Methods for Biofilm Disruption

Abstract

A composition for disrupting biofilms, comprising a 2-hydroxycarboxylic acid. There is a need for alternative treatments for disrupting and / or removing bacterial biofilms; or at least to provide a biofilm disrupting agent to complement additional bacterial treatments. The present invention solves this problem by providing an improved or alternative method for bacterial biofilm disruption and removal. The 2-hydroxycarboxylic acid is hydrophilic and preferably has a stereochemistry, with one enantiomer of the stereochemistry being preferred.

Description

[Technical field]

[0001] Technical Field The present invention relates to compositions comprising hydrophilic 2-hydroxycarboxylic acids for disrupting bacterial biofilms, and methods of disrupting bacterial biofilms using said compositions. [Background technology]

[0002] Background technology Bacterial biofilms are complex surface-attached communities of bacteria held together by a self-generated polymeric matrix. The biofilm matrix consists of substances such as proteins (e.g., fibrin), polysaccharides (e.g., alginate), and extracellular DNA. The dense polysaccharide matrix forms a shield that protects the underlying colony, which can be problematic for eliminating chronic bacterial infections present throughout nature, including human disease, crop maintenance, commercial animal farms, animal disease, and biofouling, including marine biofouling, and biofouling of membranes, sensors, and plumbing.

[0003] Biofilms are effective protective matrices secreted by bacteria. Biofilms encase, and sometimes position, bacterial colonies, creating an impenetrable barrier to block hostile environments and agents, such as bactericidal or bacteriostatic antibiotics, thereby protecting the bacteria within the biofilm colony from harm. In addition to this, concentration gradients established across biofilm-encased colonies and the biofilm matrix are known to induce mechanisms that cause resistance and / or tolerance to antibiotics, and as such, such biofilm infections are a major cause of antimicrobial resistance worldwide. For example, alterations in the presence of efflux pumps across biofilm colonies can result in increased rates of cellular mutations that result in resistance and / or tolerance to antimicrobial compounds. Furthermore, the presence of extracellular DNA and the close contact between cells in the biofilm matrix facilitate horizontal gene transfer of resistance or tolerance genes that enable survival. Bacteria in biofilms can be 1000-fold more resistant to antimicrobial drugs as a result of these and other mechanisms.

[0004] Biofilm formation is detrimental in healthcare, drinking water distribution systems, food, and marine industries, etc. For example, in the food industry, pathogenic bacteria can form biofilms in processing facilities, leading to food spoilage and endangering consumer health. In hospital environments, biofilms have also been shown to persist on medical equipment surfaces, on patient tissues, or as floating aggregates causing persistent infections, and on other surfaces such as the inside of soap dispenser pumps. Furthermore, biofilm-forming bacteria contribute to life-threatening infections and diseases in humans or animals, such as cystic fibrosis (CF)-associated pneumonia, otitis media, periodontitis, sinusitis, infective endocarditis (IE), chronic wounds, eye infections, and osteomyelitis.

[0005] The enveloping biofilm polysaccharide matrix prevents direct interaction with the applied therapeutic agent (which could be an antibiotic used to aid clearance, or an agent with a different antimicrobial mode of action, e.g., bacteriophages, antiseptics, saline, chlorine-based compounds (such as bleach), iodine-based compounds, copper-based compounds, heavy metals, etc.) The continuous application of antibiotics to biofilm colonies, combined with the ability of these colonies to communicate by quorum sensing, has been linked to antibiotic resistance.

[0006] Biofilm formation can begin with the attachment of free-planing microorganisms to a surface. These first colonies initially attach to the surface by weak, reversible adhesion via van der Waals forces. If the colonies are not immediately detached from the surface, they can use cell adhesion structures, such as pili, to anchor themselves more permanently. Some species cannot attach to a surface by themselves, but sometimes can anchor themselves to a matrix, or directly to the initial colony. During this colonization, cells can communicate via quorum sensing. Once colonization begins, biofilms grow by a combination of cell division and recruitment. Alternatively, biofilms can be initiated by the attachment of bacteria to each other, to another organism, or to non-living particles to form free-planing biofilm-associated aggregates of bacteria.

[0007] Attempts to eliminate pathogenic bacterial infections with antibiotics can be hampered by the presence of antibiotic resistance, which is made more difficult by bacterial biofilms. Failure to successfully eliminate these bacteria can lead to chronic infection and damage to the surrounding mucosal tissue as a result of prolonged inflammation.

[0008] Currently, there is a lack of regulated biofilm disrupting therapies available for use. Antibiotic and vaccine therapies remain the gold standard anti-infective treatment approaches.

[0009] Disruption, dissolution, removal or other impact on the functionality of biofilm matrices or colonies is an important antimicrobial therapy strategy that can be deployed to remove or expose bacterial colonies to the immune system, hostile environments and other antimicrobial agents; or to interfere with microbial resistance and / or resistance mechanisms to antimicrobial treatments. Currently prescribed antibiofilm strategies are limited to mechanical means to remove biofilms or chemical treatments, e.g., with heavy metals, that slow biofilm activity and cause their collapse. Novel, scalable, non-toxic antibiofilm therapies are needed to remove or impact problematic biofilm colonies and infections, and to reduce resistance to additional antimicrobial therapies across human, animal and plant health; as well as industrial applications such as shipping and wind turbines.

[0010] There is a need for alternative treatments for disrupting and / or removing bacterial biofilms; or at least providing biofilm disrupting agents to complement additional bacterial treatments. The present invention seeks to provide improved or alternative methods for bacterial biofilm disruption and removal.

[0011] The preceding description of the background art is intended only to facilitate an understanding of the present invention. The discussion is not an acknowledgment or admission that any of the material referred to was or was part of the common general knowledge at the priority date of the application. Summary of the Invention

[0012] Summary of the Invention The present invention provides a composition for disrupting biofilms, comprising a hydrophilic 2-hydroxycarboxylic acid. The hydrophilic 2-hydroxycarboxylic acid preferably has a stereochemistry, with one enantiomer of the stereochemistry being preferred. The 2-hydroxycarboxylic acid of the present invention having a preferred stereochemistry is referred to herein as the "preferred enantiomer of the 2-hydroxycarboxylic acid," the "preferred enantiomer," or the "preferred 2-hydroxycarboxylic acid." In one aspect, the preferred enantiomer of the 2-hydroxycarboxylic acid is D-lactic acid.

[0013] When the preferred 2-hydroxycarboxylic acid is not D-lactic acid, the preferred 2-hydroxycarboxylic acid utilized in the present invention may be one in which, with respect to the three-dimensional orientation of the hydroxy groups, the hydroxy groups of the 2-hydroxycarboxylic acid correspond to the absolute stereochemistry of the corresponding chiral center in D-lactic acid, with the absolute stereochemistry of the chiral center being as shown below. [ka]

[0014] Preferred 2-hydroxycarboxylic acids of the above general formula are those in which the substituent R is hydrogen, halogen (F, Cl, Br, I), methyl, ethyl, n-propyl, isopropyl, cyclopropyl, n-butyl, iso-butyl, sec-butyl, tert-butyl, cyclobutyl, n-pentyl, isopentyl, neopentyl, cyclopentyl, n-hexyl, 2-methylpentyl, 3-methylpentyl, 2,3-dimethylbutyl, 2,2-dimethylbutyl, cyclohexyl, n-heptyl, 2-methylhexyl, 3-methylhexyl, 2,3-dimethylpentyl, 2,4-dimethylpentyl, 2,2-dimethylpentyl, 3,3-dimethylpentyl, 3-ethylpentyl, 2,2,3-trimethylbutyl, cycloheptyl, phenyl, benzyl, furanyl, tetrahydrofuranyl, ethenyl, vinyl, allyl. , crotyl, isopentenyl, 1-propenyl, 2-propenyl, 1-butenyl, 2-butenyl, 2-methylprop-2-enyl, hexenyl, butadienyl, pentadienyl, hexadienyl, 2,3-dimethyl-2-butenyl, heptenyl, octenyl, octatrienyl, butadienyl, 1,3-pentadienyl, 1,4-pentadienyl, cyclopentadienyl, and cyclohexadienyl, each of which substituents (except hydrogen and halogen) may be unsubstituted or substituted with one or more substituents selected from the group consisting of hydrogen, halogen, hydroxyl, methoxyl, ethoxyl, propoxyl, butoxyl, carboxylic acid, amide or ester substituents, and pharma- ceutically acceptable salts thereof.

[0015] The compositions of the invention may contain from 0.001% to 100% total 2-hydroxycarboxylic acid. The remainder of the composition may comprise carriers, diluents or excipients, and / or other active agents.

[0016] The composition of the present invention may include a 2-hydroxycarboxylic acid of unpreferred stereochemistry, such as L-lactic acid. The 2-hydroxycarboxylic acid of the present invention having unpreferred stereochemistry is referred to herein as the "unpreferred enantiomer of the 2-hydroxycarboxylic acid", the "unpreferred enantiomer" or the "unpreferred 2-hydroxycarboxylic acid". The % of unpreferred 2-hydroxycarboxylic acid may be less than 20%. Alternatively, the composition of the present invention may not contain unpreferred 2-hydroxycarboxylic acid. Thus, the composition may only include 2-hydroxycarboxylic acid of preferred stereochemistry as the 2-hydroxycarboxylic acid.

[0017] When the non-preferred 2-hydroxycarboxylic acid is not L-lactic acid, the non-preferred 2-hydroxycarboxylic acid according to the invention is one in which, with respect to the three-dimensional orientation of the hydroxy groups, the hydroxy groups of the 2-hydroxycarboxylic acid correspond to the absolute stereochemistry of the corresponding chiral center of L-lactic acid, with the absolute stereochemistry of the chiral center being as shown below. [ka]

[0018] The present invention further provides a method for disrupting a biofilm, comprising the steps of: i. A method is provided that includes administering to a biofilm a preferred enantiomer of a 2-hydroxycarboxylic acid.

[0019] The present invention further provides a method for disrupting a biofilm, comprising the steps of: i. providing a method comprising administering to a biofilm a preferred enantiomer of a 2-hydroxycarboxylic acid; Here, a preferred enantiomer of a 2-hydroxycarboxylic acid is in combination with an antibacterial compound.

[0020] The present invention further provides a method of treating or preventing a bacterial infection, comprising the steps of: i. A method is provided which comprises administering to the site of infection a preferred enantiomer of a 2-hydroxycarboxylic acid.

[0021] The present invention further provides a method for disrupting or preventing a bacterial infection, comprising the steps of: i. providing a method comprising administering to a site of infection a preferred enantiomer of a 2-hydroxycarboxylic acid; Here, a preferred enantiomer of a 2-hydroxycarboxylic acid is in combination with an antibacterial compound.

[0022] The antimicrobial compound may be an antibiotic, or an agent with a different antimicrobial mode of action, such as a bacteriophage, an antiseptic, saline, a chlorine-based compound (such as bleach), an iodine-based compound, a copper-based compound, a heavy metal, and the like.

[0023] Preferred enantiomers of the 2-hydroxycarboxylic acid compositions of the present invention may be used to disrupt biofilms on both biotic and abiotic surfaces, as well as free-floating biofilm-associated aggregates of bacteria.

[0024] The present invention further provides a composition comprising a preferred enantiomer of a 2-hydroxycarboxylic acid for treating or preventing a bacterial infection in a subject, wherein the bacterial infection is in the form of a biofilm, and wherein the composition disrupts the biofilm.

[0025] The present invention further provides a composition comprising a preferred enantiomer of a 2-hydroxycarboxylic acid in combination with an antimicrobial compound for treating or preventing an infection in a subject, wherein the infection is associated with a biofilm, and wherein the composition disrupts the biofilm.

[0026] The present invention further provides a composition comprising a preferred enantiomer of a 2-hydroxycarboxylic acid for treating or preventing microbial colonization of a surface, wherein the microbial colonization is associated with a biofilm, and wherein the composition disrupts the biofilm.

[0027] The present invention further provides a composition comprising a preferred enantiomer of a 2-hydroxycarboxylic acid in combination with an antimicrobial compound for treating or preventing microbial colonization of a surface, where the microbial colonization is associated with a biofilm and the composition disrupts the biofilm.

[0028] Preferably, the infection or colonization is a bacterial infection or colonization.

[0029] The present invention provides the use of a preferred enantiomer of a 2-hydroxycarboxylic acid in the manufacture of a composition for the disruption of a biofilm.

[0030] The present invention further provides the use of a preferred enantiomer of a 2-hydroxycarboxylic acid for the disruption of a biofilm.

[0031] The present invention provides the use of a preferred enantiomer of a 2-hydroxycarboxylic acid in the manufacture of a composition for the disruption of biofilms in combination with an antimicrobial compound.

[0032] The present invention further provides the use of a preferred enantiomer of a 2-hydroxycarboxylic acid for the disruption of biofilms in combination with an antimicrobial compound.

[0033] The preferred enantiomer of the 2-hydroxycarboxylic acid may be in the same composition as the antimicrobial compound or in a separate composition. The antimicrobial compound may be an antibiotic or an agent with a different antimicrobial mode of action, such as a bacteriophage, an antiseptic, saline, a chlorine-based compound (such as bleach), an iodine-based compound, a copper-based compound, or a heavy metal.

[0034] The present invention further provides the use of a preferred enantiomer of a 2-hydroxycarboxylic acid in the manufacture of a composition for treating or preventing an infection in a subject, wherein the infection is in the form of a biofilm and the composition disrupts the biofilm.

[0035] The present invention further provides the use of a preferred enantiomer of a 2-hydroxycarboxylic acid in combination with an antibacterial compound in the manufacture of a composition for treating or preventing an infection in a subject, wherein the infection is in the form of a biofilm and the composition disrupts the biofilm.

[0036] The present invention further provides the use of a preferred enantiomer of a 2-hydroxycarboxylic acid to treat or prevent an infection in a subject, wherein the infection is in the form of a biofilm and the composition disrupts the biofilm.

[0037] The present invention further provides the use of a preferred enantiomer of a 2-hydroxycarboxylic acid in combination with an antimicrobial compound to treat or prevent an infection in a subject, wherein the infection is in the form of a biofilm and the composition disrupts the biofilm.

[0038] The present invention further provides the use of a preferred enantiomer of a 2-hydroxycarboxylic acid in the manufacture of a composition for treating or preventing microbial colonization of a surface, wherein the microbial colonization is in the form of a biofilm, and wherein the composition disrupts the biofilm.

[0039] The present invention further provides the use of a preferred enantiomer of a 2-hydroxycarboxylic acid in combination with an antimicrobial compound in the manufacture of a composition for treating or preventing microbial colonization of a surface, wherein the microbial colonization is in the form of a biofilm and the composition disrupts the biofilm.

[0040] The present invention further provides the use of a preferred enantiomer of a 2-hydroxycarboxylic acid for treating or preventing microbial colonization of a surface, wherein the microbial colonization is in the form of a biofilm, and wherein the composition disrupts the biofilm.

[0041] The present invention further provides the use of a preferred enantiomer of a 2-hydroxycarboxylic acid in combination with an antimicrobial compound to treat or prevent microbial colonization of a surface, wherein the microbial colonization is in the form of a biofilm, and wherein the composition disrupts the biofilm.

[0042] The present invention relates to a kit for disrupting a biofilm, comprising: a) a preferred enantiomer of a 2-hydroxycarboxylic acid, and b) Providing a kit including instructions for use.

[0043] The present invention relates to a kit for disrupting a biofilm, comprising: a) a preferred enantiomer of a 2-hydroxycarboxylic acid, and b) providing a kit containing instructions for use; Here, a preferred enantiomer of a 2-hydroxycarboxylic acid is in combination with an antibacterial compound.

[0044] In the above embodiment, the preferred enantiomer of the 2-hydroxycarboxylic acid may be D-lactic acid.

[0045] In the above embodiments, preferably the infection or colonization is a bacterial infection or colonization. [Brief description of the drawings]

[0046] BRIEF DESCRIPTION OF THE DRAWINGS Further features of the present invention will be described more fully in the following description of several non-limiting embodiments thereof. This description is included solely for the purpose of illustrating the present invention and should not be understood as a limitation on the broad summary, disclosure or description of the present invention set forth above. The following description will be made with reference to the accompanying drawings. [Figure 1]Figures 1A-B are graphs of crystal violet absorbance readings of biofilm-protected microcolonies treated with the following dilutions: Figure 1A - Prebiotic test solutions (dilutions); and Figure 1B - Tobramycin dose range (ug / ml) (absorbance readings at 600 nm). [Diagram 2] Figures 2A-B are graphs of resazurin fluorescence in supernatants collected from biofilm-protected Pseudomonas aeruginosa microcolonies (WACC91) treated with (a) prebiotic test solutions and (b) tobramycin. P. aeruginosa cultures were incubated overnight at 37 °C to establish biofilm formation. They were then treated for 24 h with dilutions of prebiotic test solutions or tobramycin (see Table 2). After treatment, supernatants were removed and resazurin was added to measure the metabolic activity of P. aeruginosa released from the biofilm. A decrease in metabolic activity was observed up to 1:24 dilution of the prebiotic test solution and from 1 to 16 µg / ml tobramycin. [Diagram 3] Figure 3 shows the identification of D-lactic acid as an active biofilm disrupting agent in the prebiotic test solution. After sequential testing of fractions from HPLC, D-lactic acid was identified and narrowed down to a single peak that could be identified by mass spectrometry and nuclear magnetic resonance. After lactate identification, optical rotation showed that 95% of the isolated fraction was the D-isomer. [Figure 4] Figure 4 shows the effect of D-lactic acid on biofilm-protected microcolonies, characterized by biofilm density (green) and viability staining (red indicates death) by confocal microscopy. D-lactic acid treatment; reduces the size of biofilm-protected microcolonies; disperses biofilm-protected microcolonies; and / or enhances the efficacy of tobramycin. [Diagram 5]Figure 5 is a graph of biofilm disruption caused by lactate enantiomers against Pseudomonas aeruginosa, Staphylococcus aureus and Staphylococcus epidermidis. Lactate enantiomers were added either at time zero (0 hours) or 24 hours after biofilm maturity. [Figure 6] Figure 6 shows images of biofilm microcolony dispersion upon addition of D-lactate. Single microcolonies of P. aeruginosa WACC91 grown overnight in a 3D biofilm model and then untreated or treated with 1 mg / mL D-lactate or 1 mg / mL L-lactate for 4 h were assessed by confocal microscopy performed with "live / dead" staining. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0047] Description of the invention Detailed Description of the Invention Lactic acid is known to be bactericidal due to its ability to reduce environmental pH and by disrupting the integrity of cell membranes. These antimicrobial properties have been used in food production.

