Liposomes for inhibiting biofilm formation

Abstract

Compositions for inhibiting biofilm formation are provided. [Solution] The composition comprises: (i) a single empty liposome, the single empty liposome being (a) an empty liposome containing cholesterol, wherein the amount of cholesterol is at least 30% (weight / weight), preferably the empty liposome being selected from empty liposomes containing cholesterol and sphingomyelin, or (b) empty liposomes consisting of sphingomyelin; or (ii) an empty liposome mixture, the empty liposome mixture being (a) empty liposomes containing cholesterol, wherein the amount of cholesterol is at least 30% (weight / weight), and comprising at least one empty liposome selected from (b) empty liposomes consisting of sphingomyelin, and (c) empty liposomes containing phosphatidylcholine and sphingomyelin.

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 predominate in almost all ecosystems under adequate nutrient conditions. Biofilms can be established in a variety of host environments, attaching 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 fixation devices, intrauterine contraceptive devices, biliary stents, breast implants, contact lenses, dentures, and, in the dental field, caries and periodontitis), as well as to biotic surfaces, such as lungs, skin, bones, and teeth. This can lead to chronic airway infections, chronic obstructive pulmonary disease, native valve endocarditis, chronic otitis media, chronic sinusitis, or chronic wound infections in cystic fibrosis patients. Furthermore, biofilms promote the development of dental diseases by forming dental plaque, as both bacteria and fungi adhere to teeth and become embedded in salivary polymers and microbial extracellular products. Biofilms are also a problem in the food industry, as they can form on surfaces of food equipment within food factories and during industrial processes.

[0005] This exposure would not be important if biofilm microorganisms were simply planktonic cells attached to a surface, but biofilm microorganisms differ significantly from planktonic microorganisms. The biofilm polymer matrix forms a shield against the host's immune defenses, protecting resident bacteria and fungi from antibacterial and antifungal agents, respectively. Biofilms also function as a microenvironment for infection, promoting chronic infection and recurrence. Biofilm-related infections are chronic, persistent, and difficult to cure. Even in the case of biofilms formed on implants such as heart valves, detached biofilm cells can migrate with the bloodstream and cause infections in other organs.

[0006] Biofilms are involved in a variety of 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 infections caused by ESKAPE pathogens. The number of biofilm-associated diseases is thought to be numerous, with colitis, vaginitis, urethritis, conjunctivitis, and otitis being just a few common examples. Biofilms are also important as colonizers of medical devices, including urinary catheters, intravenous catheters, arterial catheters, and shunts, as shown. Because the bacteria and fungi present in biofilms resist antimicrobial agents and immune defense mechanisms, biofilm-associated infections tend to become chronic, such as bronchopulmonary Pseudomonas aeruginosa (P. aeruginosa) infections in cystic fibrosis (CF) patients.

[0007] In fact, biofilm bacteria are up to 1,000 times more resistant to antibiotics than planktonic bacteria. Traditionally, antibiotics have been developed to target cellular mechanisms involved in the growth and survival of planktonic bacteria, i.e., bacteria with a planktonic ecology. However, biofilm bacteria differ significantly from their planktonic counterparts. In particular, the extracellular proteins, virulence factors, and surfactants expressed by biofilm bacteria differ from those expressed by planktonic bacteria. Furthermore, the protective biofilm matrix allows bacteria to evade host immune responses, promote survival, and disseminate. Furthermore, antimicrobial resistance is an inherent property of biofilms, allowing bacteria to survive despite intact host immune defenses and frequent antibiotic treatments. Furthermore, antibiotic treatments targeting infections using only planktonic bacteria are largely ineffective against the biofilm phenotype. Effective antibiotic doses for biofilm eradication are difficult to achieve with conventional antibiotic treatment due to the toxicity and side effects of high antibiotic doses and limited renal and liver function. Furthermore, exposing bacteria within biofilms to antibiotics further increases the selection pressure associated with the development of antibiotic resistance.Similarly, fungal biofilm-associated infections are often resistant to conventional treatment due to resistance to antimicrobial agents, in part due to surface-induced upregulation of drug efflux pumps.

[0008] Current methods may consist of physical removal of the source of infection (surgical removal of catheters or orthopedic hardware, non-adherent sutures, necrotic tissue) or treatment with antimicrobial agents. However, while antibiotics and antifungals directly target biofilm-forming organisms, they have been less successful in treating biofilm-associated conditions. Current methods are not always successful in eradicating the infection, and infections involving bacteria or fungi present in biofilms often become untreatable and ultimately develop into chronic conditions. Despite the fact that the ability to form biofilms is a universal attribute of bacteria and that many medically important fungi produce biofilms, the detailed mechanisms underlying biofilm formation remain poorly understood, making the treatment and eradication of biofilm-associated infections particularly challenging. Non-bactericidal anti-biofilm drugs under development are primarily aimed at (i) preventing microbial adhesion to surfaces, (ii) influencing biofilm maturation and / or inducing its dispersal and degradation (e.g., by targeting bacterial systems and messengers that play a key role in regulating biofilm formation, in particular bacterial quorum sensing (QS), nucleotides (especially c-di-GMP), or small non-coding RNAs (sRNAs)), or (iii) preventing microbial adhesion to surfaces, for example, by damaging amyloid structures involved in biofilm formation. Treatment of biofilm-associated conditions demonstrates a significant unmet clinical need.

[0009] Tailor-made empty liposomes, such as empty liposomes composed of cholesterol (CHOL) and / or sphingomyelin (SM), and their use for the treatment of bacterial infections have recently been reported to act 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 an existing biofilm. The composition comprises (i) a single empty liposome, the single empty liposome being (a) an empty liposome comprising cholesterol, the amount of cholesterol being at least 30% (weight / weight), 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) an empty liposome mixture, the empty liposome mixture being (a) empty liposomes comprising cholesterol, the amount of cholesterol being at least 30% (weight / weight), preferably the empty liposomes being selected from (preferably consisting of) at least one empty liposome selected from (a) empty liposomes comprising cholesterol and sphingomyelin, (more preferably consisting of), (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.

[0015] In a further aspect, the present invention provides empty liposomes consisting of sphingomyelin and cholesterol, wherein the amount of cholesterol is at least 30% (wt / wt), 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.

