptococcus, hyphomycete, coccidiodes or pneumocystis.

Infections and conditions involving biofilms include those caused by gram-positive bacteria such as Streptococcus pneumoniae, Bacillus, Listeria monocytogenes, Staphylococcus, lactic acid bacteria including Lactobacillus plantarum and lactococcus lactis, Streptococcus distans, Streptococcus mutans, gram-negative bacteria such as Escherichia coli, Pseudomonas aeruginosa, Enterobacteriaceae, Salmonella, Actinobacillus pleuropneumoniae, Proteus, Acinetobacter baumannii, Shigella, Moraxella, helicobacter pylori, maltophilia, Bacillus beijerinckii, Acetobacter aceti, Legionella, cyanobacteria, Spirochaetes, Thionella viridans and Thionella viridis, Neisseria, Haemophilus influenzae, Klebsiella pneumoniae, Serratia marcescens, and mycoplasma, spiroplasia and fungi such as Candida, Aspergillus, Cryptococcus, myces, Mycospora globisporus, etc, Infection by pneumocystis. Some of the pathogens that form biofilms are ESKAPE pathogens. Infections and conditions involving biofilms include, for example, sexually transmitted diseases caused by gram-negative cocci (e.g., neisseria gonorrhoeae), meningitis (e.g., neisseria meningitidis), or respiratory symptoms (e.g., moraxella catarrhalis, haemophilus influenzae). They also include infections and disorders caused by gram-negative bacilli, such as major respiratory diseases (e.g. klebsiella pneumoniae, legionella pneumoniae, pseudomonas aeruginosa), major urological diseases (e.g. escherichia coli, proteus, enterobacter cloacae, serratia marcescens) or major gastrointestinal diseases (e.g. helicobacter pylori, salmonella enteritidis, salmonella typhi). Infections involving biofilms also include nosocomial and intensive care unit bacteraemia, secondary meningitis and ventilator-associated pneumonia caused by hospital-acquired infections (e.g., acinetobacter baumannii).

In another preferred embodiment, said condition or disease, preferably said infection, is caused by at least one ESKAPE pathogen.

In another preferred embodiment, said condition or disease, preferably said infection, is caused by enterococcus faecalis. In another preferred embodiment, said condition or disease, preferably said infection, is caused by staphylococcus aureus. In another preferred embodiment, said condition or disease, preferably said infection, is caused by klebsiella pneumoniae. In another preferred embodiment, said condition or disease, preferably said infection, is caused by acinetobacter baumannii. In another preferred embodiment, said condition or disease, preferably said infection, is caused by pseudomonas aeruginosa. In another preferred embodiment, said condition or disease, preferably said infection, is caused by enterobacter.

In another preferred embodiment, the condition or disease is caused by a bacterium, and wherein the bacterium is a gram-negative bacterium or a gram-positive bacterium. In another preferred embodiment, the condition or disease is caused by gram-negative bacteria. In another preferred embodiment, the condition or disease is caused by gram-positive bacteria.

In another preferred embodiment, the disorder or disease is caused by a bacterium selected from the group consisting of Streptococcus pneumoniae, Bacillus, Listeria monocytogenes, Staphylococcus, lactic acid bacteria (preferably Lactobacillus plantarum and lactococcus lactis), Streptococcus sobrinus, Streptococcus mutans, Escherichia coli, Pseudomonas aeruginosa, Enterobacteriaceae, Salmonella species (preferably Salmonella enteritidis, Salmonella enteritidis or Salmonella typhi), Actinobacillus pleuropneumoniae, Proteus, Shigella dysenteriae, Moraxella (preferably Moraxella catarrhalis), Shigella dysenteriae (preferably helicobacter pylori), stenotrophomonas, Bdellovibrio, Acetobacter, Legionella (preferably Legionella pneumophila), cyanobacteria, Spirochaetes (spirochaetes), Thiobacillus viridans and Thiobacillus viridans, Neisseria (preferably Neisseria gonorrhoeae or Neisseria meningitidis), Haemophilus influenzae, Salmonella viridans, Enterococcus faecalis, staphylococcus aureus, klebsiella pneumoniae, acinetobacter baumannii, serratia marcescens, enterobacter cloacae and gram-negative bacteria or gram-positive bacteria of enterobacter. In another preferred embodiment, said condition or disease, preferably said infection, is caused by haemophilus influenzae or serratia marcescens. In another preferred embodiment, said condition or disease, preferably said infection, is caused by haemophilus influenzae. In another preferred embodiment, said condition or disease, preferably said infection, is caused by serratia marcescens.