[0048] It has further been proposed that lactic acid bacteria biofilms can be used to develop protective probiotic biofilms that exclude potentially pathogenic or contaminating bacteria. The coaggregation ability of lactic acid bacteria strains is thought to hinder the ability of pathogenic species to infect the host, thus preventing colonization by foodborne pathogens. This method is based on the prevention of biofilm formation by undesirable bacteria by pre-formation of a biofilm by lactic acid bacteria.

[0049] However, the present invention has surprisingly found that one enantiomer of a 2-hydroxycarboxylic acid, referred to in this application as the "preferred enantiomer of a 2-hydroxycarboxylic acid," the "preferred enantiomer," or the "preferred 2-hydroxycarboxylic acid," is effective in disrupting bacterial biofilms. This biofilm disrupting effect is not provided by the other enantiomer of a 2-hydroxycarboxylic acid, also known as the "unpreferred enantiomer of a 2-hydroxycarboxylic acid," the "unpreferred enantiomer," or the "unpreferred 2-hydroxycarboxylic acid." It has been found that in some circumstances, the unpreferred enantiomer is not only not active in disrupting biofilms, but may potentially prevent the preferred enantiomer from having its biofilm disrupting effect.

[0050] The biofilm destruction effect is not a bactericidal effect; both enantiomers of 2-hydroxycarboxylic acids, such as lactic acid, are known to have equal bactericidal effects. In some cases, the bactericidal effect of the two enantiomers is due to their effect on pH. In the present invention, the preferred enantiomer is used at a lower concentration than would be used if the preferred enantiomer were used for its bactericidal effect.

[0051] The present invention has found that the preferred enantiomers of 2-hydroxycarboxylic acids, including D-lactic acid, are small, water-soluble anti-biofilm agents that can be used to disrupt, dissolve, remove, or otherwise affect existing biofilms, or to prevent the formation of new bacterial biofilms. Bacterial biofilm infections and contaminations can be treated by the preferred enantiomers of 2-hydroxycarboxylic acids alone through their anti-biofilm efficacy. Alternatively, the preferred enantiomers of 2-hydroxycarboxylic acids can be used in combination with other antimicrobial agents or antibacterial therapies, including antibiotics and non-antibiotic antimicrobial compounds, where the anti-biofilm activity of the preferred enantiomers of 2-hydroxycarboxylic acids enhances the bacteriostatic and bactericidal efficacy of the antimicrobial agents or antibacterial therapies. composition

[0052] The present invention provides a composition for disrupting biofilms, the composition comprising a preferred enantiomer of a 2-hydroxycarboxylic acid, such as D-lactic acid.

[0053] In general, most lactic acid bacteria mainly produce L-lactic acid. Without being bound by any theory, it is believed that bacteria present in biofilms have evolved to avoid or modify L-lactic acid to prevent the effect of organic acids on the matrix of biofilms. Bacteria cannot avoid or modify the D-isomer of lactic acid, and therefore can have a biofilm decomposition effect.

[0054] Highly pure L-lactic acid can be produced by a wide range of microorganisms, including bacteria, fungi, algae and cyanobacteria, whereas most D-lactic acid producing microorganisms produce either a racemic mixture or other organic acids such as acetic or succinic acid (Alexandri et al. (2019) Food Tech & Biotech 57(3):293-304).

[0055] The compositions of the present invention may contain 0.001% to 100% of the preferred enantiomer of a 2-hydroxycarboxylic acid, such as D-lactic acid. The compositions may contain 0.001%, 0.01%, 0.1%, 1%, 10%, 20% 30%, 40% 50%, 60%, 70% 80% or 90% of the preferred enantiomer of a 2-hydroxycarboxylic acid. The remainder of the composition may contain carriers, diluents or excipients, and / or other active agents. For example, ophthalmic compositions may contain 0.001% to 1% of the preferred enantiomer of a 2-hydroxycarboxylic acid. Washes and soaks, dressings and wipes, topical, inhalation or oral compositions may contain 0.2% to 100% of the preferred enantiomer of a 2-hydroxycarboxylic acid.

[0056] The composition of the present invention may comprise a non-preferred enantiomer of 2-hydroxycarboxylic acid, the % of the non-preferred enantiomer of 2-hydroxycarboxylic acid being less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, less than 5% or less than 1%. More preferably, the % of the non-preferred enantiomer of 2-hydroxycarboxylic acid is less than 20%, less than 18%, less than 16%, less than 14%, less than 12%, less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2% or less than 1%. The % of the non-preferred enantiomer of 2-hydroxycarboxylic acid may be less than 20%.

[0057] Alternatively, the composition of the present invention may not contain the non-preferred enantiomer of the 2-hydroxycarboxylic acid. Thus, the composition may contain only the preferred enantiomer of the 2-hydroxycarboxylic acid as the 2-hydroxycarboxylic acid. For example, the composition may contain only D-lactic acid as the lactic acid. It has been found that compositions containing an enantiomerically pure amount of the preferred enantiomer of the 2-hydroxycarboxylic acid have a biofilm-disrupting effect, compositions containing an enantiomerically pure amount of the non-preferred enantiomer of the 2-hydroxycarboxylic acid have little or no biofilm-disrupting effect, and compositions containing a 1:1 ratio of the preferred enantiomer of the 2-hydroxycarboxylic acid to the non-preferred enantiomer of the 2-hydroxycarboxylic acid have a reduced or no biofilm-disrupting effect compared to administering the pure preferred enantiomer of the 2-hydroxycarboxylic acid.

[0058] The composition preferably comprises an enantiomerically enriched amount of a preferred enantiomer of a 2-hydroxycarboxylic acid.The composition may comprise a preferred enantiomer of an enantiomerically pure 2-hydroxycarboxylic acid.

[0059] Preferably, the concentration of the preferred enantiomer of the 2-hydroxycarboxylic acid used in the compositions of the invention is less than the concentration of the preferred enantiomer of the 2-hydroxycarboxylic acid required to kill bacteria.

[0060] Without being bound by any theory, it is believed that the present invention requires a 2-hydroxycarboxylic acid that has a chiral center at position 2. It is understood that when the aliphatic chain is at least 3 carbons long, the chiral center is at position 2. By this definition, glycolic acid [2-hydroxyacetic acid] is not chiral because it is less than 3 carbons in length.

[0061] The 2-hydroxycarboxylic acid may be a hydrophilic 2-hydroxycarboxylic acid. That is, the 2-hydroxycarboxylic acid is preferably soluble in water. Without being bound by any theory, it is believed that the water-soluble characteristic of the 2-hydroxycarboxylic acid aids in its biofilm penetrating and biofilm destroying activity.

[0062] In general, 2-hydroxycarboxylic acids having 7 or fewer carbons (including the carbon of the hydroxycarboxylic acid moiety) have been found to be more water-soluble. Thus, preferably, the 2-hydroxycarboxylic acids of the present invention contain 7 or fewer carbons. For example, 2-hydroxypropanoic acid (also known as lactic acid) contains 3 carbons, 2-hydroxypentanoic acid (also known as 2-hydroxyvaleric acid) contains 5 carbons, and 2-hydroxycaproic acid (also known as 2-hydroxyhexanoic acid) contains 6 carbons. More preferably, the 2-hydroxycarboxylic acids of the present invention contain 6 or fewer carbons. Most preferably, the 2-hydroxycarboxylic acids of the present invention contain 5 or fewer carbons.

[0063] Preferably, the molecular weight of the 2-hydroxycarboxylic acid is less than 800, less than 700, less than 600, or less than 500. More preferably, the molecular weight of the 2-hydroxycarboxylic acid is less than 200, less than 180, less than 150, or less than 100. For example,

[0064] Preferably, the absolute water solubility of the 2-hydroxycarboxylic acid is greater than 0.5 g / L, 0.75 g / L or 1.0 g / L at pH 5-10, pH 9-6, or more preferably pH 6-8.

[0065] Preferably, the absolute water solubility of the 2-hydroxycarboxylic acid is greater than 0.5 g / L, 0.75 g / L or 1.0 g / L at a temperature of about 20°C.

[0066] The water solubility of a 2-hydroxycarboxylic acid can alternatively be defined by its LogP (i.e., its octanol-water partition coefficient). LogP is calculated by the LogK ow Or also known as n-octanol-water partition ratio. As a rule, LogP:octanol-water<1.0=water solubility; LogP:octanol-water>1.0=hydrophobicity. Preferably, the 2-hydroxycarboxylic acids of the present invention have a LogP:octanol-water<1.0. Some compounds with LogP:<1.0 may have more than 7 carbons; however, their structure allows for water solubility despite the additional carbons. For example, 2-hydroxy-2-phenylacetic acid (also known as mandelic acid) has 8 carbons, but because of its ring structure, it has a LogP of 0.67 and is therefore water soluble.

[0067] Some 2-hydroxycarboxylic acids with special structures, such as 2-hydroxy-2-phenylacetic acid (also known as mandelic acid), which contains a six-membered ring and a total of eight carbons, may still be water-soluble and have a LogP:octanol-water<1.0. Such 2-hydroxycarboxylic acids are considered suitable for the present invention.

[0068] The 2-hydroxycarboxylic acids can have substituents such as hydrogen, halogen, hydroxyl, methoxyl, ethoxyl, propoxyl, butoxyl, carboxylic acid, amide or ester substituents.

[0069] The 2-hydroxycarboxylic acid may be a 2-hydroxycarboxylic acid that is pharma- ceutically acceptable. More preferably, the 2-hydroxycarboxylic acid is GRAS ("generally recognized as safe") as defined in sections 201(s) and 409 of the United States Federal Food, Drug, and Cosmetic Act or equivalent regulations.

[0070] The 2-hydroxycarboxylic acid may be selected from the group of acids, or esters, salts, amides, or other derivatives, consisting of lactic acid, glycolic acid (2-hydroxyacetic acid), tartaric acid (2,3-dihydroxysuccinic acid), mandelic acid, 1-hydroxycyclohexane-1-carboxylic acid, 2-hydroxy-2-(tetrahydrofuran-2-yl)acetic acid, 2-hydroxy-2-(2-furanyl)ethanoic acid, 2-hydroxy-2-phenylpropionic acid, 2-hydroxy-2-methylpropionic acid, 2-hydroxy-2-methylbutanoic acid, 2-hydroxybutanoic acid, 2-hydroxypentanoic acid, or mixtures thereof.

[0071] The 2-hydroxycarboxylic acid may be selected from the list including 2-hydroxypropanoic acid (also known as lactic acid), 2-hydroxypentanoic acid (also known as 2-hydroxyvaleric acid), 2-hydroxybutyric acid, 2-hydroxyacetic acid (also known as glycolic acid), 2-hydroxyhexanoic acid (also known as 2-hydroxycaproic acid), and 2-hydroxy-2-phenylacetic acid (also known as mandelic acid).

[0072] The 2-hydroxycarboxylic acid may be selected from the list including D-lactic acid, 2-hydroxypentanoic acid, 2-hydroxybutyric acid, 2-hydroxyhexanoic acid, (2)-hydroxyphenylacetic acid and 2-hydroxy-2-phenylacetic acid. Preferred 2-hydroxycarboxylic acids include those in Table 1 and / or Table 2. Table 1: Solubility of selected 2-hydroxycarboxylic acids [Table 1-1-1] [Table 1-1-2] Table 2: Solubility of selected 2-hydroxycarboxylic acids [Table 2-1]

[0073] Preferably, the 2-hydroxycarboxylic acid has both less than 7 carbons and a LogP:octanol-water<1.0.

[0074] Preferably, the 2-hydroxycarboxylic acid of the present invention is lactic acid, which ashes the two enantiomers. Preferably, the preferred enantiomer of the 2-hydroxycarboxylic acid is D-lactic acid, also known as (2R)-2-hydroxypropanoic acid and (R)-lactic acid. [ka]

[0075] The preferred enantiomer of the 2-hydroxycarboxylic acid may be synthesized via a cell-free route (e.g., enzyme-catalyzed reaction) from supernatants derived from cultured bacteria such as from Lactobacillus, multi-strain Gram-negative probiotic bacterial strains, or from natural sources such as 2-hydroxy-2-phenylacetic acid (mandelic acid) from almonds, olive oil and beer.

[0076] When the preferred enantiomer of the 2-hydroxycarboxylic acid is D-lactic acid, it can be synthesized via a cell-free route (e.g., enzyme-catalyzed reaction) from supernatants derived from cultured bacteria, such as from Lactobacillus or from multi-strain Gram-negative probiotic bacterial strains.

[0077] The D-lactic acid of the present invention is preferably a monomer. Therefore, it is preferable that the D-lactic acid is not polylactic acid (also known as PLA, poly D-lactic acid [PDLA], or poly L-lactic acid [PLLA]) or a similar polymer. PLA is a polymer obtained by ring-opening polymerization of monomer lactide (cyclic dimer of lactic acid).

[0078] The method of the present invention further provides a preferred enantiomer of a 2-hydroxycarboxylic acid for co-administration in combination with an antimicrobial compound, the preferred enantiomer of the 2-hydroxycarboxylic acid increasing the activity of the antimicrobial compound against microorganisms in a biofilm. The preferred enantiomer of the 2-hydroxycarboxylic acid may be in the same composition as the antimicrobial compound or in a separate composition. The antimicrobial compound may be an antibiotic or an agent with a different antimicrobial mode of action, such as a bacteriophage, an antiseptic, saline, a chlorine-based compound (such as bleach), an iodine-based compound, a copper-based compound, or a heavy metal. The biofilm may be an infection of a subject or a colonization of a surface, such as an abiotic surface. The biofilm may be a free-floating biofilm-associated aggregate of bacteria.

[0079] By "increasing activity" it is meant that disruption of the biofilm with a preferred enantiomer of a 2-hydroxycarboxylic acid allows the antimicrobial compound to more easily penetrate the microorganisms within the biofilm (because the microorganisms are loosened or released from the biofilm matrix) and have their antimicrobial effect.

[0080] Preferably, the pH of the compositions of the invention is about 6.5-8.0, more preferably about 7.0 and 7.4. It has previously been found that bacteria become more resistant to antibacterial therapy as the pH decreases. The preferred pH helps to avoid bacterial resistance to the effects of the preferred enantiomer of the 2-hydroxycarboxylic acid. A buffering agent may be added to adjust the pH level of the composition. Preferably, the compositions of the invention contain tris(hydroxymethyl)aminomethane (TRIS, also known as THAM or tromethamine) or phosphate buffered saline (PBS) as a buffering agent. The effectiveness of the preferred enantiomer of the 2-hydroxycarboxylic acid at a pH of about 6.5-8.0 is a further indication that the effect on bacterial biofilms is a result of the activity of the 2-hydroxycarboxylic acid itself on the biofilm, and not just the effect of the low pH killing the bacteria. Biofilm

[0081] The term "biofilm" refers to any syntrophic consortium of microorganisms (collections of microbial cells, especially bacterial cells, that are irreversibly associated (not removed by gentle rinsing) with each other and / or with a surface) in which the cells are attached to each other and often to a surface as well. These adherent cells become embedded within a slimy extracellular matrix composed of extracellular polymeric substances (EPS). Cells within a biofilm produce EPS components, which are typically polymeric assemblies of extracellular polysaccharides, proteins, lipids and DNA. Bacterial cells growing within a biofilm are physiologically distinct from planktonic cells (free-floating or swimming) of the same organism. Cells, especially bacterial cells, that switch to a biofilm mode of growth undergo a phenotypic shift in behavior in which a large set of genes is differentially regulated. Biofilms can form on a wide variety of surfaces, including living tissues, indwelling medical devices, industrial or drinking water system piping, or natural water systems.

[0082] Once a biofilm is established by the secretion of EPS by bacteria, the predominant microorganisms within the biofilm may change such that the bacterial load decreases and other organisms, such as fungi and viruses, predominate. The present invention can still be used to disrupt such biofilms because the preferred enantiomers of the 2-hydroxycarboxylic acids of the present invention act on the biofilm itself and not on the microorganisms in the biofilm.

[0083] Without being bound by any theory, it is believed that the preferred enantiomers of the 2-hydroxycarboxylic acids of the present invention act in three ways. First, the preferred enantiomers of the 2-hydroxycarboxylic acids inhibit the formation of bacterial biofilms. Second, the preferred enantiomers of the 2-hydroxycarboxylic acids destroy bacterial biofilms once they have formed. Third, the preferred enantiomers of the 2-hydroxycarboxylic acids can increase the activity of antimicrobial compounds against microorganisms in biofilms. The inhibition and destruction of biofilms can occur in the absence of any additional antimicrobial compounds, unlike the activity of any antimicrobial compounds.

