Liposomes for inhibiting biofilm formation

Abstract

The present invention relates to a composition, the composition being (i) a single empty liposome, the single empty liposome being (a) an empty liposome comprising cholesterol, the amount of cholesterol being at least 30% (wt / wt), preferably the empty liposome being selected from empty liposomes comprising (more preferably consisting of) cholesterol and sphingomyelin, or (b) empty liposomes consisting of sphingomyelin, or (ii) a mixture of empty liposomes, the mixture of empty liposomes being (a) empty liposomes comprising cholesterol, the amount of cholesterol being at least 30% (wt / wt), preferably the empty liposomes being selected from empty liposomes comprising (more preferably consisting of) cholesterol and sphingomyelin, (b) empty liposomes consisting of sphingomyelin, and (c) phosphatidylcholine. and empty liposomes comprising (preferably consisting of) at least one empty liposome selected from empty liposomes comprising (preferably consisting of) a lipid or phospholipid selected, independently of one another, from cholesterol, sphingomyelin, ceramide, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, diacylglycerol, and phosphatidic acid containing one or more saturated or unsaturated fatty acids having more than 4 carbon atoms and up to 28 carbon atoms, for use in a method for preventing or reducing biofilm formation or for eradicating or reducing an existing biofilm. [Selection diagram] None

Description

[Technical field]

[0001] The present invention relates to empty liposomes or empty liposome mixtures of defined lipid composition for preventing or reducing biofilm formation or for eradicating or reducing existing biofilms. The present invention further relates to the treatment or prevention of such biofilm formation by using the empty liposomes or empty liposome mixtures of the present invention alone or in combination with standard antimicrobial treatments. [Background technology]

[0002] References: Davies, Nat.Rev.Drug Discov.(2003)2:114-122;Bjarnsholt et al., Nat.Rev.Drug Discov.(2013)12:791-808;Wu et al., International Journal of Oral Science 7(2015);Rasamiravaka et al., BioMed Research International Volume(2015);Bhattacharya et al., Expert Rev Anti Infect Ther.(2015)13(12);Peneysan et al., Molecules(2015)20;Rabin et al., Future Med.Chem.(2015)7(4).

[0003] Microorganisms can occur as planktonic organisms (particularly planktonic bacteria or fungi) or as biofilms (particularly biofilm bacteria or fungi). Typically, such biofilms consist of a dense, highly organized community of microorganisms encased in a secreted polymeric matrix composed of proteins, extracellular DNA, amyloid fibrils, and polysaccharides, which form a highly hydrated, polar mixture that contributes to the overall structural framework and architecture of the biofilm.

[0004] Compared to planktonic microorganisms, biofilm microorganisms are dominant in almost all ecosystems under sufficient nutrient conditions. Biofilms, therefore, attach to abiotic surfaces such as implant surfaces (catheters, artificial blood vessels, artificial heart valves, cardiac pacemakers, cerebrospinal fluid shunts, urinary catheters, peritoneal dialysis catheters, artificial joints and orthopedic fixtures, intrauterine contraceptives, biliary stents, breast implants, contact lenses, dentures, and in the dental area such as dental caries and periodontitis), and also attach to biotic surfaces such as the lungs, skin, bone, teeth, etc., and are thus established in various host environments, leading to, for example, chronic airway infections, chronic obstructive pulmonary disease, native valve endocarditis, chronic otitis media, chronic rhinosinusitis, or chronic wound infections in patients with cystic fibrosis. Also, as both bacteria and fungi attach to teeth and become embedded in salivary polymers and microbial extracellular products, biofilms form dental plaque, thus promoting the development of dental diseases. Biofilms can occur on the surfaces of food equipment in food factories and during industrial processes, so biofilms are also a problem in the food industry.

[0005] If biofilm microorganisms were simply planktonic-like cells attached to the surface, this exposure would not be important, but biofilm microorganisms are very different from planktonic microorganisms. The biofilm polymer matrix forms a shield against the host's immune defenses, protecting the deep-seated bacteria and fungi from antibacterial and antifungal agents, respectively. Also, biofilms function as a microenvironment for infection, promoting chronic infections and recurrence. Biofilm-related infections are chronic, persistent, and difficult to cure. In the case of biofilms formed on implants such as heart valves, there is a risk that the detached biofilm cells may move with the bloodstream and cause infections in other organs.

[0006] Biofilms are involved in various infectious diseases and are formed by both Gram-positive and Gram-negative bacteria, as well as mycoplasmas, spiroplasmas, and fungi. Biofilms are also involved in infectious diseases caused by ESKAPE pathogens. The number of diseases associated with biofilms is considered to be very large, and colitis, vaginitis, urethritis, conjunctivitis, and otitis are just a few examples. Biofilms are also important as colonizing bacteria on medical devices, including urinary catheters, intravenous catheters, arterial catheters, shunts, etc., as shown. Bacteria and fungi present in biofilms are resistant to antibacterial agents and immune defense mechanisms, so biofilm-related infectious diseases tend to become chronic, such as Pseudomonas aeruginosa (P. aeruginosa) infections in the bronchopulmonary system of patients with cystic fibrosis (CF).

[0007] In fact, biofilm bacteria are up to 1000 times more resistant to antibiotics than planktonic bacteria. Traditionally, antibiotics have been developed to target the cellular mechanisms involved in the growth and survival of free-floating bacteria, i.e., bacteria in the planktonic-like ecology. However, biofilm bacteria are very different from their planktonic-like counterparts. In particular, the extracellular proteins, virulence factors, and surfactants expressed by biofilm bacteria are different from those expressed by planktonic bacteria. Also, the protective biofilm matrix enables the avoidance of the host immune response, the promotion of survival, and the seeding of bacteria. Furthermore, antibiotic resistance is an inherent property of biofilms. Thus, bacteria can survive despite intact host immune defenses and frequent antibiotic treatments. Additionally, antibiotic treatments targeting infections using only planktonic bacteria are mostly ineffective against the biofilm phenotype. Effective antibiotic dosages for biofilm eradication are often not achievable with conventional antibiotic administration due to the toxicity and side effects of high-dose antibiotics and due to limitations in renal and hepatic function. Also, exposing the bacteria within biofilms to antibiotics further increases the selective pressure associated with the development of antibiotic resistance. Similarly, fungal biofilm-related infections are often ineffective with conventional treatments due to resistance to antimicrobial agents and, in part, due to surface-inducible upregulation of drug efflux pumps.

[0008] Current methods may consist of the physical removal of the source of infection (surgical removal of catheters or orthopedic hardware, non-absorbable sutures, necrotic tissue) or treatment with antibacterial agents. However, while antibiotics and antifungal agents directly target the organisms that form biofilms, they have had limited success in treating biofilm-related conditions. Current methods do not always succeed in eradicating infections, and infections involving bacteria or fungi present in biofilms often become untreatable and ultimately progress to a chronic state. The ability to form biofilms is a universal attribute of bacteria, and despite the fact that many medically important fungi produce biofilms, the detailed mechanisms underlying biofilm formation are not yet fully understood, making the treatment and eradication of biofilm-related infections particularly challenging. The non-bactericidal antibiofilm drugs under development are mainly aimed at (i) avoiding the attachment of microorganisms to surfaces, (ii) influencing the maturation of biofilms and / or inducing their dispersion and degradation (e.g., by targeting bacterial systems and messengers that play important roles in the regulation of biofilm formation, particularly bacterial quorum sensing (QS), nucleotides (especially c-di-GMP) or small non-coding RNAs (sRNA)), or (iii) avoiding the attachment of microorganisms to surfaces, for example by damaging amyloid structures involved in biofilm formation. The treatment of biofilm-related conditions demonstrates a significant unmet clinical need.

[0009] Tailored empty liposomes, such as those composed of cholesterol (CHOL) and / or sphingomyelin (SM), and their use for the treatment of bacterial infections have recently been reported to function as traps for bacterial toxins (WO 2013 / 186286; Henry BD et al., Nat Biotechnol 2015;33(1):81-88; Azeredo da Silveira, S and Perez, A, Expert Rev. Anti Infect. Ther. 2015;13(5):531-533).

[0010] Accordingly, one object of the present invention is to provide compositions with anti-biofilm activity and compositions for the treatment of biofilm-associated conditions and diseases. Summary of the Invention

[0011] We have surprisingly found that the empty liposomes of defined lipid composition and empty liposome mixtures of defined lipid composition according to the present invention can prevent or reduce biofilm formation and eradicate or reduce existing biofilms. In particular, we have surprisingly found that the empty liposomes of the present invention and mixtures thereof have been demonstrated to reduce biofilm formation by Pseudomonas aeruginosa. Thus, the empty liposomes of defined lipid composition and empty liposome mixtures according to the present invention can prevent or reduce biofilm formation or eradicate or reduce existing biofilms, and thus can prevent and treat conditions and diseases such as acute and chronic bacterial infections or cystic fibrosis.

[0012] Furthermore, the destruction or weakening of biofilms, or the avoidance or reduction of biofilm formation, by a single empty liposome or empty liposome mixture of the present invention will lead to improved effectiveness of simultaneously or subsequently applied antimicrobial agents, particularly biocides (antibacterial agents), especially antibiotics, or antifungal agents. This is a highly synergistic effect, since freed planktonic bacteria or fungi are much more susceptible (up to 1000 times) to antimicrobial agents and antibiotics. Simultaneous or subsequent antibiotic or antifungal treatment can eliminate the planktonic organisms, preventing the spread of infection to other parts of the body. Freed organisms are also much more susceptible to the host's immune defense mechanisms and can therefore be more effectively eliminated by the host's own immune system.

[0013] Furthermore, the inventive compositions and empty liposomes according to the present invention are believed to be non-toxic, representing a further beneficial and advantageous feature of the present invention.

[0014] Thus, in a first aspect, the present invention provides a composition for use in a method for preventing or reducing biofilm formation or for eradicating or reducing existing biofilms. The composition is (i) a single empty liposome, the single empty liposome being (a) an empty liposome containing cholesterol, the amount of cholesterol being at least 30% (weight / weight), preferably the empty liposome being an empty liposome containing (more preferably consisting of) cholesterol and sphingomyelin, or (b) an empty liposome consisting of sphingomyelin, a single empty liposome selected therefrom, or (ii) an empty liposome mixture, the empty liposome mixture being (a) an empty liposome containing cholesterol, the amount of cholesterol being at least 30% (weight / weight), preferably the empty liposome being an empty liposome containing (more preferably consisting of) cholesterol and sphingomyelin, (b) an empty liposome consisting of sphingomyelin, and (c) an empty liposome containing (preferably consisting of) phosphatidylcholine and sphingomyelin, an empty liposome mixture containing (preferably consisting of) at least one empty liposome selected therefrom. At least one empty liposome is independently selected from empty liposomes containing (preferably consisting of) a lipid or phospholipid selected from cholesterol, sphingomyelin, ceramide, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, diacylglycerol, and phosphatidic acid containing one or two or more saturated or unsaturated fatty acids having more than 4 carbon atoms and up to 28 carbon atoms.

[0015] In a further aspect, the present invention provides an empty liposome consisting of sphingomyelin and cholesterol for use in a method for preventing or reducing biofilm formation or for eradicating or reducing existing biofilms, preferably in a mammal, more preferably in a human, the amount of cholesterol being at least 30% (weight / weight).

[0016] In a further aspect, the present invention provides empty liposomes composed of sphingomyelin for use in a method for preventing or reducing biofilm formation, or eradicating or reducing existing biofilms, preferably in mammals, more preferably in humans.