[0016] In a further aspect, the present invention provides empty liposomes comprised of sphingomyelin 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.

[0017] In yet another aspect, the present invention provides an empty liposome mixture comprising: (a) first empty liposomes comprised of sphingomyelin and cholesterol, wherein the amount of cholesterol is at least 30% (wt / wt); and (b) a second empty liposome comprised of sphingomyelin, 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.

[0018] In yet another aspect, the present invention provides a method for reducing biofilm formation or eradicating or reducing an existing biofilm, 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 an at-risk subject, 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 a mammal, preferably a human, in need thereof. In yet another aspect, the present invention provides a method for preventing or reducing biofilm formation or eradicating or reducing an existing biofilm, 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 the concomitant administration of standard antibacterial therapeutics. Furthermore, the present invention relates to preventing or reducing biofilm formation or eradicating or reducing existing biofilms, 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, prior to, following, along with, or simultaneously with standard antibacterial treatment for an infection.

[0019] Further aspects and embodiments of the invention will become apparent as the specification continues. [Brief explanation of the drawings]

[0020] [Figure 1] Inhibition of Pseudomonas aeruginosa multidrug-resistant strain 6077 biofilms by empty liposome mixtures of the present invention using a bacterial density of 1 x 10 cells / well. The graph shows the percentage of biofilm inhibition compared to biofilms formed in the absence of empty liposomes. DETAILED DESCRIPTION OF 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] The term "empty liposome" as used herein refers to a liposome, preferably an artificial liposome, 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, and consisting of one or more phospholipid bilayers, typically and preferably unilamellar vesicles and multilamellar vesicles, more preferably small unilamellar vesicles (SUVs). In a preferred embodiment, the term "empty liposome" as used herein typically and preferably refers to a liposome that does not incorporate any drug, typically and preferably refers to a liposome that does not incorporate any pharmaceutical agent. "Incorporated" as used herein and when referring to the empty liposomes of the present invention typically and preferably means encapsulated within the cavity of the liposome, within the latent 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 a liposome consisting of sphingomyelin and cholesterol or consisting of sphingomyelin, according to the present invention, and further exclusively comprises 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 originates from the synthesis of the empty liposome of the present invention, typically and preferably, the water-soluble inorganic compound is an inorganic salt, preferably selected from NaCl, KCl, and MgCl, and the water-soluble organic molecule is a buffer, preferably selected from glucose and HEPES. Typically and preferably, the water-soluble inorganic compound and / or the water-soluble organic molecule are incorporated into the inventive empty liposome of the present invention due to their presence during the production of the empty liposome of the present invention. In another preferred embodiment, the term "empty liposome" as used herein typically and preferably refers to a liposome consisting of sphingomyelin and cholesterol or consisting of sphingomyelin, according to the present invention, said empty liposome not containing any antioxidants.In a further preferred embodiment, the term "empty liposome" as used herein typically and preferably refers to a liposome consisting of sphingomyelin and cholesterol or consisting of sphingomyelin according to the present invention, and further exclusively comprises 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 originates from the synthesis of the empty liposome of the present invention, typically and preferably, the water-soluble inorganic compound is an inorganic salt, preferably selected from NaCl, KCl, and MgCl, and the water-soluble organic molecule is a buffer, preferably selected from glucose and HEPES. The empty liposome consisting of sphingomyelin and cholesterol or consisting of sphingomyelin according to the present invention does not contain an antioxidant.

[0026] The term "biofilm," as used herein, refers to a population of bacterial and / or fungal cells attached to one another or to surfaces, interfaces, including biotic and abiotic surfaces and interfaces, or associated with tissue or slime. This definition includes aggregates and floccules of small and large microorganisms, as well as adherent populations within the pore spaces of porous media. The term "anti-biofilm activity," or simply "anti-biofilm," as used herein, refers to the prevention or reduction of biofilm formation, or the eradication or reduction of an existing biofilm, the at least partial liberation of microorganisms, preferably bacteria, or the at least partial dissolution of a biofilm. The term "biofilm bacteria," as used herein, refers to non-planktonic bacteria present in a biofilm.

[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] The terms "inhibit," "destroy," "reduce," and "eradicate" as used herein with respect to biofilms or biofilm formation refer to complete or partial inhibition (preferably greater than 20%, more preferably greater than 30%, more preferably greater than 50%, even more preferably greater than 90%, even more preferably greater than 95%, or even greater than 99%) of biofilm formation and / or progression, typically and preferably in terms of the number of remaining cells, and also include within its scope the reversal of biofilm progression or processes associated with biofilm formation and / or progression. With respect to the latter, this refers to the disappearance of an existing biofilm at a rate faster than an untreated biofilm or a biofilm treated with a compound known not to affect biofilm stability. Furthermore, inhibition may be permanent or temporary. In terms 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, biofilm inhibition 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 a surgically invasive device or an implantable device, for example, but not by way of limitation, as defined in the European Commission, Directorate-General for Health and Consumer Protection, Division B, Unit B2, "Cosmetics and Medical Devices" guidelines on the application of European Union Directive 93 / 42 / EEC on medical devices.

[0030] The terms "treat," "treatment," or "therapy" as used herein refer to achieving a desired physiological effect. The effect may be therapeutic in terms of partially or completely curing a disease or condition and / or symptoms caused by the disease or condition. The term refers to inhibiting a disease or condition, i.e., arresting its progression, or ameliorating a disease or condition, i.e., causing regression of the disease or condition. The term "treat" or "treatment," as used herein in the context of ex vivo methods of the present invention or in the context of treating a surface (such as 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 a surface selected from a medical device, or a surface intended for contact with water or an aqueous solution) with a composition of the present invention, includes, but is not limited to, contacting, coating, and applying the aforementioned inventive composition to said surface in said ex vivo methods of the present invention.

[0031] As used herein, the term "prophylaxis" refers to a measure of preventing or delaying the onset of a disease or condition and / or symptoms resulting from the disease or condition.