[0084] When an existing biofilm is disrupted, the microorganisms in the biofilm may experience one or more of the following effects: -High vulnerability of the microorganism to agents of the host immune system, other microorganisms, or other antimicrobial agents, including but not limited to antibiotics and bacteriophages; -A reduced ability of microorganisms within the biofilm to develop resistance to antimicrobial agents (whether exogenous or host-derived); -Reduced growth of microorganisms in biofilms; -reduced adhesion of microorganisms to surfaces on which biofilms are formed; -Decreased rate of formation of the extracellular polymeric substance (EPS) matrix; - Reduction in the viscosity of the EPS matrix.

[0085] When inhibition of biofilm formation occurs, the microorganisms in the biofilm may experience one or more of the following effects: -reduced growth of biofilm-forming bacteria before or during biofilm formation; - high vulnerability of the microorganism to agents of the host immune system or other antimicrobial agents; -A reduced ability of microorganisms within the biofilm to develop resistance to antimicrobial agents (whether exogenous or host-derived); - Reduced adhesion of bacteria to surfaces on which biofilms form; -Decreased rate of formation of extracellular polymeric substances (EPS) matrix during biofilm formation; -Decreased viscosity of the EPS matrix during biofilm formation.

[0086] Preferably, the treatment regimen of the present invention causes the destruction of biofilm structure, and the biofilm optical density at 600 nm (OD600) is reduced by 70% or more, indicating significant destruction of biofilm compared to the control. An example of this measurement is provided in the Examples herein. The destruction of biofilm structure can be determined using crystal violet staining.

[0087] Preferably, the biofilm is produced by bacteria, although other organisms such as viruses, fungi or archaea may also be involved or present in the biofilm. These additional organisms may dominate the microbial population of the biofilm.

[0088] Biofilm-forming bacteria can be gram-positive or gram-negative. For example, biofilm-producing bacteria can be of the Enterobacteriaceae family, or from the following genera: Bacillus species, Clostridium species, Campylobacter species, Pseudomonas species, Streptococcus species, Actinobacillus species, Staphylococcus species, Escherichia species, Acinetobacter species, Klebsiella species, Aeromonas species, Enterococcus species, Legionella species, and Salmonella species, Shigella species, Ga The biofilm-forming bacteria may be selected from the species Rdnerella, Haemophilus, Helicobacter, Moraxella, Mycobacterium, Neisseria, Anaerococcus, Atopobium, Bacteroides, Leptotrichia, Mobiluncus, Peptostreptococcus, Prevotella, Sneathia, Sphingomonas, Nitrospira, Mycobacterium, or Hyphomicrobium and Vibrio. The biofilm-forming bacteria may be cyanobacteria or nontuberculous mycobacteria (NTM).

[0089] Bacteria that produce biofilms include Pseudomonas aeruginosa, Streptococcus pneumoniae, Streptococcus mutans, Actinobacillus pleuropneumoniae, Staphylococcus epidermidis, Escherichia coli, Staphylococcus aureus, Acinetobacter baumannii, Aeromonas hydrophila, Aeromonas salmonicida, Clostridium difficile, Enterococcus faecium, Gardnerella vaginalis, Haemophilus influenzae, Helicobacter pylori, Moraxella bovis, Moraxella catarrhalis, Mycobacterium avium, Mycobacterium intracellulare;, Mycobacterium tuberculosis, Neisseria gonorrhoeae, Neisseria meningitidis, Streptococcus pyogenes, and Vibrio cholerae.

[0090] For example, Bacillus, Clostridium, and Pseudomonas species are often found in gas and oil pipelines. Sphingomonas, Nitrospira, Legionella, Mycobacterium, or Hyphomicrobium species often colonize water pipes and air conditioning units. Eubacteria, including cyanobacteria, may form the basis of biofilm attachment on ship hulls.

[0091] Biofilm formation in bacterial vaginosis caused by or associated with Gardnerella vaginalis and other taxa, such as Anaerococcus, Atopobium, Bacteroides, Leptotrichia, Mobiluncus, Peptostreptococcus, Prevotella, Sneathia and Clostridia species, can be treated with the biofilm disrupting compositions of the present invention.

[0092] The biofilm-producing bacteria can be an antibiotic-resistant strain of the bacteria listed above. For example, the biofilm-producing bacteria can be an antibiotic-resistant Pseudomonas aeruginosa or an antibiotic-resistant Staphylococcus aureus (e.g., MRSA).

[0093] Biofilm-producing bacteria may be associated with conditions or diseases such as cystic fibrosis-associated pneumonia, periodontitis, diabetic ulcers, otitis media, periodontitis, sinusitis, infective endocarditis, eye infections, glue ear, bacterial vaginosis, and osteomyelitis.

[0094] Alternatively, biofilm-producing bacteria may be associated with industrial and non-biological surfaces such as hydroelectric turbines, oil and gas pipelines, air conditioning units, soap dispensers, industrial water systems, ship hulls, etc.

[0095] In a further example, bacterial biofilms may be associated with medical devices and instruments, such as bags and indwelling medical devices used to store blood donations.

[0096] Bacterial biofilms may also be associated with food production and storage: for example, the biofilms that are disrupted may form on food preparation surfaces, storage containers, or on the food itself.

[0097] Bacterial biofilms may further be associated with materials and equipment related to animal housing or husbandry.

[0098] As used herein, "treat" or "treatment" refers to inhibiting a disease or condition, i.e., stopping or reducing its development or at least one clinical or subclinical symptom thereof. "Treat" or "therapy" further refers to alleviating a disease or condition, i.e., causing at least one regression of a disease or condition or at least one clinical or subclinical symptom thereof. The benefit to the subject being treated is statistically significant or at least perceptible to the subject and / or the physician. In the context of biofilm destruction, the term treatment includes (i) a reduction in matrix density; and / or (ii) the dispersion of bacteria and their return to planktonic form.

[0099] The term "disruption" refers to one or more of a decrease in the viscosity of the extracellular polymeric substance (EPS) matrix of the biofilm, a decrease in the production of the EPS matrix, or a decrease in the attachment of bacteria to the surface on which the biofilm is formed. Disruption can be due to one of these factors, or two or three of these factors. Disruption can be the cause or result of the bacteria of the biofilm dispersing and reverting to planktonic forms or smaller aggregates, reducing the bacterial efficacy for building antibiotic resistance. Destruction method

[0100] The present invention further provides a method for disrupting a biofilm, comprising the steps of: i. A method is provided that includes administering to a biofilm a preferred enantiomer of a 2-hydroxycarboxylic acid.

[0101] The present invention further provides a method for disrupting a biofilm, comprising the steps of: i. providing a method comprising administering to a biofilm a preferred enantiomer of a 2-hydroxycarboxylic acid; Preferred herein are 2-hydroxycarboxylic acids in combination with antibacterial compounds.

[0102] The present invention further provides a method of treating or preventing an infectious disease, comprising: i. A method is provided which comprises administering to the site of infection a preferred enantiomer of a 2-hydroxycarboxylic acid.

[0103] The present invention further provides a method for preventing or destroying an infectious disease, comprising the steps of: i. providing a method comprising administering to a site of infection a preferred enantiomer of a 2-hydroxycarboxylic acid; Here, a preferred enantiomer of a 2-hydroxycarboxylic acid is in combination with an antibacterial compound.

[0104] The antimicrobial compound may be an antibiotic, or an agent with a different antimicrobial mode of action, such as a bacteriophage, an antiseptic, saline, a chlorine-based compound (such as bleach), an iodine-based compound, a copper-based compound, or a heavy metal.

[0105] The term "infection" includes microbial growth on both biological and non-biological surfaces and tissues. For example, an infection can be the growth of Pseudomonas in lung tissue or the growth of Legionella in a water pipe.

[0106] The compositions of the present invention may contain 0.001% to 100% of the preferred enantiomer of the 2-hydroxycarboxylic acid. For example, ophthalmic compositions may contain 0.001% to 1% of the preferred enantiomer of the 2-hydroxycarboxylic acid. Washes and soaks, dressings and wipes, topical, inhalation or oral compositions may contain 0.2% to 100% of the preferred enantiomer of the 2-hydroxycarboxylic acid. The remainder of the composition may include carriers, diluents or excipients, and / or other active agents. Preferably, the preferred enantiomer of the 2-hydroxycarboxylic acid is D-lactic acid.

[0107] The composition of the present invention may contain a non-preferred enantiomer of 2-hydroxycarboxylic acid, and the % of the non-preferred enantiomer of 2-hydroxycarboxylic acid may be less than 20%. Alternatively, the composition of the present invention may not contain a non-preferred enantiomer of 2-hydroxycarboxylic acid. Thus, the composition may contain only a preferred enantiomer of 2-hydroxycarboxylic acid as the 2-hydroxycarboxylic acid.

[0108] The present invention further provides a composition comprising a preferred enantiomer of a 2-hydroxycarboxylic acid for treating or preventing an infectious disease in a subject, wherein the infectious disease is associated with a biofilm, and wherein the composition disrupts the biofilm.

[0109] The present invention further provides a composition comprising a preferred enantiomer of a 2-hydroxycarboxylic acid in combination with an antimicrobial compound for treating or preventing an infection in a subject, wherein the infection is associated with a biofilm, and wherein the composition disrupts the biofilm.

[0110] The present invention further provides a composition comprising a preferred enantiomer of a 2-hydroxycarboxylic acid for treating or preventing microbial colonization of a surface, wherein the microbial colonization is associated with a biofilm, and wherein the composition disrupts the biofilm.

[0111] The present invention further provides a composition comprising a preferred enantiomer of a 2-hydroxycarboxylic acid in combination with an antimicrobial compound for treating or preventing microbial colonization of a surface, where the microbial colonization is associated with a biofilm and the composition disrupts the biofilm.

[0112] The efficacy of a method of treating or preventing infection in a subject by administering a preferred enantiomer of a 2-hydroxycarboxylic acid may be established by determining results other than the state of the biofilm itself. For example, efficacy may be determined by an increase in the FEV1 / FVC ratio, also called the Tiffeneau-Pinelli index, in a subject with obstructive or restrictive lung disease following administration of a preferred enantiomer of a 2-hydroxycarboxylic acid. In another example, the efficacy of a treatment using a preferred enantiomer of a 2-hydroxycarboxylic acid against an infection in the mammary gland may be established by a reduction in visible inflammation of the outer surface of the mammary gland tissue, or a reduction in swelling, heat, pain and / or redness. In either the lung example or the mammary gland tissue example, a further test may be a microbiological evaluation of biological fluids (e.g., sputum, breast milk) from the site of infection to determine the microbial load. If the preferred enantiomer of a 2-hydroxycarboxylic acid is disrupting the biofilm, it should reduce the microbial load at the site of infection. surface

[0113] Preferred enantiomers of the 2-hydroxycarboxylic acid compositions of the present invention can be used to disrupt biofilms on both biotic and abiotic surfaces.

[0114] Biofilms may also contain additional pathogens, such as viruses, fungi, and archaea, that are associated in the matrix of the biofilm. Biofilms can adhere to almost any type of surface, such as organic surfaces (biotic or non-biotic), plastics, metals, glass, soil particles, wood, and food. The present invention may be used to destroy biofilms on such surfaces.

[0115] Biological surfaces that may host biofilms that can be disrupted using the compositions of the invention include: pulmonary mucosal surfaces (particularly the lungs of subjects with cystic fibrosis or bronchiectasis); chronic wounds (including ulcers such as diabetic ulcers); skin surfaces (particularly those with skin dysbiosis, acne, skin pruritus, warts, psoriasis and atopic dermatitis), vaginal linings (including bacterial vaginosis and vulvovaginal candidiasis), gastrointestinal surfaces (e.g., small intestinal bacterial overgrowth, Clostridium difficile infection, gastric ulcers), middle ear surfaces (particularly those with otitis media); periodontal surfaces (particularly those with periodontitis); sinus surfaces (particularly those infected with sinusitis); cardiac surfaces (particularly those with infective endocarditis), ocular surfaces (particularly those with postoperative endophthalmitis, microbial keratitis, infectious crystalline keratopathy), other ocular surfaces (particularly those associated with scleral buckle insertion, conjunctival plug insertion, and lacrimal duct intubation) and bone surfaces (particularly those with osteomyelitis).

[0116] The presence of biofilms is common in the food industry and is of concern as pathogenic bacteria can be associated with biofilms.

[0117] Medical non-living surfaces that may host biofilms that can be disrupted using the compositions of the present invention include indwelling medical devices, such as urinary catheters; intravenous catheters; intrauterine devices; prostheses (including artificial hips, knees, heart valves, etc.; cochlear implants; intraocular lenses; breast implants, etc.); vascular access devices; endotracheal tubes; tracheotomy, enteral feeding tubes; wound drains; ear vents; soap dispensers, and surface medical devices such as contact lenses, orthodontic retainers and mouth guards.

[0118] Industrial surfaces that may host biofilms include hydroelectric turbines, oil and gas pipelines, air conditioning units, soap dispensers, industrial water systems, ship hulls, dairy equipment, etc. Bacterial biofilms can form on materials and equipment associated with animal housing or husbandry. delivery

[0119] The compositions of the present invention may be provided, for example: as topical compositions such as ointments, creams, foams, adhesives or lotions, eye ointments and eye or ear drops, or nasal sprays; as impregnated dressings, patches and wipes; as internal topical compositions such as pessaries and suppositories; as inhalation compositions such as sprayed or dry powder compositions; as injectable preparations for intravenous and local injection (e.g. into an abscess) or as oral preparations in the form of tablets, liquid capsules, oral gels, etc. The compositions may also be in the form of irrigation or soaking solutions.

[0120] As used herein, the term "therapeutically effective amount" means an amount of a composition that, when administered according to a desired dosing or application regimen, is sufficient to at least partially achieve a desired effect, or to slow the development of a biofilm or inhibit the progression of a biofilm, or to partially or completely halt the development or progression of a biofilm.

[0121] As used herein, the term "prophylactically effective amount" means an amount of a composition that, when administered according to a desired dosing or application regimen, is sufficient to at least partially prevent or delay the development of a biofilm.

[0122] Based on the above, one skilled in the art will understand that a single subject or surface can be treated using multiple different treatments and administration means. For example, a subject already receiving a medication (such as an intravenous antibiotic) may benefit from oral delivery, inhalation, or topical application of the composition of the present invention. Some subjects may be administered only the present composition comprising the preferred enantiomer of the 2-hydroxycarboxylic acid by oral administration, inhalation, or topical application. For example, a subject may have symptoms of cystic fibrosis, may be diagnosed with a lung infection, or may have symptoms of a medical condition, which may benefit from administration to the subject of a preferred enantiomer of the 2-hydroxycarboxylic acid, an inhalation composition comprising a preferred 2-hydroxycarboxylic acid. Alternatively, a subject may have a localized infection, such as a chronic wound, an eye infection, or periodontitis, and may be administered a preferred enantiomer of the 2-hydroxycarboxylic acid locally. A subject may have a disruption ("dysbiosis") in the intestine (e.g., small intestine) that may benefit from oral administration of a preferred enantiomer of the 2-hydroxycarboxylic acid composition of the present invention. These dysbiosis may contribute to intestinal pathogen-induced disorders, inflammatory bowel disease (IBS), Crohn's disease, ulcerative colitis or colorectal cancer.

[0123] The composition of the present invention can be used on non-living surfaces, such as in the form of wet wipes, as a washing or soaking solution for non-living surfaces such as indwelling equipment and water pipes, as well as a cleaning composition for pipes, food preparation machines and medical devices. If the composition is applied to a non-living surface, the D-lactic acid wipe, wash or soak can be followed by conventional detergent washing, UV sterilization or bleaching.

[0124] The compositions of the present invention can also be used diagnostically. In one embodiment, for example, a subject may receive a dose of the compositions of the present invention as part of a procedure for diagnosing a biofilm-associated infection (such as a lung infection), and one of a number of symptoms of the subject improves in response to the composition.

[0125] According to certain embodiments, the composition is administered periodically until treatment is obtained. In one preferred embodiment, the composition is administered to a subject in need of such treatment using a dosing regimen selected from the group consisting of hourly, every 2 hours, every 3 hours, once a day, twice a day, three times a day, four times a day, five times a day, once a week, twice a week, once every other week, and once a month. However, other application schedules may be utilized in accordance with the present invention. Preferably, the composition of the treatment regimen is administered to the subject one to five times a day, more preferably once or twice a day.

[0126] When administration is to a non-living surface, the composition can be applied to the surface periodically until removal or destruction of biofilm is obtained.In one preferred embodiment, the composition is applied to the surface hourly, every 2 hours, every 3 hours, once a day, twice a day, three times a day, four times a day, five times a day, once a week, twice a week, once every two weeks and once a month.However, other application schedules may be utilized according to the present invention.The composition can be applied to the indwelling device before insertion and after removal.

[0127] Preferably, the composition is administered, for example, orally, topically (ophthalmic, buccal and sublingual, rectal, vaginal, intranasal) or by aerosol administration. When the composition is delivered to a non-living surface, the composition can be administered as a wash, a soaking solution or a wipe. The mode of administration or application is preferably suitable for the form in which the composition is prepared. The mode of administration for the most effective response can be empirically determined, and the means of administration or application described below are given as examples and in no way limit the method of delivery of the composition of the present invention.

[0128] The composition of the present invention may contain pharmaceutically acceptable non-toxic excipients and carriers as necessary.As used herein, "pharmaceutical carrier" refers to the pharmaceutically acceptable solvent, suspending agent, excipient or vehicle for delivering compound to a subject.Carrier may be liquid or solid, and is selected with consideration of the planned mode of administration.