In another preferred embodiment, said condition or disease, preferably said infection, is caused by at least one ESKAPE pathogen selected from the group consisting of enterococcus faecalis, staphylococcus aureus, klebsiella pneumoniae, acinetobacter baumannii, pseudomonas aeruginosa and enterobacter, or is caused by haemophilus influenzae or serratia marcescens.

In another preferred embodiment, the condition or disease is caused by a gram-negative or gram-positive bacterium selected from the group consisting of streptococcus pneumoniae, bacillus, listeria monocytogenes, staphylococcus, lactic acid bacteria (preferably lactobacillus plantarum and lactococcus lactis), streptococcus sobrinus, streptococcus mutans, escherichia coli, pseudomonas aeruginosa, enterobacteriaceae, salmonella enterica, actinobacillus pleuropneumoniae, proteus.

In another preferred embodiment, the disorder or disease is caused by a gram-negative bacterium selected from the group consisting of pseudomonas aeruginosa, escherichia coli, enterobacteriaceae, salmonella enteritidis, actinobacillus pleuropneumoniae, and proteus.

In another highly preferred embodiment, the condition or disease is caused by Pseudomonas aeruginosa. In another very preferred embodiment, the condition or disease is caused by a gram-negative bacterium, wherein the gram-negative bacterium is pseudomonas aeruginosa. In another very preferred embodiment, the use is a method for preventing or reducing the formation of a bacterial biofilm, typically and preferably on a surface, wherein the bacteria is pseudomonas aeruginosa.

Pseudomonas aeruginosa biofilms can act as a reservoir for disease recurrence (resurvoirs), preventing its complete cure. Biofilms of such opportunistic pathogens are implicated in both chronic infections, such as pulmonary infections in cystic fibrosis patients (80% of CF patients are chronically infected with pseudomonas aeruginosa), and in life-threatening refractory hospital-acquired infections, including ventilator-associated pneumonia, wounds in burn patients, post-operative wound infections, and skin and soft tissue infections. It also relates to contamination of medical instruments, devices and tools (Rabin et al, Future med. chem.7(4) 2015; Tran et al, PLOS Pathogens 10(11) 2014).

In another preferred embodiment, the condition or disease is caused by a gram-positive bacterium selected from the group consisting of streptococcus pneumoniae, bacillus, listeria monocytogenes, staphylococcus, lactic acid bacteria (preferably lactobacillus and lactococcus lactis), streptococcus sobrinus and streptococcus mutans.

In another very preferred embodiment, the condition or disease is caused by staphylococcus aureus. In another very preferred embodiment, the use is a method for preventing or reducing biofilm formation in a bacterium, typically and preferably on a surface, wherein the bacterium is staphylococcus aureus.

S. aureus biofilms are a major cause of multiple infections and present challenges to conventional anti-infective methods, especially antibiotic therapy. Many clinical isolates of staphylococcus aureus are methicillin-resistant (MRSA) or multidrug-resistant. Resistance to antibiotic treatment is further enhanced with increased tolerance of biofilm-forming staphylococcus aureus to antibiotics that are still susceptible to few MRSA. Vancomycin is the most commonly used drug for s.aureus biofilm-associated infections, but the increased tolerance of biofilms to vancomycin indicates that the use of combination therapy is necessary.

Staphylococcus aureus biofilms are commonly associated with implant-related infections (prosthetic orthopedic implants, heart valves, pacemakers, and vascular catheters), chronic wounds, osteomyelitis, cystic fibrosis, pulmonary infections, and endocarditis. For example, cystic fibrosis lung infection is associated with the presence of biofilms and chronic, long-term, persistent increased mortality of bacteria. In the case of staphylococcus aureus, these infections are usually caused by MRSA. Staphylococcus aureus is also a major cause of infective endocarditis by infecting the endocardium or heart prosthesis surface. Aureus endocarditis is increasing with the use of surgical procedures involving the implantation of artificial cardiovascular devices such as heart valves prostheses, grafts, hemodialysis catheters and pacemakers (Bhattacharya et al, Expert Rev Anti infection ther.13(12) 2015).

In a further preferred embodiment, the method for preventing or reducing biofilm formation or for eliminating or reducing existing biofilms according to the invention further comprises administering to a mammal in need thereof, preferably to a human in need thereof, a therapeutically effective amount of a composition of the invention, a single empty liposome of the invention or a mixture of the invention.

