EA041951B1 - COMPOSITION AND METHOD FOR BIOFILM TREATMENT - Google Patents

Description

Related Applications

The present application claims the priority and benefits of US Patent Application 15/612571, filed June 2, 2017; U.S. Provisional Application 62/438189, filed December 22, 2016; U.S. Provisional Application 62/438,198, filed December 22, 2016; U.S. Provisional Application 62/438,202, filed December 22, 2016; and U.S. provisional application 62/438204, filed December 22, 2016.

The contents of each of the above applications are hereby incorporated by reference in their entirety.

The technical field to which the invention belongs

The invention generally relates to compositions of acetic acid and hypochlorous acid for the treatment of biofilms, in particular biofilms on tissue and in wounds, and other biofilm infections.

State of the art

Microbial infections that form biofilms can pose a serious health threat. Scientists estimate that up to 80% of all infections affecting mammals are biofilm infections. Biofilms represent a particular challenge in the treatment of wounds. Medical professionals have long tried to treat biofilms in wounds, including chronic wounds and wounds associated with eczema. Bacteria such as S. aureus and P. aeruginosa form biofilms on wound surfaces and in wounds. These infections are difficult to treat and interfere with wound healing. It has been noted, for example, that 93.5% of chronic leg ulcers contain S. aureus and 52.2% contain P. aeruginosa, with ulcers containing P. aeruginosa characterized by larger wounds and a slower healing rate; see Gjodsbol et al., 2006, International Wound Journal 3(3):225-31; and also see Fazli et al., 2009, Journal of Clinical Microbiology 47(12):4084-89.

Disinfectant compositions of the prior art have various disadvantages. Some cannot penetrate deep enough into the wound to completely destroy the biofilm that has become entrenched below the surface. Others dissipate too quickly and cannot act on the biofilm long enough to be effective. Still others do not have sufficient product stability, requiring local production, making them more difficult to use and less available. Other antimicrobial compositions are tissue irritants or otherwise incompatible with topical use.

The essence of the invention

Disinfectant compositions comprising hypochlorous acid and acetic acid, as described herein, can be used to treat biofilms in or on tissue, ranging from simple topical disinfectants to wounds or other skin injuries. For example, wounds are often susceptible to microbial infection, including biofilms that form on and below the surface of the wound that interfere with healing and can lead to chronic disease. Compositions containing hypochlorous acid and acetic acid can be used to treat tissue biofilms. The concentrations of HOCl and HAc are balanced to achieve a synergistic effect in which the antimicrobial properties of the composition are higher than would be expected based on the antimicrobial properties of each component individually. An HOCl-balanced acetic acid composition provides higher antimicrobial activity than acetic acid alone, often by several orders of magnitude. In addition, hypochlorous acid modulates the toxicity of acetic acid and provides an analgesic effect, allowing higher concentration formulations to be applied to the skin or other tissues without unwanted side effects or patient discomfort.

The disclosed compositions can be used to treat various types of tissue, both on the surface and below the surface. A component such as hypochlorous acid is effective against biofilms at the surface level or biofilms immediately below the surface, such as a wound, while acetic acid penetrates the wound or tissue more deeply. This document describes various compositions containing different concentrations of each acidic component for the treatment of various types of tissues and wounds. Acetic acid concentrations greater than about 0.25% can be used to penetrate below the wound surface. Some compositions thus contain acetic acid at concentrations of about 1.0%, about 2.0% or higher, up to about 5.0%. Similarly, hypochlorous acid is present in various concentrations in the compositions depending on the type of tissue being treated. For example, hypochlorous acid at a concentration of 80-250 ppm. can be used to treat root biofilm, while only 5-60 ppm is required for a mouthrinse formulation. The compositions according to the invention are particularly effective due to the synergistic balance between hypochlorous acid and acetic acid, which provides a dual action of the compositions on the surface, immediately below the surface and deeper subsurface treatment of tissue infected with biofilm. As a rule, hypochlorous acid is able to act quickly on or close to the surface; whereas acetic acid lasts longer and can therefore act below the surface of the wound.

Compositions according to the invention may be presented as a gel or cream, which provides a longer contact time with the tissue. For the treatment of wounds, gels and cream compositions also help keep wounds hydrated to promote healing. In addition, one or both components of the compositions according to the invention can be enclosed in a controlled or delayed release nanoparticle.

