BR112021008550A2 - compositions for the treatment of biofilms without inducing antimicrobial resistance - Google Patents

Abstract

COMPOSITIONS FOR THE TREATMENT OF BIOFILMS WITHOUT INDUCING ANTIMICROBIAL RESISTANCE. The compositions of disinfection containing hypochlorous acid and acetic acid are useful for the treatment of biofilms in or on tissue, including biofilms related to wounds or other skin traumas. The compositions are useful for treating a variety of tissue types, both on the surface. and below the fabric surface. Compositions are provided for the treatment of biofilms without inducing antimicrobial resistance.

Description

COMPOSITIONS FOR THE TREATMENT OF BIOFILMS WITHOUT INDUCING ANTIMICROBIAL RESISTANCE RELATED ORDERS

[0001] This application claims priority and benefit of US Provisional Patent Application Serial No. 62/755,104, filed November 2, 2018, the contents of which are incorporated by reference into this document in its entirety. Field of Invention

[0002] The present invention relates generally to methods for determining the chemotypic profile of cannabis plant material, including uses thereof. background

[0003] Microbial infections that produce biofilms can cause serious health problems. Scientists estimate that up to 80% of all infections that affect mammals are biofilm infections. Bacteria that grow in biofilms cause chronic infections that are characterized by persistent inflammation and tissue damage. Chronic infections are infections that persist despite antibiotic therapy and the host's innate and adaptive immune and inflammatory responses and which, unlike colonization, are characterized by an immune response and persistent pathology. (Hpiby N, Bjarnsholt T, Givskov M, Molin S, Ciofu O. Antibiotic resistance of bacterial biofilms. Int J Antimicrob Agents [Internet]. April 2010 [cited 14 July 2014]; 35(4):322- 32).

[0004] Bacteria that attach to a surface and grow as a biological film are protected from death by antibiotics. Reduced sensitivity to antibiotics contributes to the persistence of biofilm infections. The persistence of, for example, infections by S. aureus and P. aeruginosa is due to biofilm formation. (Int J Med Microbiol. July 2002; 292(2): 107-13. Stewart PS 1).

[0005] Antibiotic and antimicrobial resistance in bacteria is a growing problem. The accelerated development of antibiotic resistant bacteria has been intensified by the widespread use of antibiotics (Science, 264:360-374 (1994) Antibiotic resistance can spread rapidly to other bacteria, including bacteria of different species. Some strains of bacteria are susceptible to just one antibiotic and it may only be a matter of time before some bacteria are no longer eradicated by any antibiotics. (Peter David Ringel, Di Hu, Marek Basler. The Role of Type VI Secretion System Effectors in Target Cell Lysis and Subsequent Horizontal Gene Transfer. Cell Reports, 2017; 21 (13): 3927 DOI: 10.1016/j.celrep.2017.12.2020).

[0006] This public health epidemic is also due to the emergence of pathogens simultaneously resistant to several antibiotics, thus reducing the possibility of an effective treatment (WHO Global Strategy for Containment of Antimicrobial Resistance. Geneva, World Health Organization, 2001, WHO/ CDS/CSR/DRS/2001.2). Without new antimicrobial compositions, it is becoming increasingly difficult to treat biofilm infections because of the resistant nature of biofilm and antimicrobial resistance.

[0007] Prior art compositions have several shortcomings. Some prior art compositions promote antimicrobial resistance when trying to treat biofilm infections, making biofilm treatment increasingly difficult. Other prior art compositions are unable to eradicate biofilm without prolonged exposure to the affected problem, which induces antimicrobial resistance. And other prior art compositions are incapable of treating mature biofilms. summary

[0008] Antimicrobial compositions comprising hypochlorous acid and an organic acid such as acetic acid, as described herein, are useful for the treatment of biofilms in or on tissue without inducing antimicrobial resistance, ranging from simple topical disinfectants to wounds or other skin trauma. For example, wounds are often susceptible to microbial infection, including antimicrobial resistant biofilms that form on the surface and below the surface of a wound, which impede healing and can lead to chronic conditions or persistent infection. Compositions comprising hypochlorous acid and acetic acid are useful for treating biofilms in tissues and not inducing antimicrobial resistance. The concentrations of HOCl and HAc are balanced in order to achieve a synergistic effect, resulting in the antimicrobial capabilities of the composition being greater than would be expected based on the antimicrobial properties of each component alone, while not inducing antimicrobial resistance. HOCl and HAc compositions are also effective in defending against antimicrobial resistance. Acetic acid provides an important buffering capacity that allows for optimal performance of hypochlorous acid, especially in environments where the tendency is to bring the pH to homeostatic levels. For example, in the oral cavity, the natural pH is around 7.4 and acetic acid provides a buffering capacity in that environment to allow optimal HOCl activity. In addition, hypochlorous acid modulates the toxicity of acetic acid and provides an analgesic effect, allowing stronger compositions to be applied to the skin or other tissue without adverse side effects, patient discomfort, or antimicrobial resistance. Finally, acetic acid is particularly effective against anaerobic bacteria such as Pseudomona. In addition to acetic acid, other organic acids such as ascorbic acid, lactic acid, formic acid, malic acid, citric acid, uric acid and other carboxylic acids or sulfonic acids. In addition, hypochlorous acid and acetic acid compositions enhance the antimicrobial properties of other microbial treatments.

[0009] Acetic acid concentrations of about 0.1% to about 5% are useful. Hypochlorous acid concentrations greater than 5 ppm are useful in treating biofilm infections. Some compositions have hypochlorous acid concentrations of about 5 ppm, greater than 25 ppm, greater than 50 ppm, up to about 2500 ppm. The compositions of the invention are particularly effective in biofilm reduction due to the synergistic balance between hypochlorous acid and acetic acid, which gives the compositions the dual surface effect, just below the surface, and deeper treatment of the biofilm at the subsurface. In general, hypochlorous acid is able to act quickly on or near the surface; while acetic acid takes longer to act and therefore can act below the tissue surface. The synergistic effect of the hypochlorous acid and acetic acid compositions of the invention on biofilms also reduces the likelihood of antimicrobial resistance and cross-resistance.

[00010] The application of the compositions revealed in the infected area helps in the treatment of bacterial infection, including biofilms, without creating resistance to antimicrobials and antibiotics.

[00011] The compositions of the invention are effective against immature, young and mature biofilm. The application of the compositions on the infected area helps in the treatment of biofilm classified as immature, young and mature and does not induce microbial resistance.

[00012] The compositions of the invention can be provided as a gel or cream, which allows a longer contact time with the tissue, without inducing antimicrobial resistance and cross-resistance. For wound care, the compositions in gels and creams also help to keep the wound hydrated, promoting healing. Furthermore, one or both components of the compositions of the invention can be encapsulated in a nanoparticle for controlled or delayed release.

[00013] The compositions described herein can be combined with various excipients and vehicles to facilitate topical administration. Hypochlorous acid (and organic acid) products can take the form of gels, creams, lotions, sprays, liquids, foams, powders and other delivery formulations known in the art. Alternatively, the compositions can be supplied incorporated in fibrous cloths or handkerchiefs or wound dressings.

[00014] In certain aspects, the invention includes a composition made of acetic acid and hypochlorous acid. Acetic acid is present in concentrations greater than about 0.05%, and preferably less than about 5.0%. A preferred concentration of hypochlorous acid is between about 5 ppm and about 2500 ppm. In some embodiments, the particular concentrations of acetic acid and hypochlorous acid depend on the intended exposure time of the composition to the area of treatment.

[00015] In some embodiments, acetic acid is present in a concentration sufficient to penetrate below the surface of a tissue. In some embodiments, the acetic acid concentration is greater than about 0.5%, and preferably greater than about 0.25%, and in some embodiments, it is about 0.50% or greater. Acetic acid can be encapsulated in a nanoparticle for controlled or delayed release. In some modalities, hypochlorous acid is present in a concentration sufficient to treat the biofilm on the surface of a wound and just below the wound. The composition can further include a gel, cream, ointment or oil.

[00016] In related aspects, the invention involves a method for protection against antimicrobial resistance. The methods include applying to a fabric a composition comprising acetic and hypochlorous acid in an amount sufficient to protect against antimicrobial resistance.

[00017] In related aspects, the invention involves methods for treating a biofilm in or on the,

tissue. The methods include applying to a tissue a composition comprising acetic acid in a concentration sufficient to penetrate the skin and hypochlorous acid in an amount sufficient to remove the biofilm on and just below the tissue surface and not inducing antimicrobial resistance.

[00018] Acetic acid may be present in sufficient amounts to remove the biofilm under the skin surface without inducing antimicrobial resistance. Acetic acid can be present in an amount of about 0.5% to about 5.0% and hypochlorous acid can be present in a concentration of about 5 ppm to about 2500 ppm.

[00019] In related aspects, a method for treating biofilm in tissue involves applying a nanoparticle comprising acetic acid to a tissue site to protect against antimicrobial resistance. The nanoparticle can be lipid soluble. Brief Description of Drawings

[00020] Figure 1 is a schematic showing an exemplary system for the production of hypochlorous acid according to the methods of the invention.

[00021] Figure 2 is a schematic showing an enlarged view of the mixing device shown in Figure 1.

[00022] Figure 3 is a schematic that shows an internal view of the mixing chamber of the mixing device.

[00023] Figure 4 is a schematic showing a front view of the members that divide the mixing chamber into a plurality of subchambers. This view shows the openings in the limbs.

[00024] Figure 5 is a schematic showing a valve configured with metering sensors to change from a waste line to a product collection line.

[00025] Figure 6 is a schematic showing the valve in line with the waste line and product collection line.

[00026] Figure 7 is a schematic showing another exemplary system for producing hypochlorous acid according to the methods of the invention. This system is configured for automated use with buffered deionized water. The plug can be included in the inlet water or can be introduced through an injection port. The buffer can also be mixed during the mixing process using NaOH in NaOCl or separately injected and acetic acid or other similar acids or bases.

[00027] Figure 8 is a graph of a calibration curve showing the HOCl concentration (ppm) calculated indirectly in relation to the conductivity.

[00028] Figure 9 is a graph that shows a spectrophotometric analysis of the HOCl produced. The gases generally produced during the production of HOCl are ClO2, Cl2O and Cl2, all of which are detectable in the visible range as yellow or yellow-red. The graph does not show any absorption of colored gases in the HOCl produced.