[0129] The compositions of the present invention may be selected from the group consisting of immediate release compositions, delayed release compositions, controlled release compositions and rapid release compositions.

[0130] The compositions of the present invention may further comprise an anti-inflammatory agent, such as a corticosteroid.

[0131] The compositions described herein can be formulated by including such dosage forms in oil-in-water emulsion or water-in-oil emulsion.In such compositions, the immediate release dosage form is a continuous phase, and the delayed release dosage form is a discontinuous phase.The compositions can also be manufactured in a manner for delivery of three dosage forms as described hereinabove.For example, an oil-in-water-in-oil emulsion can be provided with oil being the continuous phase containing the immediate release component, water dispersed in the oil containing the first delayed release dosage form, and oil dispersed in the water containing the third delayed release dosage form.

[0132] The compositions described herein may be in the form of a liquid composition. The liquid composition may include a solution including a therapeutic agent dissolved in a solvent. Generally, any solvent may be used in which the therapeutic agent dissolves and has the desired effect that can be administered to a subject. Generally, any concentration of the therapeutic agent may be used that has the desired effect. In some variations, the composition is a solution that is an unsaturated, saturated, or supersaturated solution. The solvent may be a pure solvent or may be a mixture of liquid solvent components. In some variations, the solution formed is an in-situ gelling composition. The types of solvents and solutions that may be used are well known to those familiar with such drug delivery techniques.

[0133] The composition may or may not contain water. Preferably, the composition contains water, i.e., is aqueous. In another preferred embodiment, the composition does not contain a preservative.

[0134] Pharmaceutical or veterinary compositions may be formulated in accordance with conventional pharmaceutical or veterinary practice (see, e.g., Remington: The Science and Practice of Pharmacy, 20th ed., 2000, ed; A. R. Gennaro, Lippincott Williams & Wilkins, Philadelphia, and Encyclopedia of Pharmaceutical Technology, eds; J. Swarbrick and J. C. Boylan, 1988-1999, Marcel Dekker, New York; Remington's Pharmaceutical Sciences, 18th ed., Mack Publishing Company, Easton, Pennsylvania, USA).

[0135] In general, examples of suitable carriers, excipients and diluents include, but are not limited to, water, saline, ethanol, dextrose, glycerol, lactose, dextrose, sucrose, sorbitol, mannitol, starch, gum acacia, calcium phosphate, alginate, tragacanth, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water syrup, methylcellulose, methyl and propyl hydroxybenzoate, polysorbate, talc magnesium stearate, mineral oil or combinations thereof.The composition can further comprise lubricants, pH buffers, wetting agents, emulsifiers and suspending agents, preservatives, sweeteners or flavoring agents.

[0136] The composition may be in the form of a controlled release composition and may include degradable or non-degradable polymers, hydrogels, organogel, or other physical constructs that modify the release of D-lactic acid. It is understood that such compositions may include additional inactive ingredients that are added to provide desired color, stability, buffering capacity, dispersion, or other known desirable characteristics. Such compositions may further include liposomes such as emulsions, foams, micelles, insoluble monolayers, liquid crystals, phospholipid dispersions, lamellar layers, etc. Liposomes for use in the present invention may be formed from standard vesicle-forming lipids, generally including neutral and negatively charged phospholipids and sterols, such as cholesterol. Cleaning and Soaking Compositions

[0137] The compositions of the present invention containing the preferred enantiomers of 2-hydroxycarboxylic acids can be used to wash and / or soak devices such as indwelling medical devices or water piping systems. Washing or soaking such devices with compositions containing the preferred enantiomers of 2-hydroxycarboxylic acids helps to sterilize or clean the devices by helping to destroy any biofilm present on the device surface, thus making it easier to remove and / or kill microorganisms present in the biofilm. The destruction preferably releases the microorganisms from the biofilm polysaccharide matrix that encapsulates them.

[0138] In one form, the composition used for washing or soaking may contain from 0.1% to 100% of a preferred enantiomer of the 2-hydroxycarboxylic acid. The remainder of the composition may include carriers, diluents or excipients, and / or other active agents.

[0139] In one form, the composition used for washing or soaking may contain the unpreferred enantiomer of 2-hydroxycarboxylic acid, and the % of the unpreferred enantiomer of 2-hydroxycarboxylic acid may be less than 20%. Alternatively, the composition of the present invention may not contain the unpreferred enantiomer of 2-hydroxycarboxylic acid. Thus, the composition may contain only the preferred enantiomer of 2-hydroxycarboxylic acid as the 2-hydroxycarboxylic acid. Bandages and Wipes

[0140] The composition of the present invention comprising the preferred enantiomer of 2-hydroxycarboxylic acid can be provided in the form of an impregnated dressing or bandage, or a moistened wipe. The dressing can be in a form that can be applied to a wound or topical surface having a biofilm and left in situ for hours, days or weeks. The wipe can be used to destroy, for example, a biofilm on a wound or mucosal surface, such as a vaginal or rectal surface, or on an abiotic surface. The wipe can be used hourly, daily, weekly, or as needed. For example, the wipe can be used after each urination or defecation to destroy the formation of a biofilm or to destroy a biofilm already present on the vaginal or rectal mucosal tissue. Alternatively, the wipe can be used on a food preparation surface before use.

[0141] In one form, the composition used in the bandage or wipe may contain from 0.2% to 100% of a preferred enantiomer of the 2-hydroxycarboxylic acid. The remainder of the composition may include carriers, diluents or excipients, and / or other active agents.

[0142] In one form, the composition used in the dressing or wipe may include the unpreferred enantiomer of the 2-hydroxycarboxylic acid, and the % of the unpreferred enantiomer of the 2-hydroxycarboxylic acid may be less than 20%. Alternatively, the composition of the present invention may not contain the unpreferred enantiomer of the 2-hydroxycarboxylic acid. Thus, the composition may only include the preferred enantiomer of the 2-hydroxycarboxylic acid as the 2-hydroxycarboxylic acid. inhalation delivery

[0143] The compositions of the invention can be delivered via nebulized delivery or via an aerosol device, which is particularly suited for biofilm-associated respiratory and ear-nose-and-throat (ENT) diseases.

[0144] Preferably, the composition is administered to a subject in need thereof about once per day to about 6 times per day, more preferably about once or twice per day.

[0145] Alternatively, the composition can be administered to the subject in need by continuous inhalation via nebulizer.The nebulized composition can be delivered for 24 hours, 12 hours, 8 hours, 6 hours, 4 hours, 2 hours or 1 hour, and each of these deliveries (except for 24 hours and 12 hours) can be repeated several times within 24 hours.

[0146] In one form, the composition delivered by nebulization or aerosol delivery may contain from 0.2% to 100% of a preferred enantiomer of the 2-hydroxycarboxylic acid, the remainder of the composition may include carriers, diluents or excipients, and / or other active agents.

[0147] In one form, the composition delivered by spray or aerosol delivery may include the unpreferred enantiomer of the 2-hydroxycarboxylic acid, and the % of the unpreferred enantiomer of the 2-hydroxycarboxylic acid may be less than 20%. Alternatively, the composition of the present invention may not contain the unpreferred enantiomer of the 2-hydroxycarboxylic acid. Thus, the composition may only include the preferred enantiomer of the 2-hydroxycarboxylic acid as the 2-hydroxycarboxylic acid.

[0148] A subject may typically receive a dose of 5-500 mg / ml of the preferred enantiomer of a 2-hydroxycarboxylic acid (+20% or +10%). This dose is typically nebulized or administered by at least one, and preferably several, "puffs" from an aerosol device. For example, a subject may receive 5 mg / ml to 500 mg / ml of the preferred enantiomer of a 2-hydroxycarboxylic acid in a single dose per day, or in several doses per day.

[0149] The total daily dose is preferably administered at least once a day, but may be divided into two or more doses per day.Some subjects may benefit from a period of "loading" the subject with the preferred enantiomer of 2-hydroxycarboxylic acid, with higher or more frequent doses followed by a reduced or maintenance dose over a period of several days or weeks.Because cystic fibrosis, COPD, etc. are typically chronic conditions, it is expected that subjects will undergo such therapy for a long period of time.

[0150] There is a wide range of mechanical devices designed for pulmonary delivery of therapeutic products, including, but not limited to, nebulizers, metered dose inhalers and powder inhalers, all of which are well known to those skilled in the art.Some specific examples of commercially available devices suitable for carrying out the present invention are the Ultravent nebulizer manufactured by Mallinckrodt, Inc., St. Louis, Missouri; the Acorn II nebulizer manufactured by Marquest Medical Products, Englewood, Colorado; the Ventolin metered dose inhaler manufactured by Glaxo Inc., Research Triangle Park, North Carolina; and the Spinhaler powder inhaler manufactured by Fisons Corp., Bedford, Massachusetts.

[0151] All such devices require the use of a composition suitable for the delivery of the preferred enantiomer of the 2-hydroxycarboxylic acid. Typically, each composition is specific to the type of device used and may include the use of an appropriate propellant material in addition to the usual diluents, adjuvants and / or carriers useful in therapy.

[0152] Regardless of the form of the drug composition, it is preferred to produce droplets or particles for inhalation in the range of about 0.1 μm to 12 μm, or about 0.25 μm to 6 μm, preferably 1 μm to 6 μm, more preferably about 2 μm to 4 μm. Alternatively, the particles may be 0.1 μm to 1.0 μm, 0.2 μm to 0.9 μm, 0.3 μm to 0.8 μm, 0.4 μm to 0.7 μm, or 0.5 μm. By creating inhaled particles with a relatively narrow range of sizes, it is possible to further increase the efficiency of the drug delivery system and improve the reproducibility of administration. Thus, it is preferred that the particles not only have a size in the range of 0.1 μm to 12 μm or 2 μm to 6 μm or about 3 to 4 μm, but also that the average particle size is within a narrow range such that 80% or more of the particles delivered to the subject have a particle diameter within +20%, preferably within +10%, more preferably within +5% of the average particle size.

[0153] "Particle size" is a concept introduced to compare the size of solid particles, liquid particles (droplets). For droplets and aerosols, terms such as "aerodynamic diameter" and "mass median aerodynamic diameter (MMAD)" are used. The following definitions are provided:

[0154] "Aerodynamic diameter" is the diameter of a unit density sphere that has the same terminal settling velocity as the particle in question. It is used to predict where such particles will deposit in the airways.

[0155] "Mass median aerodynamic diameter" is the geometric mean aerodynamic diameter. 50% of the particles (by weight) will be smaller than the MMAD and 50% will be larger.

[0156] During a particle sizing experiment, the suspension contains a myriad of particles of various sizes in motion. When the particle sizing machine analyzes these particles, a particle distribution curve is formed, which covers the entire particle size range starting from the smallest particles (which may be 1 nm) to the largest particles (which may be 100 μm). In the particle size distribution curve, a cumulative frequency is calculated for the particles. D 10 refers to a particular particle diameter at which 10% of the particles in a suspension have a diameter less than or equal to that of the particular particle diameter.

[0157] D 50 :D 10 Similarly, D 50 is the cutoff diameter for 50% of the particle population in a composition and refers to a particular particle diameter at which 50% of the particles in the suspension have a diameter less than or equal to that of the particular particle diameter.

[0158] D 90 :D 90 is the cutoff diameter for 90% of the particle population in a composition and refers to a particular particle diameter at which 90% of the particles in suspension have a diameter less than or equal to that of the particular particle diameter.

[0159] The term "respiratory tract" should be taken to mean the system of cells and organs that function in respiration, and in particular the organs, tissues and cells of the respiratory tract include the lungs, nose, nasal passages, paranasal sinuses, nasopharynx, larynx, trachea, bronchi, bronchioles, respiratory bronchioles, alveolar ducts, alveolar sacs, alveoli, pneumocytes (type 1 and type 2), ciliated mucosal epithelium, mucosal epithelium, squamous epithelial cells, mast cells, goblet cells, and intraepithelial dendritic cells.

[0160] In one aspect of the invention, a method of treating or preventing biofilm development in the lungs of a subject comprises administering a therapeutically or prophylactically effective concentration of a preferred enantiomer of a 2-hydroxycarboxylic acid in one or more doses of 1-1000 mg / ml, more preferably 5-500 mg / ml.

[0161] In one aspect of the invention, a method of treating biofilm development in the lungs of a subject comprises administering a therapeutically effective concentration of an inhalation composition comprising a preferred enantiomer of a 2-hydroxycarboxylic acid in one or more doses of 1-1000 mg / kg / day, more preferably 6-600 mg / kg / day.

[0162] In one aspect of the invention, a method of preventing biofilm development in the lungs of a subject is by administering a prophylactically effective concentration of an inhalation composition comprising a preferred enantiomer of a 2-hydroxycarboxylic acid in one or more doses of 1-1000 mg / ml, more preferably 5-500 mg / ml.

[0163] The compositions of the present invention can be administered to a subject using a disposable package and a portable handheld battery-operated device such as the AERx device (US Pat. No. 5,823,178, Aradigm, Hayward, Calif.). Alternatively, the compositions of the present invention can be administered using a mechanical (non-electronic) device. Other inhalation devices can also be used to deliver the compositions, including conventional jet nebulizers, ultrasonic nebulizers, soft mist inhalers, dry powder inhalers (DPIs), metered dose inhalers (MDIs), condensation aerosol generators, and other systems.

[0164] For use as an aerosol, the compound of the present invention in solution or suspension may be packaged in a pressurized aerosol container with a suitable propellant, for example, a hydrocarbon propellant such as propane, butane or isobutane with conventional adjuvants.Dry powder inhalers are systems that can be operated with a pressurized air source to generate dry powder particles of pharmaceutical composition compressed into a very small volume.For inhalation, the system has multiple chambers or blisters, each of which contains a single dose of pharmaceutical composition and a selection element for releasing the single dose.

[0165] The aerosol may be generated by forcing the drug through pores in a membrane having a size in the range of about 0.25-6 μm (U.S. Patent No. 5,823,178). When the pores have this size, the particles that escape through the pores to generate the aerosol have a diameter in the range of 0.5-12 μm. The drug particles may be released with an airflow intended to keep the particles within this size range. The generation of small particles may be facilitated by the use of a vibration device that provides a vibration frequency in the range of about 800 to about 4000 kilohertz. Those skilled in the art will recognize that some adjustments can be made to parameters such as the size of the pores through which the drug is released, the vibration frequency, the pressure, and other parameters based on the density and viscosity of the composition, keeping in mind that the objective of some embodiments is to provide aerosolized particles having a diameter in the range of about 0.5-12 μm. Local delivery

[0166] The compositions of the present invention can be delivered topically. Topical administration can include administering a therapeutically effective amount of a preferred enantiomer of a 2-hydroxycarboxylic acid directly to the skin, eye or mucosal surface of a subject. Preferably, the preferred enantiomer of a 2-hydroxycarboxylic acid is applied topically to the skin, mucosa (oral, nasal, vaginal, rectal) or eye of a subject. Use can include administering a therapeutically effective amount of a preferred enantiomer of a 2-hydroxycarboxylic acid to the skin, mucosa (oral, nasal, vaginal, rectal) or eye of a subject.

[0167] The compositions of the invention may be administered topically. Thus, use herein contemplates compositions adapted for direct application to the skin.

[0168] In one form, a composition used for topical delivery may contain from 0.2% to 100% of a preferred enantiomer of a 2-hydroxycarboxylic acid, the remainder of the composition may include carriers, diluents or excipients, and / or other active agents.

[0169] In one form, the composition used for topical delivery may include the unpreferred enantiomer of 2-hydroxycarboxylic acid, and the % of the unpreferred enantiomer of 2-hydroxycarboxylic acid may be less than 20%. Alternatively, the composition of the present invention may not contain the unpreferred enantiomer of 2-hydroxycarboxylic acid. Thus, the composition may only include the preferred enantiomer of 2-hydroxycarboxylic acid as the 2-hydroxycarboxylic acid.

[0170] The composition may be in a form selected from the group including suspension, emulsion, liquid, cream, oil, lotion, ointment, gel, hydrogel, paste, plaster, roll-on liquid, skin patch, spray, glass bead dressing, synthetic polymer dressing and solid.For example, the composition of the present invention may be provided in the form of a water-based composition or ointment based on an organic solvent such as oil.Alternatively, the composition of the present invention may be applied by a liquid spray comprising at least a solvent in which the film-forming component and the preferred enantiomer of the 2-hydroxycarboxylic acid are dispersed or solubilized.

[0171] The compositions of the present invention may be provided in a form selected from the group including, but not limited to, a rinse, a shampoo, a lotion, a gel, a leave-on formulation, a wash-off formulation, and an ointment.

[0172] Depending on the preferred treatment regimen, various topical delivery systems may be suitable for administering the composition of the present invention.Topical compositions can be prepared by dissolving or combining the preferred enantiomer of the 2-hydroxycarboxylic acid of the present invention in an aqueous or non-aqueous carrier.In general, any liquid, cream or gel or similar substance that does not appreciably react with any other active ingredient that may be introduced into the compound or composition and is non-irritating is suitable.A suitable non-sprayable viscous, semi-solid or solid form that includes a carrier that is compatible with topical application and preferably has a dynamic viscosity greater than water can also be used.

[0173] Suitable compositions are well known to those skilled in the art and include, but are not limited to, solutions, suspensions, emulsions, creams, gels, ointments, powders, liniments, salves, aerosols, transdermal patches, and the like, optionally sterilized or mixed with auxiliary agents such as preservatives, stabilizers, emulsifiers, humectants, fragrances, colorants, odor control agents, thickening agents, such as natural gums, etc. Particularly preferred topical compositions include ointments, creams, or gels.