To prevent or reduce biofilm formation or to eliminate or reduce existing biofilms, the single empty liposomes of the invention or the empty liposome mixtures of the invention may be administered in the form of intravenous, intramuscular, intraperitoneal or subcutaneous injections. Injectable solutions are prepared by standard procedures known in the art, for example as suspensions of liposomes in sterile physiological saline. It is also contemplated that a single empty liposome of the invention or a mixture of empty liposomes of the invention may be used as an aerosol for the treatment of respiratory infections, or in the form of a formulation useful for sublingual or buccal administration, intraocular or intravitreal administration, and topical administration (e.g., eye drops, toothpaste, topical liquid suspensions of the skin or oral cavity, etc.). Methods for preparing such formulations from liposomes, such as the empty liposomes of the present invention, are known in the art. A prophylactic measure based on a single empty liposome of the invention or a mixture of empty liposomes of the invention will help to prevent a condition or disease associated with a biofilm, for example, preventing biofilm development in a patient who will undergo, is undergoing, or has undergone an implantation procedure, or a patient suffering from a chronic infection, or in an environment otherwise conducive to the development of a condition or disease associated with a biofilm.

In a further preferred embodiment, the method for preventing or reducing biofilm formation or for eliminating or reducing existing biofilms according to the invention further comprises administering to a mammal in need thereof, preferably to a human in need thereof, a therapeutically effective amount of a composition of the invention, a single empty liposome of the invention, a mixture of empty liposomes of the invention, wherein the composition of the invention, the single empty liposome of the invention or the mixture of empty liposomes of the invention is not administered in combination with any other drug, and thus is typically administered alone, wherein further typically and preferably the composition of the invention, the single empty liposome of the invention or the mixture of empty liposomes of the invention is not co-administered at the same time or at any time up to 4 weeks before and after, preferably at any time up to 2 weeks before and after.

In another preferred embodiment of the present invention, said use is in combination with an antimicrobial agent, wherein preferably said antimicrobial agent is an antibiotic, antifungal, antitoxic, antiseptic or antimicrobial agent and combinations thereof, and wherein further preferably said antimicrobial agent is an antibiotic.

The present invention also relates to a method for preventing or reducing biofilm formation or for eliminating or reducing existing biofilm, preferably to a method for preventing or reducing biofilm formation, for treating a condition or disease, preferably a bacterial infection, wherein the method comprises: a therapeutically effective amount of a single empty liposome of the invention or a mixture of empty liposomes of the invention is administered to a mammal, preferably a human, in need thereof, before, after, together or concurrently with the administration of an antimicrobial agent, preferably with the standard antibiotic treatment of bacterial infections.

This preferred aspect and embodiment of the invention aims to improve the therapeutic effect, promote the growth of plankton, thus eliminating the extra community level of resistance provided by the biofilm, promote the level of traditional antibiotics targeting pathogens to cells, and also allow for a reduction in antibiotic dosage. Moreover, since the single empty liposomes and empty liposome mixtures of the present invention have been described as toxin neutralizers, their effect on the toxin will simultaneously counteract bacterial toxicity and allow the immune defense system to exert an appropriate response against the pathogen.

In other preferred embodiments of the invention, the method further comprises administering a therapeutically effective amount of a composition of the invention, a single empty liposome or a mixture of empty liposomes of the invention to a mammal, preferably a human, in need thereof, before, after, together or concurrently with the administration of the antimicrobial agent. Wherein preferably the antimicrobial agent is an antibiotic, antifungal, antitoxin, antitoxic, antiseptic or combination thereof, and wherein further preferably the antimicrobial agent is an antibiotic. In another preferred embodiment, the method further comprises administering a therapeutically effective amount of a composition of the invention, a single empty liposome of the invention, or a mixture of empty liposomes to a mammal, preferably a human, in need thereof, before, after, together with, or concurrently with administration of standard antibiotic therapy or standard of care.

In other preferred embodiments of the invention, the method further comprises administering an effective amount of the composition of the invention, the single empty liposomes of the invention or the empty liposome mixture of the invention to an animal in need thereof, preferably a food animal, such as cattle or sheep or pig, without the need for any other drug, and therefore typically used alone, or before, after, together or concurrently with an antimicrobial agent, wherein preferably the antimicrobial agent is an antibiotic, an antifungal agent, a toxin agent, an antitoxic agent, an antiseptic agent, or a combination thereof, and wherein further preferably the antimicrobial agent is an antibiotic. In another preferred embodiment, the method further comprises administering an effective amount of a composition of the invention, a single empty liposome of the invention, or a mixture of empty liposomes of the invention to an animal in need thereof, preferably a food animal, such as a bovine or ovine, before, after, together with, or concurrently with standard antibiotic treatment or administration.