[00029] Figure 10 is a graph that shows the amount (parts per million (ppm)) of HOCl initially produced (T = 0) and its stability over time.

[00030] Figure 11 is a graph showing how the pH of the HOCl product has changed over time.

[00031] Figure 12 is a graph showing the oxidation and reduction (redox) of the HOCl product over time.

[00032] Figure 13 shows an antimicrobial composition that includes an aqueous solution of hypochlorous acid encapsulated in a nanoparticle.

[00033] Figure 14 is an illustration of a method for making an antimicrobial composition that includes an aqueous solution of hypochlorous acid encapsulated in a nanoparticle.

[00034] Figures 15-21 provide data on the reduction of various biofilms when exposed to acetic acid and hypochlorous acid compositions at different concentrations compared to commercially available biofilm treatments. Figure 22 is a graph showing the killing effect of various concentrations of HOCl with 1% or 4% acetic acid.

[00035] Figure 23 is a graph showing the killing effect of HOCl at 200 ppm and 500 ppm with 1%, 2% or 4% acetic acid. Detailed Description

[00036] The treatment of bacterial infection and biofilm is achieved by means of a synergistic composition comprising an organic acid such as acetic acid and hypochlorous acid. The acetic acid component is particularly effective in penetrating tissue, while hypochlorous acid is particularly effective in treating the biofilm on the outer surface of the tissue. Acetic acid can penetrate up to 2 mm or more below the surface of a wound to treat difficult-to-access biofilms.

[00037] Preferred compositions of the invention comprise hypochlorous acid in synergistic combination with acetic acid. It has been found that a balanced composition in which an amount of acetic acid biocide (or an organic acid equivalent) optimized with hypochlorous acid achieves the maximum therapeutic benefit, whether applied to a surface or in a manner designed to penetrate the skin.

For example, for surface applications, the relative amount of acetic acid to hypochlorous acid is less than for penetrative applications.

According to the invention, surface bacterial contamination or biofilm is sufficiently treated by hypochlorous acid with a lower amount of acetic acid; but for applications that require deep penetration (eg wounds), the amount of acetic acid must be increased.

In this case, hypochlorous acid is used to moderate the toxicity of the acetic acid to the surrounding tissue, while allowing the acetic acid to attack the biofilm.

Furthermore, it has been found that the synergistic combination of acetic acid and hypochlorous acid selectively kills the harmful biofilm while preserving the beneficial biofilm.

The compositions of the invention comprise balanced concentrations of acetic acid and hypochlorous acid to selectively kill harmful biofilm.

Concentrations are also balanced with regard to the application for which they are intended (eg surface treatment of a tooth or skin versus deep tissue treatment of a wound or tooth root). The patent application provides guidance on the synergistic effects of various combinations of acetic acid and hypochlorous acid.

The person skilled in the art can determine, based on the information provided in this specification, the relative amounts of acetic acid and hypochlorous acid required for the treatment of any bacterial infection or biofilm formation.

[00038] The treatment of superficial and subdermal infections offers a dual action treatment that is particularly needed for wound care. Chronic wounds and eczema are infested by biofilm that affects the subterranean parts of the wound. These wounds often have S. aureus infections that normally exist on or near the surface and prevent the wound from closing and healing. In addition, P. aeruginosa infection is often present, which is usually deeper under the surface of the wound. When a deep infection is present, it is important to keep the wound open and hydrated as it heals from the inside out, to prevent the wound from closing before the deeper infection is healed. Treating only the superficial infection causes the wound to close and trap the biofilm deeper within the tissue, which can lead to sepsis and other complications.

[00039] A feature of the invention is also observed in the modulation of hypochlorous acid and acetic acid. For example, increasing the concentration of HOCl or acetic acid, while keeping the other constant, increases the bacteria killing effect of the combination. Furthermore, increasing the concentration of acetic acid increases the killing effect in a “dirty” sample, that is, one that contains organics (such as blood, sputum and other organic compounds). Thus, in a dirty sample, the acetic acid concentration is more important to modulate. Figure 22 shows the killing effect of various concentrations of

HOCl with 1% or 4% acetic acid. Figure 23 shows that the biggest jump in the killing effect occurs between 1% and 2% acetic acid. Both Figures 22 and 23 are the result of applying various concentrations of HOCl and acetic acid to a wound simulation model of Pseudomonas aeruginosa infection.

[00040] There are numerous applications of the invention to prevent and/or treat biofilm. For example, a common problem with diabetic foot ulcers, for example, is that they close on the surface, creating an open cavity below. The cavity will contain pus as part of the immune system's response, which consists of debris, bacteria and white blood cells. Pus contained in a closed compartment, especially in a foot ulcer, can help spread the infection and potentially cause sepsis. In a properly treated open wound, however, pus will be released into the wound bed and dressing. Bacteria that survive the pus environment inside a closed wound can spread the infection. Therefore, removing the S. aureus infection without first or simultaneously removing the P. aeruginosa infection may not completely eradicate the biofilm infection, leading to early closure and possibly sepsis.

[00041] Antimicrobial solutions, such as the compositions provided herein, reduce infection in the deep areas of the wound bed and allow healing of the wound from the inside out, so that the surface does not heal faster than the internal wound. Acetic acid is present in an amount sufficient to disinfect a biofilm under the wound bed, and hypochlorous acid is present in an amount sufficient to disinfect the wound surface. The composition therefore allows for complete disinfection of the wound to prevent premature closure and entrapment of a biofilm under the surface of a closed wound.

[00042] The disclosed compositions are particularly effective because the balance of concentrations of hypochlorous acid and acetic acid allows the treatment of the biofilm at the surface level and also of the sub-surface biofilm. Precise balance depends on the treatment site and the amount of surface penetration desired. Hypochlorous acid can be present at about 10 ppm to about 500 ppm or more. Different uses and types of fabric may require higher or lower concentrations. Acetic acid can be present at about 0.25% to about 2.0% or more, and preferably about 1.0%. By balancing the two components, the composition can have the dual effect of treating on the surface and below the surface of the tissue or wound.

[00043] Compositions consistent with the present disclosure can be created for a variety of uses. For example, a composition with a relatively low concentration of acetic acid (as low as about 0.05%) and about 5-60 ppm of hypochlorous acid is a composition useful as a mouthwash to combat infections in dental tissue. The lower concentration of acetic acid is sufficient in mouthwash compositions because the microbial infection does not tend to penetrate deeply into the tissue.

[00044] For wound care, on the other hand, a composition may include a higher concentration of acetic acid (about 1.0%, about 2.0%, about 3.0%, about 4, 0% or about 5.0%) to more effectively treat the deeper tissue biofilm.

[00045] Other uses may require more or less of each component. For example, a composition with a hypochlorous acid concentration of about 80-250 ppm is useful for flushing the bladder, a treatment that is often necessary for patients with a urinary catheter to prevent infection or blockage. A composition with a hypochlorous acid concentration of about 15-60 ppm is sufficient for the treatment of an infected lung.

[00046] In certain modalities, the composition is in the form of a gel, which allows longer contact times with the wound. A rinse with a solution may not be enough, as the contact time of the antiseptic will be too short. In many cases, to completely remove a biofilm, the composition must be in contact with it for an extended period of time, ranging from a few seconds to several minutes, to an hour or more. The composition can be supplied in a gel or cream, which resists evaporation or immediate dispersion. Gels, creams, ointments, oils and other similar vehicles for topical administration are known in the art.

[00047] In addition, for wound care, compositions in gel form have the benefit of keeping the wound site moist. It is important to keep the wound hydrated during and after treatment with the compositions of the invention. The compositions disclosed are primarily water (generally 95% or more), allowing the wound to remain hydrated while the antiseptic ingredients in the composition fight infection in the wound and prevent new infections from setting in. Maintaining hydration also prevents the wound from closing prematurely and trapping the biofilm within the tissue. Acetic acid is readily formulated into a gel because acetic acid is not overly reactive. Other organic acids can also be used, and those that are less reactive are desirable.

[00048] Slow release compositions can also be used. In some compositions, acetic acid can be encapsulated in fat-soluble nanoparticles, which allow the acetic acid to be transported down to the surface of a wound before being released from the nanoparticle. Administration with nanoparticles of different properties allows acetic acid to be released slowly over time, prevents dispersion and provides other administration benefits. Acetic acid diffuses freely, is water soluble and has high vapor pressure. These properties make it more difficult to control where acetic acid goes. Acetic acid encapsulated in nanoparticles allows the composition to be controlled more precisely. Nanoparticles are described in more detail below and shown in FIGS. 13 and 14.

[00049] In some embodiments, the composition includes some acetic acid that is free from nanoparticles and some acetic acid that is encapsulated in nanoparticles. Alternatively, slow release formulations can be used alone or in combination with other instant-on formulations. For example, a wound can be treated with a composition of acetic acid and hypochlorous acid and then treated with a composition primarily of acetic acid encapsulated in nanoparticles to provide continuous release of acetic acid into the deep parts of the wound after the initial treatment. Production of hypochlorous acid compositions

[00050] The basis of the compositions and methods of the invention is the protonation of the hypochlorite ion (OCl-) using HCl or acetic acid (HAc) and NaOCl as an example, the protonation is performed by introducing an acid (for example, HCl) into the solution, which results in the following reaction occurring: or .

[00051] The hypochlorous acid in aqueous solution partially dissociates into the hypochlorite anion (OCl), therefore, in aqueous solution there is always a balance between the hypochlorous acid and the anion (OCl). This balance is pH dependent and at higher pH the anion dominates. In aqueous solution, hypochlorous acid is also in equilibrium with other chlorine species, in particular chlorine gas, Cl2 and various chlorine oxides. At acidic pH, chlorine gases become increasingly dominant, while at neutral pH the different balances result in a solution dominated by hypochlorous acid. Thus, it is important to control exposure to air and pH in the production of hypochlorous acid.

[00052] In addition, the concentration of protons (H+) affects the stability of the product. The invention recognizes that the concentration of protons can be controlled using an acid that has a lower capacity at a given pH to donate a proton (i.e., the acid can provide buffering capacity). For example, running the process with acetic acid rather than hydrochloric acid is optimal when the desired pH of the final solution is approximately the pKa of acetic acid. This can be achieved by mixing water proportions of 250X or greater, which means 1 part proton donor at 100% concentration (eg HCl or acetic acid) to 250 parts water.