[0032] The term "effective amount" as used herein, in the context of prophylaxis or treatment, refers to an amount of an active ingredient, typically and preferably a composition of the present invention, sufficient to produce a beneficial or desired result when administered or applied to a subject or patient, or to a surface, such as the surface of a medical device (typically and preferably intended for insertion into a subject's body). More specifically, the term "therapeutically effective amount" as used herein refers to an amount of an active ingredient, such as a composition of the present invention, sufficient to produce a beneficial or desired result 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 of the present invention can be readily determined by one of skill in the art. In the context of the present invention, a "therapeutically effective amount" is one that prevents or reduces biofilm formation, eradicates or reduces an existing biofilm, or produces an objectively measured change in one or more parameters associated with the treatment of a condition or disease associated with biofilm formation or the existing biofilm. Preferably, the condition or disease is caused by bacteria present in the biofilm, and preferably, the condition or disease is selected from infectious diseases. More preferably, the infection is a respiratory tract infection, 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. Of course, the therapeutically effective amount will vary depending on the particular subject and condition being treated, the subject's weight and age, 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" or "animal" or "patient" or "mammal" refer to any subject for whom diagnosis, prognosis, prevention or treatment is desired, particularly a mammalian subject, such as a human or a domestic mammal such as a dog, cat or horse, or a food animal such as a cow, sheep or pig, preferably 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 of the present invention comprises (preferably consists of) a single empty liposome, which is an empty liposome composed of sphingomyelin.

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

[0042] In another embodiment, the composition for use comprises (preferably consists of) an empty liposome mixture selected from (a) first empty liposomes comprising cholesterol, where the amount of cholesterol is at least 30% (w / w), (b) second empty liposomes consisting of sphingomyelin, and optionally (c) empty liposomes comprising (preferably consisting of) phosphatidylcholine and sphingomyelin, and optionally (d) at least one empty liposome comprising (preferably consisting of) a lipid or phospholipid 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.

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

[0044] In one embodiment, the composition comprises (preferably consists of) an empty liposome mixture comprising (preferably consisting of): (a) first empty liposomes comprised of sphingomyelin and cholesterol, the first empty liposomes comprising at least 30% (w / w) cholesterol, (b) second empty liposomes comprised of sphingomyelin, and (c) third 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.

[0045] In a preferred embodiment, the composition comprises (preferably consists of) an empty liposome mixture comprising (a) first empty liposomes comprising cholesterol and sphingomyelin, the first empty liposomes having a cholesterol content of at least 30% (w / w), and (b) second empty liposomes comprising sphingomyelin.

[0046] In another preferred embodiment, the composition for use in the method of the present invention comprises (preferably consists of) an empty liposome mixture. The empty liposome mixture comprises (a) first empty liposomes composed of sphingomyelin and cholesterol, the first empty liposomes having a cholesterol content of at least 30% (wt / wt), and (b) second empty liposomes composed of sphingomyelin. Preferably, the cholesterol content of the first empty liposomes is 30% to 70% (wt / wt), and more preferably, the cholesterol content of the first empty liposomes is 35% to 60% (wt / wt). In another preferred embodiment, the cholesterol content of the first empty liposomes is 45% to 55% (wt / wt), and even more preferably, the cholesterol content of the first empty liposomes 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 an existing biofilm, preferably in a mammal, more preferably a human, said first empty liposomes comprise (preferably consist of) 50% (wt / wt) sphingomyelin and 50% (wt / wt) 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 of the present invention comprises a mixture of empty liposomes, including (a) first empty liposomes composed of sphingomyelin and cholesterol, the first empty liposomes containing at least 30% (wt / w), preferably 30% to 70% (wt / w), cholesterol, and (b) second empty liposomes containing sphingomyelin. Preferably, the first empty liposomes contain 35% to 60% (wt / w), more preferably 45% to 55% (wt / w), and even more preferably, the first empty liposomes contain 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.

[0049] 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% (wt / wt) of the first 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% (wt / wt) of the second empty liposomes. Still more 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.

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

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

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

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

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

[0062] In a more preferred embodiment, the empty liposomes used in or for use in the present invention are liposomes, preferably artificial liposomes, having an average diameter of 20 nm to 10 μm, preferably 20 to 500 nm, and more preferably 20 nm to 200 nm. 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, within the latent 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 are composed 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 comprising sphingomyelin and cholesterol for use in a method for preventing or reducing biofilm formation or eradicating or reducing an existing biofilm, preferably in a mammal, more preferably in a human, wherein the amount of cholesterol is at least 30% (wt / wt). In another preferred embodiment, the empty liposomes for use in the method of the present invention comprise (preferably consist of) sphingomyelin and cholesterol, and the amount of cholesterol is 30% to 70% (wt / wt), more preferably, the amount of cholesterol in the empty liposomes is 35% to 60% (wt / wt). In another preferred embodiment, the amount of cholesterol in the empty liposomes is 45% to 55% (wt / wt), again more preferably, the amount of cholesterol in the empty liposomes 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 an existing biofilm, preferably in a mammal, more preferably a human, the empty liposomes comprise (preferably consist of) 50% (wt / wt) sphingomyelin and 50% (wt / wt) cholesterol.

[0064] In a further aspect, the present invention provides empty liposomes comprised of sphingomyelin 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.

[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 an existing biofilm, preferably in a mammal, more preferably a human, the empty liposome mixture comprising: (a) first empty liposomes composed of sphingomyelin and cholesterol, the first empty liposomes having a cholesterol content of at least 30% (wt / wt); and (b) second empty liposomes composed of sphingomyelin. Preferably, the cholesterol content of the first empty liposomes is 30% to 70% (wt / wt), more preferably, 35% to 60% (wt / wt). In another preferred embodiment, the cholesterol content of the first empty liposomes is 45% to 55% (wt / wt), and even more preferably, the cholesterol content of the first empty liposomes 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 an existing biofilm, preferably in a mammal, more preferably a human, the first empty liposomes comprise (preferably consist of) 50% (wt / wt) sphingomyelin and 50% (wt / wt) cholesterol. 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 the empty liposome mixture comprises 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 the empty liposome mixture comprises about 50% (wt / wt) of the first empty liposomes and about 50% (wt / wt) of the second empty liposomes.