[0017] In yet another aspect, the present invention provides an empty liposome mixture comprising: (a) a first empty liposome composed of sphingomyelin and cholesterol, wherein the amount of cholesterol is at least 30% (weight / weight), and (b) a second empty liposome composed of sphingomyelin, for use in a method for preventing or reducing biofilm formation, or eradicating or reducing existing biofilms, preferably in mammals, more preferably in humans.

[0018] In yet another aspect, the present invention provides a method for reducing biofilm formation or eradicating or reducing existing biofilms, the method comprising administering 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 to a mammal, preferably a human, in need thereof. In yet another aspect, the present invention provides a method for preventing biofilm formation in at-risk subjects, the method comprising administering an 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 to an at-risk mammal, preferably a human. In yet another aspect, the present invention provides a method for preventing or reducing biofilm formation or eradicating or reducing existing biofilms, the method comprising administering 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 to a mammal, preferably a human, in need thereof without co-administering a standard antibacterial therapeutic agent. Further, the present invention relates to preventing or reducing biofilm formation or eradicating or reducing existing biofilms, and comprises administering 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 to a mammal, preferably a human, in need thereof before standard antibacterial treatment of an infectious disease, after standard antibacterial treatment of an infectious disease, together with standard antibacterial treatment of an infectious disease, or simultaneously with a standard antibacterial therapeutic agent of an infectious disease.

[0019] As the specification proceeds, further aspects and embodiments of the present invention will become apparent.

Brief Description of the Drawings

[0020] [Figure 1] Inhibition of Pseudomonas aeruginosa multi-drug resistant strain 6077 biofilm by the empty liposome mixture of the present invention using a bacterial density of 1×107 cells / well. The graph shows the percentage of biofilm inhibition compared to the biofilm formed in the absence of empty liposomes.

Modes for Carrying Out the Invention

[0021] Unless otherwise defined, 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.

[0022] As used herein, the term "about" means + / - 10%. For example, about 50% means 45% to 55%. Preferably, as used herein, the term "about" means + / - 5%. For example, about 50% means 47.5% to 52.5%.

[0023] When the terms "a" or "an" are used herein, unless otherwise indicated, they mean "at least one." In particular, the use of the terms "a" or "an" in connection with the single empty liposome, the first empty liposome, and the second empty liposome to describe empty liposomes and empty liposome mixtures according to the present invention typically and preferably refers to a single empty liposome and an empty liposome mixture comprising the first empty liposome and the second empty liposome.

[0024] Thus, in a first aspect, the present invention provides a composition for use in a method for preventing or reducing biofilm formation or for eradicating or reducing an existing biofilm. The composition comprises (preferably consists of) (i) a single empty liposome, wherein the single empty liposome is (a) an empty liposome comprising cholesterol, wherein the amount of cholesterol is at least 30% (weight / weight), preferably the empty liposome is selected from empty liposomes comprising (more preferably consisting of) cholesterol and sphingomyelin, or (b) empty liposomes consisting of sphingomyelin, or (ii) a mixture of empty liposomes, wherein the mixture of empty liposomes is (a) empty liposomes comprising cholesterol, wherein the amount of cholesterol is at least 30% (weight / weight), preferably the empty liposome is selected from empty liposomes comprising (more preferably consisting of) cholesterol and sphingomyelin, (b) empty liposomes consisting of sphingomyelin, and (c) empty liposomes comprising (preferably consisting of) phosphatidylcholine and sphingomyelin. At least one empty liposome is independently selected from empty liposomes comprising (preferably consisting of) a lipid or phospholipid selected from cholesterol, sphingomyelin, ceramide, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, diacylglycerol, and phosphatidic acid containing one or more saturated or unsaturated fatty acids having more than 4 carbon atoms and up to 28 carbon atoms.

[0025] As used herein, the term "empty liposome" refers to liposomes having an average diameter of 20 nm to 10 μm, preferably 20 to 500 nm, more preferably 20 nm to 400 nm, and even more preferably 40 nm to 400 nm or 20 nm to 200 nm, consisting of one or more phospholipid bilayers, typically and preferably unilamellar vesicles and multilamellar vesicles, more preferably small unilamellar vesicles (SUVs), preferably artificial liposomes. In a preferred embodiment, the term "empty liposome" as used herein typically and preferably refers to liposomes that do not incorporate any drug, typically and preferably liposomes that do not incorporate any pharmaceutical. "Incorporated / incorporate", when used herein and when referring to the empty liposomes of the present invention, typically and preferably means encapsulated / encapsulating within the cavity of the liposome, or within the potential bilayer of the liposome, or as part of the membrane layer of the liposome. In another preferred embodiment, the term "empty liposome" as used herein typically and preferably refers to liposomes consisting of sphingomyelin and cholesterol, or consisting of sphingomyelin, according to the present invention, and further exclusively containing water-soluble inorganic compounds and / or water-soluble organic molecules. Typically and preferably, the water-soluble inorganic compounds and / or water-soluble organic molecules are derived from the synthesis of the empty liposomes of the present invention, typically and preferably, the water-soluble inorganic compounds are inorganic salts preferably selected from NaCl, KCl, MgCl2, and the water-soluble organic molecule is a buffer, preferably the water-soluble organic molecule is selected from glucose and HEPES. Typically and preferably, the water-soluble inorganic compounds and / or water-soluble organic molecules are incorporated into the inventive empty liposomes of the present invention due to their presence during the production of the empty liposomes of the present invention. In another preferred embodiment, the term "empty liposome" as used herein typically and preferably refers to liposomes consisting of sphingomyelin and cholesterol, or consisting of sphingomyelin, according to the present invention, and the empty liposomes do not contain antioxidants.In a more preferred embodiment, the term "empty liposome" as used herein typically and preferably refers to a liposome composed of sphingomyelin and cholesterol or consisting of sphingomyelin, and further exclusively contains a water-soluble inorganic compound and / or a water-soluble organic molecule. Typically and preferably, the water-soluble inorganic compound and / or the water-soluble organic molecule are derived from the synthesis of the empty liposomes of the present invention. Typically and preferably, the water-soluble inorganic compound is preferably an inorganic salt selected from NaCl, KCl, and MgCl2, and the water-soluble organic molecule is a buffer, and preferably the water-soluble organic molecule is selected from glucose and HEPES. The empty liposomes of the present invention composed of sphingomyelin and cholesterol or consisting of sphingomyelin do not contain antioxidants.

[0026] As used herein, the term "biofilm" refers to a population of bacterial cells and / or fungal cells that are attached to each other, attached to a surface, biological and abiotic surfaces and interfaces including interfaces, or associated with tissues or mucus. This definition also includes aggregates of small and large microorganisms, as well as floccules, and also adherent populations within the pore spaces of porous media. The term "anti-biofilm activity" or simply "anti-biofilm" as used herein, when used herein, refers to the prevention or reduction of biofilm formation, or the eradication or reduction of existing biofilms, the at least partial release of microorganisms, preferably bacteria, or the at least partial dissolution of biofilms. The term "biofilm bacteria" as used herein, when used herein, refers to non-planktonic-like bacteria present in biofilms.

[0027] As used herein, the term "ESKAPE pathogen" refers to a pathogen selected from Enterococcus faecalis, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp.

[0028] As used herein, the terms "inhibit", "destroy", "reduce", and "eradicate" with respect to biofilms or biofilm formation typically and preferably mean complete or partial inhibition of biofilm formation and / or progression (preferably greater than 20%, more preferably greater than 30%, more preferably greater than 50%, more preferably greater than 90%, even more preferably greater than 95%, or even greater than 99%) from the perspective of the number of remaining cells, and within that range, include reversal of biofilm progression or processes related to biofilm formation and / or progression. With respect to the latter, it means disappearance of existing biofilms at a faster rate than untreated biofilms or biofilms treated with compounds known not to affect the stability of the biofilm. Further, the inhibition may be permanent or temporary. From the perspective of temporary inhibition, biofilm formation and / or progression may be inhibited for a time sufficient to produce the desired effect (e.g., at least 5 days, preferably at least 10 days). Preferably, the inhibition of the biofilm is complete and / or permanent (preferably with no viable bacteria present) ("eradication").

[0029] As used herein, the term "medical device intended for insertion into the body of a subject" refers to surgically invasive devices or implantable devices such as, but not limited to, those defined in the "Cosmetics and Medical Devices" guidelines regarding the application of Council Directive 93 / 42 / EEC of the European Communities on medical devices, Directorate-General for Health and Consumer Protection, Directorate B, Unit B2

[0030] As used herein, the terms "treating", "treatment" or "therapy" refer to obtaining a desired physiological effect. The effect may be therapeutic in terms of partially or completely curing a disease or condition and / or the symptoms resulting from the disease or condition. The terms refer to inhibiting a disease or condition, i.e., arresting its progression, or to alleviating a disease or condition, i.e., causing regression of the disease or condition. The term "treating" or "processing" as used herein in the context of an ex vivo method according to the invention, or in the context of treating a surface (such as a surface selected from the surface of a mammalian cell, tissue or structure, or the surface of a plant cell, tissue or structure, or the surface of an edible plant, or the surface of a medical device, or a surface for the purpose of contact with water or an aqueous solution) with a composition of the invention, includes but is not limited to contacting, coating and applying the composition of the invention to the surface in the ex vivo method according to the invention or to the surface as described above.

[0031] As used herein, the term "prevention" refers to means for preventing or delaying the occurrence of a disease or condition and / or the symptoms resulting from the disease or condition.

[0032] As used herein, the term "effective amount" refers to the amount of an active ingredient, typically and preferably of a composition according to the invention, which, when administered or applied to a subject or patient, or to a surface such as the surface of a medical device (typically and preferably for the purpose of insertion into the body of the subject), in the context of prevention or treatment, results in a beneficial or desirable outcome. More specifically, the term "therapeutically effective amount" as used herein refers to the amount of an active ingredient, such as a composition according to the invention, which is sufficient to result in a beneficial or desirable outcome when administered to a subject or patient. A therapeutically effective amount can be administered in one or more administrations, applications or 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 one that prevents or reduces biofilm formation, or eradicates or reduces an existing biofilm, or results in an objectively measurable change in one or more parameters related to the treatment of a condition or disease associated with said biofilm formation or said existing biofilm. Preferably, said condition or disease is caused by bacteria present in the biofilm, and preferably, said condition or disease is selected from infectious diseases. More preferably, said infectious disease is a respiratory infection, a sexually transmitted infection, meningitis, a urinary tract infection, a gastrointestinal disorder, native valve endocarditis, colitis, vaginitis, urethritis, conjunctivitis, otitis (preferably otitis media), cystic fibrosis, ventilator-associated pneumonia, bacteremia or a wound infection. Of course, a therapeutically effective amount will vary depending on the particular subject and condition being treated, the weight and age of the subject, the severity of the disease state, the particular composition selected, the dosing regimen to be followed, the timing of administration, the mode of administration, etc., all of which can be readily determined by one of ordinary skill in the art.

[0033] As used herein, the terms "subject", "animal", "patient" or "mammal" refer to any subject for which diagnosis, prognosis, prevention or treatment is desired, particularly a mammalian subject, such as a human, or a domestic mammalian animal such as a dog, cat, horse, or a food animal such as a cow, sheep, pig, and preferably refers to a human.