[00053] In certain embodiments, the manufacturing methods of HOCl involve mixing in water in an air-free environment, a compound that generates a proton (H+) in water and a compound that generates a hypochlorite anion (OCl-) in water to thereby produce air-free Hypochlorous acid. The water can be tap water or purified water such as water purchased from a water purification company such as Millipore (Billerica, MA). Generally, the pH of water is maintained from about 4.5 to about 9 during the method, however the pH can go above and below this range during the production process. Conducting the methods of the invention in an air-free environment prevents the accumulation of chlorine gases during the production process. Furthermore, conducting the methods of the invention in an air-free environment further stabilizes the HOCl produced.

[00054] Any compound that produces a hypochlorite anion (OCl-) in water can be used. Exemplary compounds include NaOCl and Ca(OCl)2. In particular embodiments, the compound is NaOCl. Any compound that produces a proton (H+) in water can be used with the methods of the invention. Exemplary compounds are acids such as acetic acid, HCl and H2SO4. In particular embodiments, the compound is HCl. In preferred embodiments, the compound is acetic acid because it is a weaker acid with a preferred pKa for HCl, which means that it donates less protons during the reaction than HCl and is better able to maintain the preferred pH level.

[00055] The mixture can be conducted in any type of container or chamber or fluidic system. In certain embodiments, a fluidic system 100, as shown in Figure 1, is used to carry out the methods of the invention. System 100 includes a series of interconnected tubes 101a-c with a plurality of mixing devices 102 and 103 in line with the plurality of tubes 101a-c. Tubes and mixing devices can be interconnected using seals so that all air can be purged from the system, allowing the methods of the invention to be performed in an air-free environment. In certain embodiments, the methods of the invention are also conducted under pressure. The production of HOCl in an air-free environment under pressure allows the production of HOCl that does not interact with gases in the air (eg oxygen) that can destabilize the HOCl produced.

[00056] Tubes 101a-c generally have an inside diameter ranging from about 5mm to about 50mm, more preferably from about 17mm to about 21mm. In specific embodiments, tubes 101a-c have an inside diameter of about 21 mm. Tubes 101a-c generally are from about 10 cm to about 400 cm in length, more preferably from about 15 cm to about 350 cm. In certain embodiments, tubes 101a-c are the same length. In other embodiments, tubes 101a-c are different lengths. In specific embodiments, tube 101a is about 105 cm in length, tube 101b is about 40 cm long, and tube 101c is about 200 cm long.

[00057] The tubes and mixers can be made of any inert material, so that the material of the tubes and mixers does not get involved with the reaction that takes place within the fluidic system. Exemplary materials include PVC-U. Tubes are commercially available from Georg Ficher AB. Tubes and mixers can be configured to have a linear arrangement so that tubes and mixers are laid out in a straight line. Alternatively, pipes and mixers can have a non-linear arrangement so that water must flow through bends and bends throughout the process. System 100 shows a non-linear configuration of tubes 101a-c and mixers 102 and 103.

[00058] Tube 101a is an inlet tube that receives the water that will flow through the system. Generally, the water in pipes 101a-c is under a pressure of at least about 0.1 bar, such as, for example, 0.2 bar or greater, 0.3 bar or greater, 0.4 bar or greater, 0.5 bar or greater, 0.7 bar or greater, 0.9 bar or greater, 1.0 bar or greater, 1.2 bar or greater, 1.3 bar or greater, or 1.5 bar or greater. At such pressures, a turbulent water flow is produced, thus the reactants are introduced into a highly turbulent water flow that facilitates an initial mixing of the reactants with the water before further mixing in mixing devices 102 and 103.

[00059] In order to control the pH during the production process, the inlet water must have a buffering capacity in the range of pH 3.5-9.0, more preferably 6.0 and 8.0, to facilitate the addition of the compounds that generate the proton and the compound that generates the hypochlorite anion. Dissolved salts and other molecules found in most tap water give tap water a buffering capacity in the pH range of 5.5-9.0 and therefore tap water is suitable water to be used with methods of the invention.

[00060] In certain embodiments, deionized water with the addition of known buffering agents to produce a water with a buffering capacity in the range of pH 3.5-9.0 is used. In the example a buffer in this particular range is phosphate buffer. For greater control and consistency of the process, using formulated deionized water may be preferable to using tap water because tap water can change between locations and also over time. Furthermore, the use of deionized water with known additives also ensures a stable pH of the incoming water stream. This process is discussed in more detail below.

[00061] In particular embodiments, an initial pH of water prior to addition of the proton generating compounds or the hypochlorite anion generating compound is at least about 8.0, including 8.1 or greater, 8.2 or greater, 8.3 or greater, 8.4 or greater, 8.5 or greater, 8.6 or greater, 8.7 or greater, 8.8 or greater, 8.9 or greater, 9.0 or greater, 9.5 or greater, 10.0 or greater, 10.5 or greater, or 10.8 or greater. In specific modalities, the pH of the water before the addition of the compound that generates the proton or the compound that generates the hypochlorite anion is 8.4.

[00062] The methods of preparation of HOCl include introducing into water the compound that generates the proton and the compound that generates the hypochlorite anion in any order (for example, simultaneously or sequentially) and in any way (aqueous form, solid form, etc. .). For example, the compound that generates the proton and the compound that generates the hypochlorite anion are aqueous solutions and are introduced into water sequentially, for example, the compound that generates the proton can be introduced into the water first and the compound that generates the hypochlorite anion can be introduced into water secondly.

[00063] The system 100 is configured for sequential introduction of reagents to the water flow and the process is described in this document in which the compound that generates the proton is introduced into the water first and the compound that generates the hypochlorite anion is introduced into the water at second place. In certain embodiments, the proton generating compound and the hypochlorite anion generating compound are introduced into the water in small aliquots, for example, from about 0.1 ml to about 0.6 ml. Iterative and minute titrations allow you to control the pH despite additions of acid (compound that generates the proton) and alkali (compound that generates the hypochlorite anion). In certain embodiments, no more than about 0.6 ml of the amount of compound that generates the proton is introduced into water at a single point in time. In other embodiments, no more than about 0.6 ml of the amount of compound that generates the hypochlorite anion is introduced into the water at a single point in time.

[00064] To introduce the reagents into the water, the tube 101a includes an injection port 104 and the tube 101b includes an injection port 105. The injection ports 104 and 105 allow the introduction of reagents into the water flow. In this modality, the aqueous compound that generates the proton is introduced into the water in tube 101a via the injection port 104. The compound that generates the proton is introduced by an infusion pump that is sealably connected to the port 104. flow rate and, therefore, the amount of the compound that generates the proton introduced into the water at any time is controlled. The infusion pump can be controlled automatically or manually. The rate of introduction of the proton-generating compound into the water is based on the quality of the inlet water (conductivity and pH level) and the pressure and flow of the inlet water. In certain embodiments, the pump is configured to introduce approximately 6.5 liters per hour of hydrochloric acid into the water. The introduction can be in continuous or intermittent infusion. As the water flows through the tubes in a turbulent way, there is an initial mixture of the compound that generates the proton with the water through the introduction of hydrochloric acid in the water.

[00065] Further mixing occurs when water enters the first mixing device 102. FIG. 2 shows an enlarged view of the mixing device 102 shown in FIG. 1. In the illustrated embodiment, the mixing device includes a length of about 5.5 cm and a diameter of about 5 cm. One of skill in the art will recognize that these are exemplary dimensions and the methods of the invention may be conducted with mixing devices having dimensions other than the exemplified dimensions. Mixing device 102 includes a fluid inlet 106 that sealably mates with tube 101a and a fluid outlet 107 that sealably mates with tube 101b. In this way, water can enter the mixing chamber 108 of the device 102 of the tube 101a and exit the chamber 108 of the device 102 through the tube 101b.

[00066] The mixing device 102 is configured to produce a plurality of fluid vortices within the device. An exemplary device configured in such a way is shown in FIG. 3, which is a figure providing an internal view of chamber 108 of device 102. Chamber 108 includes a plurality of members 109, the members being spaced apart and secured within chamber 108 perpendicular to the inlet and outlet to form a plurality of subchambers 110. Each member 109 includes at least one opening 111 that allows fluid to flow therethrough. FIG. 4 shows a front view of the members 109 so that the openings 111 can be seen. The size of the openings will depend on the water flow and pressure in the system.

[00067] Any number of members 109 can be set in chamber 108, the number of members 109 set in chamber 108 will depend on the amount of mixing desired. FIG. 4 shows four members 109a-d that are attached to the chamber to produce four subchambers 10a-d. The members 109 can be spaced a uniform distance within the chamber 108, producing subchambers 110 of uniform size. Alternatively, members 109 can be spaced at different distances within chamber 108, producing subchambers 110 of different size. The members 109 are of such a size that they can be attached to an inner wall within the chamber 108. In this way, water cannot flow around the members and can only pass through the openings 111 in each member 109 to move through the device. of mixing 102. Generally, the members will have a diameter of from about 1 cm to about 10 cm. In specific embodiments, the members have a diameter of about 3.5 cm.

[00068] A fluidic vortex is produced within each 110a-d subchamber. The vortices result from the flow of water through the openings 111 in each member 109. The methods of the invention allow any arrangement of the openings 111 over each member 109. FIG. 4 illustrates non-limiting examples of different arrangements of openings 111 within a member 109. The openings can be of any shape. FIG. 4 illustrates circular apertures 111. In certain embodiments, all apertures 111 are located at the same location on the members 109. In other embodiments, the apertures 111 are located at different locations on the members 109. Within a single member 109, all the apertures 111 can have the same diameter. Alternatively, within a single member 110, at least two of the openings 111 are of different sizes. In other embodiments, all openings 111 within a single member 110 are of different sizes.

[00069] In certain embodiments, openings 111 in one member 110 have a first size and openings 111 in a different member 110 have a different second size. In other embodiments, openings 111 in at least two different members 110 are the same size. The size of the openings will depend on the water flow and pressure in the system. Exemplary aperture diameters are from about 1 mm to about 1 cm. In specific embodiments, the openings have a diameter of about 6 mm.