The disclosed compositions and methods of treatment are effective against biofilms wherever they are, in a wound, on or in tissue, on non-tissue surfaces, or in preoperative or post-traumatic conditions. Application of the compositions to the site of skin injury helps fight microbial infection by preventing and treating bacterial infection, including biofilms. Compositions according to the invention can be used in first aid or in surgical settings, for skin treatment before, during or after surgery. Disinfecting the area before the surgical incision not only helps to disinfect the area of the operation, but also reduces the chance of infection spreading to other tissues during the operation. Compositions of hypochlorous and acetic acids can also be used to remove and clean damaged tissues.

The compositions described herein may be combined with various excipients and carriers to facilitate topical application. Products containing hypochlorous acid (and organic acid) may take the form of gels, creams, lotions, sprays, liquids, foams, powders, and other delivery forms known in the art. In the alternative, the compositions may be included in tissue or fibrous wipes or wound dressings.

Because the disclosed compositions are capable of penetrating tissues, they are also effective in combating biofilms below the surface of a wound, in subcutaneous tissue, such as at the root of a tooth, surgical site, or deep wound.

In some aspects, the invention includes a composition consisting of acetic acid and hypochlorous acid. Acetic acid is present in concentrations greater than about 0.1% and preferably greater than about 0.25%. The preferred concentration of hypochlorous acid is from about 10 to about 1000 ppm. In various embodiments, the specific concentrations of acetic acid and hypochlorous acid depend on the intended area of treatment and how deep below the surface of the tissue the treatment is required.

In some embodiments, the acetic acid is present in a concentration sufficient to penetrate below the surface of the fabric. In some embodiments, the concentration of acetic acid is greater than about 0.5%, and preferably greater than about 1.0%, and in some embodiments, it is greater than or equal to about 2.0%. The acetic acid can be nanoparticulated for controlled or delayed release. In some embodiments, the implementation of hypochlorous acid is present in a concentration sufficient to treat the biofilm on the surface and just below the surface of the wound. The hypochlorous acid concentration may be from about 20 to about 200 ppm. The composition may further include a gel, cream, ointment or oil.

In related aspects, the invention includes a method of treating biofilm in or on tissue. The methods include applying to the fabric a composition containing acetic acid in a concentration sufficient to penetrate the skin and hypochlorous acid in an amount sufficient to remove biofilm on the surface of the tissue and immediately below the surface of the tissue.

Acetic acid may be present in amounts sufficient to remove the biofilm below the surface of the skin. Acetic acid may be present in an amount of from about 0.1 to about 2.0%, and hypochlorous acid may be present in a concentration of from about 10 to about 1000 ppm.

In some embodiments, the tissue to be treated is bladder tissue and the hypochlorous acid concentration is about 80-250 ppm. In other embodiments, the tissue to be treated is lung tissue and the hypochlorous acid concentration is about 15-60 ppm. In other embodiments, the tissue to be treated is dental tissue and the concentration of hypochlorous acid is about 5-60 ppm and the concentration of acetic acid is from about 0.05 to about 5.0%.

In some embodiments, the biofilm to be treated is formed at the site of the wound, on the surface, and below the surface of the wound. The concentration of acetic acid for wound treatment may be from about 0.5% to about 5.0%.

In related aspects, a method of treating a biofilm in a tissue comprises applying a nanoparticle comprising acetic acid to a tissue site where biofilm formation is suspected. The nanoparticle may be fat soluble.

- 2 041951

Brief description of the drawings

Fig. 1 is a diagram showing an exemplary system for producing hypochlorous acid according to the methods of the invention.

Fig. 2 is a diagram showing an enlarged view of the mixing device shown in FIG. 1.

Fig. 3 is a diagram showing an inside view of the mixing chamber of the mixing device.

Fig. 4 is a diagram showing a front view of the elements that divide the mixing chamber into a plurality of sub-chambers. This view shows holes in the elements.

Fig. 5 is a diagram showing a valve provided with sensors for switching from a discharge line to a product collection line.

Fig. 6 is a diagram showing a valve in the discharge line and the product collection line.