[0034] In a preferred embodiment, the at least one empty liposome selected from empty liposomes comprising (preferably consisting of) lipids or phospholipids independently selected from cholesterol, sphingomyelin, ceramide, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, diacylglycerol, and phosphatidic acid containing one or more saturated or unsaturated fatty acids having more than 4 carbon atoms and up to 28 carbon atoms is (a) an empty liposome comprising cholesterol, wherein the amount of cholesterol is at least 30% (w / w), and preferably the empty liposome is selected from empty liposomes comprising (more preferably consisting of) cholesterol and sphingomyelin, (b) empty liposomes consisting of sphingomyelin, and (c) empty liposomes comprising (preferably consisting of) phosphatidylcholine and sphingomyelin.

[0035] In a preferred embodiment, the (i) single empty liposome is selected from (a) an empty liposome comprising cholesterol and sphingomyelin, wherein the amount of cholesterol is at least 30% (weight / weight), or (b) an empty liposome consisting of sphingomyelin. 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), and (b) a second empty liposome consisting of sphingomyelin.

[0036] In a preferred embodiment, the (i) single empty liposome is selected from (a) an empty liposome composed of sphingomyelin and cholesterol, wherein the amount of cholesterol is at least 30% (weight / weight), or (b) an empty liposome composed of sphingomyelin. The empty liposome mixture includes (a) a first empty liposome composed of sphingomyelin and cholesterol, wherein the amount of cholesterol is at least 30% (weight / weight), and (b) a second empty liposome composed of sphingomyelin.

[0037] In a preferred embodiment, the composition comprises (preferably consists of) a single empty liposome selected from (a) empty liposomes comprising cholesterol and sphingomyelin, wherein the amount of cholesterol is at least 30% (wt / wt).

[0038] In a preferred embodiment, the composition for use in the methods of the present invention comprises (preferably consists of) a single empty liposome selected from (a) an empty liposome composed of sphingomyelin and cholesterol, wherein the amount of cholesterol is at least 30% (w / w), or (b) an empty liposome composed of sphingomyelin.

[0039] In another preferred embodiment, the composition for use in the method of the present invention comprises (preferably consists of) a single empty liposome. The single empty liposome is composed of sphingomyelin and cholesterol, with the amount of cholesterol being at least 30% (wt / wt). Preferably, the amount of cholesterol in the empty liposome is 30% to 70% (wt / wt), and more preferably, the amount of cholesterol in the empty liposome is 35% to 60% (wt / wt). In another preferred embodiment, the amount of cholesterol in the empty liposome is 45% to 55% (wt / wt), and even more preferably, the amount of cholesterol in the empty liposome is about 50% (wt / wt). Thus, in a highly preferred embodiment of the present invention, for use in a method for preventing or reducing biofilm formation or eradicating or reducing existing biofilms, preferably in mammals, more preferably in humans, the single empty liposome comprises (preferably consists of) 50% (wt / wt) sphingomyelin and 50% (wt / wt) cholesterol.

[0040] In another preferred embodiment, the composition for use in the method according to the present invention comprises (preferably, consists of) a single empty liposome. The single empty liposome is an empty liposome consisting of sphingomyelin.

[0041] In one embodiment, the composition comprises (preferably, consists of) a mixture of empty liposomes. The mixture of empty liposomes comprises (a) a first empty liposome containing cholesterol, the amount of cholesterol being at least 30% (weight / weight), (b) a second empty liposome consisting of sphingomyelin, and (c) a third empty liposome containing (preferably, consisting of) phosphatidylcholine and sphingomyelin.

[0042] In another embodiment, the composition for use comprises (preferably, consists of) a mixture of empty liposomes. The mixture of empty liposomes comprises (a) a first empty liposome containing cholesterol, the amount of cholesterol being at least 30% (weight / weight), (b) a second empty liposome consisting of sphingomyelin, optionally (c) an empty liposome containing (preferably, consisting of) phosphatidylcholine and sphingomyelin, and further optionally (d) at least one empty liposome selected from at least one empty liposome containing a lipid or phospholipid selected independently of each other from cholesterol, sphingomyelin, ceramide, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, diacylglycerol, and phosphatidic acid containing one or two or more saturated or unsaturated fatty acids having more than 4 carbon atoms and up to 28 carbon atoms.

[0043] In one embodiment, the composition comprises (preferably, consists of) an empty liposome mixture. The empty liposome mixture comprises (preferably, consists of): (a) a first empty liposome consisting of sphingomyelin and cholesterol, wherein the amount of cholesterol is at least 30% (weight / weight); (b) a second empty liposome consisting of sphingomyelin; and (c) a third empty liposome comprising (preferably, consisting of) phosphatidylcholine and sphingomyelin.

[0044] In one embodiment, the composition comprises (preferably, consists of) an empty liposome mixture. The empty liposome mixture comprises (preferably, consists of): (a) a first empty liposome consisting of sphingomyelin and cholesterol, wherein the amount of cholesterol is at least 30% (weight / weight); (b) a second empty liposome consisting of sphingomyelin; and (c) a third empty liposome comprising (preferably, consisting of) a lipid or phospholipid selected from cholesterol, sphingomyelin, ceramide, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, diacylglycerol, and phosphatidic acid containing one or two or more saturated or unsaturated fatty acids having more than 4 carbon atoms and up to 28 carbon atoms.

[0045] In a preferred embodiment, the composition comprises (preferably, consists of) an empty liposome mixture. The empty liposome mixture comprises (preferably, consists of): (a) a first empty liposome comprising cholesterol and sphingomyelin, wherein the amount of cholesterol is at least 30% (weight / weight); and (b) a second empty liposome consisting of sphingomyelin.

[0046] In another preferred embodiment, the composition for use in the method according to the present invention comprises (preferably, consists of) an empty liposome mixture. The empty liposome mixture comprises (a) a first empty liposome consisting of sphingomyelin and cholesterol, the amount of cholesterol being at least 30% (weight / weight), and (b) a second empty liposome consisting of sphingomyelin. Preferably, the amount of cholesterol in the first empty liposome is 30% to 70% (weight / weight), and more preferably, the amount of cholesterol in the first empty liposome is 35% to 60% (weight / weight). In another preferred embodiment, the amount of cholesterol in the first empty liposome is 45% to 55% (weight / weight), and even more preferably, the amount of cholesterol in the first empty liposome is about 50% (weight / weight). Thus, in a highly preferred embodiment of the present invention, preferably for use in a method for preventing or reducing biofilm formation in a mammal, more preferably a human, or for eradicating or reducing an existing biofilm, the first empty liposome comprises (preferably, consists of) 50% (weight / weight) sphingomyelin and 50% (weight / weight) cholesterol.

[0047] In another preferred embodiment, the composition for use in the method of the present invention comprises a mixture of empty liposomes, the mixture comprising: (a) first empty liposomes composed of sphingomyelin and cholesterol, the amount of cholesterol being at least 30% (wt / w), preferably 30% to 70% (wt / w), and (b) second empty liposomes composed of sphingomyelin. Preferably, the amount of cholesterol in the first empty liposomes is 35% to 60% (wt / w), more preferably 45% to 55% (wt / w), and even more preferably, the amount of cholesterol in the first empty liposomes is about 50% (wt / w). Thus, in a highly preferred embodiment of the present invention, for use in a method for preventing or reducing biofilm formation, or eradicating or reducing an existing biofilm, preferably in a mammal, more preferably a human, the first empty liposome of the empty liposome mixture consists of 50% (wt / wt) sphingomyelin and 50% (wt / wt) cholesterol.

[0048] In another preferred embodiment, the composition for use in the method according to the present invention consists of an empty liposome mixture. The empty liposome mixture comprises (a) a first empty liposome consisting of sphingomyelin and cholesterol, wherein the amount of cholesterol is at least 30% (weight / weight), preferably 30% to 70% (weight / weight), and (b) a second empty liposome consisting of sphingomyelin. Preferably, the amount of cholesterol in the first empty liposome is 35% to 60% (weight / weight), more preferably, the amount of cholesterol in the first empty liposome is 45% to 55% (weight / weight), and even more preferably, the amount of cholesterol in the first empty liposome is about 50% (weight / weight). Thus, in a highly preferred embodiment of the present invention, for use in a method for preventing or reducing biofilm formation, or eradicating or reducing existing biofilms, preferably in mammals, more preferably in humans, the first empty liposome of the empty liposome mixture consists of 50% (weight / weight) sphingomyelin and 50% (weight / weight) cholesterol.

[0049] In another preferred embodiment, the empty liposome mixture comprises at least 1%, preferably at least 5%, more preferably at least 10%, even more preferably at least 20%, and even more preferably at least 30% (weight / weight) of the first empty liposome, and up to 99%, preferably up to 95%, more preferably up to 90%, even more preferably up to 80%, and even more preferably up to 70% (weight / weight) of the second empty liposome. Even more preferably, the empty liposome mixture comprises at least 40% (weight / weight) of the first empty liposome and up to 60% (weight / weight) of the second empty liposome.

[0050] In another preferred embodiment, the empty liposome mixture comprises at least 1%, preferably at least 5%, more preferably at least 10%, still more preferably at least 20%, still more preferably at least 30% (w / w) of the second empty liposomes and up to 99%, preferably up to 95%, more preferably up to 90%, still more preferably up to 80%, still more preferably up to 70% (w / w) of the first empty liposomes. Still more preferably, the empty liposome mixture comprises at least 40% (w / w) of the second empty liposomes and up to 60% (w / w) of the first empty liposomes.

[0051] In another preferred embodiment, the empty liposome mixture contains 1 to 99%, preferably 5 to 95%, more preferably 10 to 90%, again more preferably 20 to 80%, again more preferably 30 to 70% (weight / weight) of the first and second empty liposomes. Still more preferably, the empty liposome mixture contains 40 to 60% (weight / weight) of the first and second empty liposomes.

[0052] In another preferred embodiment, the empty liposome mixture comprises at least 30% (wt / wt) of the first empty liposomes and at most 70% (wt / wt) of the second empty liposomes, preferably at least 40% (wt / wt) of the first empty liposomes and at most 60% (wt / wt) of the second empty liposomes. In a further preferred embodiment, the empty liposome mixture comprises at least 45% (wt / wt) of the first empty liposomes and at most 55% (wt / wt) of the second empty liposomes, preferably about 50% (wt / wt) of the first empty liposomes and about 50% (wt / wt) of the second empty liposomes.

[0053] In another preferred embodiment, the empty liposome mixture comprises at least 30% (weight / weight) of the second empty liposomes and at most 70% (weight / weight) of the first empty liposomes, preferably the empty liposome mixture comprises at least 40% (weight / weight) of the second empty liposomes and at most 60% (weight / weight) of the first empty liposomes. In a more preferred embodiment, the empty liposome mixture comprises at least 45% (weight / weight) of the second empty liposomes and at most 55% (weight / weight) of the first empty liposomes, preferably the empty liposome mixture comprises about 50% (weight / weight) of the second empty liposomes and about 50% (weight / weight) of the first empty liposomes.

[0054] In another preferred embodiment, the empty liposome mixture comprises at least 20% (weight / weight) of the first and the second empty liposomes, preferably the empty liposome mixture comprises at least 30% (weight / weight) of the first and the second empty liposomes. In a more preferred embodiment, the empty liposome mixture comprises at least 40% (weight / weight) of the first and the second empty liposomes.

[0055] In another preferred embodiment, the empty liposome mixture comprises at least 20% (weight / weight) of each of the first and the second empty liposomes, preferably the empty liposome mixture comprises at least 30% (weight / weight) of each of the first and the second empty liposomes. In a more preferred embodiment, the empty liposome mixture comprises at least 40% (weight / weight) of each of the first and the second empty liposomes.