[0174] Ointments are generally prepared using either (1) an oleaginous base, i.e., composed of fixed oils or hydrocarbons, e.g., white petroleum, mineral oil, or (2) an absorbent base, i.e., composed of anhydrous substances or substances capable of absorbing water, e.g., anhydrous lanolin. Typically, after formation of the base, whether oleaginous or absorbent, the preferred 2-hydroxycarboxylic acid is added in an amount to give the desired concentration.

[0175] Creams are oil / water emulsions. They consist of an oil phase (internal phase) that typically contains fixed oils, hydrocarbons, etc., waxes, petroleum oils, mineral oils, etc., and an aqueous phase (continuous phase) that contains water and any water-soluble substances, e.g. added salts. The two phases are stabilized by the use of emulsifiers, e.g. surfactants such as sodium lauryl sulfite; hydrophilic colloids such as acacia colloidal clay, veegum, etc. Upon formation of the emulsion, the preferred enantiomer of the 2-hydroxycarboxylic acid can be added in an amount to achieve the desired concentration.

[0176] The gel comprises a base selected from an oil base, water, or an emulsion-suspension base. A gelling agent is added to the base to form a matrix in the base and increase its viscosity. Examples of gelling agents are hydroxypropyl cellulose, acrylic acid polymers, etc. Typically, the preferred enantiomer of the 2-hydroxycarboxylic acid is added to the composition at a desired concentration prior to the addition of the gelling agent.

[0177] Topical delivery compositions for application to mucosal surfaces (e.g., oral, vaginal, nasal or rectal mucosal surfaces) or skin wounds may contain 5% to 100% of a preferred enantiomer of a 2-hydroxycarboxylic acid. The remainder of the composition may include carriers, diluents or excipients, and / or other active agents. In one embodiment, higher dosages may be used in medically managed situations and lower dosages may be used for non-life threatening wounds treated at home.

[0178] In one form, the composition used for application to mucosal surfaces and / or skin wounds may include the non-preferred enantiomer of the 2-hydroxycarboxylic acid, and the % of the non-preferred enantiomer of the 2-hydroxycarboxylic acid may be less than 20%. Alternatively, the composition of the present invention may not contain the non-preferred enantiomer of the 2-hydroxycarboxylic acid. Thus, the composition may only include the preferred enantiomer of the 2-hydroxycarboxylic acid as the 2-hydroxycarboxylic acid.

[0179] The compositions of the present invention may be administered by topical ocular delivery. Preferably, the ophthalmic composition contains 0.001% to 1% of a preferred enantiomer of the 2-hydroxycarboxylic acid.

[0180] Ocular delivery includes delivery to the sclera, retina, intraocular fluids, periocular tissues, etc. For example, delivery can be topical (cream, gel, ointment, spray, eye drops), intraocular implant, or other means.

[0181] Ocular delivery may also include injecting the preferred enantiomer of 2-hydroxycarboxylic acid into the sclera, intraocular cavity, or the area behind the eye. Compositions suitable for ocular injection include, as appropriate, sterile aqueous solutions (if water-soluble) or dispersions, and sterile powders for extemporaneous preparation of sterile injectable solutions or dispersions. Alternatively, the compounds of the present invention are, in certain embodiments, encapsulated in liposomes and delivered in injectable solutions to aid their transport across cell membranes. Alternatively, or in addition, such preparations contain components of self-assembling pore structures to facilitate transport across cell membranes. Oral Delivery Compositions

[0182] The compositions of the present invention may be administered by oral delivery.

[0183] In one form, the orally delivered composition may contain 5% to 100% of a preferred enantiomer of the 2-hydroxycarboxylic acid, the remainder of the composition may include carriers, diluents or excipients, and / or other active agents.

[0184] In one form, the composition delivered orally may include the unpreferred enantiomer of the 2-hydroxycarboxylic acid, and the % of the unpreferred enantiomer of the 2-hydroxycarboxylic acid may be less than 20%. Alternatively, the composition of the present invention may not contain the unpreferred enantiomer of the 2-hydroxycarboxylic acid. Thus, the composition may only include the preferred enantiomer of the 2-hydroxycarboxylic acid as the 2-hydroxycarboxylic acid.

[0185] Subjects may typically be administered a dose of 0.1 mg / kg / day to 2 g / kg / day of the preferred enantiomer (+20% or +10%) of the 2-hydroxycarboxylic acid.

[0186] Despite the various pHs of the digestive system (acidic in the stomach, alkaline in the small intestine), the effectiveness of the preferred enantiomers of 2-hydroxycarboxylic acids against biofilms remains. The pH of the surrounding fluid does not affect the biofilm-disrupting ability of the preferred enantiomers of 2-hydroxycarboxylic acids. This is because 2-hydroxycarboxylic acids have been shown to remain stable in the gastrointestinal tract and maintain their functionality, which has been observed in many studies investigating the acid resistance of lactic acid bacteria (e.g. Wang et al., 2018, Archives of Microbiology, 200, 195-201) and lactic acidosis (metabolic acidosis resulting from over-fermentation in the digestive tract).

[0187] The oral compositions of the present invention can be delivered with immune enhancing or regulating agents. Examples of immune enhancing or regulating agents include specific immune stimulants (such as vaccines and antigens) and non-specific agents, such as bee products (including propolis and honey), probiotics and prebiotics, hormones, vitamins (vitamin c, vitamin d), minerals (zinc oxide), antioxidants (including glutathione), interferons (including INF-alpha), interleukins (including interleukin 10), colchicine, thalidomide and imiquimod. The immune booster can be delivered simultaneously with the composition comprising the preferred enantiomer of 2-hydroxycarboxylic acid or after the composition comprising the preferred enantiomer of 2-hydroxycarboxylic acid. subject

[0188] The subject may be any subject capable of infection by bacteria, viruses, fungi or archaea. The subject may be a mammal or bird. Preferably, the subject is selected from the group comprising humans, canines, birds, porcines, bovines, ovines, equines, rodents, mustelids, lagomorphs and felines. Most preferably, the subject is selected from the group comprising humans, bovines, porcines, equines, felines, rodents, mustelids, lagomorphs and canines. The subject may be a companion animal, a livestock animal or an animal of agricultural importance. Most preferably, the subject is a human. Excipients

[0189] The above-exemplified forms of the compositions described herein can be prepared by methods well known to those skilled in the art of composition science. In addition, the compositions described herein may optionally include other excipients to aid in the preparation and / or administration of the compositions described herein. In one embodiment, the compositions further include one or more pharma- ceutically or physiologically acceptable carriers, diluents, or excipients. Non-limiting examples of such excipients are well known in the art and include flavorings, colorings, palatability agents, antioxidants, viscosity adjusters, isotonicity agents, drug carriers, sustained release agents, comfort enhancers, emulsifiers, solubilization aids, lubricants, binders, and other stabilizing agents to aid in the preparation and / or administration of the compositions.

[0190] The compositions of the present invention are intended for use in pharmaceutical or veterinary applications, or in cleaning systems such as medical instruments, water pipes or food preparation equipment.

[0191] Preferably, the compositions are sterile. In addition to or instead of sterilization, the compositions of the present invention may contain a pharma- ceutically acceptable preservative to minimize the possibility of microbial contamination. In another embodiment, the compositions of the present invention are stable. A pharma- ceutically acceptable preservative may be used in the compositions to enhance the stability of the composition. However, it should be noted that any preservative must be selected for safety, as the treated tissue may be sensitive to irritants. Preservatives suitable for use herein include those that protect the solution from contamination by pathogens, including, but are not limited to, phenylethyl alcohol, benzalkonium chloride or benzoic acid, or a benzoate salt such as sodium benzoate and phenylethyl alcohol. In certain embodiments, the compositions of the present invention include about 0.001% to about 10.0% w / w benzalkonium chloride, or about 0.01% v / w phenylethyl alcohol. Preservatives may also be present in an amount of about 0.001% to about 1%, preferably about 0.002% to about 0.02%, more preferably 0.02% w / w.

[0192] The compositions provided herein may also contain from about 0.001% to about 90%, or from about 0.001% to about 50%, or from about 0.001% to about 25%, or from about 0.001% to about 10%, or from about 0.001% to about 1% of one or more emulsifying agents, wetting agents, or suspending agents.Such agents as used herein include, but are not limited to, polyoxyethylene sorbitan fatty esters or polysorbates, including polyethylene sorbitan monooleate (polysorbate 80), polysorbate 20 (polyoxyethylene (20) sorbitan monolaurate), polysorbate 65 (polyoxyethylene (20) sorbitan tristearate), polyoxyethylene (20) sorbitan monooleate, polyoxyethylene (20) sorbitan monopalmitate, polyoxyethylene (20) sorbitan monostearate; lecithin; agar; carrageenan; locust bean gum; guar gum; tragacanth; acacia; xanthan gum; karaya gum; pectin; amidated pectin; ammonium phosphatides; microcrystalline cellulose; methylcellulose; hydroxypropylcellulose; hydroxypropylmethylcellulose; ethylmethylcellulose; carboxymethylcellulose; sodium, potassium and calcium salts of fatty acids; monosodium salts of fatty acids; and the like. - and diglycerides;Acetate esters of mono- and diglycerides of fatty acids;Lactate esters of mono- and diglycerides of fatty acids;Citrate esters of mono- and diglycerides of fatty acids;Tartrate esters of mono- and diglycerides of fatty acids;Mono- and diacetyltartaric acid esters of mono- and diglycerides of fatty acids;Mixed acetate and tartaric acid esters of mono- and diglycerides of fatty acids;Sucrose esters of fatty acids;Sucroglycerides;Polyglycerol esters of fatty acids esters; polyglycerol esters of polycondensed fatty acids of castor oil; propane-1,2-diol esters of fatty acids; sodium stearoyl-21 actylate; calcium stearoyl-2-lactylate; stearoyl tartrate; sorbitan monostearate; sorbitan tristearate; sorbitan monolaurate; sorbitan monooleate; sorbitan monopalmitate; quillaja extract; polyglycerol esters of dimerized fatty acids of soybean oil; oxidatively polymerized soybean oil; and pectin extract.

[0193] The composition of the present invention may contain about 0.001% to about 5% (by weight) of a humectant to inhibit drying of the mucous membrane and prevent irritation. For example, any of a variety of pharma- ceutically acceptable humectants may be used, including sorbitol, propylene glycol, polyethylene glycol, glycerol, or mixtures thereof.

[0194] The present invention encompasses variations of the above compositions since the amount of each compound may vary by +5%, +7.5%, +10%, +15%, +17.5%, or +20%.

[0195] The present invention encompasses compositions in which the relative percentages of the active ingredient and / or each excipient are independently different from those specified above. In one form of the invention, the relative percentages of the active ingredient and / or each excipient vary independently from those specified above by up to 50%. In one form of the invention, the relative percentages of the active ingredient and / or each excipient vary independently from those specified above by up to 40%. In one form of the invention, the relative percentages of the active ingredient and / or each excipient vary independently from those specified above by up to 30%. In one form of the invention, the relative percentages of the active ingredient and / or each excipient vary independently from those specified above by up to 20%. In one form of the invention, the relative percentages vary independently from those specified above by up to 10%. In one form of the invention, the relative percentages of the active ingredient and / or each excipient vary independently from those specified above by up to 5%. In one aspect of the invention, the relative percentages vary independently by up to 10% from those specified above. In one aspect of the invention, the relative percentages of the active ingredient and / or each excipient vary independently by up to 2% from those specified above.

[0196] As will be appreciated by those skilled in the art, the sum of the percentages of excipients and active agents cannot exceed 100, and the above variations are subject to this limitation. As will be appreciated by those skilled in the art, the sum of the percentages of excipients and active agents may be less than 100, since the forms of the invention contain ingredients other than those specified.

[0197] The above variations are percentage variations in relative proportions. As an example, a 20% variation in the relative proportion of an ingredient (excipient or active agent) specified at 1% means that the relative proportion of that ingredient can be between 0.8% and 1.2%. Inhalation Delivery Vehicles

[0198] Compositions suitable for use in a nebulizer (either jet or ultrasonic) typically contain a preferred enantiomer of the 2-hydroxycarboxylic acid suspended in water or a non-aqueous solvent. The composition may also contain a buffer and a simple sugar (e.g., for stabilization and adjustment of osmotic pressure). Nebulizer compositions may also contain surfactants to reduce or prevent surface-induced aggregation of the preferred enantiomer of the 2-hydroxycarboxylic acid caused by atomization of the solution in forming the aerosol. The use of liposomes, microcapsules or microspheres, inclusion complexes, or other types of carriers is also contemplated.

[0199] Compositions for use with metered dose inhalers generally comprise a fine powder containing the preferred enantiomer of 2-hydroxycarboxylic acid suspended in a propellant with a surfactant. The propellant may be any conventional material used for this purpose, such as chlorofluorocarbons, hydrochlorofluorocarbons, hydrofluorocarbons, or hydrocarbons, including trichlorofluoromethane, dichlorodifluoromethane, dichlorotetrafluoroethanol, and 1,1,1,2-tetrafluoroethane, or combinations thereof. Suitable surfactants include sorbitan trioleate and soybean lecithin. Oleic acid may also be useful as a surfactant.

[0200] Compositions for dispensing from powder inhalation devices comprise a finely divided dry powder containing the preferred enantiomer of the 2-hydroxycarboxylic acid and may also contain a bulking agent such as lactose, sorbitol, sucrose or mannitol in an amount to facilitate dispersion of the powder from the device, e.g., 50-90% (by weight) of the composition. The preferred enantiomer of the 2-hydroxycarboxylic acid should be most advantageously prepared in particulate form having an average particle size of less than 10 microns, most preferably 0.5-5 microns, for most effective delivery to the distal lung.

[0201] In one embodiment, the compositions of the invention may include preservatives, suspending agents, wetting agents, tonicity agents and / or diluents. The compositions provided herein may include from about 0.01% to about 90%, or from about 0.01% to about 50%, or from about 0.01% to about 25%, or from about 0.01% to about 10%, or from about 0.01% to about 5% of one or more pharmacologically suitable suspensions that are physiologically acceptable when administered by inhalation. Pharmacologically suitable fluids for use herein include, but are not limited to, polar solvents (including, but not limited to, compounds containing hydroxyl or other polar groups). Solvents include, but are not limited to, water or alcohols, such as ethanol, isopropanol, and glycols, including propylene glycol, polyethylene glycol, polypropylene glycol, glycol ethers, glycerol, and polyoxyethylene alcohols. Polar solvents also include protic solvents, including, but not limited to, water, aqueous saline solutions containing one or more pharma-ceutically acceptable salts, alcohols, glycols, or mixtures thereof. In an alternative embodiment, water for use in the present compositions should meet or exceed applicable regulatory requirements for use in inhaled medications.

[0202] In one embodiment, the compositions described herein may be aqueous and contain 0-90% water. In other embodiments, the aqueous compositions described herein may contain 20-80% water. In yet other embodiments, the aqueous compositions may contain 50-70% water. The water may further comprise fresh water, distilled water, sterile water, demineralized water or deionized water. Alternatively, the compositions may be non-aqueous and contain no water or a negligible amount of water (e.g., less than 1%, less than 0.1%, less than 0.01%).

[0203] The compositions of the present invention may further comprise an adjuvant such as a bronchodilator, an anti-inflammatory agent, a surfactant, aspirin or ethyl alcohol.

[0204] Bronchodilators optionally used in the compositions of the present invention include, but are not limited to, β2-adrenergic receptor agonists (e.g., albuterol, bambuterol, salbutamol, salmeterol, formoterol, alloformoterol, levosalbutamol, procaterol, indacaterol, carmoterol, mirveterol, procaterol, terbutaline, etc.) and antimuscarinics (e.g., trospium, ipratropium, glycopyrronium, aclidinium, etc.). Combinations of drugs may also be used.

[0205] Anti-inflammatory drugs that may be optionally used in the compositions of the present invention include, but are not limited to, inhaled corticosteroids (e.g., beclomethasone, budesonide, ciclesonide, fluticasone, etiprednol, mometasone, etc.), leukotriene receptor antagonists and leukotriene synthesis inhibitors (e.g., montelukast, zileuton, ibudilast, zafirlukast, pranlukast, amelvant, tipelukast, etc.), cyclooxygenase inhibitors (e.g., ibuprofen, ketoprofen, ketorolac, indomethacin, naproxen, zaltoprofen, lornoxicam, meloxicam, celecoxib, lumiracoxib, etoricoxib, piroxicam, ampiroxicam, cinnoxicam, diclofenac, felbinac, lornoxicam, mesalazine, trifludal, tinoridine, iguratimod, pamicogrel, etc.). Combinations of drugs may also be used. Aspirin may be added to act as an anti-inflammatory agent.

[0206] Surfactants encompassed by the present invention include, but are not limited to, synthetic surfactants (Exosurf®), dipalmitoyl phosphatidylcholine and oleic acid. Combinations of drugs may be used. Antioxidants such as glutathione and vitamin E, zinc and the zinc salt of EDTA may be added.

[0207] Ethyl alcohol vapor can act as a defoamer in the lungs, making phlegm more liquid, aiding breathing, and reducing pulmonary edema. Ethanol can be added to the compositions of the present invention at 0.5% to 60%, more preferably 1 to 40%, 1 to 20%, or 1 to 10%. Ethanol can be added at 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, or 60%.