[00070] The solution enters the mixing device 102 through the inlet 106, which is sealably coupled to the tube 101a. The solution enters chamber 108 and turbulent mixing occurs in each of the subchambers 110 ad as the solution passes through members 109a-d through openings 111 in each member 109a-d. After mixing in the final subchamber 110d, water exits chamber 108 through fluidic outlet 107 which is sealably coupled to tube 101b.

[00071] The compound that generates the hypochlorite anion is then introduced into the solution that is flowing through the tube 101b through the injection port 105. The compound that generates the hypochlorite anion is introduced by an infusion pump that is sealably connected to port 105. In this way, the flow, and therefore the amount, of the compound that generates the hypochlorite anion introduced into the water at any time is controlled. The infusion pump can be controlled automatically or manually. The rate of introduction of the compound that generates the hypochlorite anion into the water is based on the properties of the solution (conductivity and pH level) and the pressure and flow of the solution. In certain embodiments, the pump is configured to introduce about 6.5 liters per hour of the compound that generates the hypochlorite anion into the solution. The introduction can be in continuous or intermittent infusion. As the solution is flowing through the tubes in a turbulent manner, there is an initial mixing of the compound that generates the hypochlorite anion with the solution, by introducing the compound that generates the hypochlorite anion into the solution.

[00072] Further mixing occurs when the solution enters the second mixing device 103. The mixing device 103 includes all of the features discussed above with respect to the mixing device 102. The mixing device 103 can be configured the same or different from the mixing device. mixture 102, for example, with the same or different number of subchambers, the same or different diameter of openings, the same or different sizes of subchambers, etc. However, like mixing device 102, mixing device 103 is configured to produce a fluid vortex within each subchamber.

[00073] The solution enters the mixing device 103 through an inlet in the device, which is sealably coupled to the tube 101b. The solution enters the mixing chamber and turbulent mixing occurs in each sub-chamber of the mixing device as the solution passes through the chamber members through the openings in each member. After mixing in the final subchamber, water exits the chamber through the fluid outlet in the mixing device which is sealably coupled to tube 101c.

[00074] At this point, the reaction was completed and HOCl was formed. Production is controlled in-line by measuring pH and conductivity. pH is used in combination with conductivity based on a pre-calibrated relationship between conductivity and HOCl concentration measured spectrophotometrically. Measured conductivity is a measure of the solvent's ability to conduct an electrical current. Comparing the same matrix with different known concentrations of HOCl and OCl-, a calibration curve (FIG. 8) was established which is used in combination with the pH meter to regulate the titrations and control the process.

[00075] Tube 101c can be connected to a switching valve 112 that switches between a waste line 113 and a product collection line 114. Shown in FIGS. 5 and

6. Valve 112 includes pH meter and conductivity measuring device. These devices measure the concentration (ppm), purity and pH of the HOCl being produced and provide feedback to alter these properties of the HOCl being produced. Once the HOCl that is produced in tube 101c meets the required concentration, purity, and pH, valve 112 switches from waste line 113 to product collection line 114 to collect the desired product.

[00076] The HOCl that has been produced air-free is collected and bottled in an air-free manner. The placement of liquids into a bottle in an air-free manner is known in the art. An exemplary method includes placing an inflatable container (such as a balloon) in a bottle. The inflatable vessel is connected directly to collection line 114 and the HOCl is pumped into the inflatable vessel in the bottle without ever being exposed to air. Another method involves filling the bottles under vacuum. Another method of airless filling involves filling the bottles in an environment of an inert gas that does not interact with the HOCl, such as an argon environment.

[00077] The hypochlorous acid produced is air-free and will have a pH of about 4.5 to about 7.5. However, the pH of the produced HOCl can be adjusted after the production process by adding acid (eg HCl) or alkali (eg NaOCl) to the hypochlorous acid produced. For example, a pH between about 4.5 and about 7 is particularly suitable for the reprocessing application of heat-sensitive medical instruments. Other applications, such as use in non-medical environments, for example in poultry and fish processing and agricultural and petrochemical uses in general, bacterial biofilm breakage and water treatment, may require different pH levels.

[00078] The process can be performed manually or in an automated way. The fluidic systems described in this document can be operationally connected to a computer that controls the production process. The computer may be a PCL logic controller system. The computer opens and closes the water inlet, wastewater outlet, and product outlet valves according to feedback received from sensors in the system (eg, conductivity, pH, and concentration of the product (ppm) that is produced). The computer can also store values for pressures and amounts of water and can adjust them according to feedback received from the sensors regarding the properties of the HOCl being produced. The computer can also control the infusion pumps that inject reagents into the water for the production process.

[00079] The process can be performed iteratively in which the tube 101c can be connected to a second fluidic system and the HOCl produced is then drained through the second system, where the process described above is repeated with the initial solution being HOCl instead of Water. In this way, an increased yield of HOCl is produced. Any number of fluidic systems can be interconnected with methods of the invention.

[00080] FIG. 7 is a schematic showing another exemplary system 200 for producing hypochlorous acid according to the methods of the invention. System 200 is configured to regulate the pH of incoming water and injection buffer for stability. In system 200, water is introduced into tube 201a. Tube 201a has a 208 pH meter connected to it. The 208 pH meter measures the pH of the incoming water. The pH meter 208 is connected to the injection port 202. The injection port 202 allows the introduction of at least one buffering agent into the incoming water. The buffering agent is introduced by an infusion pump that is sealably connected to port 202. In this way, the flow rate, and therefore the amount, of buffering agent introduced into the water at any given time is controlled. The infusion pump can be controlled automatically or manually. The rate of introduction of the buffering agent into the water is based on the quality of the inlet water (conductivity and pH level), the composition of the buffer, and the pressure and flow of the inlet water. The introduction can be in continuous or intermittent infusion. Since water is flowing through tube 201a turbulently, there is an initial mixing of the buffering agent with the water after the introduction of the buffering agent into the water. This initial mixture may be sufficient to properly adjust the properties of the incoming water.

[00081] In certain embodiments, additional mixing of water and buffer is carried out before conducting the HOCl production process. In these embodiments, further mixing occurs when water with buffering agent enters the first mixing device 203. The mixing device 203 includes all of the features discussed above with respect to the mixing device 102. The mixing device 203 is configurable the same or different to the mixing device 202, for example, with the same or different number of subchambers, the same or different diameter of openings, the same or different sizes of subchambers, etc. However, like mixing device 102, mixing device 103 is configured to produce a fluid vortex within each subchamber.

[00082] The solution enters the mixing device 203 through an inlet in the device, which is sealably coupled to the tube 201a. The solution enters the mixing chamber and turbulent mixing occurs in each sub-chamber of the mixing device as the solution passes through the chamber members through the openings in each member. After mixing in the final subchamber, water exits the chamber through the fluid outlet in the mixing device which is sealably coupled to tube 202b. The water has a pH of at least about 8.0, preferably 8.4, and a buffering capacity of pH 5.5-9.0.

[00083] The process is now conducted as described above for the production of HOCl. The proton-generating compound is then introduced into the water flowing through tube 201b through injection port 204. The proton-generating compound is introduced by an infusion pump that is sealably connected to port 204. This way, the flow, and therefore the amount, of the compound that generates the proton introduced into the water at any time is controlled. The infusion pump can be controlled automatically or manually. The rate of introduction of the compound that generates the proton into the water is based on the properties of the water (conductivity and pH level), the composition of the buffer, and the pressure and flow of the water.

[00084] In certain embodiments, the pump is configured to introduce from about 6.5 liters per hour to about 12 liters per hour of the compound that generates the proton into the water. The introduction can be in continuous or intermittent infusion. As the water flows through the tubes in a turbulent way, there is an initial mixture of the compound that generates the proton with the water through the introduction of hydrochloric acid in the water.

[00085] Further mixing occurs when the solution enters the second mixing device 205. The mixing device 205 includes all of the features discussed above with respect to the mixing device 102. The mixing device 205 may be configured the same or different from the mixing device. mixture 202, for example, with the same or different number of subchambers, the same or different diameter of openings, the same or different sizes of subchambers, etc. However, like mixing device 203, mixing device 205 is configured to produce a fluid vortex within each subchamber.

[00086] The solution enters the mixing device 205 through an inlet in the device, which is sealably coupled to the tube 201b. The solution enters the mixing chamber and turbulent mixing occurs in each sub-chamber of the mixing device as the solution passes through the chamber members through the openings in each member. After mixing in the final subchamber, water exits the chamber through the fluid outlet in the mixing device which is sealably coupled to tube 201c.

[00087] The compound that generates the hypochlorite anion is then introduced into the solution that is flowing through tube 201c through the injection port 206. The compound that generates the hypochlorite anion is introduced by an infusion pump that is sealably connected to port 206. In this way, the flow, and therefore the amount, of the compound that generates the hypochlorite anion introduced into the water at any time is controlled. The infusion pump can be controlled automatically or manually. The rate of introduction of the compound that generates the hypochlorite anion into the water is based on the properties of the solution (conductivity and pH level) and the pressure and flow of the solution. In certain embodiments, the pump is configured to introduce about 6.5-12 liters per hour of the compound that generates the hypochlorite anion into the solution. The amount introduced depends on the desired concentration of HOCl (ppm) and the flow of water in the tubes. The introduction can be in continuous or intermittent infusion. As the solution is flowing through the tubes in a turbulent manner, there is an initial mixing of the compound that generates the hypochlorite anion with the solution, by introducing the compound that generates the hypochlorite anion into the solution.

[00088] Further mixing occurs when the solution enters the second mixing device 207. The mixing device 207 includes all of the features discussed above with respect to the mixing device 102. The mixing device 207 can be configured the same or different from the mixing devices. mixture 205 or 203, for example, with the same or different number of subchambers, the same or different diameter of openings, the same or different sizes of subchambers, etc. However, like mixing devices 205 and 203, mixing device 207 is configured to produce a fluid vortex within each subchamber.

[00089] The solution enters the mixing device 207 through an inlet in the device, which is sealably coupled to the tube 201c. The solution enters the mixing chamber and turbulent mixing occurs in each sub-chamber of the mixing device as the solution passes through the chamber members through the openings in each member. After mixing in the final subchamber, water exits the chamber through the fluid outlet in the mixing device which is sealably coupled to tube 201d.

[00090] At this point, the reaction was completed and HOCl was formed. The HOCl produced can be measured and collected as described above. Tube 201d can be connected to a changeover valve that switches between a waste line and a product collection line. The valve includes a pH meter and a conductivity measuring device. These devices measure the concentration, purity and pH of the HOCl being produced and provide feedback to alter these properties of the HOCl being produced. Once the HOCl is being produced in the 201d tube and meets the required concentration, purity and pH, the valve switches from the waste line to the product collection line to collect the desired product.