Fig. 7 is a diagram showing another exemplary system for producing hypochlorous acid according to the methods of the invention. This system is designed for automated use with buffered deionized water. The buffer may be included either in the supply water or may be introduced through the inlet. The buffer can also be mixed during the mixing process using NaOH in NaOCl or introduced separately, as well as acetic acid or other similar acids or bases.

Fig. 8 is a graph of a calibration curve showing the HOCl concentration (ppm) calculated indirectly as a function of electrical conductivity.

Fig. 9 is a graph showing a spectrophotometric analysis of the resulting HOCl. Gases commonly produced during the production of HOCl are ClO ₂ , Cl ₂ O and Cl ₂ , all of which are detectable in the visible range as yellow or yellow-red. The plot does not show absorption from non-ferrous gases in the resulting HOCl.

Fig. 10 is a graph showing the amount (parts per million (ppm)) of HOCl initially produced (T=0) and its stability over time.

Fig. 11 is a graph showing how the pH of the HOCl product changed over time.

Fig. 12 is a graph showing the oxidation and reduction (redox potential) of the HOCl product over time.

Fig. 13 shows an antimicrobial composition that includes an aqueous solution of hypochlorous acid enclosed in a nanoparticle.

Fig. 14 is an illustration of a method for preparing an antimicrobial composition that includes an aqueous solution of hypochlorous acid enclosed in a nanoparticle.

In FIG. 15-21 show data on the reduction of various biofilms with compositions of acetic acid and hypochlorous acid at various concentrations compared to commercially available biofilm treatments.

Detailed description

Treatment of bacterial infection and biofilm is achieved with a synergistic composition of acetic acid and hypochlorous acid. A component such as acetic acid is particularly effective in penetrating tissues, while hypochlorous acid is particularly effective in treating biofilm on the outer surface of tissue. Acetic acid can penetrate up to 2 mm or more below the wound surface, allowing treatment of biofilms otherwise difficult to reach.

Compositions according to the invention include hypochlorous acid in synergistic combination with acetic acid. A balanced composition in which the biocidal amount of acetic acid (or equivalent organic acid) is optimized with hypochlorous acid has been found to achieve maximum therapeutic efficacy, whether applied to the surface or in a manner intended to penetrate the skin. For example, for surface applications, the ratio of acetic acid to hypochlorous acid is lower than for penetrating applications. According to the invention, surface bacterial contamination or biofilm is sufficiently treatable with hypochlorous acid with less acetic acid; but for applications requiring deep penetration (such as wounds), the amount of acetic acid must be increased. In this case, hypochlorous acid is used to reduce the toxicity of acetic acid to surrounding tissues by allowing the acetic acid to act on the biofilm. In addition, a synergistic combination of acetic acid and hypochlorous acid has been found to selectively destroy harmful biofilm while maintaining beneficial biofilm. Compositions according to the invention contain balanced concentrations of acetic acid and hypochlorous acid to selectively kill harmful biofilm. The concentrations are also balanced with regard to the application for which they are intended (for example, treatment of the surface of the tooth or skin versus treatment of the deep tissues of the wound or root of the tooth). This application provides guidance on the synergistic action of various combinations of acetic acid and hypochlorous acid. One skilled in the art, based on the information provided herein, will be able to determine the relative amounts of acetic acid and hypochlorous acid needed to treat any bacterial infection or biofilm formation.

Treatment of both superficial and subcutaneous infections provides a dual action treatment that is especially required for wound healing. Chronic wounds and eczema are affected by a biofilm that affects the subsurface of the wound. These wounds are often infected with S. aureus, which is usually present on or near the surface and prevents the wound from closing and healing. In addition, P. aeruginosa infection is often present and tends to be located deeper under the surface of the wound. In the presence of a deep infection, it is important to keep the wound open and hydrated while healing from the inside, to prevent the wound from closing until the deeper infection has healed. Treatment of the infection only at the surface level leads to the fact that the wound closes and traps a deeper biofilm inside the tissue, which can lead to sepsis and other complications.

A common problem with diabetic foot ulcers, for example, is that they close at the surface, forming an open cavity below the surface. The cavity contains 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 foot ulcers, may contribute to the spread of infection and potentially cause sepsis. However, with a properly dressed open wound, pus will flow into the wound bed and dressing. Bacteria that survive in the purulent environment inside a closed wound can spread infection. Thus, clearing the S. aureus infection without first or simultaneously clearing the P. aeruginosa infection may not completely clear the biofilm infection, leading to early closure and possibly sepsis.