[0056] In another preferred embodiment, the empty liposome mixture consists of the first empty liposomes and the second empty liposomes.

[0057] In a further preferred embodiment, the empty liposome mixture consists of the first empty liposomes and the second empty liposomes, the empty liposome mixture consisting of at least 40% (wt / wt) of the first empty liposomes and at most 60% (wt / wt) of the second empty liposomes, and preferably the empty liposome mixture consisting of about 50% (wt / wt) of the first empty liposomes and about 50% (wt / wt) of the second empty liposomes.

[0058] In a further preferred embodiment, the empty liposome mixture consists of the first empty liposomes and the second empty liposomes, the empty liposome mixture consisting of at least 40% (wt / wt) of the second empty liposomes and at most 60% (wt / wt) of the first empty liposomes, and preferably the empty liposome mixture consisting of about 50% (wt / wt) of the second empty liposomes and about 50% (wt / wt) of the first empty liposomes.

[0059] In another preferred embodiment, the empty liposome mixture consists of 1 to 99%, preferably 5 to 95%, more preferably 10 to 90%, again more preferably 20 to 80%, again more preferably 30 to 70% (weight / weight) of the first and second empty liposomes. Still more preferably, the empty liposome mixture consists of 40 to 60% (weight / weight) of the first and second empty liposomes.

[0060] In a more preferred embodiment, the empty liposome mixture comprises the first empty liposomes and the second empty liposomes. The empty liposome mixture comprises at least 40% (wt / wt) of the first empty liposomes and up to 60% (wt / wt) of the second empty liposomes, preferably about 50% (wt / wt) of the first empty liposomes and about 50% (wt / wt) of the second empty liposomes. The amount of cholesterol in the first empty liposomes is 45% to 55% (wt / wt), preferably about 50% (wt / wt).

[0061] In a more preferred embodiment, the empty liposome mixture consists of the first empty liposome and the second empty liposome. The empty liposome mixture consists of at least 40% (weight / weight) of the second empty liposome and at most 60% (weight / weight) of the first empty liposome. Preferably, the empty liposome mixture consists of about 50% (weight / weight) of the second empty liposome and about 50% (weight / weight) of the first empty liposome. The amount of cholesterol in the first empty liposome is 45% - 55% (weight / weight), and preferably the amount of cholesterol in the first empty liposome is about 50% (weight / weight).

[0062] In a more preferred embodiment, the empty liposomes used in or for use in the present invention have an average diameter of 20 nm to 10 μm, preferably 20 to 500 nm, and more preferably 20 nm to 200 nm, and are preferably artificial liposomes. The empty liposomes used in or for use in the present invention are liposomes that do not encapsulate any drug. The empty liposomes used in or for use in the present invention do not contain any drug. The empty liposomes used in or for use in the present invention do not incorporate other drugs. As used herein, "incorporated" typically and preferably means encapsulated within the cavity of the liposome, or within the potential bilayer of the liposome, or as part of the membrane layer of the liposome. The liposomes used in or for use in the present invention consist of one or more phospholipid bilayers and are typically and preferably unilamellar vesicles and multilamellar vesicles. Small unilamellar vesicles (SUVs) are most preferred.

[0063] In a further aspect, the present invention provides empty liposomes consisting of sphingomyelin and cholesterol for use in a method for preventing or reducing biofilm formation, or eradicating or reducing existing biofilms, preferably in mammals, more preferably in humans, wherein the amount of cholesterol is at least 30% (weight / weight). In another preferred embodiment, said empty liposomes for use in the method according to the present invention comprise (preferably consist of) sphingomyelin and cholesterol, and the amount of cholesterol is 30% to 70% (weight / weight), more preferably, the amount of cholesterol in said empty liposomes is 35% to 60% (weight / weight). In another preferred embodiment, the amount of cholesterol in said empty liposomes is 45% to 55% (weight / weight), and even more preferably the amount of cholesterol in said empty liposomes is about 50% (weight / weight). Thus, in a highly preferred embodiment of the present invention, for use in a method for preventing or reducing biofilm formation, or eradicating or reducing existing biofilms, preferably in mammals, more preferably in humans, said empty liposomes comprise (preferably consist of) 50% (weight / weight) sphingomyelin and 50% (weight / weight) cholesterol.

[0064] In a further aspect, the present invention provides empty liposomes consisting of sphingomyelin for use in a method for preventing or reducing biofilm formation, or eradicating or reducing existing biofilms, preferably in mammals, more preferably in humans.

[0065] In yet another aspect, the present invention provides an empty liposome mixture for use in a method for preventing or reducing biofilm formation, or eradicating or reducing existing biofilms, preferably in mammals, more preferably in humans. The empty liposome mixture comprises: (a) a first empty liposome consisting of sphingomyelin and cholesterol, wherein the amount of cholesterol is at least 30% (weight / weight); and (b) a second empty liposome consisting of sphingomyelin. Preferably, the amount of cholesterol in the first empty liposome is from 30% to 70% (weight / weight), more preferably from 35% to 60% (weight / weight). In another preferred embodiment, the amount of cholesterol in the first empty liposome is from 45% to 55% (weight / weight), and even more preferably, the amount of cholesterol in the first empty liposome is about 50% (weight / weight). Thus, in a highly preferred embodiment of the present invention, for use in a method for preventing or reducing biofilm formation, or eradicating or reducing existing biofilms, preferably in mammals, more preferably in humans, the first empty liposome comprises 50% (weight / weight) sphingomyelin and 50% (weight / weight) cholesterol (preferably consists of). In another preferred embodiment, the empty liposome mixture comprises at least 30% (weight / weight) of the first empty liposome and at most 70% (weight / weight) of the second empty liposome, preferably the empty liposome mixture comprises at least 40% (weight / weight) of the first empty liposome and at most 60% (weight / weight) of the second empty liposome. In a more preferred embodiment, the empty liposome mixture comprises at least 45% (weight / weight) of the first empty liposome and at most 55% (weight / weight) of the second empty liposome, preferably the empty liposome mixture comprises about 50% (weight / weight) of the first empty liposome and about 50% (weight / weight) of the second empty liposome.

[0066] In yet another aspect, the present invention provides an empty liposome mixture for use in a method for preventing or reducing biofilm formation, or eradicating or reducing existing biofilms, preferably in mammals, more preferably in humans. The empty liposome mixture comprises: (a) a first empty liposome consisting of sphingomyelin and cholesterol, wherein the amount of cholesterol is at least 30% (weight / weight); and (b) a second empty liposome consisting of sphingomyelin. Preferably, the amount of cholesterol in the first empty liposome is from 30% to 70% (weight / weight), more preferably from 35% to 60% (weight / weight). In another preferred embodiment, the amount of cholesterol in the first empty liposome is from 45% to 55% (weight / weight), and even more preferably, the amount of cholesterol in the first empty liposome is about 50% (weight / weight). Thus, in a highly preferred embodiment of the present invention, for use in a method for preventing or reducing biofilm formation, or eradicating or reducing existing biofilms, preferably in mammals, more preferably in humans, the first empty liposome comprises 50% (weight / weight) sphingomyelin and 50% (weight / weight) cholesterol (preferably, consists of). In another preferred embodiment, the empty liposome mixture comprises at least 30% (weight / weight) of the second empty liposome and at most 70% (weight / weight) of the first empty liposome, preferably the empty liposome mixture comprises at least 40% (weight / weight) of the second empty liposome and at most 60% (weight / weight) of the first empty liposome. In a more preferred embodiment, the empty liposome mixture comprises at least 45% (weight / weight) of the second empty liposome and at most 55% (weight / weight) of the first empty liposome, preferably the empty liposome mixture comprises about 50% (weight / weight) of the second empty liposome and about 50% (weight / weight) of the first empty liposome.

[0067] In yet another aspect, the present invention provides an empty liposome mixture for use in a method for preventing or reducing biofilm formation, or eradicating or reducing existing biofilms, preferably in mammals, more preferably in humans. The empty liposome mixture comprises: (a) a first empty liposome consisting of sphingomyelin and cholesterol, wherein the amount of cholesterol is at least 30% (weight / weight); and (b) a second empty liposome consisting of sphingomyelin. Preferably, the amount of cholesterol in the first empty liposome is 30% to 70% (weight / weight), more preferably 35% to 60% (weight / weight). In another preferred embodiment, the amount of cholesterol in the first empty liposome is 45% to 55% (weight / weight), and even more preferably, the amount of cholesterol in the first empty liposome is about 50% (weight / weight). Thus, in a highly preferred embodiment of the present invention, for use in a method for preventing or reducing biofilm formation, or eradicating or reducing existing biofilms, preferably in mammals, more preferably in humans, the first empty liposome comprises 50% (weight / weight) sphingomyelin and 50% (weight / weight) cholesterol (preferably, consists of). In another preferred embodiment, the empty liposome mixture comprises 1 to 99%, preferably 5% to 95%, more preferably 10 to 90%, even more preferably 20 to 80%, even more preferably 30 to 70% (weight / weight) of the first and second empty liposomes, and even more preferably, the empty liposome mixture comprises 40% to 60% (weight / weight) of the first and second empty liposomes.

[0068] Thus, in another aspect, the present invention provides a composition for use in a method for preventing or reducing biofilm formation or eradicating or reducing existing biofilms, preferably in mammals, more preferably in humans, the composition comprising (preferably consisting of) an empty liposome mixture. The empty liposome mixture comprises (preferably consists of): (a) a first empty liposome consisting of sphingomyelin and cholesterol, wherein the amount of cholesterol is at least 30% (weight / weight); and (b) a second empty liposome consisting of sphingomyelin. Preferably, the amount of cholesterol in the first empty liposome is 30% to 70% (weight / weight), more preferably, the amount of cholesterol in the first empty liposome is 35% to 60% (weight / weight). In another preferred embodiment, the amount of cholesterol in the first empty liposome is 45% to 55% (weight / weight), and even more preferably, the amount of cholesterol in the first empty liposome is about 50% (weight / weight). Thus, in a highly preferred embodiment of the present invention, for use in a method for preventing or reducing biofilm formation or eradicating or reducing existing biofilms, preferably in mammals, more preferably in humans, the first empty liposome comprises (preferably consists of) 50% (weight / weight) sphingomyelin and 50% (weight / weight) cholesterol. In another preferred embodiment, the empty liposome mixture comprises 1 to 99%, preferably 5% to 95%, more preferably 10 to 90%, even more preferably 20 to 80%, even more preferably 30 to 70% (weight / weight) of the first and second empty liposomes, and even more preferably, the empty liposome mixture comprises 40% to 60% (weight / weight) of the first and second empty liposomes.

[0069] Therefore, in a highly preferred embodiment of the present invention, for use in a method for preventing or reducing biofilm formation, or eradicating or reducing existing biofilms, preferably in mammals, more preferably in humans, the empty liposome mixture comprises (preferably consists of): (i) a first empty liposome consisting of about 50% (weight / weight) sphingomyelin and about 50% (weight / weight) cholesterol; and (ii) a second empty liposome consisting of (100%) sphingomyelin. The empty liposome mixture comprises at least 45% (weight / weight) of the first empty liposome and at most 55% (weight / weight) of the second empty liposome, and preferably the empty liposome mixture comprises about 50% (weight / weight) of the first empty liposome and about 50% (weight / weight) of the second empty liposome.