[00091] In another modality, a deionizer is placed in line with the incoming water. The deionizer deionizes the water and then a buffering agent is added to the deionized water. The production process is then conducted as described for embodiments of system 200 to produce water having a pH of at least about 8, for example 8.4, and a buffering capacity of pH 6-8.

[00092] The HOCl produced by the above process can be used in many different applications, for example,

medical, food service, food retail, agricultural, wound care, laboratory, hospitality, dental, delignification or floral industries. wound care

[00093] In certain embodiments, the compositions of the invention are used for wound care. Wound care involves treating damaged or wounded skin, including abrasions, lacerations, ruptures, punctures or burns. Specific treatments for wound care involve treating biofilms. Biofilms can form when free-floating microorganisms such as bacteria and fungi attach to a surface. Biofilms are known to impair skin wound healing and reduce topical antibacterial effectiveness in healing or caring for infected wounds. Other common health conditions related to biofilms include urinary tract infections, middle ear infections, chronic wounds and the formation of dental plaque. Cystic fibrosis, native valve endocarditis, otitis media, periodontitis, and chronic prostatitis also involve microorganisms that produce biofilms. Microorganisms commonly associated with biofilms include Candida albicans, coagulase-negative staphylococci, Enterococcus, Klebsiella pneumoniae, Pseudomonas aeruginosa, Staphylococcus aureus, and others.

[00094] Biofilms are often resistant to traditional antimicrobial treatments and therefore pose a serious health risk. Biofilm resistance makes traditional antibiotic and antimicrobial treatments ineffective. As biofilms can greatly reduce susceptibility to antibiotics and disinfectants, treatments are needed that are capable of breaking down the biofilms but that are not too toxic to the patient.

[00095] Methods are provided for administering a composition to an individual in need of treatment for a biofilm associated infection. Methods of the invention include prophylaxis, therapy or cure of a biofilm associated infection. The methods include administering one or more unit doses of a composition in a therapeutically or prophylactically effective amount for treating an existing biofilm-associated infection or preventing the onset of a biofilm-associated infection in the individual. In some embodiments, the spread of a biofilm-associated infection to another location in the individual is inhibited. In various embodiments, the composition can be administered parenterally, orally, locally or topically. The compositions can be applied by intravenous, intramuscular or subcutaneous injection. In the methods of the invention, the compositions can be administered in a pharmaceutically acceptable vehicle, examples of which are discussed below.

[00096] Treatment includes killing microbes that inhabit the biofilm or removing a biofilm, inhibiting biofilm formation and disrupting an existing biofilm. The compositions disclosed herein are particularly effective for treating microbial biofilms in or on a wound. The composition may be in the form of a topically administerable wound care composition comprising a hypochlorous acid and an acetic acid compound. The composition can be combined with an additional antimicrobial agent.

[00097] The compositions of the invention can be administered topically to a subject, for example, by direct application or spreading of the composition on the epidermal or epithelial tissue of the subject. The composition can be formulated as a liquid, powder, lotion, cream, gel, oil, ointment, gel, solid, semi-solid formulation, or aerosol spray. Such formulations can be produced in a conventional manner using suitable vehicles which are well known to one of skill in the art.

[00098] Carriers suitable for topical administration preferably remain in place on the skin as a continuous film and resist removal by perspiration or immersion in water. The vehicle can include pharmaceutically acceptable emollients, emulsifiers, thickening agents, solvents and the like.

[00099] The composition can be provided as part of a wound dressing, in which the composition is provided within the dressing or on the wound contact surface thereof. A wound dressing may be intended to be applied to a wound to be treated and which comprises a substrate comprising compositions according to the invention. Such a dressing is particularly convenient because it applies the composition of the invention to the wound to be treated and, at the same time, provides a dressing for it. The wound dressing can, for example, be fibrous, a foam, a hydrocolloid, a collagen, a film, a sheet of hydrogel or a combination thereof. The wound dressing may take the form of a layered dressing in which one or more layers of the dressing are formed at least in part or from one or among; natural fibers, alginate, chitosan, chitosan derivatives, cellulose, carboxymethylcellulose, cotton, Rayon, Nylon, acrylic, polyester, polyurethane foam, hydrogels, hydrocolloids, polyvinyl alcohol, starch, a starch film, collagen, hylaronic acid and its derivatives , biodegradable materials and other materials known in the art. The methods of the invention can further comprise negative pressure therapy for wounds, as is known in the art. These therapies involve applying negative pressure to the wound, such as with a vacuum dressing.

[000100] The composition can be administered in a single daily dose or in multiple doses, for example 2, 3, 4 or more doses per day. The total daily amount of the composition can be from about 0.01 mg, 0.1 mg, 1 mg, 2 mg, etc., up to about 1000 mg. In some embodiments, the total daily amount administered is about 0.01 mg to about 1 mg, about 1 mg to about 10 mg, about 10 mg to about 100 mg, about 100 mg to about 500mg or about 500mg to about 1000mg. The actual dosage may vary depending on the specific composition administered, the mode of administration, the type or location of the biofilm being treated and other factors known in the art. In some embodiments, a dosage may also be selected to provide a predetermined amount of composition per kilogram of patient weight.

[000101] The use of the compound in conjunction with another known antimicrobial treatment may increase the effectiveness of the antimicrobial agent. In some embodiments, the methods of the invention further comprise administering (simultaneously or sequentially with the compositions of the invention) one or more doses of an antibiotic substance, including, but not limited to, ciprofloxacin, ampicillin, azithromycin, cephalosporin, doxycycline, acid fusidic, gentamycin, linezolid, levofloxacin, norfloxacin, ofloxacin, rifampicin, tetracycline, tobramycin, vancomycin, amikacin, deftazidime, cefepime, trimethoprim/sulfamethoxazole, piperacillin/tazobactamine/cholesterol merenopenene, chlorophenol, ortrenophenol, atrenophenol, atrenophenol, atrenophenol or atrenophenol. In some embodiments, the methods of the invention further comprise administering one or more doses of an antibiotic substance from a class of antibiotics, including, but not limited to, aminoglycosides, carbacephem, carbapenems, first-generation cephalosporins, second-generation cephalosporins , third-generation cephalosporins, fourth-generation cephalosporins, glycopeptides, macrolides, monobactam, penicillins, polypeptides, quinolones, sulfonamides, tetracyclines, lincosamides and oxazolidinones. In some embodiments, the methods of the invention comprise administering a non-antibiotic antimicrobial substance, including, but not limited to sertraline, racemic and stereoisomeric forms of thioridazine, benzoyl peroxide, taurolidine and hexitidine. Biofilm treatment in other tissues

[000102] The compositions of the invention can be used to treat biofilms that affect various parts of the body or are fixed on various surfaces. In some embodiments, the methods of the invention comprise administering a therapeutically effective composition to a subject in need thereof for treating a biofilm-associated infection in the bladder, kidney, heart, middle ear, sinus, skin, lung, a joint, subcutaneous tissue, soft tissue, vascular tissue, and/or the eye. In other embodiments, a therapeutically effective amount of the composition is administered to a subject in need thereof for the treatment of one or more of the following conditions associated with the biofilm: urinary tract infection; chronic bacterial vaginosis; prostatitis; bacterial infection due to diabetes such as diabetic skin ulcer; pressure ulcer; venous catheter-associated ulcer; or a surgical wound (eg, a surgical site infection). In some embodiments, the biofilm is on an individual's skin. In some modalities, biofilm is associated with a wound, including abrasions, lacerations, ruptures, perforations, burns, and chronic wounds. In some modalities, the biofilm is below the surface of the skin, in the subcutaneous tissue, such as a deep tissue wound or surgical site infection. Biofilm treatment on non-tissue surfaces

[000103] Other applications for the treatment of biofilms are also planned. For example, the composition of the invention has application for the treatment of microbial biofilms on surfaces, for example surfaces in hospitals (such as operating rooms or patient care rooms) as well as other surfaces (for example, domestic work surfaces) . The invention also encompasses the treatment of biofilms that form on implanted prostheses and medical devices.

[000104] As is known in the art, implanted medical devices are susceptible to the formation of biofilms, including fungal biofilms and bacterial biofilms. Methods and compositions of the invention can also be used to treat biofilms that form on the surfaces of implanted medical devices, such as catheters and prostheses. The compositions of the invention can be applied to a pre-implantation medical device. Alternatively, the medical device can comprise a reservoir containing the composition so that the composition can be released in a controlled manner after implantation. Methods for treating implanted medical devices can be found in US Patents 5,902,283 and 6,589,591 and in US Patent Publication 2005/0267543, each of which is incorporated herein by reference in their entirety. Dental treatment

[000105] In another embodiment of the invention, a method is provided for the treatment of an orally associated biofilm, such as dental plaque. The invention provides methods for preventing oral plaque, treating oral plaque infection, treating tooth hypersensitivity, sterilizing a root canal or treating a dental disease.

[000106] The methods of the invention comprise contacting an oral surface, such as teeth, gums, gums or tongue, with a therapeutically effective amount of the composition. Some methods of the invention comprise preventing an orally associated biofilm by administering a prophylactically effective amount of the composition to a subject. The composition can be formulated as a dentifrice, as a toothpaste, for treating or preventing dental plaque. In other modalities, the biofilm may be located on the tongue, oral mucosa or gums. In some embodiments, the composition is formulated as a mouthwash. In some embodiments, the composition is formulated as a paint, foam, gel or varnish, for example, in a fluoride-containing composition. In one embodiment, the composition is in the form of a gel or foam in a mouthguard that a patient wears for several minutes for a fluoride treatment. In other embodiments, the composition is placed in contact with adhesive strips, which can be applied to teeth or another oral surface. The composition may comprise a liquid polymer formulation, which is a composition that is preferably applied topically to a surface, such as a tooth, to the skin, to a mucous membrane, and which dries as a film adhering to that surface. , in a manner that resists removal under normal conditions, such as eating or brushing, for applications to teeth and oral mucosa, or normal washing and abrasion, when applied to the skin. Alternatively, the composition can be applied to bandages, dressings, gauze, brushes, implants, etc. and can dry to a film before administering it to a patient. mastitis treatment

[000107] In another embodiment of the invention, compositions and methods are provided for the treatment of mastitis. Mastitis is inflammatory tissue in the breast or udder of a mammal. It is often associated with bacterial infections, such as Pseudomonas aeruginosa, Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus agalactiae, Streptococcus uberis, and others. Some of the bacteria known to cause mastitis also form biofilms, but not all mastitis results from the formation of biofilms.