In other preferred embodiments of the invention, the method further comprises administering a therapeutically effective amount of a composition of the invention, a single empty liposome of the invention or a mixture of empty liposomes of the invention to an animal in need thereof, preferably a food animal, such as a cow or sheep or pig, without any other drug, and therefore typically used alone, or before, after, together with or concurrently with the administration of an antimicrobial agent, wherein preferably the antimicrobial agent is an antibiotic, antifungal agent, antimicrobial agent, antitoxin agent, antitoxic agent, antiseptic agent or a combination thereof, and wherein further preferably the antimicrobial agent is an antibiotic. In another preferred embodiment, the method further comprises administering a therapeutically effective amount of a composition of the invention, a single empty liposome of the invention or a mixture of empty liposomes of the invention to an animal in need thereof, preferably a food animal, such as a bovine or ovine, before, after, together with or concurrently with standard antibiotic treatment.

In other preferred embodiments of the invention, the method further comprises contacting or coating a therapeutically effective amount of the composition of the invention, the single empty liposomes of the invention or the empty liposome mixture of the invention on a surface, preferably a plant surface, or without any other drug, thus generally used alone, or before, after, together or concurrently with the use of an antimicrobial agent, wherein preferably the antimicrobial agent is an antibiotic, antifungal, antitoxin, antitoxic, antiseptic or a combination thereof, and wherein further preferably the antimicrobial agent is an antibiotic.

It will be appreciated that empty liposomes as defined above and mixtures of empty liposomes as defined above may be used together or in combination with other compounds, prodrugs or drugs, if desired. For example, other compounds, prodrugs or drugs may be added to prepare standard pharmaceutical compositions. It is also contemplated to add a drug or drug-like compound, or further to add known or novel liposomes that incorporate a drug or drug-like compound within the liposome.

The drugs and prodrugs under consideration are in particular standard antimicrobial or antifungal therapeutic or antitoxin or antitoxic agents known to the person skilled in the art. In addition, drugs such as antibiotics are especially contemplated. Such antibiotics are, for example, carbapenems, such as imipenem/cilastatin, meropenem, ertapenem and doripenem; first generation cephalosporins, such as cephalexin and cephalexin; second generation cephalosporins such as cefuroxime, cefaclor and cefprozil; third-generation cephalosporins, such as ceftazidime, ceftriaxone, cefixime, cefdinir, cefditoren, cefotaxime, cefpodoxime and cefotiam; fourth generation cephalosporins, such as cefepime; cephalosporins of passage 5, such as ceftaroline fosamil and cefpirome; glycopeptides such as vancomycin, teicoplanin and telavancin; macrolides such as clarithromycin, azithromycin, dirithromycin, erythromycin, roxithromycin, triamcinolone, telithromycin, spectinomycin, and spiramycin; penicillins, such as amoxicillin, flucloxacillin, oxacillin, carbenicillin, and piperacillin; penicillin combinations such as amoxicillin/clavulanate, piperacillin/tazobactam, ampicillin/sulbactam, and ticarcillin/clavulanate; quinolones, such as ciprofloxacin (e.g., the ciprofloxacin liposome of Aradigm) and moxifloxacin; drugs against mycobacteria, such as rifampicin (rifampicin, referred to as rifampicin in the united states), clofazimine, dapsone, kemptycin, cycloserine, ethambutol, ethion, isoniazid, pyrazinamide, rifabutin, rifapentine, and streptomycin; other antibiotics, such as metronidazole, aspipramine, chloramphenicol, fosfomycin, fusidic acid, linezolid, mupirocin, bantamycin, quinupristin/dalfopristin, rifaximin, thiamphenicol, tigecycline, and imidazole; aminoglycosides, e.g. amikacin, gentamicin, kanamycin, neomycin, netilmicin, tobramycin (e.g. Fluidosomes from Axentis)^TMTobramycin) and paromomycin; sulfonamidesDrugs such as mafenib, sulphamidoimidazolidine, sulphacetamide, sulphadiazine, silver sulphadiazine, sulphamethoxazole, sulphamide, sulfasalazine, sulphaisoxazole, trimethoprim and trimethoprim (trimetrexazole, TMP-SMX); tetracyclines, such as, for example, norcycline, doxycycline, minocycline, oxytetracycline, and tetracycline; lincosamides such as clindamycin and lincomycin; and lipopeptides such as daptomycin.