[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 an existing biofilm, preferably in a mammal, more preferably a human, the empty liposome mixture comprising: (a) first empty liposomes composed of sphingomyelin and cholesterol, the first empty liposomes having a cholesterol content of at least 30% (wt / wt); and (b) second empty liposomes composed of sphingomyelin. Preferably, the cholesterol content of the first empty liposomes is 30% to 70% (wt / wt), more preferably, 35% to 60% (wt / wt). In another preferred embodiment, the cholesterol content of the first empty liposomes is 45% to 55% (wt / wt), and even more preferably, the cholesterol content of the first empty liposomes 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 an existing biofilm, preferably in a mammal, more preferably a human, the first empty liposomes comprise (preferably consist of) 50% (wt / wt) sphingomyelin and 50% (wt / wt) cholesterol. In another preferred embodiment, the empty liposome mixture comprises at least 30% (wt / wt) of the second empty liposomes and at most 70% (wt / wt) of the first empty liposomes, preferably the empty liposome mixture comprises at least 40% (wt / wt) of the second empty liposomes and at most 60% (wt / wt) of the first empty liposomes. In a further preferred embodiment, the empty liposome mixture comprises at least 45% (wt / wt) of the second empty liposomes and at most 55% (wt / wt) of the first empty liposomes, preferably the empty liposome mixture comprises about 50% (wt / wt) of the second empty liposomes and about 50% (wt / wt) of the first empty liposomes.

[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 an existing biofilm, preferably in a mammal, more preferably a human, the empty liposome mixture comprising: (a) first empty liposomes composed of sphingomyelin and cholesterol, the first empty liposomes having a cholesterol content of at least 30% (wt / wt); and (b) second empty liposomes composed of sphingomyelin. Preferably, the cholesterol content of the first empty liposomes is 30% to 70% (wt / wt), more preferably, 35% to 60% (wt / wt). In another preferred embodiment, the cholesterol content of the first empty liposomes is 45% to 55% (wt / wt), and even more preferably, the cholesterol content of the first empty liposomes is about 50% (wt / wt). Thus, in a highly preferred embodiment of the present invention, the first empty liposomes comprise (preferably consist of) 50% (wt / wt) sphingomyelin and 50% (wt / wt) cholesterol 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. In another preferred embodiment, the empty liposome mixture comprises 1 to 99%, preferably 5% to 95%, more preferably 10 to 90%, again more preferably 20 to 80%, again more preferably 30 to 70% (wt / wt) of the first and second empty liposomes, and again more preferably the empty liposome mixture comprises 40% to 60% (wt / wt) 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 an existing biofilm, preferably in a mammal, more preferably a human, comprising (preferably consisting of) an empty liposome mixture. The empty liposome mixture comprises (preferably consisting of) (a) first empty liposomes composed of sphingomyelin and cholesterol, the first empty liposomes having a cholesterol content of at least 30% (wt / wt), and (b) second empty liposomes composed of sphingomyelin. Preferably, the cholesterol content of the first empty liposomes is 30% to 70% (wt / wt), more preferably, 35% to 60% (wt / wt). In another preferred embodiment, the cholesterol content of the first empty liposomes is 45% to 55% (wt / wt), and even more preferably, the cholesterol content of the first empty liposomes is about 50% (wt / wt). Thus, in a highly preferred embodiment of the present invention, the first empty liposomes comprise (preferably consist of) 50% (wt / wt) sphingomyelin and 50% (wt / wt) cholesterol 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. In another preferred embodiment, the empty liposome mixture comprises 1 to 99%, preferably 5% to 95%, more preferably 10 to 90%, again more preferably 20 to 80%, again more preferably 30 to 70% (wt / wt) of the first and second empty liposomes, and again more preferably the empty liposome mixture comprises 40% to 60% (wt / wt) of the first and second empty liposomes.

[0069] 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 empty liposome mixture comprises (preferably consists of): (i) first empty liposomes consisting of about 50% (wt / wt) sphingomyelin and about 50% (wt / wt) cholesterol, and (ii) second empty liposomes consisting of (100%) sphingomyelin, the empty liposome mixture comprising 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.

[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 an existing biofilm, preferably in a mammal, more preferably a human, comprising (preferably consisting of) an empty liposome mixture, said empty liposome mixture comprising (preferably consisting of) (i) first empty liposomes consisting of about 50% (wt / w) sphingomyelin and about 50% (wt / w) cholesterol, and (ii) second empty liposomes consisting of (100%) sphingomyelin. The empty liposome mixture comprises at least 45% (wt / w) of the first empty liposomes and up to 55% (wt / w) of the second empty liposomes, preferably about 50% (wt / w) of the first empty liposomes and about 50% (wt / w) of the second empty liposomes.

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

[0072] Liposomes are prepared according to art-known methods such as extrusion, sonication, or microfluidization (e.g., high-pressure homogenization). 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 saline (0.9% NaCl), Krebs' solution, or Tyrode's solution, followed by sonication to produce liposomes. If necessary, liposome size can be controlled by extrusion through a membrane filter with a fixed pore size. Separately prepared liposomes of different lipid compositions are typically mixed in the required ratio, preferably immediately before application. As another example, liposomes can be produced using standard liposome hydration, extrusion, and diafiltration processes. Lipids are dissolved in a solvent such as ethanol with mixing, and the lipid solution is then added to PBS buffer with mixing to spontaneously form and fully hydrate heterogeneously sized liposomes. 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 solvent from the process fluid and finally concentrated and / or diluted with PBS buffer to a target concentration of total lipid.

[0073] The lipid surface (bilayer) of liposomes forms spontaneously in aqueous media, thus trapping water and other water-soluble inorganic and organic molecules (which may be present during liposome production) within the liposome interior. The empty liposomes used in this study are liposomes produced in a buffer containing water and simple organic or inorganic molecules (e.g., NaCl, KCl, MgCl, glucose, HEPES, and / or CaCl).

[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 in a method for eradicating or reducing an existing biofilm, 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.

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

[0079] In another preferred embodiment, the use is in a method for 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 in a method for 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 a specific embodiment, the plant cells are derived from leaves, roots, flowers, fruits, or other edible structures of the plant, and the tissues or structures are selected from 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 in a method for 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 lung, muscle, bone or skin cell or tissue, and the structure is a tooth.