[0208] The present invention also relates to the use of the preferred enantiomer of a 2-hydroxycarboxylic acid in combination with other drugs given by inhalation. These other drugs may include nucleotide sequences that may be incorporated into a suitable delivery vector, such as a plasmid or viral vector.Other drugs include therapeutic nucleotide sequences (DNA, RNA, siRNA), enzymes that decrease the viscoelasticity of mucus, e.g. DNase and other mucolytic agents, chemicals that upregulate chloride ion channels or increase the flow of ions across cells, nicotine, P2Y2 agonists, elastase inhibitors, e.g. alpha-1 antitrypsin (AAT), N-acetylcysteine, antibiotics and cationic peptides, e.g. lantibiotics, specifically duramycin, short-acting bronchodilators (e.g. albuterol or indacaterol). β2-adrenergic receptor agonists such as β2-adrenergic receptor agonists (e.g., ipatropium bromide), K channel openers, long-acting bronchodilators (e.g., formoterol, salmeterol), steroids (e.g., budesonide, fluticasone, triamcinolone, beclomethasone, ciclesonide, etc.), xanthines, leukotriene antagonists (e.g., montelukast sodium), phosphodiesterase 4 inhibitors, adenosine receptor antagonists, various other anti-inflammatory drugs (e.g., Syk kinase inhibitors, (AVE-0950), tryptase inhibitors (AVE-8923 and AVE-5638), tachykinin antagonists (AVE-5883), inducible nitric oxide synthase inhibitors (GW-274150), etc.), transcription factor decoys, TLR-9 agonists, antisense oligonucleotides, siRNA, DNA, CGRP, lidocaine, inverse β2-agonists, anti-infective oxidative therapy, cytokine modulators (e.g., CCR3 receptor antagonists (GSK-766994, DPC-168, AZD-3778), TNF-α production inhibitors (LMP- 160 and YS-TH2), and IL-4 antagonists (AVE-0309), small molecule inhibitors of IgE, cell adhesion molecule (CAM) inhibitors, small molecules targeting the VLA4 receptor or integrin α.4.β.1 (e.g., R-411, PS-460644, DW-908e, and CDP-323), immunomodulators including those that block T cell signaling by inhibition of calcineurin (tacrolimus), heparin neutralizers (talactoferrin α), cytoplasmic PLA2 inhibitors (Efipladib), or combinations thereof.If a subject in need has CF, they may also be administered standard medications in combination with the compositions of the invention, such as ivacaftor, pulmozyme, mannitol, or other approved drugs in accordance with standard practice. Topical Delivery Vehicles

[0209] The compositions of the present invention comprising the preferred enantiomer of the 2-hydroxycarboxylic acid can be delivered to a topical surface. For example, the topical formulation can be for topical application to the skin, mucous membranes (oral, nasal, vaginal, rectal) or eyes of a subject.

[0210] The compositions of the present invention may contain water (aqueous) or may be non-aqueous.

[0211] The compositions of the present invention may also contain small amounts of conventional additives, such as viscosity modifiers, e.g., xanthan gum, and preservatives, e.g., phenoxyethanol or benzyl alcohol, including mixtures thereof. For some therapeutic agents, it may be necessary to incorporate a buffering agent to maintain the appropriate pH.

[0212] Suitable preservatives for use in such compositions or medicaments include, for example, phenoxyethanol and other preservatives conventionally used in pharmaceutical formulations, particularly creams. Suitable preservatives include methyl hydroxybenzoate, chlorocresol, sorbic acid and benzoic acid.

[0213] The compositions of the present invention can be prepared by conventional pharmaceutical techniques. Thus, ointments and creams are conveniently prepared by mixing the components that make up the vehicle together at elevated temperature, preferably 60-70°C, until an emulsion is formed. The mixture is then cooled to room temperature and the preferred enantiomer of the 2-hydroxycarboxylic acid is added along with any other ingredients, which may then be stirred to ensure adequate dispersion.

[0214] The therapeutic agent is dissolved in the components that make up the vehicle, and then other ingredients are added to produce liquid preparations such as nasal drops, eye drops, etc. The resulting solution or suspension is dispensed into glass or plastic bottles, or into single-dose packs such as soft gelatin capsules, which are then heat-sealed.

[0215] Artificial tear vehicles may be used in ophthalmic compositions that contain the preferred enantiomer of the 2-hydroxycarboxylic acid delivery. More viscous artificial tears use higher concentrations of thickening agents, such as Celluvisc®, a high viscosity carboxymethylcellulose (CMC) and Refresh Liquigel®, a blend of 0.35% high viscosity CMC and 0.65% low viscosity CMC.

[0216] Gelling agents may be used in compositions containing the preferred enantiomer of 2-hydroxycarboxylic acid delivered to the eye. Such agents may be instilled as liquids and then triggered into a gel phase almost immediately. Timoptic gel (gellan gum), AzaSite® (polycarbophil, poloxamer), and Besivance® (polycarbophil, poloxamer), 0.3% alginate Keltrol® are examples of such agents. Another gelling agent is polycarbophil poloxamer gel (e.g., Durasite®).

[0217] Ocular carriers are, in various embodiments, solvents or dispersion media containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, etc.), suitable mixtures thereof, and vegetable oils. Proper fluidity is maintained, for example, but not limited to, by the use of a coating such as lecithin, by controlling the required particle size in the case of dispersions, and by the use of surfactants. Prolonged absorption of injectable compositions is brought about in certain embodiments by the use in the composition of agents that delay absorption, for example, aluminum monostearate and gelatin.

[0218] Nasal delivery of the preferred enantiomer of the 2-hydroxycarboxylic acid is also contemplated. Nasal delivery allows the passage of the preferred enantiomer of the 2-hydroxycarboxylic acid into the bloodstream immediately after administration of the therapeutic product to the nose, without the need for deposition of the preferred enantiomer in the lungs. Compositions for nasal delivery include those that contain dextran or cyclodextran. Oral Delivery Vehicles

[0219] The compositions of the present invention that contain the preferred enantiomer of 2-hydroxycarboxylic acid can be delivered orally. For example, oral formulations can be for localized application to a specific part of the gastrointestinal system (such as the esophagus, stomach, or large or small intestine) or for systemic application, and can be delivered in a number of forms, including simple solutions, syrups, suspensions, tablets, or capsules.

[0220] The compositions of the present invention may contain water (aqueous) or may be non-aqueous.

[0221] The compositions of the present invention may also contain small amounts of conventional additives, such as preservatives, solubilizers (including ethanol), complexing agents (such as cyclodextrin), flow improvers and lubricants (including colloidal silica), flavor enhancers and compressibility improvers (including mixtures thereof). For some therapeutic agents, it may be necessary to incorporate a buffer to maintain the appropriate pH.

[0222] The oral compositions of the present invention can be delivered with immune enhancing or regulating agents. Examples of immune enhancing or regulating agents include specific immune stimulants (such as vaccines and antigens) and non-specific agents, such as bee products (including propolis and honey), probiotics and prebiotics, hormones, vitamins (vitamin c, vitamin d), minerals (zinc oxide), antioxidants (including glutathione), interferons (including INF-alpha), interleukins (including interleukin 10), colchicine, thalidomide and imiquimod. The immune booster can be delivered simultaneously with the preferred enantiomer of the 2-hydroxycarboxylic acid composition or after the preferred enantiomer of the 2-hydroxycarboxylic acid composition.

[0223] Without being bound by any theory, it is believed that administration of a preferred enantiomer of a 2-hydroxycarboxylic acid can sufficiently disrupt biofilms in the body such that co-administration or subsequent administration of an immune enhancing agent allows the body to eliminate the bacteria in the disrupted biofilm without the use of additional agents such as antibiotics. stability

[0224] Preferably, the compositions of the present invention are stable. As used herein, the stability of the compositions provided herein refers to the length of time that at a given temperature, more than 80%, 85%, 90% or 95% of the initial amount of antibiotic-lactic acid is present in the composition. For example, the compositions provided herein can be stored at about 15°C to about 30°C and remain stable for at least 1, 2, 12, 18, 24 or 36 months. The compositions can also be suitable for administration to a subject in need thereof after storage at 250°C for more than 1, 2, 12, 18, 24 or 36 months. In another alternative embodiment, using Arrhenius kinetics, more than 80%, or more than 85%, or more than 90%, or more than 95% of the initial amount of active agent (e.g., preferred enantiomer of 2-hydroxycarboxylic acid) remains after the composition is stored at about 15°C to about 30°C for more than 1, 2, 12, 18, 24 or 36 months.

[0225] As used herein, a statement that a composition is stable during "long-term storage" means that the composition is suitable for administration to a subject in need thereof if it has an estimated shelf life of greater than 1, 2 or 3 months of use at 25° C. and greater than 1, 2 or 3 years of storage at 5° C. In certain embodiments herein, an estimated >80% or >85% or >90% or >95% of the preferred enantiomer of the 2-hydroxycarboxylic acid remains after such storage using Arrhenius kinetics. Antibiotics

[0226] Other active agents may also be incorporated into the compositions of the present invention, for example additional antimicrobial agents such as antibacterial agents, antifungal agents, and the like.

[0227] The additional active agent provided with the preferred enantiomer of the 2-hydroxycarboxylic acid of the present invention may be an active agent that kills bacteria, but is not an antibiotic. For example, the additional active agent may be a chlorine-based compound (such as bleach), an iodine-based compound, a copper-based compound, or the like. The antibacterial compound may be an agent with a different antibacterial mode of action, such as a bacteriophage, an antiseptic, saline, a chlorine-based compound (such as bleach), an iodine-based compound, a copper-based compound, or a heavy metal.

[0228] The composition may further comprise benzoyl peroxide or an antibiotic such as erythromycin, clindamycin, doxycycline or meclocycline.

[0229] Additional antimicrobial agents that may be used include, but are not limited to, silver compounds (e.g., silver chloride, silver nitrate, silver oxide), silver ions, silver particles, iodine, povidone / iodine, chlorhexidine, 2-p-sulfanylanilinoethanol, 4,4'-sulfinyldianiline, 4-sulfanilamide salicylic acid, acetosulfone, acetosulfone, amikacin, amoxicillin, amphotericin B, ampicillin, apalcillin, apicycline, apramycin, arbekacin, aspaxicillin, azidanphenicol, azithromycin, aztreona. , bacitracin, bambermycin(s), biapenem, brodimoprim, butirocin, capreomycin, carbenicillin, carbomycin, carumonam, cefadroxil, cefamandole, cefatrizine, cefbuperazone, cefclidin, cefdinir, cefditoren, cefepime, cefetamet, cefixime, cefinenoxime, cefminox, cefodizime, cefonicid, cefoperazone, ceforanide, cefotaxime, cefotetan, cefotiane, cefozopran, cefpimizole, cefpiramide, cefpirome, cef Prozil, cefroxadine, ceftazidime, cephalam, ceftibuten, ceftriaxone, cefuzonam, cephalexin, cephaloglycin, cephalosporin C, cephaphazine, chloramphenicol, chlortetracycline, ciprofloxacin, clarithromycin, clinafloxacin, clindamycin, chromocycline, colistin, cyclacillin, dapsone, demeclocycline, diathymosulfone, dibekacin, dihydrostreptomycin, dirithromycin, doxycycline, enoxacin, enviomycin, episilicin , erythromycin, flomoxef, fortimycin(s), gentamicin(s), glucosulfone solasulfone, gramicidin S, gramicidin(s), grepafloxacin, guamecycline, hetacillin, imipenem, isepamicin, josamycin, kanamycin(s), leucomycin(s), lincomycin, lomefloxacin, rucimycin, lymecycline, meclocycline, meropenem, methacycline, micronomycin, midecamycin(s), minocycline, moxalactam, mupirocin,Nadifloxacin, natamycin, neomycin, netilmicin, norfloxacin, oleandomycin, oxytetracycline, p-sulfanylbenzylamine, panipenem, paromomycin, pazufloxacin, penicillin N, pipacycline, pipemidic acid, polymyxin, primycin, quinacillin, ribostamycin, rifamide, rifampin, rifamycin SV, rifapentine, rifaximin, ristocetin, ritipenem, rokitamycin, rolitetracycline, rosaramycin, roxithromycin, salazosulfapyrimidine, sancycline, sisomicin, sparfloxacin, spectinomycin, spiramycin, streptomycin, succisulfone, sulfachrysoidine, sulfaloxic acid, sulfamidokaidin, sulfani. tetracycline, tetroxoprim, thiamphenicol, thiazole sulfone, thiostrepton, ticarcillin, tigemonam, tobramycin, tosufloxacin, trimethoprim, trospectomycin, trovafloxacin, tubeactinomycin, vancomycin, azaserine, candicidin(s), chlorphenesin, dermostatin(s), filipin, fungichromin, mepartricin, nystatin, oligomycin(s), ciprofloxacin, norfloxacin, ofloxacin, pefloxacin, enoxacin, rosofloxacin, amifloxacin, fleroxacin, temafloxacin, lomefloxacin, perimycin A or tubercidin. use

[0230] The present invention provides the use of a preferred enantiomer of a 2-hydroxycarboxylic acid, a preferred 2-hydroxycarboxylic acid, in the manufacture of a composition for the disruption of a biofilm.

[0231] The present invention further provides the use of a preferred enantiomer of a 2-hydroxycarboxylic acid for the disruption of a biofilm.

[0232] The present invention provides the use of a preferred enantiomer of a 2-hydroxycarboxylic acid in the manufacture of a composition for the disruption of biofilms in combination with an antimicrobial compound.

[0233] The present invention further provides the use of a preferred enantiomer of a 2-hydroxycarboxylic acid for the disruption of biofilms in combination with an antimicrobial compound.

[0234] The present invention further provides the use of a preferred 2-hydroxycarboxylic acid in the manufacture of a composition for treating or preventing an infection in a subject, wherein the infection is associated with a biofilm, and wherein the composition disrupts the biofilm.

[0235] The present invention further provides the use of a preferred enantiomer of a 2-hydroxycarboxylic acid in combination with an antimicrobial compound in the manufacture of a composition for treating or preventing an infection in a subject, wherein the infection is associated with a biofilm, and wherein the composition disrupts the biofilm.

[0236] The present invention further provides the use of a preferred enantiomer of a 2-hydroxycarboxylic acid to treat or prevent an infection in a subject, wherein the infection is associated with a biofilm, and wherein the composition disrupts the biofilm.

[0237] The present invention further provides the use of a preferred enantiomer of a 2-hydroxycarboxylic acid in combination with an antimicrobial compound to treat or prevent an infection in a subject, wherein the infection is associated with a biofilm and the composition disrupts the biofilm.

[0238] The present invention further provides the use of a preferred enantiomer of a 2-hydroxycarboxylic acid to disrupt or prevent the formation of a biofilm on an abiotic surface.

[0239] The present invention further provides the use of a preferred enantiomer of a 2-hydroxycarboxylic acid in combination with an antimicrobial compound to disrupt or prevent the formation of biofilms on abiotic surfaces.

[0240] The present invention further provides the use of a preferred enantiomer of a 2-hydroxycarboxylic acid in the manufacture of a composition for treating or preventing microbial colonization of a surface, wherein the microbial colonization is associated with a biofilm, and wherein the composition disrupts the biofilm.

[0241] The present invention further provides the use of a preferred enantiomer of a 2-hydroxycarboxylic acid in combination with an antimicrobial compound in the manufacture of a composition for treating or preventing microbial colonization of a surface, wherein the microbial colonization is associated with a biofilm and the composition disrupts the biofilm.

[0242] The present invention further provides the use of a preferred enantiomer of a 2-hydroxycarboxylic acid to treat or prevent microbial colonization of a surface, wherein the microbial infection is associated with a biofilm and the composition disrupts the biofilm.

[0243] The present invention further provides the use of a preferred enantiomer of a 2-hydroxycarboxylic acid in combination with an antimicrobial compound to treat or prevent microbial colonization of a surface, where the microbial colonization is associated with a biofilm and the composition disrupts the biofilm.

[0244] The preferred enantiomer of the 2-hydroxycarboxylic acid may be in the same composition as the antibacterial compound or in a separate composition. The antibacterial compound may be an antibiotic or an agent with a different antibacterial mode of action, such as a bacteriophage, an antiseptic, saline, a chlorine-based compound (such as bleach), an iodine-based compound, a copper-based compound, or a heavy metal. The biofilm may be an infection of a subject or a colonization of a surface, such as an abiotic surface. The biofilm may be a free-floating biofilm-associated aggregate of bacteria.

[0245] When the preferred enantiomer of the 2-hydroxycarboxylic acid is in a composition separate from the antibacterial compound, the preferred enantiomer of the 2-hydroxycarboxylic acid may be administered at the same time as the antibacterial compound or may be administered at a different time than the antibacterial compound.

[0246] For the above uses, the preferred enantiomer of the 2-hydroxycarboxylic acid may be D-lactic acid.Preferably, the infection or colonization is an infection or colonization by a bacteria. kit

[0247] The present invention relates to a kit for disrupting a biofilm, comprising: a) a preferred enantiomer of a 2-hydroxycarboxylic acid, and b) Providing a kit including instructions for use.

[0248] The present invention relates to a kit for disrupting a biofilm, comprising: a) a preferred enantiomer of a 2-hydroxycarboxylic acid, and b) providing a kit containing instructions for use; Here, a preferred enantiomer of a 2-hydroxycarboxylic acid is in combination with an antibacterial compound.

[0249] The preferred enantiomer of the 2-hydroxycarboxylic acid of the kit may be D-lactic acid.

[0250] When the components of the kit are provided in one or more liquid solutions, the liquid solution can be an aqueous solution, for example a sterile aqueous solution. For in vivo use, the expression construct can be formulated into a pharma-ceutically acceptable injectable composition. In this case, the container means can itself be an inhaler, syringe, pipette, eye dropper, or other such device, from which the composition can be applied to the affected area of ​​an animal, such as the lungs, injected into an animal, or even applied to and mixed with other components of the kit.