Other drugs contemplated for use are, for example, anti-inflammatory drugs comprising corticosteroids (glucocorticoids), such as hydrocortisone (Cortisol), cortisone, prednisone, prednisolone, methylprednisolone, dexamethasone, betamethasone, triamcinolone, beclomethasone, fludrocortisone acetate, deoxycorticosterone acetate, budesonide, desonide and fluocinonide; non-steroidal anti-inflammatory drugs, such as salicylates, e.g., aspirin (acetylsalicylic acid), diflufenican, and salicylate; propionic acid derivatives such as ibuprofen, dexibuprofen, naproxen, fenoprofen, ketoprofen, dexketoprofen, flurbiprofen, oxabloxine and loxoprofen; acetic acid derivatives such as indomethacin, tolmetin, sulindac, etodolac, ketorolac, diclofenac, and nabumetone; enolic acid (oxicam) derivatives such as piroxicam, meloxicam, tenoxicam, droxicam, lornoxicam and isoxicam; fenamic acid derivatives (fenamate salts), such as mefenamic acid, meclofenamic acid, flufenamic acid and toluenesulfonic acid. Selective COX-2 inhibitors (coxibs), such as celecoxib; and other drugs such as licofelone.

Other drugs contemplated are, for example, vasopressors and vasoconstrictors, such as vasopressin, oxymetazoline, phenylephrine, propylhexanediamine, pseudoephedrine, epinephrine, norepinephrine, dopamine, and antihistamines.

Other types of drugs are also contemplated, such as paracetamol (an analgesic), nystatin, amphotericin B, polyenes, azoles and triazoles, nucleoside analogues, echinocandin, pneumococci (an antifungal infection), bupivacaine (post-operative pain control), morphine (an analgesic drug)Drugs), Verteporfm (ophthalmic disease), estradiol (climacteric disorder),a compound is provided.

In such combination therapy, the empty liposomes and liposome mixtures of the invention can be considered as adjuvants and the corresponding therapeutic methods as adjuvant therapy.

In another preferred embodiment of the invention, the method is an ex vivo method, wherein preferably the method comprises contacting a surface, preferably a surface of a medical device, with any one of the compositions of the invention.

In another aspect, the present invention provides a method for preventing or reducing biofilm formation, preferably bacterial biofilm formation, on a surface or for eliminating or reducing existing biofilm on a surface, the method comprising contacting the surface or existing biofilm with any one of the compositions of the present invention.

In another aspect, the present invention provides a method for preventing or reducing biofilm formation, preferably bacterial biofilm formation, on a surface or for eliminating or reducing existing biofilm on a surface, wherein the method comprises coating the surface or existing biofilm with any one of the compositions of the present invention.

In another aspect, the present invention provides a method for preventing or reducing biofilm formation, preferably bacterial biofilm formation, on a surface or for eliminating or reducing existing biofilm on a surface, comprising treating the surface or existing biofilm with any one of the compositions of the present invention.

In another aspect, the invention provides an ex vivo method for preventing or reducing biofilm formation, preferably bacterial biofilm formation, on a surface or for eliminating or reducing existing biofilm on a surface, comprising contacting the surface or existing biofilm with any of the compositions of the invention. Thus, the inventive method is not a method of treatment of the human or animal body by therapy.

In another aspect, the present invention provides a method for preventing or reducing biofilm formation, preferably bacterial biofilm formation, on a surface or for eliminating or reducing existing biofilm on a surface, wherein the method comprises coating the surface or existing biofilm with any one of the compositions of the present invention.

In another aspect, the invention provides an ex vivo method for preventing or reducing biofilm formation, preferably bacterial biofilm formation, on a surface or for eliminating or reducing existing biofilm on a surface, comprising treating the surface or existing biofilm with any one of the compositions of the invention. Thus, the inventive method is not a method of treatment of the human or animal body by therapy.

Methods of coating surfaces with biologically or pharmaceutically active compounds or compositions are well known in the art. For example, the non-biological surfaces described above, i.e., the surfaces of medical devices, can be coated by incorporating the compositions of the present invention into a film-forming component and made into an anti-biofilm coating that can be used to inhibit biofilm formation on the surfaces of medical devices. The film-forming component may comprise one or more resins, such as, but not limited to, one or more hydrolyzable, soluble, or insoluble resins. For example, the resin may be one or more of a glycol resin, an acrylic resin, a chlorinated rubber resin, an epoxy resin, a silicone resin, a polyester resin, a polyurethane resin, a fluoropolymer resin, and other resins known to those skilled in the art.