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

[0083] In another preferred embodiment, said surface is a typical and preferably non-living surface selected from medical devices or surfaces intended for contact with water or aqueous solutions.

[0084] In another preferred embodiment, the surface is a typical, and preferably inanimate, surface of a medical device, typically, and preferably, intended for insertion into the body of a subject, the medical device being selected from pacemakers, pacemaker leads, catheters, stents, vascular prostheses, artificial heart valves, cardiac pacemakers, cerebrospinal fluid shunts, urinary catheters, peritoneal dialysis catheters, artificial joints and orthopedic fixation devices, intrauterine contraceptive devices, biliary stents, breast implants, contact lenses, and dentures.

[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 strain or a pump, preferably the surface is made of stainless steel or polypropylene.

[0086] In certain preferred embodiments, 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), including, but not limited to, a clamp, an infusion cannula, a skin closure device, a temporary filler material, 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 the pericardium, a carotid shunt, an ablation catheter, a neurological catheter, a cortical electrode, or a brachytherapy device.

[0087] In certain preferred embodiments, the medical devices intended for insertion into the body of a subject are implantable devices or long-term surgically invasive devices (>30 days), such as artificial joint replacements, ligaments, shunts, stents and valves (e.g., pulmonary valves), nails and plates, intraocular lenses, internal occlusion 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 maxillofacial implants, viscoelastic surgical instruments specifically for anterior segment surgery, bridges and crowns, dental fillings and pins, dental alloys, ceramics and polymers, prosthetic heart valves, aneurysm clips, vascular grafts and stents, central vascular catheters, spinal stents, CNS electrodes, cardiovascular sutures, permanent and retrievable vena cava filters, septal closure devices, intra-aortic balloon pumps, external left ventricular assist devices, and the like.

[0088] In the food processing industry, biofilms cause chronic bacterial contamination of food processing equipment such as pasteurizer pipes and tubing.

[0089] In the marine industry, marine fouling is typically described as involving several stages, beginning with an initial stage of bacterial adhesion, which initiates biofilm formation, followed by secondary settlement of self-adhering macroalgae (e.g., Enteromorpha spp., Hymenophora spp.) and protozoan (e.g., Vorticella spp., Zoothamnium spp.) spores. Finally, tertiary settlement occurs, i.e., the settlement of macrofouling organisms, such as tunicates, mollusks, and sessile cnidarians. Biofilm formation thus provides a substrate for biofouling of submerged surfaces, such as ship hulls, boat propellers, cages, underwater dock structures, underwater structures on offshore oil platforms, undersea mines, buoys, undersea cables, pipes and filters in power plant cooling systems, and desalination plants.

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

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

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

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

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

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

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

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

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

[0103] Biofilm-related infections and conditions include those caused by Gram-positive bacteria (e.g., Pneumococcus, Bacillus spp., Listeria monocytogenes, Staphylococcus spp., Lactobacillus plantarum and Lactococcus lactis), Streptococcus sobrinus, and Streptococcus mutans) and Gram-negative bacteria (e.g., Escherichia coli, Pseudomonas aeruginosa, Enterobacteriaceae, Salmonella spp., Actinobacillus pleuropneumoniae, Proteus mirabilis, and Acinetobacter baumannii). These include infections caused by bacteria (e.g., Shigella, Moraxella, Helicobacter, Stenotrophomonas, Bdellovibrio, Acetobacter, Legionella, Cyanobacteria, Spirochetes, Green Sulfur and Green Non-Sulfur Bacteria, Neisseria, Haemophilus influenzae, Klebsiella pneumoniae, Serratia marcescens), and infections caused by mycoplasmas, spiroplasmas, and fungi (e.g., Candida, Aspergillus, Cryptococcus, Trichosporon, Coccidioides, Pneumocystis). Some pathogens that form biofilms are ESKAPE pathogens. Biofilm-associated infections and conditions include those caused by gram-negative cocci that cause sexually transmitted diseases (e.g., Neisseria gonorrhoeae), meningitis (e.g., Neisseria meningitidis), or respiratory symptoms (e.g., M. catarrhalis, Haemophilus influenzae). It also includes infections and conditions caused by Gram-negative bacilli that primarily cause respiratory problems (e.g., Klebsiella pneumoniae, Legionella pneumophila, Pseudomonas aeruginosa), urinary problems (e.g., Escherichia coli, Proteus mirabilis, Enterobacter cloacae, Serratia marcescens), or gastrointestinal problems (e.g., Helicobacter pylori, Salmonella enteritidis, Salmonella typhi). Biofilm-associated infections also include nosocomial infections (e.g., Acinetobacter baumannii) that cause bacteremia, secondary meningitis, and ventilator-associated pneumonia in hospitals and intensive care units.

[0104] In another preferred embodiment, said condition or disease, preferably said 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 is a gram-negative or gram-positive bacterium. In another preferred embodiment, the condition or disease is caused by a gram-negative bacterium. In another preferred embodiment, the condition or disease is caused by a gram-positive bacterium.

[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 Pneumococcus, Bacillus, Listeria monocytogenes, Staphylococcus, Lactobacillus (preferably Lactobacillus plantarum and Lactococcus lactis), Streptococcus sobrinus, Streptococcus mutans, Escherichia coli, Pseudomonas aeruginosa, Enterobacteriaceae, Salmonella, 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, 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, and the bacterium is Pseudomonas aeruginosa.

[0112] Pseudomonas aeruginosa biofilms act as a reservoir for disease recurrence and prevent complete healing. Biofilms of this opportunistic pathogen are involved in both chronic infections, such as lung infections in cystic fibrosis patients (80% of CF patients are chronically infected with P. aeruginosa), and difficult, life-threatening hospital-acquired infections, such as ventilator-associated pneumonia, postoperative wound infections, and skin and soft tissue infections in burn patients. They are also involved in contaminating medical equipment, devices, 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 a Gram-positive bacterium selected from Streptococcus pneumoniae, Bacillus, Listeria monocytogenes, Staphylococcus, Lactobacillus (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 in a method for preventing or reducing bacterial biofilm formation, typically and preferably on a surface, wherein the bacterium is Staphylococcus aureus.