Antimicrobial solutions, such as the compositions provided herein, reduce infection in deep areas of the wound bed and allow the wound to heal from the inside out so that the surface does not heal faster than the inside wound. Acetic acid is present in an amount sufficient to disinfect the biofilm beneath the wound layer, and hypochlorous acid is present in an amount sufficient to disinfect the surface of the wound. Thus, the composition provides complete disinfection of the wound to prevent premature closure and trapping of biofilm under the surface of the closed wound.

The disclosed compositions are particularly effective because the optimal selection of concentrations of hypochlorous and acetic acids allows the treatment of biofilm at the surface level, as well as biofilm below the surface. The exact balance depends on the area to be treated and the level of penetration required through the surface. Hypochlorous acid may be present in an amount of from about 10 to about 500 ppm. or more. Different applications and tissue types may require higher or lower concentrations. Acetic acid may be present in an amount of from about 0.25 to about 2.0% or more, and preferably about 1.0%. With the selection of concentrations of the two components, the composition can have a dual effect in the treatment of the surface and subsurface of the tissue or wound.

Compositions according to the present description can be created for various applications. For example, a composition with a relatively low content of acetic acid (up to about 0.05%) and about 5-60 ppm. hypochlorous acid is a composition suitable as a mouthrinse to fight infection in dental tissue. A lower concentration of acetic acid is sufficient in mouthrinse formulations because the microbial infection usually does not penetrate deep into the tissue.

For wound healing, on the other hand, the 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 biofilm deep within. fabrics.

Other uses may require more or less of each component. For example, a composition with a hypochlorous acid concentration of about 80-250 ppm. may be used to flush the bladder, a treatment often required in patients with a urinary catheter to prevent infection or blockage. Composition with a concentration of hypochlorous acid of about 15-60 ppm. is sufficient to treat an infected lung.

In some embodiments, the implementation of the composition is in the form of a gel, which provides a longer time of contact with the wound. Flushing with a solution may not be sufficient, as the contact time of the antiseptic will be very short. In many cases, in order to completely remove the biofilm, the composition must be in contact with it for an extended period of time ranging from a few seconds to several minutes, up to an hour or more.

The composition may be presented as a gel or cream which prevents rapid evaporation or dispersal. Gels, creams, ointments, oils and other similar topical carriers are known in the art.

In addition, for the treatment of wounds, gel compositions have the advantage of retaining moisture at the wound site. It is important to maintain wound hydration during and after treatment with the compositions of the invention. The disclosed compositions are primarily water (typically 95% or more) to keep the wound moist while the antiseptic components of the composition fight infection in the wound and prevent the spread of new infections. Maintaining a hydrated state also prevents premature wound closure and entrapment of the biofilm into the tissue. Acetic acid can be easily incorporated into the gel because acetic acid is not too reactive. Other organic acids may also be used, with those acids being less reactive being preferred.

Sustained release compositions may also be used. In some compositions, the acetic acid may be encapsulated in lipid-soluble nanoparticles that allow the acetic acid to be transported to the subsurface of the wound before it is released from the nanoparticles. The introduction of nanoparticles with different properties allows the slow release of acetic acid, prevents its dispersion and provides other advantages during the introduction. Acetic acid is freely diffusible, soluble in water and has a high vapor pressure. These properties increase the difficulty of controlling where the acetic acid will go. Nano-encapsulated acetic acid allows more precise control of the composition. Nanoparticles are described in more detail below and shown in FIG. 13 and 14.

In some embodiments, the composition includes some acetic acid that is free of nanoparticles and a small amount of acetic acid that is encapsulated in nanoparticles. Alternatively, sustained release formulations may be used alone or in combination with other immediate dosage forms. For example, a wound can be treated with a composition of acetic acid and hypochlorous acid, and then treated with a composition consisting primarily of acetic acid encapsulated in nanoparticles to ensure continuous release of acetic acid into the deep areas of the wound after initial treatment.

Preparation of Hypochlorous Acid Compositions.