[000108] Mastitis can occur in any mammal such as humans, cows (dairy cattle) and other animals. Mastitis is a particular problem for dairy cattle. In cattle, the condition occurs when white blood cells are released into the mammary gland, usually in response to bacteria in the teat canal. Cows that are repeatedly infected must be euthanized to prevent the spread of infection in the herd. The loss of milk from infected cows and the loss of entire cows and herds due to infection results in huge economic losses for the dairy industry worldwide. In the United States, for example, mastitis is estimated to cost the dairy industry up to $2 billion a year.

[000109] Methods are provided for administering a composition to a mammal in need of treatment for mastitis. Methods of the invention include prophylaxis, therapy or cure for mastitis. In some modalities, the spread of mastitis to another room or to another animal is inhibited.

[000110] The formulations, dosages and routes of administration discussed above are applicable to these embodiments of the invention. For example, in various embodiments, the composition can be administered parenterally, orally, locally or topically. The compositions can be applied by intravenous, intramuscular or subcutaneous injection. The compositions can be applied by infusion through a teat channel, as is known in the art. In the methods of the invention, the compositions can be administered in a pharmaceutically acceptable vehicle, which can include emollients, emulsifiers, thickening agents, solvents and the like.

[000111] The composition can be administered in a single daily dose or in multiple doses, for example 2, 3, 4 or more doses per day. The total daily amount of composition can be from about 0.01 mg, 0.1 mg, 1 mg, 2 mg, etc., up to about 1000 mg. In some embodiments, the total daily amount administered is about 0.01 mg to about 1 mg, about 1 mg to about 10 mg, about 10 mg to about 100 mg, about 100 mg to about 500mg or about 500mg to about 1000mg. The actual dosage may vary depending on the specific composition administered, the mode of administration and other factors known in the art. The composition can be administered in conjunction with another known antimicrobial treatment, such as an antibiotic.

[000112] The compositions can be administered topically to the udder of a cow by directly applying or spreading the composition on the udder or teat. The composition can be formulated as a liquid, powder, lotion, cream, gel, oil, ointment, gel, solid, semi-solid formulation, or aerosol spray. The methods of the invention can further comprise dipping a teat into the composition. Teat dip can be used to treat an already infected udder or to prophylactically prevent the development of mastitis. The composition can be applied immediately before milking, immediately after milking, or both. Theta dipping methods are known in the art and are described in more detail in US Patent 4,113,854, as well as in US Patent Publications 2003/0235560 and 2003/0113384, each of which is incorporated herein by reference in its entirety. . The methods can further comprise the use of a teat sealant to create a physical barrier to the teat orifice after administration of the composition.

[000113] In other modalities, the compositions can be provided through intramammary infusion. Intramammary infusion involves forcing the antibiotic up through the teat canal into the udder. The infusion liquid can comprise a composition disclosed herein in combination with a pharmaceutically acceptable carrier such as canola oil. Before infusion, the teat is cleaned, for example, with a cotton swab soaked in alcohol. An antibiotic infusion device can include a cannula sized and shaped to fit the teat canal. The cannula can be inserted fully or partially through the narrow channel. Methods for infusion are known in the art and are described, for example, in US Patents 4,983,634 and

5,797,872, all of which are incorporated herein by reference.

[000114] The methods of the invention may further comprise administering antibiotics in conjunction with the compositions of the invention, or in sequential doses before or after administration of the compositions. Wounds and surgical uses

[000115] The compositions can also be applied to prevent and treat biofilm in other types of living tissue. Tissue includes, for example, skin, mucous membranes, wounds or ostomies. As described above, the composition is useful for wound care. Wounds include bed sores, chronic wounds, burns, pressure sores, diabetes wounds and other forms of skin trauma. Wounds are often susceptible to biofilm formation, which impedes healing and can lead to chronic illness. Hypochlorous acid and acetic acid compositions can be used for debridement and cleaning of damaged tissue.

[000116] The compositions can also be used in a surgical environment, to treat the skin before or after an operation. The composition prevents infection that would lead to biofilm formation. Sometimes an area that requires surgery, such as a traumatic wound, may already be at risk for having developed a biofilm. The hypochlorous acid composition can be used to disinfect the area before the surgical incision, which would not only help treat the biofilm, but would also decrease the likelihood of it spreading to other tissue during surgery. The compositions can be used to disinfect any surface within a surgical operating field. Other medical uses

[000117] In addition to wound care, the HOCl compositions of the invention can also be used for treating non-traumatic tissues. They can be used for bladder irrigation, to prevent or treat bladder infections or catheter-associated urinary tract infections, and the like. They can be used in a similar way to treat infections in the aerodigestive tract, such as sinus and lung infections, or infections in the oral cavity, pharynx, paranasal sinuses, nasosinusal tract, larynx, piriform sinus, or esophagus. They can be used to fight microbial growth that leads to infection and to reduce allergens that cause adverse immune responses. The compositions can also be administered to the gastrointestinal tract, including stomach, intestines and colon, to combat microbial infections such as gastroenteritis Clostridium difficile infection and small intestine bacterial overgrowth (SIBO).

[000118] In various other modalities, the compositions can be administered in the form of drops to combat eye infections or can be used to clean or store contact lenses to prevent bacterial growth and biofilm formation. In other embodiments, the composition can be used as an oral rinse or mouthwash to combat biofilm buildup in the oral cavity, or it can be used to clean or store dentures.

[000119] In addition to use as an antiseptic to treat or prevent biofilms on living tissue, the compositions can be used as a disinfectant on other surfaces, such as for use in healthcare facilities, food preparation, kitchen utensils and the like. The compositions can be used to disinfect countertops, hospital beds or food preparation surfaces.

[000120] The compositions can be used to disinfect medical devices and surgical instruments, for example. Medical devices are generally initially supplied sterile but may require additional or subsequent cleaning and disinfection or sterilization. Reusable medical devices, in particular, must be sterilized or disinfected before being reused. The compositions can be applied to the medical device using any known technique. For example, the composition can be applied by wiping or spraying onto the surface of the device, spraying an aerosol or mist form of the composition onto the device, dipping the device into a container containing a volume of the composition, or placing the device in a flow of the composition , like from a faucet. Additionally or alternatively, medical devices and surgical instruments can also be stored submerged in the composition and removed at the time of use.

[000121] Treatment with a composition of hypochlorous acid disclosed in this document can be done in addition to other known techniques, such as autoclaving. Alternatively, the composition can be applied in lieu of autoclaving. As heat sterilization is not useful for all devices (for example, some devices contain delicate parts or electronics that cannot withstand high temperatures), hypochlorous acid compositions are a useful alternative, providing an effective way to sterilize or disinfect such devices.

[000122] The compositions can also be used to disinfect implants and prostheses before introducing them into the body. Such devices include orthopedic implants, wires, screws, rods, artificial discs, prosthetic joints, soft tissue fillers, pacemakers, intrauterine devices, coronary stents, ear tubes, artificial lenses, dental implants and many others known in the art.

[000123] The various modalities and uses described above involve a variety of methods of administration, as would be understood in the art.

[000124] Hypochlorous acid compositions are particularly effective for transdermal treatment due to the small size of the HOCl molecule. Hypochlorous acid is able to penetrate the epithelium and wound surfaces and therefore can usually reach the deeper tissue layers without the need for injection. This is particularly useful for biofilm infection that forms below the top layer of skin. Unlike many other antimicrobial treatments, acetic acid and similar organic acids can penetrate the deeper layers of the skin without requiring an invasive application mechanism.

[000125] In some embodiments, however, it may be desirable to prevent HOCl from penetrating the skin and therefore the compositions may be combined with excipients, vehicles, emulsifiers, polymers or other ingredients, examples of which are discussed in the patent application US 2016/0271171, which is incorporated herein by reference in its entirety.

[000126] In addition to topical use, in which the compositions can be sprayed, rubbed or rubbed into the skin, in other embodiments the compositions can be injected into a specific tissue that requires treatment. The compositions can be taken in the form of capsules for administration to the gastrointestinal tract. They can be supplied in slow release or delayed release capsules. The compositions may be provided as a suppository for insertion into the rectum or vagina.

[000127] In other embodiments, the compositions may be provided as a nasal spray for the treatment of the aerodigestive tract, which may include the treatment of allergic reactions, sinus infections, and the like. The nasal spray can be in the form of a drop, aerosol, gel or powder. The buffered hypochlorous acid and acetic acid composition can be combined with one or more of a decongestant or an anti-inflammatory or antihistamine agent as needed. The composition can be aerosolized with a nasal spray dispenser as is known in the art.

[000128] The nasal spray may also include a pharmaceutically acceptable vehicle, such as a diluent, to facilitate administration to the nasal mucosa. The vehicle can be an aqueous vehicle such as saline. The composition can be isotonic, having the same osmotic pressure as blood and tear fluid. Suitable pharmaceutically acceptable non-toxic carriers are known to those skilled in the art. Various carriers may be particularly suitable for different formulations of the composition, for example if it is to be used as drops or as a spray, a nasal suspension, a nasal ointment, a nasal gel or other nasal form. Other additives, excipients, emulsifiers, dispersing agents, buffering agents, preservatives, wetting agents, consistency aids and gelling agents may also be included. Preferably, additives should be chosen which impart the desired characteristics without reducing the stability of the hypochlorous acid. Additives can help to uniformly administer the composition over the mucosa or to reduce or delay the rate of absorption of the composition.

[000129] The composition can be delivered by various devices known in the art for administering drops, droplets and sprays. The nasal spray composition can be delivered by a dropper, pipette or dispenser. Fine droplets, sprays and aerosols can be delivered from an intranasal pump dispenser or pressure bottle. The composition can also be inhaled by means of a metered dose inhaler, such as a dry powder inhaler or a nebulizer. Controlled release with nanoparticle encapsulation

[000130] Stable aqueous solutions of hypochlorous acid and/or acetic acid can be encapsulated in nanoparticles that allow the controlled release of acid from the nanoparticles. Controlled release allows persistent antimicrobial protection.