[0115] Staphylococcus aureus biofilms are a major cause of concern for multiple infections, challenging traditional anti-infective approaches, particularly antibiotic treatment. Many clinical isolates of Staphylococcus aureus are either methicillin-resistant (MRSA) or multidrug-resistant. Resistance to antibiotic treatments is further amplified by the increasing resistance of biofilm-forming S. aureus bacteria to the few antibiotics to which MRSA remains susceptible. Vancomycin is the most commonly administered drug for S. aureus biofilm-associated infections, but the increasing resistance of biofilms to vancomycin has necessitated the use of combination treatment approaches.

[0116] Staphylococcus aureus biofilms are commonly implicated in implant-associated infections (prosthetic orthopedic implants, heart valves, pacemakers, and vascular catheters), chronic wounds, osteomyelitis, pulmonary cystic fibrosis infections, and endocarditis. For example, pulmonary cystic fibrosis infections are associated with the presence of biofilms and chronic, long-term bacterial survival with increased mortality. In the case of S. aureus, these infections are often caused by MRSA. S. aureus is also a major cause of infection of the endocardium, or artificial cardiac surfaces, leading to infective endocarditis. S. aureus endocarditis is increasing with the increased use of surgical procedures, including the implantation of artificial cardiovascular devices such as prosthetic 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 of the present invention for preventing or reducing biofilm formation or eradicating or reducing an existing biofilm further comprises administering to a mammal, preferably a human, in need thereof 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.

[0118] To prevent or reduce biofilm formation or eradicate or reduce existing biofilms, the single empty liposomes of the present invention or the empty liposome mixtures of the present invention can be administered intravenously, intramuscularly, intraperitoneally, or subcutaneously. Injections are prepared using standard methods known in the art, such as a suspension of liposomes in sterile saline. The single empty liposomes of the present invention or the empty liposome mixtures of the present invention can also be administered as aerosols for treating respiratory tract infections, or as formulations useful for sublingual or buccal administration, intraocular or intravitreal administration, and topical application (e.g., eye drops, toothpaste, topical liquid suspensions for skin or mouth, etc.). Preparation of such formulations from liposomes, such as the empty liposomes of the present invention, is known in the art. A prophylactic treatment based on the single empty liposomes of the present invention or the empty liposome mixtures of the present invention may be useful for preventing biofilm-related conditions or diseases, such as biofilm progression in patients who are about to undergo, have undergone, or have undergone implant surgery, or who suffer from chronic infections, or in other environments that promote the progression of biofilm-related conditions or diseases.

[0119] In a further preferred embodiment, the method of the present invention for preventing or reducing biofilm formation or eradicating or reducing an existing biofilm further comprises administering a therapeutically effective amount of the composition of the present invention, the single empty liposome 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 liposome of the present invention, or the mixture of empty liposomes of the present invention is not administered in combination with any other drug, and thus is typically administered alone. Furthermore, typically and preferably, the composition of the present invention, the single empty liposome of the present invention, or the mixture of empty liposomes of the present invention is not administered in combination at the same time or at any time within a maximum of four weeks or so (preferably a maximum of two weeks).

[0120] In a further preferred embodiment of the present invention, said use is in combination with an antimicrobial agent, preferably said antimicrobial agent is an antibiotic, antifungal agent, antitoxin, antipathogenic agent, antiseptic or a combination thereof, more preferably said antimicrobial agent is an antibiotic.

[0121] The present invention also relates to a method for preventing or reducing biofilm formation or eradicating or reducing an existing biofilm, preferably for treating a condition or disease, preferably a bacterial infection, comprising administering a therapeutically effective amount of 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, after, together with, or simultaneously with an antibacterial agent, preferably together with a standard antibiotic treatment for the bacterial infection.

[0122] This preferred aspect and embodiment of the present invention aims to improve treatment efficacy, eliminate the additional community-level resistance caused by biofilms by promoting planktonic growth, facilitate targeting of pathogens at the cellular level by conventional antibiotics, and allow for a reduction in antibiotic dosage. Furthermore, as the single empty liposomes and empty liposome mixtures according to the present invention have already been described as toxin neutralizers, their action against toxins not only disarms bacteria but also allows the immune defense system to generate an appropriate response against pathogens.

[0123] In a further preferred embodiment of the present invention, the method further comprises administering to a mammal, preferably a human in need thereof, a therapeutically effective amount of the composition of the present invention, the single empty liposome of the present invention, or the 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, antifungal, antitoxin, antipathogenic, 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, preferably a human in need thereof, a therapeutically effective amount of the composition of the present invention, the single empty liposome of the present invention, or the mixture of empty liposomes of the present invention, before, after, in combination with, or simultaneously with standard antibiotic therapy and treatment.

[0124] In a further preferred embodiment of the present invention, the method further comprises administering to an animal in need thereof, preferably a food-producing animal such as a cow, sheep, pig, etc., 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, without any other drug, and therefore typically alone, or before, after, in combination with, or simultaneously with an antibacterial agent. Preferably, the antibacterial agent is an antibiotic, antifungal, antitoxin, antipathogenic agent, 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 a food-producing animal such as a cow or sheep, 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, before, after, in combination with, or simultaneously with a standard antibiotic therapy or administration.

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

[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, the single empty liposome of the present invention or the mixture of empty liposomes of the present invention, without any other drug in combination, and thus typically alone, or before, after, in conjunction with or simultaneously with an antibacterial agent. Preferably, said antibacterial agent is an antibiotic, antifungal, antitoxin, antipathogenic agent, antiseptic or a combination thereof, and more preferably, said antibacterial agent is an antibiotic.

[0127] It is understood that the empty liposomes defined above and the empty liposome mixtures defined above may be used together or in combination with additional compounds, prodrugs, or drugs, if necessary. For example, additional compounds, prodrugs, or drugs can be added to prepare standard pharmaceutical compositions. It is also possible to add drugs or drug-like compounds, or to add additional known or novel liposomes incorporating drugs or drug-like compounds inside the liposomes.