The basis of the compositions and methods of the invention is the protonation of hypochlorite ion (OCl) ^- . Using HCl or acetic acid (HAc) and NaOCl as an example, protonation is achieved by introducing an acid (eg HCl) into the solution, which results in the following reaction:

110^^^^0,,..^11^.11.,^/^0.^^^^^ ^{or H} A ^s (aq) + № ^OS1 (aq) ^ ^HOS1 (aq) + No ^A1 (aq) ).

Hypochlorous acid in an aqueous solution partially dissociates to form hypochloritanion (OCl) ^- , thus, in an aqueous solution there is always an equilibrium between hypochlorous acid and anion (OCl) ^- . This equilibrium is pH dependent and at higher pH the anion dominates. In aqueous solution, hypochlorous acid is also in equilibrium with other chlorine compounds, in particular gaseous chlorine, Cl ₂ , and various oxides of chlorine. At acidic pH, chlorine-containing gases become increasingly dominant, while at neutral pH, a different equilibrium results in hypochlorous acid predominating in the solution. Thus, when preparing hypochlorous acid, it is important to control contact with air and pH.

In addition, the concentration of protons (H) ⁺ affects the stability of the product. It is known that the concentration of protons can be controlled by using an acid which, at a given pH, has a lower ability to donate a proton (ie, the acid can provide buffering capacity). For example, running a process with acetic acid instead of hydrochloric acid is optimal when the desired pH of the final solution is approximately equal to the pKa of acetic acid. This can be achieved by mixing ratios in water of 250x or more, which means 1 part proton donor at 100% concentration (eg HCl or acetic acid) to 250 parts water.

In some embodiments, methods for producing HOCl include mixing in water, in an air-free environment, a compound that forms a proton (H) ⁺ in water and a compound that forms a hypochlorite anion (OCl) ^- in water, thus obtaining not containing air hypochlorous acid. The water may be tap water or purified water such as water purchased from a water treatment company such as Millipore (Billerica, MA). Usually the pH of the water during the process is maintained at a level of from about 4.5 to about 9, however, during the production process, the pH can be above and below the specified range. Carrying out the processes of the invention in an airless environment prevents the accumulation of chlorine-containing gases during the production process. In addition, carrying out the processes of the invention in an airless environment further stabilizes the HOCl produced.

Any compound that forms hypochlorite anion (OCl) ^- in water can be used. Examples of compounds include NaOCl and Ca(OCl) ₂ . In specific embodiments, such a compound is NaOCl. Any compound that forms a proton (H) ⁺ in water can be used in accordance with the methods of the invention. Examples of compounds are acids such as acetic acid, HCl and H2SO4. In specific embodiments, such a compound is HCl. In preferred embodiments, the compound is acetic acid because it is a weaker acid with a preferred pKa than HCl, meaning it donates fewer protons during the reaction than HCl and is better able to maintain the preferred pH.

The mixing may be carried out in any type of vessel or chamber or fluid system. In some embodiments, the fluid system 100 shown in FIG. 1 is used to carry out the methods of the invention. System 100 includes a series of communicating pipes 101a-c with a plurality of mixing devices 102 and 103 in line with a plurality of pipes 101a-c. Pipes and mixing devices can be connected to each other by seals so that all air can be removed from the system, allowing the methods according to the invention to be carried out in an airless environment. In some embodiments, the methods of the invention are also carried out under pressure. Airless and pressurized production of HOCl produces HOCl that does not react with gases in the air (such as oxygen) that can reduce the stability of the resulting HOCl.

The pipes 101a-c typically have an internal diameter ranging from about 5 to about 50 mm, more preferably from about 17 to about 21 mm. In certain embodiments, pipes 101a-c have an internal diameter of approximately 21 mm. The pipes 101a-c typically have a length of from about 10 cm to about 400 cm, more preferably from about 15 to about 350 cm. In some embodiments, the pipes 101a-c are the same length. In other embodiments, the pipes 101a-c are of different lengths. In certain embodiments, conduit 101a is approximately 105 cm long, conduit 101b is approximately 40 cm long, and conduit 101c is approximately 200 cm long.

The pipes and mixers can be made from any inert material, so that the material of the pipes and mixers does not participate in the reaction in the fluid system. Examples of materials include PVC. The pipes are manufactured by Georg Ficher AB. Pipes and mixers can be made with the possibility of a linear arrangement, while the pipes and mixers are located in a straight line. Alternatively, pipes and mixers may be non-linear, with water flowing through bends and curves during the process. System 100 shows a non-linear configuration of pipes 101a-c and mixers 102 and 103.