[0070] Thus, in another aspect, the present invention provides a composition for use in a method for preventing or reducing biofilm formation, or eradicating or reducing existing biofilms, preferably in mammals, more preferably in humans, the composition comprising (preferably consisting of) an empty liposome mixture. The empty liposome mixture comprises (preferably consists of): (i) a first empty liposome consisting of about 50% (weight / weight) sphingomyelin and about 50% (weight / weight) cholesterol; and (ii) a second empty liposome consisting of (100%) sphingomyelin. The empty liposome mixture comprises at least 45% (weight / weight) of the first empty liposome and at most 55% (weight / weight) of the second empty liposome, and preferably the empty liposome mixture comprises about 50% (weight / weight) of the first empty liposome and about 50% (weight / weight) of the second empty liposome.

[0071] In a more preferred embodiment, the first empty liposome composed of about 50% (weight / weight) sphingomyelin and about 50% (weight / weight) cholesterol, and the second empty liposome composed of (100%) sphingomyelin have an average diameter of 20 to 500 nm, preferably 20 nm to 400 nm, and more preferably 40 nm to 400 nm. In a more preferred embodiment, the first empty liposome composed of about 50% (weight / weight) sphingomyelin and about 50% (weight / weight) cholesterol, and the second empty liposome composed of (100%) sphingomyelin have a pH of 6.6 to 8.0. In a more preferred embodiment, the first empty liposome composed of about 50% (weight / weight) sphingomyelin and about 50% (weight / weight) cholesterol, and the second empty liposome composed of (100%) sphingomyelin have a polydispersity index of <0.45. In a more preferred embodiment, the first empty liposome composed of about 50% (weight / weight) sphingomyelin and about 50% (weight / weight) cholesterol, and the second empty liposome composed of (100%) sphingomyelin have a zeta potential of -25 to +2 mV.

[0072] Liposomes are manufactured according to methods of extrusion or sonication or microfluidization (e.g., high-pressure homogenization) known in the art. For example, lipids are mixed with an organic solvent such as chloroform. The chloroform is evaporated and the dried lipid film is hydrated with an aqueous solution such as physiological saline (0.9% NaCl), Krebs solution, or Tyrode's solution, and further sonicated to generate liposomes. If necessary, the size of the liposomes can be controlled by extrusion through a membrane filter with a fixed pore size. Liposomes of different lipid compositions generated individually are typically and preferably mixed in the required proportions immediately prior to application. As another example, liposomes are prepared using the standard processes of liposome hydration, extrusion, and diafiltration. The lipids are dissolved while being mixed with a solvent such as ethanol, and then the lipid solution is added while being mixed with PBS buffer to spontaneously form liposomes of non-uniform size and fully hydrate them. The resulting vesicles are repeatedly extruded through a series of polycarbonate track-etched membranes until the desired particle size is achieved as measured by dynamic light scattering. After extrusion, the process fluid is diafiltered against 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.

[0073] The lipid surface (bilayer) of liposomes is spontaneously formed in an aqueous solvent and thus traps water as well as other water-soluble inorganic and organic molecules (which may be present during liposome formation) inside the liposomes. The empty liposomes used in this study are liposomes generated in a buffer containing water and simple organic or inorganic molecules (e.g., NaCl, KCl, MgCl2, glucose, HEPES, and / or CaCl2).

[0074] In a highly preferred embodiment of the present invention, a composition for use in a method for preventing or reducing biofilm formation, or eradicating or reducing existing biofilms, preferably in a mammal, more preferably in a human, and even more preferably for use in a method for preventing or reducing biofilm formation in a human, comprises (preferably consists of) an empty liposome mixture comprising: (a) first empty liposomes comprised of sphingomyelin and cholesterol, the first empty liposomes comprising about 50% (wt / wt) cholesterol; and (b) second empty liposomes comprised of sphingomyelin. Preferably, the empty liposome mixture comprises at least 40% (wt / wt) of the first empty liposomes and up to 60% (wt / wt) of the second empty liposomes. More preferably, the empty liposome mixture comprises about 50% (wt / wt) of the first empty liposomes and about 50% (wt / wt) of the second empty liposomes.

[0075] In another preferred embodiment, the use is in a method for preventing or reducing biofilm formation. Preferably, the use is in a method for preventing or reducing biofilm formation on a surface, preferably the surface is the surface of a mammalian, preferably human, cell, tissue or structure, more preferably the cell or tissue is a lung, muscle, bone or skin cell or tissue, and the structure is a tooth.

[0076] In another preferred embodiment, the use is in a method for preventing or reducing biofilm formation in a mammal, preferably a human, and even more preferably, the use is in a method for preventing or reducing biofilm formation on a surface in a mammal, preferably a human.

[0077] In another preferred embodiment, the use is for a method of eradicating or reducing an existing biofilm, preferably the use is for a method of preventing or reducing biofilm formation on a surface. Preferably, the surface is the surface of a mammalian, preferably human, cell, tissue or structure, more preferably, the cell or tissue is a cell or tissue of the lung, muscle, bone or skin, and the structure is a tooth.

[0078] In another preferred embodiment, the use is for a method of preventing or reducing biofilm formation on a surface, or for a method of eradicating or reducing an existing biofilm on a surface.

[0079] In another preferred embodiment, the use is for a method of preventing or reducing biofilm formation on a surface, preferably the surface is the surface of a mammalian cell, tissue or structure.

[0080] In another preferred embodiment, the use is for a method of preventing or reducing biofilm formation on a surface, preferably the surface is the 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 fruits or plums. In certain embodiments, the plant cells are derived from the leaves, roots, flowers, fruits or other edible structures of the plant, and the tissue or structure is selected from the leaves, roots, flowers, fruits or other edible structures of the plant. In another preferred embodiment, the surface is the surface of an edible plant.

[0081] In another preferred embodiment, the use is for a method of eradicating or reducing an existing biofilm on a surface. Preferably, the surface is the surface of a mammalian, preferably human, cell, tissue or structure, more preferably, the cell or tissue is a cell or tissue of the lung, muscle, bone or skin, and the structure is a tooth.

[0082] In another preferred embodiment, the surface is the surface of a wound dressing material.

[0083] In another preferred embodiment, the surface is a typical and preferably non-biological surface selected from medical devices, or a surface intended for contact with water or an aqueous solution.

[0084] In another preferred embodiment, the surface is a typical and preferably non-biological surface of a medical device typically intended for insertion into the body of a subject, and the medical device is selected from a pacemaker, a pacemaker lead, a catheter, a stent, an artificial blood vessel, an artificial heart valve, a cardiac pacemaker, a cerebrospinal fluid shunt, a urinary catheter, a peritoneal dialysis catheter, an artificial joint and orthopedic fixation devices, an intrauterine contraceptive device, a bile duct stent, a breast implant, a contact lens, and a denture.

[0085] In another preferred embodiment, the surface is a surface intended for contact with water or an aqueous solution. The surface intended for contact with water or an aqueous solution is the surface of a ship hull, a pipe, a filter, a strainer, or a pump, and preferably the surface is made of stainless steel or polypropylene.

[0086] In a particular preferred embodiment, the medical device intended for insertion into the body of a subject is a surgically invasive device intended for short-term use (>60 minutes, <30 days), such as a clamp, an infusion cannula, a skin suturing device, a temporary filler, a tissue stabilizer used in cardiac surgery, a cardiac catheter, a cardiac output probe, a temporary pacemaker lead, a chest catheter for cardiac drainage such as a pericardium, a carotid shunt, an ablation catheter, a nervous system catheter, a cortical electrode, or a small source therapy device, etc., but not limited thereto.

[0087] In certain preferred embodiments, the medical device intended for insertion 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), pins and plates, intraocular lenses, internal occluding devices (such as vascular occlusion devices), tissue augmentation implants, peripheral vascular catheters, peripheral vascular grafts and stents, penile implants, non-absorbable sutures, bone cements and craniofacial implants, viscoelastic surgical instruments for anterior eye surgery, bridges and crowns, dental fillings and pins, dental alloys, ceramics and polymers, artificial heart valves, aneurysm clips, artificial blood vessels and stents, central vascular catheters, spinal stents, CNS electrodes, cardiovascular sutures, permanent and retrievable inferior vena cava filters, septal occluding devices, intra-aortic balloon pumps, external left ventricular assist devices, and the like.

[0088] In the case of the food processing industry, biofilms cause chronic bacterial contamination in food processing equipment such as pasteurization pipes and tubes.

[0089] In the case of the marine industry, marine fouling is usually described as including several stages, starting with the formation of a biofilm at the initial stage of bacterial attachment, followed by the secondary colonization of spores of large algae (e.g., Enteromorpha prolifera, Ulva pertusa) and protozoa (e.g., Balanus amphitrite, Zoothamnium species) that attach to it. Finally, the tertiary colonization, i.e., the attachment of macrofoulers such as tunicates, mollusks, sessile cnidarians, etc. occurs. Thus, biofilm formation provides the substrate for biofouling of underwater surfaces such as ship hulls, boat propellers, cages, underwater dock structures, underwater structures of offshore oil platforms, undersea mines, buoys, submarine cables, pipes and filters in the cooling systems of power plants, desalination plants, and the like.

[0090] In another preferred embodiment, said use is in a method for preventing or reducing bacterial biofilm formation on a surface.

[0091] The bacteria living in the biofilm can be any bacteria, i.e., Gram-negative bacteria, Gram-positive bacteria, mycoplasmas, and spiroplasmas. Within these groups, there are bacteria associated with animal cells, plant cells, or artificial surfaces. In certain embodiments, the bacteria that generate the biofilm discussed herein and / or the bacteria living in the biofilm are Gram-negative bacteria or Gram-positive bacteria.

[0092] In another preferred embodiment, the use is for the prevention or treatment of a condition or disease, preferably the condition or disease is caused by the bacteria present in the biofilm.

[0093] In another preferred embodiment, the use is typically and preferably for the prevention of a condition or disease, more preferably the condition or disease is caused by the bacteria present in the biofilm. In another preferred embodiment, the use is for the treatment of a condition or disease, preferably the condition or disease is caused by the bacteria present in the biofilm.

[0094] In another preferred embodiment, the use is for the prevention or treatment of a condition or disease, and the condition or disease is an infectious disease. In another preferred embodiment, the use is typically and preferably for the prevention of a condition or disease, and the condition or disease is an infectious disease. In another preferred embodiment, the use is for the treatment of a condition or disease, and the condition or disease is an infectious disease.

[0095] In another preferred embodiment, the use is for the prevention or treatment of a condition or disease of a mammal, preferably a human, and preferably the condition or disease is an infectious disease. In another preferred embodiment, the use is for the treatment of a condition or disease of a mammal, preferably a human, and preferably the condition or disease is an infectious disease.

[0096] In another preferred embodiment, the condition or disease is selected from infectious diseases. Preferably, the infectious disease is a respiratory infectious disease, a sexually transmitted disease, meningitis, a urinary tract infection, a gastrointestinal disease, native valve endocarditis, colitis, vaginitis, urethritis, conjunctivitis, otitis (preferably otitis media), cystic fibrosis, ventilator-associated pneumonia, bacteremia or a wound infection.

[0097] In another preferred embodiment, the condition or disease is a respiratory infectious disease, a sexually transmitted disease, meningitis, a urinary tract infection, a gastrointestinal disease, native valve endocarditis, colitis, vaginitis, urethritis, conjunctivitis, otitis (preferably otitis media), cystic fibrosis, ventilator-associated pneumonia, bacteremia or a wound infection.

[0098] In another preferred embodiment, the condition or disease is an infectious disease.