[000131] FIG. 13 shows an antimicrobial composition 1301 comprising an aqueous solution 1303 of hypochlorous acid encapsulated in a nanoparticle 1305. The aqueous solution 1303 of hypochlorous acid is made by a method described herein to produce a solution in which the acid is stable. Stable hypochlorous acid solution 1303 is then encapsulated in nanoparticle 1305. The nanoparticle allows for the gradual release of hypochlorous acid. Although not shown, acetic acid can also be encapsulated in a nanoparticle for controlled release.

[000132] The nanoparticle can be any type of nanoparticle that provides controlled release of acid from the nanoparticle.

The nanoparticle can comprise a polymer, such as an organic polymer.

Examples of suitable polymers for controlled release nanoparticles include acrylic acid, carrageenan, cellulosic polymers (eg, ethyl cellulose or hydroxypropyl cellulose), chitosan, cyclodextrins, gelatin, guar gum, high amylase starch, hyaluronic acid, locust bean gum , pectin, polyacrylamide, poly(D,L-lactide-co-glycolide acid), poly(lactic acid), poly(xylitol adipate salicylate), polyanhydride, poly(ethylene oxide), poly(ethyleneimine), ester of polyglycerol of a fatty acid, polysaccharides, polyvinyl alcohol, povidone, sodium alginate and xanthan gum.

For details on using polymers to form controlled-release nanoparticles, see Binnebose, et al., PLOS Negl Trop Dis 9:e00047l3 (2015); Campos, et al., Scientific Reports 5:13809 (2015); Dasgupta et al., Mol.

Pharmaceutics 12:3479-3489; Gao, et al., The Journal of Antibiotics 64:625-634, (2011); Lee, et al., International Journal of Nanomedicine 11:285-297 (2016); and US Patent no. 8,449,916 (incorporated by reference). The nanoparticle may contain an aluminosilicate (such as a zeolite, eg analcime, chabazite, clinoptilolite, heulandite, leucite, montmorillonite, natrolite, phillipsite or stilbite), calcium ammonium nitrate, hydroxyapatite (eg urea-modified hydroxyapatite), metal oxide, polyphosphate or silicon compound (eg silicon dioxide). The nanoparticle can contain lipids, that is, it can be a lipid nanoparticle.

The nanoparticle can include a liposome.

For details on the use of liposomes to form controlled-release nanoparticles, see Weiniger et al., Anaesthesia 67:906-916 (2012). The liposome may be multilamellar. The nanoparticle can contain a gel, sol-gel, emulsion, colloid or hydrogel. For details on the use of hydrogels to form controlled-release nanoparticles, see Grijalvo et al., Biomater. Sci. 4:555 (2016). The nanoparticle can contain a combination of shapes, such as a hydrogel encapsulated in a liposome. The nanoparticle may have a shell-core structure. The nanoparticle can be biodegradable. Compositions of the invention can include an antimetabolic agent. The antimetabolic agent can be a metal ion. For example, the antimetabolic agent can be zinc, copper or silver.

[000133] A nanoparticle that allows the controlled release of hypochlorous acid or acetic acid allows acid diffusion to occur more slowly than acid diffusion from an equal volume of the same aqueous acid solution that is not encapsulated in a nanoparticle . Controlled release of hypochlorous acid or acetic acid may be due to the permeability characteristics of the nanoparticle, for example, a nanoparticle that is partially or poorly permeable to acid. A controlled-release nanoparticle can be a nanoparticle that releases acid due to the degradation of the nanoparticle or the compromise of its structural integrity in a time-dependent manner. The release of acid from the nanoparticle can be triggered by environmental conditions such as pH, temperature, light, pressure, redox conditions or the presence of a certain chemical.

[000134] FIG. 14 is an illustration of a method 1401 of preparing an antimicrobial composition that includes a 1403 aqueous solution of hypochlorous acid encapsulated in a nanoparticle 1405. The method involves mixing 1411 in water in a chamber 1413 from which air has been purged from a compound 1415 which generates a proton (H+) in water and a compound 1417 which generates a hypochlorite anion (OCl-) in water. Mixture 1411 produces an air-free aqueous solution 1403 of hypochlorous acid. Solution 1403 is then encapsulated 1421 into a nanoparticle 1405. Encapsulation can be carried out in an air-free environment to produce a composition that is substantially free of air. Acetic acid and hypochlorous acid compositions for biofilm treatment

[000135] The developed formulations of acetic acid and hypochlorous acid are superior for treating biofilms on surfaces including skin or other tissue. The compositions use a balanced formula where the combination of acetic acid and hypocolorous acid provides greater disinfectant qualities than either substance alone. Indeed, the present invention recognizes that the particular combinations disclosed provide greater disinfecting power than would be expected from the addition of acetic acid and hypochlorous acid. In other words, compositions were considered greater than the sum of their parts. These benefits are shown in the attached data in FIGS. 15-21, which demonstrate how the balanced compositions of acetic acid and hypochlorous acid provide improved disinfection capabilities against biofilms and outperform all other products on the market. Performance differences are shown over a wide range of concentrations.

[000136] Furthermore, since acetic acid is toxic in high concentrations, the prior art has not taught its use on skin or other tissue, except in small amounts. Some of the disclosed compositions contain acetic acid at 2% or more and, when in combination with HOCl, have proven to be safe and effective for the treatment of skin and other tissues. HOCl in these compositions was found to have an acetic acid modulating effect. This allows the compositions to take advantage of the antimicrobial properties of acetic acid without causing tissue damage. In addition, HOCl has an analgesic function, so it also allows higher concentrations of HAc to be used on the skin or other tissue without causing excessive pain or discomfort to the patient.

[000137] FIG. 15, for example, shows a comparison of various concentrations of HOCl and acetic acid against other commercially available antimicrobial compositions. Eight different treatments were tested, as listed along the x-axis. Each composition was exposed to a S. aureus biofilm cultured on a 24-hour filter, and the reduction in biofilm was measured in colony-forming units per milliliter (cfu/ml) and reported on a logarithmic scale along the y-axis. Biofilm reduction measurements were recorded at 3 hours and 6 hours. Each column therefore has two bars and shows the effect of each composition on the biofilm over time.

[000138] The first three columns show the results of 200 ppm HOCl with three different concentrations of acetic acid (0.25%, 1.0% and 2.0%, respectively). The fourth column shows 1% acetic acid alone. The next four columns show commercially available antimicrobial products: Prontosan; Octenillin; Pyrisept; and Microdacin, which is a composition of hypochlorous acid.

[000139] The results show that all three combinations of acetic acid and hypochlorous acid were more effective against the biofilm than any of the other compositions. At 3 hours, test composition A (HOCl 200 ppm and Hac 0.25%) performed approximately as well as Prontosan, the current market leader in biofilm treatment. It also far outperformed Hac 1% or other commercially available products. After 6 hours, however, composition A showed much greater effectiveness than even Protosan.

[000140] Meanwhile, test composition B (HOCl 200 ppm and Hac 1.0%) was even more effective in biofilm treatment. Comparison of composition B with 1% Hac (in the fourth column) shows the unexpected benefit of adding HOCl. Despite having the same acetic acid concentration, composition B far outperforms 1% Hac alone in 3 hours and 6 hours.

[000141] Composition C (HOCl 200 ppm and Hac 2.0%) showed by far the greatest reduction in biofilm among the compositions tested. At 3 and 6 hours, it was several orders of magnitude more effective than commercially available products.

[000142] These data show that, in addition to being more effective in reducing biofilm than any of the commercially available products, compositions containing acetic acid and hypochlorous acid were more effective than acetic acid alone (Hac 1%) or acid hypochlorous alone (microdacin), and these superior results cannot be explained merely by the additive effect of the two components. Without being tied to any particular mechanism, the data show that the combination of acetic acid and hypochlorous acid provides a synergistic effect that allows the composition to be more effective than it would otherwise be predicted based on the effectiveness of each component alone.

[000143] FIGS. 16-19 show the effects of various HOCl and HAc compositions on P. aeruginosa biofilms. FIG. 16 shows a comparison of compositions with 1% acetic acid and varying concentrations of HOCl. Five different treatments were tested with HOCl at concentrations of 0ppm, 50ppm, 100ppm, 150ppm and 200ppm. Each composition was exposed to a P. aeruginosa biofilm cultured on a 24-hour filter, and the reduction in biofilm was measured in colony-forming units per milliliter (cfu/ml) and reported on a logarithmic scale along the y-axis. Biofilm reduction measurements were recorded at 2 hours and 4 hours.

[000144] As shown in the graph, the 2 hour reduction was greater at higher HOCl concentrations, with a particularly significant peak at 150 ppm. Within 4 hours, the peak occurs at even lower concentrations of HOCl.

[000145] FIG. 17 shows the effects on P. aeruginosa of different compositions where the HOCl concentration is kept at 100 ppm and the acetic acid percentage varies from 25% to 2%. FIG. 18 shows the effects as HOCl and HAc increase.

[000146] FIGS. 19-21 show different compositions of HOCl and HAc against S. aureus and P. aeruginosa under various conditions. The figures show the superior results obtained with the combinations of hypochlorous acid and acetic acid, which demonstrate the synergistic effect of these two compounds.

[000147] The various formulations disclosed can be effective for the treatment of biofilm infections in different tissue types. For example, the composition of 200 ppm HOCl and 0.25% Hac is useful for topical applications such as hand sanitizers or mouthwashes. This composition is more effective than other commercially available products in treating surface level biofilms as shown in FIG. 15. To treat deeper tissue penetration, or to clear particularly bad biofilm infections or invasive biofilms that have penetrated below the surface, a higher percentage of HAc can be used, such as the 200 ppm HOCl formulation with 2% Hac. This composition is useful for treating infected wounds, preventing biofilm in wounds, treating eczema or treating other infections. This formulation has been found to be effective in combating biofilms that have formed on the roots of teeth.

[000148] FIGS. 20-21 show additional data supporting the unexpected effectiveness of acetic acid and hypochlorous acid compositions on various biofilms, particularly compared to prior art and commercially available compositions. As the figures make clear, various compositions that balance HOCl and HAc concentrations in different ways provide a variety of disinfectant compositions that can target different types of biofilms on different types of tissue.