Pipe 101a is an inlet pipe that receives water that will flow through the system. Typically, the water in pipes 101a-c is at a pressure of at least about 0.1 bar, such as, for example, 0.2 bar or more, 0.3 bar or more, 0.4 bar or more, 0.5 bar, or greater than, 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 these pressures, a turbulent flow of water is created, so the reactants enter the turbulent flow of water, which facilitates the initial mixing of the reactants with water before subsequent mixing in the mixing devices 102 and 103.

For pH control during the production process, the feed water should have a buffering capacity between pH 3.5-9.0, more preferably between 6.0 and 8.0, to facilitate the addition of compounds that form a proton and a compound that forms hypochlorite anion. Dissolved salts and other molecules normally present in tap water give tap water a buffering capacity in the range of pH 5.5-9.0 and thus tap water is the appropriate water to be used in accordance with the methods of the invention.

In some embodiments, deionized water is used with the addition of known buffering agents to produce water having a buffering capacity in the pH range of 3.5-9.0. One example of a buffer in this particular range is phosphate buffer. For better process control and consistency, the use of treated deionized water may be preferred over the use of tap water, as tap water may vary by location and also over time. In addition, the use of deionized water with known additives also guarantees a stable pH value of the incoming water stream. This process is discussed in more detail below.

In specific embodiments, the initial pH of the water prior to addition of any of the compounds that form a proton or the compound that forms a hypochlorite anion is at least about 8.0, including 8.1 or more, 8.2 or more, 8, 3 or more, 8.4 or more, 8.5 or more, 8.6 or more, 8.7 or more, 8.8 or more, 8.9 or more, 9.0 or more, 9.5 or more greater than, 10.0 or greater, 10.5 or greater, or 10.8 or greater. In certain embodiments, the pH of the water prior to addition of any of the compounds that form a proton or the compound that forms the hypochlorite anion is 8.4.

Methods for producing HOCl include introducing a compound that forms a proton and a compound that forms a hypochlorite anion into water in any order (eg, simultaneously or sequentially) and in any manner (aqueous form, solid form, etc.). For example, a compound that forms a proton and a compound that forms a hypochlorite anion are aqueous solutions, and they are introduced into water sequentially, for example, a compound that forms a proton can be introduced into water first, and a compound that forms hypochlorite anion , can be introduced into the water second.

- 6 041951

The system 100 is configured to sequentially introduce reactants into a water stream, and a process is described herein in which a compound that forms a proton is introduced into the water first, and a compound that forms the hypochlorite anion is introduced into the water second. In some embodiments, the proton-forming compound and the hypochloritanion-forming compound are introduced into the water in small aliquots, eg, from about 0.1 to about 0.6 ml. Repeated and accurate titrations allow pH to be adjusted despite the addition of an acid (a compound that forms a proton) and an alkali (a compound that forms the hypochlorite anion). In some embodiments, an amount of a compound that forms a proton is introduced into the water, not greater than about 0.6 ml, at one time point. In other embodiments, no more than about 0.6 ml of a compound that forms the hypochlorite anion is introduced into the water at one time point.

To introduce chemicals into the water, pipe 101a includes an inlet 104 and pipe 101b includes an inlet 105. Injections 104 and 105 allow the chemicals to be introduced into the water stream. In this embodiment, the aqueous compound that forms the proton is introduced into the water in the pipe 101a through the inlet 104. The compound that forms the proton is administered by means of an infusion pump that is hermetically connected to the orifice 104. In this way, the flow rate is controlled and therefore , the amount of a compound that forms a proton, introduced into the water at any given time. The infusion pump can be controlled automatically or manually. The rate at which the compound that forms the proton is introduced into the water depends on the quality of the incoming water (electrical conductivity and pH), as well as the pressure and flow of the incoming water. In some embodiments, the pump allows approximately 6.5 L/hr of hydrochloric acid to be injected into the water. Administration may be by continuous infusion or intermittently. Since the water flows through the pipes in a turbulent mode, there is an initial mixing of the compound that forms the proton with water when hydrochloric acid is introduced into the water.