[0099] In another preferred embodiment, the condition or disease is selected from a respiratory infectious disease, a sexually transmitted disease, meningitis, a urinary tract infection, a gastrointestinal disease, native valve endocarditis, colitis, vaginitis, urethritis, conjunctivitis, otitis, preferably otitis media, cystic fibrosis, ventilator-associated pneumonia, bacteremia or a wound infection.

[0100] In another preferred embodiment, the condition or disease is a respiratory tract infection. In another preferred embodiment, the condition or disease is a sexually transmitted infection. 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 (chronic) wound infection, typically and preferably, chronic wound infection.

[0101] In another preferred embodiment, the condition or disease is a chronic respiratory tract infection, preferably the chronic respiratory tract infection is cystic fibrosis, chronic obstructive pulmonary disease or chronic rhinosinusitis. In another preferred embodiment, the condition or disease is cystic fibrosis.

[0102] In another preferred embodiment, the condition or disease is caused by bacteria, mycoplasma, spiroplasma or fungi. 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 condition or disease is caused by spiroplasma. In another preferred embodiment, the condition or disease is caused by fungi, preferably the fungi are selected from Candida, Aspergillus, Cryptococcus, Trichosporon, Coccidioides or Pneumocystis.

[0103] Infections and conditions involving biofilms include those caused by Gram-positive bacteria (such as Streptococcus pneumoniae, Bacillus spp., Listeria monocytogenes, Staphylococcus spp., lactic acid bacteria (such as Lactobacillus plantarum and Lactococcus lactis), Streptococcus sobrinus, Streptococcus mutans, etc.), those caused by Gram-negative bacteria (such as Escherichia coli, Pseudomonas aeruginosa, Enterobacteriaceae, Salmonella spp., Actinobacillus pleuropneumoniae, Proteus mirabilis, Acinetobacter baumannii, Shigella spp., Moraxella, Helicobacter, Stenotrophomonas, Delftia, acetic acid bacteria, Legionella, cyanobacteria, Spirochaeta, green sulfur bacteria and green non-sulfur bacteria, Neisseria, Haemophilus influenzae, Klebsiella pneumoniae, Serratia marcescens, etc.), and those caused by Mycoplasma, Spiroplasma and fungi (such as Candida, Aspergillus, Cryptococcus, Trichosporon, Coccidioides, Pneumocystis, etc.). Some pathogens that form biofilms are ESKAPE pathogens. Infections and conditions involving biofilms include, for example, those caused by Gram-negative cocci (such as Neisseria gonorrhoeae) that cause sexually transmitted infections, Gram-negative cocci (such as Neisseria meningitidis) that cause meningitis, or Gram-negative cocci (such as Moraxella catarrhalis, Haemophilus influenzae) that cause respiratory symptoms. Also included are infections and conditions caused by Gram-negative bacilli that are mainly the cause of respiratory disorders (such as Klebsiella pneumoniae, Legionella pneumophila, Pseudomonas aeruginosa), Gram-negative bacilli that are mainly the cause of urinary disorders (such as Escherichia coli, Proteus mirabilis, Enterobacter cloacae, Serratia marcescens), or Gram-negative bacilli that are mainly the cause of gastrointestinal disorders (such as Helicobacter pylori, Salmonella enteritidis, Salmonella typhi). Infections involving biofilms also include nosocomial infections (such as Acinetobacter baumannii) that cause bacteremia, secondary meningitis and ventilator-associated pneumonia in hospitals and intensive care units.

[0104] In another preferred embodiment, the condition or disease, preferably the infection, is caused by at least one ESKAPE pathogen.

[0105] In another preferred embodiment, the condition or disease, preferably the infection, is caused by Enterococcus faecalis. In another preferred embodiment, the condition or disease, preferably the infection, is caused by Staphylococcus aureus. In another preferred embodiment, the condition or disease, preferably the infection, is caused by Klebsiella pneumoniae. In another preferred embodiment, the condition or disease, preferably the infection, is caused by Acinetobacter baumannii. In another preferred embodiment, the condition or disease, preferably the infection, is caused by Pseudomonas aeruginosa. In another preferred embodiment, the condition or disease, preferably the infection, is caused by Enterobacter spp.

[0106] In another preferred embodiment, the condition or disease is caused by bacteria, and the bacteria are Gram-negative bacteria or Gram-positive bacteria. 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.

[0107] In another preferred embodiment, the condition or disease is caused by bacteria selected from the group consisting of Streptococcus pneumoniae, Bacillus spp., Listeria monocytogenes, Staphylococcus spp., Lactobacillus (preferably Lactobacillus plantarum and Lactococcus lactis), Streptococcus sobrinus, Streptococcus mutans, Escherichia coli, Pseudomonas aeruginosa, Enterobacteriaceae, Salmonella spp. (preferably Salmonella enterica, Salmonella enteritidis or Salmonella typhi), Actinobacillus pleuropneumoniae, Proteus mirabilis, Shigella spp., Moraxella (preferably Moraxella catarrhalis), Helicobacter pneumoniae, Proteus mirabilis, Shigella spp., Moraxella spp. (preferably Moraxella catarrhalis), Helicobacter pneumoniae, Proteus mirabilis, Proteus spp. ... In another preferred embodiment, the condition or disease, preferably the infection, is caused by a Gram-negative or Gram-positive bacterium selected from Helicobacter (preferably Helicobacter pylori), Stenotrophomonas, Bleedinger, Acetobacter, Legionella (preferably Legionella pneumophila), Cyanobacteria, Spirochetes, Green sulfur and green non-sulfur bacteria, Neisseria (preferably Neisseria gonorrhoeae or Neisseria meningitidis), Haemophilus influenzae, Enterococcus faecalis, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Serratia marcescens, Enterobacter cloacae, and Enterobacter species. In another preferred embodiment, the condition or disease, preferably the infection, is caused by Haemophilus influenzae or Serratia marcescens. In another preferred embodiment, the condition or disease, preferably the infection, is caused by Haemophilus influenzae. In another preferred embodiment, the condition or disease, preferably the infection, is caused by Serratia marcescens.

[0108] In another preferred embodiment, said condition or disease, preferably said infection, is caused by at least one ESKAPE pathogen selected from Enterococcus faecalis, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa and Enterobacter spp., or is caused by Haemophilus influenzae or Serratia marcescens.

[0109] In another preferred embodiment, the condition or disease is caused by a Gram-negative or Gram-positive bacterium selected from Streptococcus pneumoniae, Bacillus spp., Listeria monocytogenes, Staphylococcus spp., lactic acid bacteria (preferably Lactobacillus plantarum and Lactococcus lactis), Streptococcus sobrinus, Streptococcus mutans, Escherichia coli, Pseudomonas aeruginosa, Enterobacteriaceae, Salmonella spp., Actinobacillus pleuropneumoniae, and Proteus mirabilis.

[0110] In another preferred embodiment, the condition or disease is caused by a Gram-negative bacterium selected from Pseudomonas aeruginosa, Escherichia coli, Enterobacteriaceae, Salmonella spp., Actinobacillus pleuropneumoniae, and Proteus mirabilis.

[0111] In another highly preferred embodiment, the condition or disease is caused by Pseudomonas aeruginosa. In another highly preferred embodiment, the condition or disease is caused by a Gram-negative bacterium, and the Gram-negative bacterium is Pseudomonas aeruginosa. In another highly preferred embodiment, the use is in a method for preventing or reducing bacterial biofilm formation, typically and preferably on a surface, wherein the bacterium is Pseudomonas aeruginosa.

[0112] Pseudomonas aeruginosa biofilms function as a reservoir for disease recurrence and prevent complete cure. The opportunistic pathogen biofilms are involved in both chronic infections such as lung infections in cystic fibrosis patients (80% of CF patients are chronically infected with Pseudomonas aeruginosa) and life-threatening nosocomial infections such as ventilator-associated pneumonia, postoperative wound infections, and skin and soft tissue infections in burn patients. They are also involved in the contamination of medical devices, equipment, and tools (Rabin et al., Future Med Chem. 7(4) 2015; Tran et al., PLOS Pathogens 10(11) 2014).

[0113] In another preferred embodiment, the condition or disease is caused by Gram-positive bacteria selected from Streptococcus pneumoniae, Bacillus spp., Listeria monocytogenes, Staphylococcus spp., lactic acid bacteria (preferably Lactobacillus plantarum and Lactococcus lactis), Streptococcus sobrinus, and Streptococcus mutans.

[0114] In another highly preferred embodiment, the condition or disease is caused by Staphylococcus aureus. In another highly preferred embodiment, the use is a use in a method for preventing or reducing bacterial biofilm formation, typically and preferably on a surface, wherein the bacteria are Staphylococcus aureus.

[0115] Staphylococcus aureus biofilms are a major concern in multiple infectious diseases and challenge conventional anti-infective approaches, particularly antibiotic therapy. Many clinical isolates of Staphylococcus aureus are either methicillin-resistant (MRSA) or multi-drug resistant. Resistance to antibiotic therapeutic agents is further amplified by the increased resistance of biofilm-forming Staphylococcus aureus bacteria to the few antibiotics to which MRSA still remains susceptible. Vancomycin is the most commonly administered drug for Staphylococcus aureus biofilm-related infectious diseases, but the increased resistance of biofilms to vancomycin has shown the need for the use of combination treatment approaches.

[0116] Staphylococcus aureus biofilms are generally involved in implant - related infections (artificial orthopedic implants, heart valves, pacemakers, and vascular catheters), chronic wounds, osteomyelitis, infections in cystic fibrosis of the lung, and endocarditis. For example, infections in cystic fibrosis of the lung are associated with the presence of biofilms and chronic, long - term bacterial survival with increased mortality. In the case of Staphylococcus aureus, these infections are often caused by MRSA. Staphylococcus aureus is also a major cause of infections of the endocardium or artificial surfaces of the heart, causing infective endocarditis. Staphylococcus aureus endocarditis has been increasing with the increasing use of surgical procedures, including the implantation of artificial cardiovascular devices such as artificial heart valves, grafts, hemodialysis catheters, and pacemakers (Bhattacharya et al., Expert Rev Anti Infect Ther. 13(12)2015).

[0117] In a further preferred embodiment, the method according to the invention for preventing or reducing biofilm formation or eradicating or reducing existing biofilms further comprises administering a therapeutically effective amount of the composition of the invention, a single empty liposome of the invention, or a mixture of empty liposomes of the invention to a mammal, preferably a human, in need thereof.

[0118] To prevent or reduce biofilm formation or to eradicate or reduce existing biofilms, the single empty liposomes of the present invention or the mixture of empty liposomes of the present invention can be applied in the form of intravenous, intramuscular, intraperitoneal or subcutaneous injection. The injection solution is prepared by standard methods known in the art, for example, as a suspension of liposomes in sterile physiological saline. It is also conceivable to apply the single empty liposomes of the present invention or the mixture of empty liposomes of the present invention as an aerosol for the treatment of airway infections, or as a formulation useful for sublingual or intraoral application, intraocular or intravitreal application, and topical application (for example, eye drops, toothpaste, topical liquid suspensions for the skin or mouth, etc.). Preparation of such formulations from liposomes such as the empty liposomes of the present invention is known in the art. Preventive measures based on the single empty liposomes of the present invention or the mixture of empty liposomes of the present invention will be useful for preventing biofilm-related conditions or diseases, for example, in patients who are scheduled to undergo, are undergoing, or have undergone implant surgery, or patients suffering from chronic infections, or for preventing biofilm progression in other environments that promote the progression of biofilm-related conditions or diseases.