Minimum Inhibitory Concentration

[000149] The minimal inhibitory concentration of the disclosed hypochlorous acid and acetic acid compositions is useful for biofilm removal on surfaces and tissues discussed herein. The minimal inhibitory concentration (MIC) was determined against 5 pathogenic bacterial strains (Acinetobacter baumannii (resistant to carbapenems), Pseudomonas aeruginosa (resistant to carbapenems), Enterococcus faecium (resistant to vancomycin), Candida spp (resistant fluconazole) and Staphyloccocus aureus) by microdilution in broth (2-fold dilution) in a 96-well microtiter plate. After incubation for 24 hours in the microtiter tray, optical density will be measured to assess growth. In addition, suspensions are streaked onto agar and controlled for next day growth. The 5 microorganisms were tested against 10 different concentrations of the antimicrobial compound HOCl and acetic acid and included growth control and sterility control.

[000150] For all organisms tested, the MIC was surprisingly low (25 ppm and 0.25% for HOCl and acetic acid, respectively), and the minimal bactericidal concentration (MBC - "minimum bactericidal concentration") was 50 ppm and 0.5% for HOCl and acetic acid, respectively. Biofilm treatment without inducing antimicrobial resistance

[000151] The stabilized hypochlorous acid and acetic acid compositions described herein are useful for both biofilm prevention and biofilm removal on all surfaces and tissues discussed herein. Because biofilms make microbes much more resistant to traditional antimicrobial agents, the microbes that make up biofilms are better able to share and modify their resistance genes and spread through the air and surroundings. As a consequence of biofilm development, a simple infection can become chronic, antibiotics and antiseptics stop working, and new strains of infections emerge.

[000152] Both acetic acid (or other organic acids) and hypochlorous acid, however, are particularly useful for the treatment and prevention of biofilms. The HAc and HOCl compositions disclosed here mimic the immune system's natural disinfectant. Therefore, the compositions are not susceptible to microbial resistance.

[000153] Tests were carried out to investigate whether Pseudomonas aeruginosa could become resistant to the disclosed compositions. The clinical isolate of P. aeruginosa was cultured and passed each day into a Miieller Hinton broth solution containing increasing concentrations of the disclosed compositions. Each day the isolate was passed (1:100 and 1:1000) in a new tube containing the same concentration or challenged with a higher concentration, this was carried out for 14 days. At the end of the experiment, P. aeruginosa was isolated from the highest concentration where growth was observed and tested for its susceptibility to 3 different antibiotics (Tobramycin, Colistin and Ciprofloxacin). As a control, the same isolate, which had not been subjected to the drug, was also tested for susceptibility to antibiotics.

Surprisingly, the results provided that the disclosed compositions were unable to induce resistance or cross-resistance against the antimicrobial substances tested. The acetic acid and hypochlorous acid compositions disclosed herein are effective in treating and preventing biofilms without inducing antimicrobial resistance. In addition, they are non-toxic, non-stinging and relieve itching. Incorporation by Reference

[000154] Any and all references and citations to other documents, such as patents, patent applications, patent publications, periodicals, books, articles, web content, which have been made throughout this disclosure, are hereby incorporated by reference in their entirety for all purposes. Equivalents

[000155] The invention can be embodied in other specific forms without departing from the spirit or its essential characteristics. The foregoing embodiments are, therefore, to be considered in all respects illustrative rather than limiting the invention described herein. Examples Example 1: Product Analysis

[000156] When the spectrophotometry is expanded to also cover the visible range, it is possible to detect colors. The gases typically produced during the production of HOCl are ClO2, Cl2O and Cl2, all of which are detectable in the visible range as yellow or yellow-red. Tzanavaras et al. (Central European J. of Chemistry, 2007, 5(1)1-12). Data in FIG. 9 illustrate that the HOCl produced by methods in the invention do not show absorption of colored gases as shown by the lack of colored substance. HOCl is known to produce a peak at 292 nm (Feng et al. 2007, J. Environ. Eng. Sci. 6, 277-284). Example 2:

[000157] The HOCl produced by the process described above was stored under thermal stress at 40 °C in order to accelerate the degradation using four different types of aqueous solutions: (1) reagent grade water (deionized water); (2) tap water; (3) reagent grade water with a phosphate buffer; and (4) phosphate-buffered tap water. The characteristics of the HOCl product were monitored after the initial reaction (T = 0); four weeks (T = 4); eight weeks (T = 8); and twelve weeks (T = 12).

[000158] FIG. 10 is a graph showing the amount (parts per million (ppm)) of HOCl initially produced (T = 0) and its stability over time. The data show that reagent grade water (deionized water) without phosphate buffer is the most stable over the twelve weeks, showing the least degradation of the product compared to the initial amount produced. Deionized water produces a much more stable product than tap water. Furthermore, and surprisingly, the data shows that phosphate buffer can negatively impact the amount of HOCl product produced.

[000159] FIG. 11 is a graph showing how the pH of the HOCl product has changed over time. In all cases, the pH decreased over time, however, for all cases, the pH remained in the range of pH = 4 to pH = 7 throughout the twelve weeks.

[000160] FIG. 12 is a graph showing the oxidizing ability of the HOCl product over time. The data show that the product retained its oxidizing capacity over the twelve weeks, regardless of the starting water. Example 3: Acetic acid compared to hydrochloric acid

[000161] Using the process described above, HOCl was produced using hydrochloric acid (HCl) and acetic acid and then stored under thermal stress at 40°C. The amount of HOCl produced initially was recorded (T = 0) and then the amount of HOCl product remaining after twelve days was recorded. Three batches of each were produced. Data for HOCl produced by HCl are shown in Table

1. Data for HOCl produced with acetic acid are shown in Table 2. Table 1: HOCl produced with HCl Quantity Number pH Quantity pH Quantity Initial batch quantity after 12 after changeover (ppm) days (ppm) 12 degradation pH days 1 192 7.12 159 5.71 17.2% 19.8% 2 183 5.88 147 4.01 19.7% 31.8% 3 189 5.21 154 3.97 18.5% 23, 8% Table 2: HOCl produced with acetic acid Quantity Number pH Quantity pH Quantity Initial batch quantity after 12 after changeover (ppm) days (ppm) 12 pH degradation days 1 205 4.62 180 4.50 12.4% 2.7% 2 205 5.33 178 5.04 13.3% 5.4% 3 207 4.07 178 3.89 13.9% 4.6%

[000162] The data show that the use of acetic acid provides greater product stability, probably due to greater pH stability. Without being limited by any theory or specific mechanism of action, it is believed that the different protonation capacity of acetic acid compared to hydrochloric acid, ie, acetic acid donates less protons to a liquid than hydrochloric acid, results in greater stability of HOCl over time. Example 4: Hand Sanitizer

[000163] Tests were performed on the hands of healthy individuals to determine the transient bacterial killing effect of the disclosed compositions.

[000164] In this experiment, 18 subjects washed their hands and dipped their fingertips in an Escherichia coli solution. Once the subjects' hands were dry, aliquots of the revealed composition (5 ml of 160 ppm HOCl, 0.13% Hac) were applied to the subjects' dry hands and rubbed into the skin for 30 minutes to encourage the removal of transient bacteria on the skin, according to the Handrub procedure PN-EN 1500:2013-07. The same was done with 3 ml of the reference alcohol 2-propanol 60% v/v.

[000165] The results provided that both the revealed composition and the reference alcohol reduced the presence of transient bacteria by a 4 log reduction.

[000166] In another experiment, aliquots of the revealed composition (3 ml of 0.03% HOCl, 0.13% Hac) were applied to each of the hands of 20 subjects. For each of the subjects, one hand was bare and the other had a surgical glove covering it. The composition was rubbed onto the hands and forearms for 5 minutes to maintain exposure to the compositions according to the Handrub to Hands procedure PN-EN 12791:2005. The same was done using 3 ml of the reference alcohol propan-1-ol, 60% v/v for 3 minutes of sustained exposure.

[000167] The killing of bacteria, Pseudomonas aeruginosa, Staphylococcus areus, and Enterococcus hirae were measured immediately after application and again 3 hours after application. The results showed that the reference alcohol reduced natural bacteria by 2.48 logs immediately after application and 2.16 logs after application. The disclosed composition reduced natural bacteria by 0.69 logs 3 hours after application and a 1.01-log reduction immediately after application.

[000168] Surprisingly, the disclosed compositions of the present invention, compared to prior art alcohol-based disinfectants, were more effective in targeting transient pathogenic bacteria without harming natural bacteria. Therefore, the disclosed compositions are effective in the targeted treatment of pathogenic biofilm infections, without harming the body's natural flora that protects the skin.

Claims (13)

1. Composition, characterized by the fact that it comprises acetic acid and hypochlorous acid, sufficient to treat a bacterial biofilm and not induce antimicrobial resistance. 2. Composition according to claim 1, characterized in that the concentration of acetic acid is between 0.05% and 5.0%. 3. Composition according to claim 1, characterized in that the concentration of acetic acid is greater than about 0.25%. 4. Composition according to claim 1, characterized in that the concentration of hypochlorous acid is between 5 ppm and 2500 ppm. 5. Composition according to claim 1, characterized in that the concentration of hypochlorous acid is approximately 25 ppm. 6. Composition according to claim 1, characterized in that the acetic acid is present in a sufficient concentration to penetrate the tissue. 7. Composition according to claim 1, characterized in that hypochlorous acid is present in a concentration sufficient to modulate a toxic property of acetic acid. 8. Composition according to claim 1, characterized in that the composition is formulated in a gel, cream, ointment or oil. 9. Composition according to claim 1, characterized in that the acetic acid is encapsulated in a nanoparticle. 10. A method of treating a bacterial infection in tissue, the method characterized in that it comprises providing the tissue with a composition comprising acetic acid and an amount of hypochlorous acid effective to modulate a toxic property of acetic acid without inducing antimicrobial resistance. 11. Method according to claim 10, characterized in that the acetic acid is in a sufficient concentration to penetrate the skin. 12. Method according to claim 11, characterized in that said acetic acid is present in an amount from about 0.05% to about 1.0% and said hypochlorous acid is present in a concentration of about 5 ppm to about 100 ppm. 13. Method according to claim 11, characterized in that the tissue is skin and in which the concentration of hypochlorous acid is about 25 ppm, and in which the concentration of acetic acid is between about 0.0625 and about 0.25%.