[0128] Contemplated drugs and prodrugs are, in particular, standard antibacterial or antifungal therapeutic agents, or antitoxins or antipathogenic agents known to those skilled in the art. Furthermore, drugs such as antibiotics are particularly contemplated. Such antibiotics include, for example, carbapenems (such as imipenem / cilastatin, meropenem, ertapenem, doripenem), first-generation cephalosporins (such as cefadroxil and cephalexin), second-generation cephalosporins (such as cefuroxime, cefaclor, and cefprozil), third-generation cephalosporins (such as ceftazidime, ceftriaxone, cefixime, cefdinir, cefditoren, cefotaxime, cefpodoxime, and ceftibuten), fourth-generation cephalosporins (such as ceftazidime, ceftriaxone, cefixime, cefdinir, cefditoren, cefotaxime, cefpodoxime, and ceftibuten), and the like. cephalosporins (such as cefepime), fifth-generation cephalosporins (such as ceftaroline fosamil and ceftobiprole), glycopeptides (such as vancomycin, teicoplanin, and telavancin), macrolides (such as clarithromycin, azithromycin, dirithromycin, erythromycin, roxithromycin, troleandomycin, telithromycin, spectinomycin, and spiramycin), penicillins (such as amoxicillin, flucloxacillin, oxacillin, and carbenicillin), and piperacillin), penicillin combinations (such as amoxicillin / clavulanic acid, piperacillin / tazobactam, ampicillin / sulbactam and ticarcillin / clavulanic acid), quinolones (such as ciprofloxacin (e.g., Aradigm's liposomal ciprofloxacin) and moxifloxacin), drugs against mycobacteria (rifampicin (called rifampin in the US), clofazimine, dapsone, capreomycin, cycloserine, ethambutol, ethiocin, Other antibiotics (such as metronidazole, arsphenamine, chloramphenicol, fosfomycin, fusidic acid, linezolid, mupirocin, platensimycin, quinupristin / dalfopristin, rifaximin, thiamphenicol, tigecycline and tinidazole), aminoglycosides (amikacin, gentamicin, kanamycin, neomycin, netilmicin,tobramycin (such as Axentis' fluidosomes™ tobramycin) and paromomycin), sulfonamides (such as mafenide, sulfonamide chrysoidine, sulfacetamide, sulfadiazine, silver sulfadiazine, sulfamethizole, sulfamethoxazole, sulfanilamide, sulfasalazine, sulfisoxazole, trimethoprim, and trimethoprim-sulfamethoxazole (cotrimoxazole, TMP-SMX)), tetracyclines (such as demeclocycline, doxycycline, minocycline, oxytetracycline, and tetracycline), lincosamides (such as clindamycin and lincomycin), and lipopeptides (such as daptomycin).

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

[0130] Further drugs that may be considered are, for example, vasopressors and vasoconstrictors such as vasopressin, oxymetazoline, phenylephrine, propylhexedrine, pseudoephedrine, epinephrine, norepinephrine, dopamine and antihistamines.

[0131] Other types of drugs are also contemplated, such as paracetamol (analgesic), nystatin, amphotericin B, polyenes, azoles and triazoles, nucleoside analogues, echinocandins, pneumocandins (anti-fungal infections), bupivacaine (post-operative pain management), morphine (analgesic), verteporfin (ophthalmological disorders), estradiol (menopausal disorders), aganocide® compounds.

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

[0133] In a further preferred embodiment of the invention, the method is an ex vivo method, and preferably the method comprises contacting a surface, preferably the surface of a medical device, with any one of the compositions of the 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 comprising 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, said method comprising covering said surface or 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 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 therapeutic treatment of the human or animal body.

[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, said method comprising covering said surface or existing biofilm with any one of the compositions of 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, comprising treating the surface or existing biofilm with any one of the compositions of the present invention. Thus, the method of the present invention is not a method for therapeutic treatment of the human or animal body.

[0140] Methods for coating surfaces with biologically or pharmaceutically active compounds or compositions are well known in the art. For example, the above-mentioned non-living surfaces, i.e., surfaces of medical devices, can be coated by blending the compositions of the present invention with a film-forming component to form an anti-biofilm coating that can be used to inhibit biofilm formation on the surface of the medical device. The film-forming component can include one or more resins, including, but not limited to, one or more hydrolyzable, soluble, or insoluble resins. 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] Example

[0142] Liposomes:

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

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

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

[0146] Liposome preparation

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

[0148] Sonication:

[0149] Lipids were individually dissolved in chloroform at a concentration of 1 mg / ml and stored at -20°C. To prepare liposomes, the chloroform solutions of individual lipids were mixed in the required proportions to produce a final solution of 50–500 μl, as specified. The chloroform was completely evaporated at 60°C for 20–50 min. 50 μl or 100 μl of Tyrode's buffer (140 mM NaCl, 5 mM KCl, 1 mM MgCl, 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 vigorously. The lipid suspension was incubated at 45°C for 20–30 min with vigorous shaking in an Eppendorf thermomixer. To generate liposomes, the final lipid suspension was sonicated 3 x 5 s at 6°C in a Bandelin Sonopuls sonicator at 70% power. The liposome preparations were left at 6°C for at least 1 hour before use in experiments.

[0150] Hydration, Extrusion and Diafiltration Process Protocol:

[0151] Alternatively, each liposome formulation was prepared by ethanol hydration and extrusion. Lipids were dissolved separately in ethanol and t-butanol with mixing at elevated temperatures (approximately 55°C). The lipid solution was then added to PBS buffer (sodium chloride, monosodium phosphate dihydrate, and disodium phosphate dihydrate dissolved in water for injection with mixing, adjusted to pH 7.0-7.4 with either hydrochloric acid (HCl) or sodium hydroxide (NaOH) as needed, and filtered through a 0.2 μm filter) with mixing for approximately 30 minutes at elevated temperatures (approximately 65°C). The resulting process fluid was then repeatedly extruded through a series of polycarbonate track-etched membranes (e.g., a LIPEX® extruder) at elevated pressures and temperatures (approximately 65°C) until the desired particle size, as measured by dynamic light scattering, was achieved. The resulting process fluid was then concentrated approximately two-fold using a 100,000 molecular weight cutoff hollow fiber cartridge and diafiltered against approximately 10 volume exchanges of PBS buffer to remove ethanol and t-butanol. At the end of diafiltration, the process fluid was concentrated by approximately 30%, allowing for subsequent dilution to the target lipid concentration. Prior to dilution, the process fluid was filtered through a 0.2 μm sterilizing-grade filter to remove larger liposomes that could clog the filter during sterile filtration. The process fluid was then diluted with PBS buffer to a target of 40 mg / mL total lipid. The final formulation was sterile filtered through two 0.2 μm sterilizing-grade filters in series and aseptically filled into glass tube vials.