[0119] In a further preferred embodiment, the method according to the present invention for preventing or reducing biofilm formation or for eradicating or reducing existing biofilms further comprises administering a therapeutically effective amount of the composition of the present invention, the single empty liposomes of the present invention or the mixture of empty liposomes of the present invention to a mammal, preferably a human, in need thereof. The composition of the present invention, the single empty liposomes of the present invention or the mixture of empty liposomes of the present invention is administered typically alone, without being combined with any other drug. Further, typically and preferably, the composition of the present invention, the single empty liposomes of the present invention or the mixture of empty liposomes of the present invention is not combined simultaneously or at any time up to approximately 4 weeks (preferably up to 2 weeks) beforehand.

[0120] In a more preferred embodiment of the present invention, said use is combined with an antibacterial agent. Preferably, said antibacterial agent is an antibiotic, an antifungal agent, an antitoxin agent, an anti-pathogenic agent, a preservative or a combination thereof, and more preferably, said antibacterial agent is an antibiotic.

[0121] The present invention also relates to a method for preventing or reducing biofilm formation, or eradicating or reducing existing biofilms, preferably for treating a condition or disease, preferably a bacterial infection, and more preferably to a method for preventing or reducing biofilm formation. The method comprises administering to a mammal, preferably a human, in need thereof, a therapeutically effective amount of a single empty liposome of the present invention or a mixture of empty liposomes of the present invention, before, after, together with, or simultaneously with an antibacterial agent, preferably together with a standard antibiotic treatment for a bacterial infection.

[0122] This preferred aspect and embodiment of the present invention aims to improve treatment efficacy, promote planktonic-like growth, eliminate further community-level resistance caused by biofilms, facilitate cell-level targeting of pathogens by conventional antibiotics, and also enable reduction of antibiotic dosages. Also, as the single empty liposomes and mixtures of empty liposomes according to the present invention have already been described as toxin neutralizing agents, their action against toxins simultaneously not only disarms the bacteria but also enables the immune defense system to generate an appropriate response against the pathogen.

[0123] In a further preferred embodiment of the present invention, the method further comprises administering to a mammal in need thereof, preferably a human, a therapeutically effective amount of the composition of the present invention, a single empty liposome of the present invention or a mixture of empty liposomes of the present invention, before, after, in combination with or simultaneously with an antibacterial agent. Preferably, the antibacterial agent is an antibiotic, an antifungal agent, an antitoxin agent, an anti-pathogenic agent, an antiseptic or a combination thereof, and more preferably, the antibacterial agent is an antibiotic. In a further preferred embodiment, the method further comprises administering to a mammal in need thereof, preferably a human, a therapeutically effective amount of the composition of the present invention, a single empty liposome of the present invention or a mixture of empty liposomes of the present invention, before, after, in combination with or simultaneously with a standard antibiotic treatment and a standard treatment.

[0124] In a further preferred embodiment of the present invention, the method further comprises administering to an animal in need thereof, preferably an edible animal such as cattle, sheep, pigs, etc., an effective amount of the composition of the present invention, a single empty liposome of the present invention or a mixture of empty liposomes of the present invention, without combination with any other drug, thus typically alone, before, after, in combination with or simultaneously with an antibacterial agent. Preferably, the antibacterial agent is an antibiotic, an antifungal agent, an antitoxin agent, an anti-pathogenic agent, an antiseptic or a combination thereof, and more preferably, the antibacterial agent is an antibiotic. In a further preferred embodiment, the method further comprises administering to an animal in need thereof, preferably an edible animal such as cattle, sheep, etc., an effective amount of the composition of the present invention, a single empty liposome of the present invention or a mixture of empty liposomes of the present invention, before, after, in combination with or simultaneously with a standard antibiotic treatment or administration.

[0125] In a further preferred embodiment of the present invention, the method further comprises administering, without combination with any other drug, thus typically alone, or before, after, in combination with, or simultaneously with an antibacterial agent, a therapeutically effective amount of the composition of the present invention, a single empty liposome of the present invention or a mixture of empty liposomes of the present invention, to an animal in need thereof, preferably a food animal such as cattle, sheep, pigs, etc. Preferably, the antibacterial agent is an antibiotic, an antifungal agent, an antitoxin agent, an anti-pathogenic agent, an antiseptic or a combination thereof, and more preferably, the antibacterial agent is an antibiotic. In a more preferred embodiment, the method further comprises administering, before, after, in combination with, or simultaneously with a standard antibacterial therapeutic agent, a therapeutically effective amount of the composition of the present invention, a single empty liposome of the present invention or a mixture of empty liposomes of the present invention, to an animal in need thereof, preferably a food animal such as cattle or sheep.

[0126] In a further preferred embodiment of the present invention, the method further comprises contacting or coating a surface, preferably a plant surface, with a therapeutically effective amount of the composition of the present invention, a single empty liposome of the present invention or a mixture of empty liposomes of the present invention, without combination with any other drug, thus typically alone, or before, after, in combination with, or simultaneously with an antibacterial agent. Preferably, the antibacterial agent is an antibiotic, an antifungal agent, an antitoxin agent, an anti-pathogenic agent, an antiseptic or a combination thereof, and more preferably, the antibacterial agent is an antibiotic.

[0127] It is understood that the empty liposomes and the mixture of empty liposomes as defined above may be used, optionally, together with or in combination with further compounds, prodrugs or drugs. For example, further compounds, prodrugs or drugs can be added to prepare standard pharmaceutical compositions. It is also conceivable to add drugs or drug-like compounds, or to add further known or novel liposomes incorporating drugs or drug-like compounds inside the liposomes.

[0128] Drugs and prodrugs to be considered are, in particular, standard antibacterial or antifungal therapeutics known to those skilled in the art, or antitoxin or anti-pathogenic agents. Further, drugs such as antibiotics are particularly contemplated. Such antibiotics include, for example, carbapenems (imipenem / cilastatin, meropenem, ertapenem, doripenem, etc.), first-generation cephalosporins (cephalodoxyl and cephalexin, etc.), second-generation cephalosporins (ceftriaxone, cefaclor, and cefprozil, etc.), third-generation cephalosporins (ceftazidime, ceftriaxone, cefixime, cefdinir, cefditoren, cefotaxime, cefpodoxime, and cefibuten, etc.), fourth-generation cephalosporins (cefepime, etc.), fifth-generation cephalosporins (ceftaroline fosamil and ceftolozane / tazobactam, etc.), glycopeptides (vancomycin, teicoplanin, and telavancin, etc.), macrolides (clarithromycin, azithromycin, dirithromycin, erythromycin, Roxithromycin, troleandomycin, telithromycin, spectinomycin, and spiramycin, etc.), penicillins (amoxicillin, flucloxacillin, oxacillin, carbenicillin, and piperacillin, etc.), combinations of penicillins (amoxicillin / clavulanic acid, piperacillin / tazobactam, ampicillin / sulbactam, and ticarcillin / clavulanic acid, etc.), quinolones (ciprofloxacin (e.g., liposomal ciprofloxacin from Aradigm) and moxifloxacin, etc.), drugs against mycobacteria (rifampicin (rifampin in the United States), clofazimine, dapsone, capreomycin, cycloserine, ethambutol, ethionamide, isoniazid, pyrazinamide, rifabutin, rifapentine, and streptomycin, etc.), other antibiotics (metronidazole, arsphenamine, chloramphenicol, fosfomycin, fusidic acid, linezolid, mupirocin, platensimycin, quinupristin / dalfopristin, rifaximin, thiamphenicol, tigecycline, and tinidazole, etc.), aminoglycosides (amikacin, gentamicin, kanamycin, neomycin, netilmicin,Tobramycin (e.g., Axentis' fluidosomes™ tobramycin) and paromomycin, etc.), sulfonamides (mafenide, sulfonamide chrysoidine, sulfacetamide, sulfadiazine, silver sulfadiazine, sulfamethizole, sulfamethoxazole, sulfanilamide, sulfasalazine, sulfisoxazole, trimethoprim, and trimethoprim-sulfamethoxazole (cotrimoxazole, TMP-SMX), etc.), tetracyclines (demeclocycline, doxycycline, minocycline, oxytetracycline, and tetracycline, etc.), lincosamides (clindamycin and lincomycin, etc.), lipopeptides (daptomycin, etc.).

[0129] Other drugs that may be considered include, for example, anti-inflammatory drugs including corticosteroids (glucocorticoids) (hydrocortisone (cortisol), cortisone, prednisone, prednisolone, methylprednisolone, dexamethasone, betamethasone, triamcinolone, beclomethasone, fludrocortisone acetate, deoxycorticosterone acetate (DOCA), aldosterone, budesonide, desonide, and fluocinonide, etc.), non-steroidal anti-inflammatory drugs, such as salicylic acid (aspirin (acetylsalicylic acid), diflunisal, and salsalate, etc.), propionic acid derivatives (ibuprofen, dexibuprofen, naproxen, fenoprofen, ketoprofen, dexketoprofen, flurbiprofen, oxaprozin, and loxoprofen, etc.), acetic acid derivatives (indomethacin, tolmetin, sulindac, etodolac, ketorolac, diclofenac, and nabumetone, etc.), enolic acid (oxicam) derivatives (piroxicam, meloxicam, tenoxicam, droxicam, lornoxicam, and isoxicam, etc.), fenamic acid derivatives (fenamic acid) (mefenamic acid, meclofenamic acid, flufenamic acid, and tolfenamic acid, etc.), COX-2 selective inhibitors (coxibs) (celecoxib, etc.), and other drugs (licoferon, etc.).

[0130] Possible additional drugs are, for example, vasopressors and vasoconstrictors such as vasopressin, oxymetazoline, phenylephrine, propylhexedrine, pseudoephedrine, epinephrine, norepinephrine, dopamine, and antihistamines.

[0131] Also, other types of drugs, such as paracetamol (analgesic), nystatin, amphotericin B, polyenes, azoles and triazoles, nucleoside analogs, echinocandins, pneumocandin (for fungal infections), bupivacaine (for postoperative pain management), morphine (analgesic), verteporfin (for ophthalmic diseases), estradiol (for menopausal disorders), aganocide (registered trademark) compounds are also considered.

[0132] In this combined treatment, the empty liposomes and liposome mixtures according to the present invention may be regarded as adjuvants, and the corresponding treatment methods may be regarded as adjuvant treatments.

[0133] In a more preferred embodiment of the present invention, the method is an ex vivo method, preferably the method includes contacting a surface, preferably the surface of a medical device, with any one of the compositions of the present invention.

[0134] In a further aspect, the present invention provides a method for preventing or reducing biofilm formation, preferably bacterial biofilm formation, on a surface, or for eradicating or reducing an existing biofilm on a surface, the method including contacting the surface or the existing biofilm with any one of the compositions of the present invention.

[0135] In a further aspect, the present invention provides a method for preventing or reducing biofilm formation, preferably bacterial biofilm formation, on a surface, or for eradicating or reducing an existing biofilm on a surface. The method includes covering the surface or the existing biofilm with any one of the compositions of the present invention.

[0136] In a further aspect, the present invention provides a method for preventing or reducing biofilm formation, preferably bacterial biofilm formation, on a surface, or for eradicating or reducing an existing biofilm on a surface, the method comprising treating the surface or the existing biofilm with any one of the compositions of the present invention.

[0137] In a further aspect, the present invention provides an ex vivo method for preventing or reducing biofilm formation, preferably bacterial biofilm formation, on a surface, or for eradicating or reducing an existing biofilm on a surface, the method comprising contacting the surface or the existing biofilm with any one of the compositions of the present invention. Thus, the method of the invention is not a method for treating the human or animal body by therapy.