[0251] In one embodiment, the kit of the invention comprises a composition comprising a preferred enantiomer of a 2-hydroxycarboxylic acid, optionally in combination with an antimicrobial agent, hi an alternative embodiment, the composition is pre-measured, pre-mixed, and / or pre-packaged.

[0252] The kit of the present invention may also include instructions designed to facilitate user compliance. Instructions as used herein refers to any label, insert, etc., and may be placed on one or more surfaces of the packaging material, or the instructions may be provided on a separate sheet, or any combination thereof. For example, in one embodiment, the kit of the present invention includes instructions for administering the composition of the present invention. In one embodiment, the instructions indicate that the composition of the present invention is suitable for disrupting biofilms. Such instructions may also include dosage instructions as well as administration instructions.

[0253] The preferred enantiomer of the 2-hydroxycarboxylic acid and suitable excipients can be packaged separately, allowing the practitioner or user to formulate the ingredients into a pharma- ceutically acceptable composition as needed. Alternatively, the antisense oligomer and suitable excipients can be packaged together, thereby requiring minimal formulation by the practitioner or user. In either case, the packaging should maintain the chemical, physical, and aesthetic integrity of the active ingredient. General

[0254] Those skilled in the art will understand that the invention described herein is susceptible to variations and modifications other than those specifically described. The invention includes all such variations and modifications. The invention also includes all of the steps, features, compositions and compounds referred to or indicated herein, individually or collectively, and any and all combinations or any two or more steps or features.

[0255] Each document, reference, patent application, or patent cited in this document is expressly incorporated herein by reference in its entirety, meaning that it should be read and considered by the reader as part of this document. It is for the sake of brevity only that documents, references, patent applications, or patents cited in this document are not repeated in this document.

[0256] Any manufacturer's instructions, descriptions, product specifications, and product sheets for any products mentioned in this specification, or in any document incorporated by reference herein, are incorporated herein by reference and may be used in the practice of this invention.

[0257] The scope of the present invention is not limited by any of the specific embodiments described herein. These embodiments are for illustrative purposes only. Functionally equivalent products, compositions and methods are clearly within the scope of the invention described herein.

[0258] The invention described herein can include one or more ranges of values ​​(e.g., size, displacement, field strength, etc.). A range of values ​​is understood to include all values ​​within the range, including the values ​​defining the range and the values ​​adjacent to the range that produce the same or substantially the same results as the values ​​immediately adjacent to the values ​​defining the boundaries to the range. Thus, unless indicated to the contrary, the numerical parameters set forth in this specification and claims are approximations that may vary depending on the desired properties sought to be obtained by the present invention. Thus, "about 80%" means "about 80%" and also means "80%". At the very least, each numerical parameter should be construed in light of the number of significant digits and ordinary rounding approaches.

[0259] Throughout this specification, unless the context requires otherwise, the word "comprise" or variations such as "comprises" or "comprising" are understood to mean the inclusion of a recited integer or group of integers but not the exclusion of any other integer or group of integers. Also, in this disclosure, particularly in the claims and / or paragraphs, it is noted that terms such as "comprises," "comprised," "comprising," and the like can have the meaning ascribed to them in U.S. patent law. For example, they can mean "includes," "included," "including," and the like. Terms such as "consisting essentially of" and "consists essentially of" have the meaning ascribed to them in U.S. patent law, for example, they allow for elements not expressly recited, but exclude elements found in the prior art or that affect a basic or novel characteristic of the invention.

[0260] Other definitions of selected terms used herein can be found in the detailed description of the present invention and can be applied throughout.Unless otherwise defined, all other scientific and technical terms used herein have the same meaning as commonly understood by those skilled in the art to which the present invention belongs.The term "active agent" can mean one active agent or can include two or more active agents.

[0261] The following examples serve to more fully describe the manner of using the above-described invention, as well as to set forth the best modes contemplated for carrying out various aspects of the invention, it being understood that these methods in no way serve to limit the true scope of the invention, but rather are presented for illustrative purposes. EXAMPLES

[0262] Working Example Further features of the present invention are more fully described in the following non-limiting examples, which are included solely for the purpose of illustrating the invention and should not be construed as a limitation on the broad description of the invention as set forth above. Example 1 Application of prebiotic-derived drugs for biofilm destruction

[0263] This study demonstrates that a prebiotic solution derived from cultures grown from a commercially available probiotic capsule disrupts biofilm-protecting bacterial microcolonies. The extent of disruption was found to be comparable to that caused by gold-standard antibiotic therapy prescribed to treat this bacterial strain. material and method Preparation of prebiotic test solutions

[0264] The powdered contents of a commercial multi-strain probiotic capsule (45 billion CFU, see Table 1 for contents) were added to 10 ml of lysogeny broth (LB) and incubated overnight at 37°C and 5% CO2 (initial culture, C0). Table 1: Bacteria present in multi-strain probiotic capsules [Table 1-2]

[0265] Cultures were grown to stationary phase. Stationary phase refers to the time in a closed culture system when the number of new cells being produced in the culture is equal to the number of cells dying. This occurs shortly after the exponential growth phase due to nutrient depletion and waste accumulation. During the stationary phase, the bacteria remain metabolically active, but overall culture growth ceases, resulting in a flattening of the growth curve.

[0266] Cultures were grown to stationary phase to induce production of secondary metabolites (metabolites produced after active growth, such as antibiotics). Substrates generated from this phase were predicted to have potential biofilm disruption capabilities. Stationary phase culture (C1) was achieved by looping from the initial culture (C0) into 5 ml of LB broth and incubated overnight at 37 °C, 5% CO2 to generate the resulting culture C1. Both the resulting multi-strain bacterial culture and the prebiotic test solution collected from C1 were evaluated for their ability to disrupt the biofilm matrix of biofilm-protecting bacterial microcolonies. Biofilm-protected bacterial microcolony culture

[0267] Laboratory strains of Pseudomonas aeruginosa (WACC91 and PA-001) were thawed (stored as 50% glycerol stocks at -70 °C ± 2 °C) and streaked onto blood agar plates. These were grown for 12-24 hours at 37 °C, 5% CO2 until isolated colonies were large enough that they could be selected to maintain further culture. Isolated colonies were looped into 5 ml of LB broth and incubated overnight at 37 °C, 5% CO2 with gentle shaking to obtain stationary phase (B0). In this case, cultures were grown to stationary phase to induce the stress response necessary to induce biofilm formation when seeded for treatment.

[0268] The B0 culture was diluted 1:1000 in Pseudomonas minimal medium containing 2% arginine (referred to herein as PMM) to obtain approximately 10 8 cfu / ml was achieved (confirmed by measuring 0.02 absorbance units at 600 nm, approximately 1.8 ul in 2 ml PMM). The diluted B0 culture was mixed well and plated at 200 ul / well into 96-well microtiter plates. Biofilm formation was complete approximately 24-48 hours after plating, as confirmed by a minimum of 1.8 absorbance units at 600 nm. Biofilm destruction treatment

[0269] The prebiotic solution harvested from C1 was applied to the B0 cultures that had achieved biofilm formation. A 10-dose dilution series of the prebiotic test solutions in PMM was prepared (Table 2) and added to each B0 culture in a total volume of 50 μl to determine the optimal dose range for biofilm disruption in the B0 cultures. Table 2: Ten-dose dilution series of prebiotic test solutions in PMM. [Table 2-2]

[0270] Twelve-fold serial dilutions of tobramycin (99% pure tobramycin sulfate powder, Research Products International) standard were prepared in PMM (8ng / ml to 16ug / ml) to act as a reference point for evaluating the biofilm disruption activity observed by the prebiotic test treatments (Table 2). Tobramycin is an antibiotic commonly used to treat chronic respiratory bacterial infections, particularly Pseudomonas aeruginosa. A total of 50ul of tobramycin standard was added to the B0 cultures that had achieved biofilm formation. The target concentration of bioactive tobramycin in the epithelial lining fluid of pediatric patients with cystic fibrosis is 20μg / ml (Rosenthal et al., 2001), while the antibiotic resistance breaking point for tobramycin is 16μg / ml, which is the maximum dose used across studies investigating tobramycin activity (Clinical and Laboratory Standards Institute, 2005). Biofilm destruction evaluation i. Crystal violet staining

[0271] After the plate wells were cleared and washed by immersion in water, biofilms were stained with 0.1% w / v crystal violet (CV) in water for each well of the microtiter plate (O'Toole 2011). After incubation at room temperature for 15 min, the microtiter plate was washed 3-4 times with water by immersion in water and blotting to remove all excess cells and dye from the plate. The plate was allowed to dry overnight. The CV staining solution was solubilized with 125ul of 30% acetic acid, and the absorbance, which indicates biofilm density (also called biofilm biomass), was quantified at 550nm after incubation at room temperature for 15 min. ii. Resazurin staining

[0272] Resazurin staining was used to measure changes in bacterial metabolic activity after treatment (Kirchner et al., 2012). 10 μl of 0.02% (v / v) resazurin (diluted in distilled water) was added to each well and the microtitre plates were incubated under aerobic conditions at 37 °C for 1-2 h with shaking at 150 rpm. Viable cells reduce the blue resazurin dye to the pink fluorescent resorufin form. After incubation, fluorescence was quantified with an excitation wavelength of 540 nm and an emission wavelength of 590 nm using an Enspire microplate reader. statistical analysis

[0273] All data presented represent a minimum of 12 independent experimental replicates containing technical duplicates unless otherwise stated. Student's t-test was used to assess significance compared to control untreated biofilms, with significance measured at p<0.05. result

[0274] The five most concentrated dilutions of the prebiotic test solution induced a 70-80% reduction in crystal violet staining of WACC91 and PA01 laboratory strains of P. aeruginosa biofilms compared to untreated biofilms (n=12 biological replicates, Figure 1). The results showed a significant reduction in biofilm density compared to untreated biofilm cultures within the same experiment (n=12, p<0.0001).

[0275] The reduction in biofilm density induced by the prebiotic test solutions was comparable to the density reduction (80-90%) observed in the same biofilm-protected P. aeruginosa microcolonies treated with the antibiotic tobramycin (16ug / ml, bioactive dose). This demonstrates that the prebiotic test solutions were able to reduce biofilm density approaching the efficacy achieved by the bioactive dose observed for tobramycin, the antibiotic most commonly prescribed to treat invasive P. aeruginosa infections.

[0276] To quantify the effect of the prebiotic solutions and tobramycin on the metabolic activity of biofilm-protected cultures, resorufin fluorescence was measured. A 90% inhibition of metabolic activity was observed for cultures treated with the prebiotic test solutions for the four most concentrated dilutions (Figure 2). This was comparable to the inhibition observed for the bioactive dose of tobramycin (16ug / ml), indicating a significant disruption of bacterial activity when treated with the prebiotic solutions.

[0277] The prebiotic test solutions were able to reduce P. aeruginosa biofilm biomass as effectively as the most commonly prescribed antibiotics for invasive P. aeruginosa infections, with a significant effect on baseline metabolic activity. Example 2 D-Lactic acid is an active biofilm disrupter

[0278] The active agents causing biofilm disruption in Example 1 were investigated by testing the biofilm disruption of biofilm-protected P. aeruginosa (WACC91) microcolonies in response to HPLC fractionation until a single peak was identified. Mass spectrometry and nuclear magnetic resonance were used to identify the content of the single peak. material and method HPLC fractionation

[0279] Bioactivity analysis of incremental ethanol solute fractions, as determined by high performance liquid chromatography (HPLC), was performed until single compound resolution was reached. Bioassays to evaluate biofilm disruption of prebiotic HPLC fractions Biofilm microcolony culture

[0280] A laboratory strain of Pseudomonas aeruginosa WACC91 was thawed, streaked onto a blood agar plate, and grown for 12-24 h at 37 °C, 5% CO2 until adequate growth was achieved to allow further culturing. An isolated colony (W0) was looped into 5 ml of LB broth and incubated overnight at 37 °C, 5% CO2 with gentle shaking to obtain stationary phase.

[0281] The W0 culture was diluted 1:1000 into Pseudomonas minimal medium containing 2% arginine (referred to herein as PMM) to obtain approximately 10 8 cfu / ml was achieved (verified by measuring 0.02 absorbance units at 600 nm, approximately 1.8 ul in 2 ml PMM). The diluted W0 cultures were mixed well and plated at 200 ul / well into 96-well microtiter plates. Biofilm formation was complete approximately 24-48 hours after plating, as determined by a minimum of 1.8 absorbance units at 600 nm. process

[0282] The HPLC fractions were added to biofilm microcolonies to assess biofilm disruption activity after overnight treatment at 37° C., 5% CO2 (50 μl added to each culture). Each HPLC fraction treatment was performed in duplicate. Assessment of biofilm destruction i. Crystal violet staining

[0283] After the plate wells were cleared and washed by immersion in water, each well of the microtiter plate was stained for biofilms with 0.1% w / v crystal violet (CV) in water (O'Toole 2011). After 15 min incubation at room temperature, the microtiter plate was washed 3-4 times with water by immersion in water and blotting to remove all excess cells and dye from the plate. The plate was allowed to dry overnight. The CV stain was solubilized with 125ul of 30% acetic acid and the absorbance, indicative of biofilm biomass, was quantified at 550nm after 15 min incubation. Bioactivity analysis

[0284] A minimum of two bioactivity assays were completed for each HPLC fraction. For untreated control samples, assay controls and technical duplicates were included in each evaluation. Nuclear magnetic resonance

[0285] Nuclear magnetic resonance (NMR) was used to identify the compounds in the final HPLC peak. result

[0286] The prebiotic test solutions were dissolved in ethanol and the aqueous and non-aqueous extracts were evaluated for biofilm bioactivity. Biofilm disruption was only observed for the aqueous extracts, precluding the identification of small protein biofilm disrupting agents.

[0287] Fractionation of the ethanolic aqueous extract by HPLC revealed biofilm disrupting activity in a single fraction (Figure 2). Mass spectrometry and nuclear magnetic resonance (NMR) of this fraction identified lactic acid as the single component of this major peak.

[0288] Optical rotation analysis of this compound showed that 95% of the content was D-lactic acid, the rarer isomeric form of lactic acid (Figure 2).

[0289] Pure grade D-lactic acid standards were prepared (Sigma ThermoFisher) and run against the prebiotic test solution dilutions used to treat biofilm-protected microcolonies (using the method in Example 1) to determine the active dose range of D-lactic acid, which was measured in the range of 0.25-1.25 mg / ml.

[0290] D-Lactic acid is the active agent in the probiotic extract that reduces P. aeruginosa biofilm density at an active dose range of 0.25-1.25 mg / ml. Example 3 D-Lactic acid biofilm disruption activity is not toxic to human cells

[0291] The bactericidal activity, cytotoxicity of D-lactate towards human cells, and the effect of pH on D-lactate biofilm disruption activity were evaluated.

[0292] The resorufin fluorescence data from Example 1 showed an effect of D-lactate on bacterial metabolic activity, but it was unclear from this data whether this was bactericidal. A bacterial bioassay designed for high-throughput screening of antibiotics was used to evaluate the bactericidal activity of D-lactate and L-lactate as outlined below. The ability of D-lactate to remain active at physiological pH without causing cytotoxicity is a key data point for demonstrating the therapeutic potential of D-lactate. material and method Bactericidal activity

[0293] Using flow cytometry (FCM) and a modified FAST analysis pipeline, the antibacterial activity of D-lactate and L-lactate was evaluated by quantifying changes in planktonic (non-biofilm) bacterial cell numbers and phenotypes of E. coli (ATCC 25922), P. aeruginosa (ATCC 27853), and S. aureus (ATCC 33592) upon processing into concentration gradients. This was performed in parallel with a conventional broth microdilution (BMD) assay. The minimum inhibitory concentration (MIC) of D-lactate and L-lactate was found to be 4 g / L for all strains.

[0294] Two-fold serial dilutions of D- or L-lactate were performed in 96-well plates in a volume of 50 μL per well, ranging from 0.008 grams / liter to 8 g / L. Each replicate dilution series had an additional control well containing 50 μL of assay medium without lactate (CAMHB) to serve as a non-exposed control.

[0295] Bacterial suspensions were prepared from cultures grown overnight in Trypticase Soya Broth (TSB). Each culture was diluted 1:1000 in Hank's Buffered Saline (HBSS), stained with 1 μL of SYTO® 9, incubated with shaking for a minimum of 8 minutes, and counted by flow cytometry. The TSB cultures were then diluted to 2 × 10 5 Adjusted to cells / mL.

[0296] A replicate dilution series in lactic acid was added with 50 µL of bacterial suspension per well in a final volume of 100 µL, with each row containing a concentration gradient ranging from 0.004 to 4 g / L. There were three organisms per plate (E. coli, P. aeruginosa, S. aureus) set up in duplicate. Plates also contained three wells containing 50 µL of CAMHB and 10 µL of 12% formalin, to which 50 µL of each bacterial suspension was added individually. These wells were representative of the suspension density at the time of inoculation.

[0297] The plates were incubated at 35.5°C. For conventional BMD, the incubation time was 24 hours. For flow cytometry, the incubation time was 3 hours. For flow cytometry, after incubation, 100 μL of HBSS containing SYTO® 9 at a concentration of 10 μM was added to each well of the plate for a final dye concentration of 5 μM. The plates were then incubated in the dark at ambient temperature for 10 minutes, loaded into the Attune™ autosampler, and data was acquired. The plates were run simultaneously on two different Attune™ cytometers. Cytotoxicity of D-Lactic Acid

[0298] D-lactate cytotoxicity was evaluated against transformed human airway epithelial cell cultures. A549 and BEAS-2B human airway epithelial cells were seeded at 60,000 cells / ml and grown to 70% confluence in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum containing 0.05% gentamicin.