[0152] The concentrations of individual lipids within liposomes are always given as weight / weight ratios. For liposomes containing sphingomyelin and cholesterol, a 1:1 (weight / weight) ratio corresponds to 50% (weight / weight) or a 35:65 molar ratio. Specifications are shown in Table 1.

[0153] [Table 1]

[0154] Example 1

[0155] Inhibition of Pseudomonas aeruginosa multidrug-resistant strain 6077 biofilms

[0156] A preferred empty liposome mixture of the present invention has been shown to reduce biofilm formation by Pseudomonas aeruginosa strain 6077, as determined by the crystal violet method. The preferred empty liposome mixture of the present invention comprises 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) in a 1:1 weight ratio (1:1 w / w; 35:65 molar ratio), and the second empty liposome is composed of SM only.

[0157] method

[0158] Briefly, P. aeruginosa strain 6077 was grown in trypticase soy broth (TSB) and LB broth containing 2% glucose at 1 × 10 per mL. 7 or 1 x 10 6The bacteria were diluted to a concentration of colony-forming units (CFUs). One hundred microliters (100 μl) of the diluted inoculum was added to wells of a 96-well flat-bottom microtiter plate. Increasing concentrations of the preferred empty liposome mixture of the present invention were added in 100 μL volumes to assess inhibition of biofilm formation. Each condition was performed in triplicate. Concentrations ranged from 75 μg / mL to 2883 μg / mL. Ciprofloxacin, gentamicin, and tobramycin were used as controls. The plates were incubated overnight at 37°C. Bacteria were then removed from the wells, and the biofilm monolayers were washed. The biofilms were fixed by incubating the plates at 60°C for 1 hour. After this incubation, the biofilms were stained with 0.4% crystal violet solution for 5 minutes and eluted with 70% ethanol. The optical density of the eluted material 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 wells to which no bacterial species was added (i.e., background noise).

[0159] result

[0160] Bacterial density is 1×10 7 For cells / well (high bacterial density setting), the preferred empty liposome mixture of the present invention alone (i.e., in the absence of antibiotics) inhibited biofilm formation at a concentration of 75 μg / mL, with a maximum inhibition of 72% (Figure 1). To avoid potential "overgrowth" that may contribute to inconsistent OD, a concentration of 1 x 10 6 Lower bacterial densities of 100 cells / well were also tested. At this lower bacterial density, the overall level of inhibition of biofilm formation by the preferred empty liposome mixture of the present invention was low (46% biofilm formation inhibition), but still fell in the middle of the dose-response curve (300-800 μg / mL). Additionally, some biofilm-disrupting activity was observed when the preferred empty liposome mixture of the present invention was applied 8 hours after bacterial inoculation using the lower bacterial density.

Claims

1. (a) a first empty liposome consisting of sphingomyelin and cholesterol, wherein the amount of cholesterol is at least 30% (wt / wt); (b) a second empty liposome comprising sphingomyelin; and (c) a third empty liposome consisting of phosphatidylcholine and sphingomyelin; an empty liposome mixture comprising 1. A composition for use in a method for preventing or reducing biofilm formation or for eradicating or reducing an existing biofilm, comprising:

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

3. The amount of cholesterol in the empty liposome (a) is 35% to 60% (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 amount of cholesterol in the empty liposome (a) is 50% (wt / wt). A composition for use according to any one of claims 1 to 4.

6. the empty liposome mixture comprises at least 20% (weight / weight) of the first and second empty liposomes; A composition for use according to any one of claims 1 to 5.

7. the empty liposome mixture comprises at least 30% (weight / weight) of the first and second empty liposomes; A composition for use according to any one of claims 1 to 6.

8. the empty liposome mixture comprises at least 40% (weight / weight) of the first and second empty liposomes; A composition for use according to any one of claims 1 to 7.

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

10. 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 9.

11. The condition or disease is selected from a respiratory tract infection, a sexually transmitted disease, meningitis, a urinary tract infection, a gastrointestinal disease, native valve endocarditis, colitis, vaginitis, urethritis, conjunctivitis, otitis, cystic fibrosis, ventilator-associated pneumonia, bacteremia, or a wound infection. A composition for use according to claim 10.

12. A composition for use as described in claim 11, wherein the otitis is otitis media.

13. The condition or disease is caused by at least one ESKAPE pathogen. A composition for use according to any one of claims 10 to 12.

14. The condition or disease is caused by a Gram-negative or Gram-positive bacterium selected from Pneumococcus, Bacillus, Listeria monocytogenes, Staphylococcus, Streptococcus sobrinus, Streptococcus mutans, Escherichia coli, Pseudomonas aeruginosa, Enterobacteriaceae, Salmonella, Actinobacillus pleuropneumoniae, Proteus mirabilis, Shigella, Moraxella, Helicobacter, Stenotrophomonas, Bdellovibrio, Acetobacter, Legionella, Cyanobacteria, Spirochetes, Green sulfur and green non-sulfur bacteria, Neisseria, Haemophilus influenzae, Enterococcus faecalis, Klebsiella pneumoniae, Acinetobacter baumannii, Serratia marcescens, and Enterobacter.

12. A composition for use according to claim 10 or 11.

15. A composition for use according to claim 14, wherein the Staphylococcus bacterium is Staphylococcus aureus, the Enterobacteriaceae bacterium is Lactobacillus, the Moraxella bacterium is Moraxella catarrhalis, the Helicobacter bacterium is Helicobacter pylori, or the Enterobacter bacterium is Enterobacter cloacae.

16. The use is in combination with an antimicrobial agent, wherein the antimicrobial agent is an antibiotic, an antifungal agent, an antitoxin, an antipathogenic agent, an antiseptic, or a combination thereof. A composition for use according to any one of claims 1 to 15.

17. The method is an ex vivo method or the method is an ex vivo method comprising contacting a surface with a composition according to any one of claims 1 to 16. A composition for use according to any one of claims 1 to 16.

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