[0138] In a further aspect, the present invention provides an ex vivo method for preventing or reducing biofilm formation, preferably bacterial biofilm formation, on a surface, or for eradicating or reducing an existing biofilm on a surface, the method comprising covering the surface or the existing biofilm with any one of the compositions according to the present invention.

[0139] In a further aspect, the present invention provides an ex vivo method for preventing or reducing biofilm formation, preferably bacterial biofilm formation, on a surface, or for eradicating or reducing an existing biofilm on a surface, the method comprising treating the surface or the existing biofilm with any one of the compositions of the present invention. Thus, the method of the present invention is not a method for treating the human or animal body by therapy.

[0140] Methods for coating surfaces with biologically or pharmaceutically active compounds or compositions are well known in the art. For example, the above-mentioned abiotic surfaces, i.e., the surfaces of medical devices, can be coated by blending the composition of the present invention with a film-forming component, resulting in an anti-biofilm coating that can be used to inhibit biofilm formation on the surface of medical devices. The film-forming component may include one or more resins, including, but not limited to, one or more hydrolyzable, soluble or insoluble resins, etc. For example, the resin can be one or more of glyptal resin, acrylic resin, chlorinated rubber resin, epoxy resin, silicone resin, polyester resin, polyurethane resin, fluoropolymer resin, and other resins known to those skilled in the art.

[0141] Examples

[0142] Liposomes:

[0143] Sphingomyelin (SM; CAS No. 85187-10-6) derived from egg yolk was purchased from Sigma (S0756), Avanti Polar Lipids (860061) or Lipoid GmbH.

[0144] Cholesterol (CHOL; CAS No. 57-88-5) derived from lanolin was purchased from Sigma (C-8667), Avanti Polar Lipids (70000) or Dishman Netherlands B.V.

[0145] According to the present invention, sphingomyelin (SM) and cholesterol (CHOL) contained in or constituting the empty liposomes of the present invention or the mixture of empty liposomes of the present invention may be obtained from the above natural sources or, alternatively, by chemical synthesis.

[0146] Liposome preparation

[0147] Unilamellar sphingomyelin: Liposomes composed of cholesterol (35:65 molar ratio) and sphingomyelin only (100%) were prepared using sonication or microfluidization (e.g., high-pressure homogenization), or according to the protocol of a hydration, extrusion, and diafiltration process.

[0148] Sonication:

[0149] The lipids were individually dissolved in chloroform at a concentration of 1 mg / ml and stored at -20°C. For liposome preparation, the chloroform solutions of the individual lipids were mixed in the required ratios to produce 50 - 500 μl of the final solution as specified. The chloroform was completely evaporated at 60°C for 20 - 50 minutes. 50 μl or 100 μl of Tyrode's buffer (140 mM NaCl, 5 mM KCl, 1 mM MgCl2, 10 mM glucose, 10 mM HEPES; pH = 7.4) containing 2.5 mM CaCl2 was added to the tube containing the dried lipid film and vortexed thoroughly. The lipid suspension was incubated at 45°C for 20 - 30 minutes while shaking vigorously with an Eppendorf Thermomixer. To generate liposomes, the final lipid suspension was sonicated at 6°C for 3 × 5 seconds with a Bandelin Sonopuls sonicator at 70% output. The liposome samples were left at 6°C for at least 1 hour before use in experiments.

[0150] Hydration, Extrusion and Diafiltration Process Protocol:

[0151] In another method, each liposome formulation was prepared by ethanol hydration and extrusion. The lipids were individually dissolved in ethanol and t-butanol while mixing at elevated temperature (about 55°C). Next, while mixing the lipid solutions at elevated temperature (about 65°C) for about 30 minutes, PBS buffer (sodium chloride, sodium dihydrogen phosphate dihydrate, and disodium hydrogen phosphate dihydrate were dissolved in distilled water for injection while mixing and adjusted to pH 7.0 - 7.4 with either hydrochloric acid (HCl) or sodium hydroxide (NaOH) as necessary, and filtered through a 0.2 μm filter) was added. Next, the resulting process fluid was repeatedly extruded through a series of polycarbonate track-etching membranes at elevated pressure and temperature (about 65°C) until the desired particle size was achieved as measured by dynamic light scattering (example of an extruder: LIPEX® extruder). Next, using a 100,000 molecular weight cut-off hollow fiber cartridge, the resulting process fluid was concentrated approximately 2-fold and diafiltered against about 10 volume exchanges of PBS buffer to remove ethanol and t-butanol. At the end of diafiltration, the process fluid was concentrated to about 30% to allow dilution to the subsequent target lipid concentration. Before dilution, the process fluid was filtered through a 0.2 μm sterile grade filter to remove larger liposomes that could potentially clog the filter during sterile filtration. Next, the process fluid was diluted with PBS buffer to a target of 40 mg / mL total lipid. The final formulation was sterile filtered through two consecutive 0.2 μm sterile grade filters and aseptically filled into glass tube vials.

[0152] The concentration of each individual lipid within the liposome is always given as a weight / weight ratio. In liposomes containing sphingomyelin and cholesterol, a 1:1 (weight / weight) ratio corresponds to 50% (weight / weight) or a 35:65 molar ratio. The specifications are shown in Table 1.

[0153]

Table 1

[0154] Example 1

[0155] Inhibition of Biofilm of Multidrug-Resistant Pseudomonas aeruginosa Strain 6077

[0156] The preferred empty liposome mixture of the present invention was demonstrated to reduce biofilm formation by Pseudomonas aeruginosa strain 6077, as revealed by the crystal violet method. The preferred empty liposome mixture of the present invention consists of a 1:1 (weight / weight - w / w) mixture of the first empty liposome and the second liposome. The first empty liposome is composed of sphingomyelin (SM) and cholesterol (CHOL) at a weight ratio of 1:1 (1:1 w / w; 35:65 molar ratio), and the second empty liposome is composed of SM only.

[0157] method

[0158] Briefly, Pseudomonas aeruginosa strain 6077 was grown in trypticase soy broth (TSB) and in LB broth containing 2% glucose at 1×10 7 or 1×10 6It was diluted to the concentration of colony forming units (CFU). 100 microliters (100 μl) of the diluted inoculum was added to the wells of a 96-well flat-bottom microtiter plate. A suitable empty liposome mixture of the present invention was added in a volume of 100 μL while increasing the concentration to evaluate the inhibition of biofilm formation. Each condition was performed three times. The concentration ranged from 75 μg / mL to 2883 μg / mL. Ciprofloxacin, gentamicin and tobramycin were used as controls. The plates were incubated at 37 °C overnight. Next, the bacteria were removed from the wells and the biofilm monolayer was washed. The biofilm was fixed by incubating the plate at 60 °C for 1 hour. After this incubation, the biofilm was stained with a 0.4% crystal violet solution for 5 minutes and eluted with 70% ethanol. The optical density of the eluted substance was measured spectrophotometrically at 600 nm. The positive control was biofilm formation in the absence of liposomes or control drugs. The negative control was the optical density of the wells to which no bacterial species was added (i.e., background noise).

[0159] result

[0160] When the bacterial density was 1×10 7 cells / well (high bacterial density setting), a suitable empty liposome mixture of the present invention inhibited biofilm formation at a concentration of 75 μg / mL alone (i.e., in the absence of antibiotics), and the maximum inhibition was 72% (Figure 1). To avoid potential "abnormal growth" that might contribute to inconsistent OD, 1×10 6 cells / well, a lower bacterial density, was also tested. At this lower bacterial density, the overall level of inhibition of biofilm formation by a suitable empty liposome mixture of the present invention was low (46% biofilm formation inhibition), but still present at intermediate concentrations (300 - 800 μg / mL) in the dose-response curve. Also, when a suitable empty liposome mixture of the present invention was applied 8 hours after bacterial inoculation using a lower bacterial density, some biofilm disruption activity was observed.

Claims

1. (a)An empty liposome containing cholesterol, wherein the amount of cholesterol is at least 30% (weight / weight), and the empty liposome contains cholesterol and sphingomyelin, the empty liposome, and (b)An empty liposome consisting of sphingomyelin, and An empty liposome mixture containing Containing A composition for use in a method for preventing or reducing biofilm formation or eradicating or reducing an existing biofilm.

2. The composition contains an empty liposome mixture, and the empty liposome mixture includes: (a) a first empty liposome consisting of sphingomyelin and cholesterol, wherein the amount of cholesterol is at least 30% (weight / weight); and (b) a second empty liposome consisting of sphingomyelin. A composition for use according to claim 1.

3. The amount of cholesterol in the empty liposome (a) is 30% to 70% (weight / weight). A composition for use according to claim 1 or 2.

4. The amount of cholesterol in the empty liposome (a) is 45% to 55% (weight / weight). A composition for use according to any one of claims 1 to 3.

5. The empty liposome mixture contains at least 20% (weight / weight) of the first and the second empty liposomes. A composition for use according to any one of claims 2 to 4.

6. The empty liposome mixture contains at least 40% (weight / weight) of the first and the second empty liposomes. A composition for use according to any one of claims 2 to 5.

7. The use is for use in a method for preventing or reducing biofilm formation on a surface or in a method for eradicating or reducing an existing biofilm on a surface. A composition for use according to any one of claims 1 to 6.

8. The use is for the prevention or treatment of a condition or disease caused by bacteria present in the biofilm. A composition for use according to any one of claims 1 to 7.

9. The condition or disease is selected from airway infections, sexually transmitted infections, meningitis, urinary tract infections, gastrointestinal diseases, native valve endocarditis, colitis, vaginitis, urethritis, conjunctivitis, otitis (including otitis media), cystic fibrosis, ventilator-associated pneumonia, bacteremia, or wound infections. The composition for use according to claim 8.

10. wherein said condition or disease is caused by at least one ESKAPE pathogen, The composition for use according to claim 8 or claim 9.

11. wherein said condition or disease is caused by a Gram-negative or Gram-positive bacterium selected from Streptococcus pneumoniae, Bacillus spp., Listeria monocytogenes, Staphylococcus spp. (including Staphylococcus aureus), Enterobacteriaceae (including Lactobacillus plantarum and / or Lactococcus lactis), Streptococcus sobrinus, Streptococcus mutans, Escherichia coli, Pseudomonas aeruginosa, Salmonella spp. (including Salmonella enteritidis and / or Salmonella typhi), Actinobacillus pleuropneumoniae, Proteus mirabilis, Shigella spp., Moraxella (including Moraxella catarrhalis), Helicobacter (including Helicobacter pylori), Stenotrophomonas, Delftia, Acetobacter, Legionella (including Legionella pneumophila), Cyanobacteria, Spirochaeta, Green sulfur bacteria and Green non-sulfur bacteria, Neisseria (including Neisseria gonorrhoeae and / or Neisseria meningitidis), Haemophilus influenzae, Enterococcus faecalis, Klebsiella pneumoniae, Acinetobacter baumannii, Serratia marcescens, and Enterobacter spp. (including Enterobacter cloacae). The composition for use according to claim 8 or 9.

12. wherein said use is combined with an antimicrobial agent, said antimicrobial agent being an antibiotic, an antifungal agent, an antitoxin agent, an anti-pathogenic agent, a preservative or a combination thereof, The composition for use according to any one of claims 1 to 11.

13. wherein said method is an ex vivo method, said method comprising contacting a surface with the composition according to any one of claims 1 to 12. The composition for use according to any one of claims 1 to 12.

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