[0299] The observed active dose range for biofilm disruption (0.5-1.25mg / ml) was used to assess cytotoxic effects by trypan blue microscopy of cell exclusion after 24 hours. Briefly, 50ul of 0.5, 1 and 1.25mg / ml D-lactate doses were added to cell cultures, which were incubated for 24 hours at 37°C in 5% CO2. After incubation, 50ul of 0.4% (w / v in ethanol) trypan blue solution was added to each well and incubated for 10 minutes at room temperature. Once trypan blue penetrates the compromised cell walls of dead cells, trypan blue positive cells are identified as dead cells which can be counted and used to quantify the overall portion of dead cells in the cultures for treatment comparisons. Effect of pH on the ability of D-lactate to disrupt biofilms Biofilm microcolony culture

[0300] A laboratory strain of Pseudomonas aeruginosa WACC91 was thawed, streaked onto a blood agar plate, and grown for 12-24 h at 37 °C, 5% CO2 until adequate growth was achieved to allow further culturing. An isolated colony (W0) was looped into 5 ml of LB broth and incubated overnight at 37 °C, 5% CO2 with gentle shaking to obtain stationary phase.

[0301] The W0 culture was diluted 1:1000 into Pseudomonas minimal medium containing 2% arginine (referred to herein as PMM) to obtain approximately 10 8 cfu / ml was achieved (verified by measuring 0.02 absorbance units at 600 nm, approximately 1.8 ul in 2 ml PMM). The diluted W0 cultures were mixed well and plated at 200 ul / well into 96-well microtiter plates. Biofilm formation was complete approximately 24-48 hours after plating, as determined by a minimum of 1.8 absorbance units at 600 nm. Treatment with D-lactic acid buffered to pH 2.4 and 7

[0302] D-Lactic acid stock solutions were buffered to pH 2.4 with 5 M hydrochloric acid and to pH 7 with 5 M sodium hydroxide before dilution with PMM for use. Assessment of biofilm destruction i. Crystal violet staining

[0303] After the plate wells were cleared and washed by immersion in water, the biofilms were stained with 0.1% w / v crystal violet in water for each well of the microtiter plate (O'Toole 2011). After 15 min incubation at room temperature, the microtiter plate was washed 3-4 times with water by immersion in water and blotting to remove all excess cells and dye from the plate. The plate was allowed to dry overnight. The CV stain was solubilized with 125ul of 30% acetic acid and the absorbance, indicative of biofilm biomass, was quantified at 550nm after 15 min incubation. statistical analysis

[0304] All data presented represent a minimum of three independent experimental replicates containing technical duplicates unless otherwise stated. Student's t-test was used to assess significance compared to control untreated biofilms, with significance measured at p<0.05. result Bactericidal activity

[0305] L- and D-lactic acid induced similar trends in phenotypic changes and overall event counts in E. coli, P. aeruginosa, and S. aureus. After correcting for time-dependent growth (some growth may occur during data acquisition), we observed a proportional increase in bacterial event counts with increasing concentrations up to 0.5 g / L for both compounds. These data suggest that both compounds have a stimulatory effect on bacterial growth from 0.008 g to 0.5 g / L.

[0306] No significant phenotypic changes were observed in E. coli, P. aeruginosa, and S. aureus up to 1 g / L, where a significant reduction in SYTO® 9 fluorescence was observed, and no change in forward scatter. This trend continued up to 2 g / L. At 2 g / L, we observed an overall reduction in total bacterial counts. This trend continued at 4 g / L, where another sharp reduction in bacterial counts was observed, consistent with a very large shift in bacterial populations, with both mean forward scatter (FSC) and SYTO® 9 fluorescence being reduced. The distribution of the populations by SYTO® 9 fluorescence increased, with clusters losing cohesion and becoming more diffuse. This change in phenotype was consistent with the occurrence of early or incomplete cell lysis, suggesting a toxic effect of the D- and L-isomers at doses above 4 g / L for E. coli, P. aeruginosa, and S. aureus. toxicity

[0307] There is no significant difference between treated and untreated control cell cultures (data not shown).

[0308] Acute toxicity data for D-lactic acid obtained from the Fisher Scientific MSDS suggests that the active dose range of 0.25-1.25 mg / ml is below the LD50 dose observed for oral, dermal and inhalation toxicity doses observed in vivo (measured in rats and rabbits) (Table 3). Table 3: Toxicity of Lactic Acid (Fisher Scientific MSDS for D-Lactic Acid) [Table 3] Effect of pH on the ability of D-lactate to disrupt biofilms

[0309] The biofilm disruption activity of D-lactate observed at pH 2.4 was maintained at pH 7 (physiological pH), indicating that the activity of D-lactate is independent of pH (data not shown). D-lactate buffered to pH 6 was used as part of the standard treatment protocol from this point on.

[0310] In summary, D-lactic acid is not bactericidal in the active biofilm-disrupting dose range (0.5-1.25 mg / ml). D-lactic acid is not cytotoxic in the active biofilm-disrupting dose range (active dose range). The biofilm-disrupting activity of D-lactic acid is not affected by pH. Example 4 D-Lactic acid disrupts bacterial biofilms derived from a variety of bacterial strains, including antibiotic-resistant and non-resistant clinical isolates

[0311] The efficacy of D-lactic acid in disrupting biofilms was tested across 24 clinical isolates from cystic fibrosis patients with known antibiotic resistance profiles. Laboratory strains of non-typable Haemophilus influenzae (NTHi) and Actinobacillus pleuropneumoniae (App), the most common pathogens of human otitis media and porcine respiratory syndrome, respectively, were included in the analysis. material and method clinical isolation

[0312] These were sourced by the researchers from a biobank of culture extracts grown from bronchoalveolar lavage fluid collected from cystic fibrosis patients held at PathWest Laboratory Services. These extracts are collected by physicians for diagnostic purposes, whereby the presence of antibiotic-resistant pathogens is identified by PathWest to inform clinical diagnostics and patient treatment pathways. Biofilm microcolony culture

[0313] The culture of interest was thawed, streaked onto a blood agar plate, and grown for 12-24 hours at 37 °C, 5% CO2 until adequate growth was achieved to allow further culturing. Isolated colonies were looped into 5 ml of LB broth and incubated overnight at 37 °C, 5% CO2.

[0314] Stock cultures were diluted 1:1000 in 1% of the appropriate medium (LB for mucoid P. aeruginosa strains; M63 medium was used for P. aeruginosa crude strain 001-11R; PMM for all other strains) to give a total concentration of approximately 10 8 cfu / ml was achieved (confirmed by measuring 0.02 absorbance units, approximately 1.8ul in 2ml of medium). The diluted working culture was mixed thoroughly and plated at 200ul / well into 96-well microtiter plates, with biofilm formation being complete approximately 24-48 hours after plating. process

[0315] D-Lactic acid was applied to all bacterial biofilm microcolonies at 0.5-1.25ug / ml for 24 hours. Treatments were completed in duplicate for all bacterial strains across the dose range used. Buffer solutions of 0.5mg / ml D-Lactic acid at pH 2.4-pH 7 were prepared using Tris and HCl to assess any pH dependency of biofilm disruption activity. Biofilm destruction evaluation i. Crystal violet staining

[0316] After the plate wells were cleared and washed by immersion in water, each well of the microtiter plate was stained for biofilms with 0.1% w / v crystal violet (CV) in water (O'Toole 2011). After 15 min incubation at room temperature, the microtiter plate was washed 3-4 times with water by immersion in water and blotting to remove all excess cells and dye from the plate. The plate was allowed to dry overnight. The CV stain was solubilized with 125ul of 30% acetic acid and the absorbance, indicative of biofilm biomass, was quantified at 550nm after 15 min incubation. ii. Resazurin staining

[0317] Resazurin staining was used to measure changes in bacterial metabolic activity after treatment (Kirchner et al., 2012). 10 μl of 0.02% (v / v) resazurin (diluted in distilled water) was added to each well and the microtiter plates were incubated under aerobic conditions at 37 °C for 1-2 h with shaking at 150 rpm. Viable cells reduce the blue resazurin dye to the pink fluorescent resorufin form. After incubation, fluorescence was quantified with an excitation wavelength of 540 nm and an emission wavelength of 590 nm using an Enspire microplate reader. result

[0318] More than 50% of all strains showed evidence of biofilm disruption after treatment with D-lactic acid. More than 65% of all strains tested showed changes in metabolic activity after treatment with D-lactic acid (Table 4). Table 4: Summary of biofilm-protected microcolonies established from clinical isolates response to D-lactate. [Table 4] Bold = % that were significantly different from control. Non-bold = % that showed a response that could not be quantified compared to control, e.g. variation not allowed by the assay for quantification.

[0319] This suggests that changes in biofilm morphology may have been observed with continued D-lactate treatment outside of the controlled conditions implemented for experimental rigor.

[0320] Biofilm disruption was evident in biofilm-protected NTHi and App microcolonies (data not shown). This is important because NTHI is commonly associated with otitis media (ear infection) and App are problematic pathogens in commercial pig farms demonstrating the broad-spectrum biofilm-disrupting activity of D-lactic acid.

[0321] D-Lactic acid reduces P. aeruginosa biofilms that are antibiotic resistant, mucoid or non-mucoid. D-Lactic acid reduces biofilms grown from other bacteria known to induce clinical disease. D-Lactic acid can be considered a broad-spectrum bacterial biofilm disruptor. Example 5 D-Lactic acid attacks biofilms and induces antibacterial activity similar to that of antibiotic therapy

[0322] Crystal violet is a widely recognized standard for quantifying biofilm density. However, data reproducibility is difficult and provides very limited data on complex systems. To circumvent these issues, we developed an agarose-based biofilm microcolony culture model designed to facilitate staining and visualization of colonies to confirm the presence of dispersed versus intact colonies, viability and biofilm density. This technique was used as a model to characterize the effect of D-lactate treatment on biofilm microcolonies compared to tobramycin. material and method Biofilm microcolony culture

[0323] WACC91 and PA-001-11R (P. aeruginosa non-antibiotic resistant and resistant cultures) were thawed, streaked onto blood agar plates, and grown for 12-24 h at 37 °C, 5% CO2 until adequate growth was achieved to allow further culturing. Isolated colonies were looped into 5 ml of LB broth and incubated overnight at 37 °C, 5% CO2.

[0324] Stock cultures were diluted 1:1000 into casting agarose composed of 1% PMM to give approximately 10 8Achieved cfu / ml (confirmed by measuring 0.02 absorbance units, approximately 1.8ul in 2ml agarose). The diluted working culture was mixed thoroughly and plated at 200ul / well across a 96-well microtiter plate or per chamber of an 8-chamber slide for confocal microscopy analysis. Biofilm formation was complete approximately 24-48 hours after plating. process

[0325] D-Lactic acid and L-Lactic acid were applied to antibiotic resistant and non-resistant P. aeruginosa bacterial biofilm colonies at 0.5-1.25mg / ml either independently or as a combined treatment for 24 hours. Tobramycin was applied to the cultures at 16ug / ml for 24 hours. Assessment of biofilm disruption using confocal microscopy

[0326] Media was removed from chamber slides and resazurin (0.0002% in PMM and arginine) was added to SYTO® 9 stain (0.5ul / ml in PMM with arginine) and incubated for 1 hour at 37°C in 5% CO2. Slides were then rinsed in PMM and fixed overnight in 10% neutral buffered formalin (NBF). The confocal was set to view resorufin at excitation 530 / 570nm, emission 580-590nm. SYTO® 9 stain at excitation 483nm, emission 503nm. result

[0327] D-lactate was more effective than L-lactate in disrupting P. aeruginosa bacterial biofilms (Figure 4). The treatment effect of tobramycin was enhanced when given together with D-lactate. By combining D-lactate with antibiotics, D-lactate was shown to cause a reversal of bacterial colony resistance to these antibiotics in user-designed 3D biofilm models. Example 6 D-Lactic acid prevents biofilm formation and disrupts mature biofilms

[0328] To verify the above results, experiments were performed with Pseudomonas aeruginosa, Staphylococcus aureus and S. epidermidis using the crystal violet staining method to evaluate biofilms. material and method Biofilm microcolony culture

[0329] The culture of interest was thawed and streaked onto an LB agar or blood agar plate and grown at 37 °C for 12-24 hours until adequate growth was achieved to allow further culturing. Isolated colonies were looped into 5 ml of LB broth and incubated overnight at 37 °C. The stock culture was then diluted 1:1000 in 1% of the appropriate medium (LB) to obtain a total concentration of approximately 10 8 Achieved cfu / ml (confirmed by measuring 0.02 absorbance units, approximately 1.8ul in 2ml of medium). The diluted working culture was mixed well and plated at 200ul / well into 96-well microtiter plates for biofilm treatment. process

[0330] D-lactic acid, L-lactic acid, D / L lactic acid or glycolic acid were then added to final concentrations of 0.55 mM, 0.055 mM or 0.0055 mM (1x, 0.1x or 0.01x) in phosphate buffered saline (final pH 7.0) either immediately after plating to assess the effect on biofilm formation, or after 24 hours of incubation at 37°C to assess the effect on mature biofilms. After addition of treatments, all plates were then incubated at 37°C for 24 hours. Biofilm destruction evaluation

[0331] After the plate wells were cleared and washed by immersion in water, each well of the microtiter plate was stained for biofilms with 0.1% w / v crystal violet (CV) in water (O'Toole 2011). After 15 min incubation at room temperature, the microtiter plate was washed 3-4 times with water by immersion in water and blotting to remove all excess cells and dye from the plate. The plate was allowed to dry overnight. The CV stain was solubilized with 125ul of 30% acetic acid and absorbance, indicating biofilm biomass, was quantified at 550nm after 15 min incubation and normalized to vehicle only. result

[0332] As shown in Figure 5, for P. aeruginosa, S. aureus and S. epidermidis, a significant reduction in biofilm volume was observed for D-lactate but not for L-lactate (compared to baseline). The effect was observed for both de novo formed biofilms (when D-lactate or L-lactate was added at t = 0) and established biofilms (when D-lactate or L-lactate was added at t = 24 h). Note: 1x = 0.55 mM for both lactic acid (D- or L-; 0.5 mg / mL) and glycolic acid.

[0333] The variable results of the racemic mixture (D / L) (depending on the time point and strain added) are due to the inefficiency of the basic crystal violet assay. However, the results demonstrate the applicability of D-LA against biofilms produced by multiple pathogens.

[0334] The results for glycolic acid (2-hydroxyacetic acid), which has antibacterial properties but is also known to be achiral, further support the proposal that the observed antibiofilm properties depend on the chirality of the molecule rather than any known antibacterial properties. Example 7 D-Lactic acid induces microcolony dispersion

[0335] Biofilm disruption caused by D-lactic acid against Pseudomonas aeruginosa WACC91 was tested. The method used was that of Example 5, except that samples were fixed for staining / confocal 4 hours after addition of D-LA or L-LA.

[0336] FIG. 6 shows that treatment with 1 mg / mL D-lactic acid for 4 hours caused dispersion of biofilm colonies and dispersion of individual bacteria compared to untreated controls or treatment with L-lactic acid. References [ka]

Claims

1. 1. A composition for disrupting biofilms, comprising: 【Chemistry 6】 A composition comprising a preferred enantiomer of a 2-hydroxycarboxylic acid having the formula:

2. The following formula: 【Chemistry 7】 10. The composition of claim 1, further comprising a non-preferred enantiomer of a 2-hydroxycarboxylic acid having the formula:

3. 3. The composition of claim 2, wherein the percentage of the unpreferred enantiomer of the 2-hydroxycarboxylic acid is less than 20%.

4. 10. The composition of claim 1, which is free of unpreferred enantiomers of the 2-hydroxycarboxylic acid.

5. 10. A composition for use in a method for disrupting biofilms, comprising the 2-hydroxycarboxylic acid of claim 1, said method comprising: i. A composition comprising the step of administering said composition to said biofilm.

6. 6. The composition of claim 5, wherein the composition is used in combination with an antibacterial compound.

7. 10. A composition for treating or preventing a microbial infection in a subject, comprising the 2-hydroxycarboxylic acid of claim 1, wherein the microbial infection is associated with a biofilm, and wherein the composition disrupts the biofilm.

8. The composition of claim 7 further comprising an antibacterial compound.

9. 10. Use of the 2-hydroxycarboxylic acid according to claim 1 in the manufacture of a composition for the disruption of biofilms.

10. The use according to claim 9, characterized in that the 2-hydroxycarboxylic acid is used together with an antibacterial compound.

11. A composition for destroying biofilms, comprising the 2-hydroxycarboxylic acid of claim 1.

12. The composition of claim 11, wherein the composition is used in conjunction with an antibacterial compound.

13. 10. Use of the 2-hydroxycarboxylic acid of claim 1 in the manufacture of a composition for treating or preventing a microbial infection in a subject, wherein the microbial infection is associated with a biofilm and the composition disrupts the biofilm.

14. The use according to claim 13, characterized in that the 2-hydroxycarboxylic acid is used together with an antibacterial compound.

15. 10. Use of a 2-hydroxycarboxylic acid according to claim 1 for disrupting or preventing the formation of biofilms on abiotic surfaces.

16. The use according to claim 15, characterized in that the 2-hydroxycarboxylic acid is used together with an antibacterial compound.

17. A kit for disrupting a biofilm, said kit comprising: a) a 2-hydroxycarboxylic acid according to claim 1, and b) Instructions for use Includes a kit.

18. 18. The kit of claim 17, wherein the 2-hydroxycarboxylic acid is used in conjunction with an antibacterial compound.

19. The composition of claim 1, wherein the 2-hydroxycarboxylic acid is D-lactic acid.

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