Subsequent mixing occurs as water enters the first mixing device 102. FIG. 2 is an enlarged view of the mixing device 102 shown in FIG. 1. In the embodiment shown, the mixing device comprises a length of approximately 5.5 cm and a diameter of approximately 5 cm. Those skilled in the art will recognize that these are exemplary dimensions, and the methods of the invention may be carried out with mixing devices having other dimensions than presented as an example. The mixing device 102 includes a fluid inlet 106 which is hermetically connected to the pipe 101a and a fluid outlet 107 which is hermetically connected to the pipe 101b. In this way, water can enter the mixing chamber 108 of the device 102 from the pipe 101a and exit the chamber 108 of the device 102 through the pipe 101b.

The mixing device 102 is configured to create a plurality of fluid vortices within the device. An exemplary device so arranged is shown in FIG. 3, which is a figure showing an interior view of chamber 108 of device 102. Chamber 108 includes a plurality of elements 109 installed at intervals and fixed within chamber 108, perpendicular to an inlet and outlet, to form a plurality of sub-chambers 110. Each element 109 includes at least one opening 111 through which fluid can flow. In FIG. 4 is a front view of the elements 109 showing the openings 111. The size of the openings will depend on the flow of water and the pressure in the system.

Any number of elements 109 can be fixed in the chamber 108, with the number of elements 109 fixed in the chamber 108 will depend on the desired intensity of mixing. In FIG. 4 shows four elements 109a-d which are fixedly mounted in the chamber to form four sub-chambers 110a-d. Elements 109 may be located at different intervals in the chamber 108, with the formation of the sub-chamber 110 of the same size. Alternatively, elements 109 may be spaced at different intervals within chamber 108 to form subchambers 110 of varying sizes. The elements 109 are sized such that they can be fixedly attached to the inner wall of the chamber 108. As a result, water cannot flow around the elements and can only flow through holes 111 in each element 109 passing through the mixing device 102. Typically, the elements will have a diameter of about 1 to about 10 cm. In certain embodiments, the elements are about 3.5 cm in diameter.

A swirling fluid flow is formed in each subchamber 110a-d. The vortices result from water flowing through openings 111 in each element 109. The methods of the invention allow for any configuration of openings 111 near each element 109. FIG. 4 shows non-limiting examples of various configurations of openings 111 in member 109. The openings may be of any shape. In FIG. 4 depicts circular holes 111. In some embodiments, holes 111 are all located at the same location on elements 109. In other embodiments, holes 111 are located at different locations on elements 109. In one element 109, all holes 111 may be the same diameter. Alternatively, in one element 110, at least two holes 111 have different sizes. In other embodiments, all holes 111 in one

- 7 041951 element 110 have different sizes.

In some embodiments, openings 111 in element 110 are of a first size and openings 111 in another element 110 are of a different, second size. In other embodiments, openings 111 in at least two different elements 110 are of the same size. The size of the holes will depend on the flow of water and the pressure in the system. Approximate hole diameters are from about 1 mm to about 1 cm. In certain embodiments, the holes have a diameter of about 6 mm.

The solution enters the mixing device 102 through the inlet 106, which is hermetically attached to the pipe 101a. The solution enters the chamber 108 and turbulent mixing occurs in each of the subchambers 110a-d as the solution passes through the elements 109a-d due to the holes 111 in each element 109a-d. After mixing in the last sub-chamber 110d, the water leaves the chamber 108 through the fluid outlet 107 which is hermetically connected to the pipe 101b.

The compound that forms the hypochlorite anion is then injected into the solution that flows through the pipe 101b through the injection hole 105. The compound that forms the hypochlorite anion is injected using an infusion pump that is hermetically connected to the hole 105. In this way, the rate is adjusted flow and, thus, the amount of compound introduced into the water, which forms the hypochlorite anion, at any given time. The infusion pump can be controlled automatically or manually. The rate of introduction of the compound that forms the hypochlorite anion into water depends on the properties of the solution (electrical conductivity and pH level), as well as the pressure and flow of the solution. In some embodiments, the pump allows approximately 6.5 L/hr of the compound that forms the hypochlorite anion to be injected into the solution. Administration may be by continuous infusion or intermittently. As the solution flows through the pipes in a turbulent manner, there is an initial mixing of the compound that forms the hypochlorite anion with the solution as the compound that forms the hypochlorite anion is introduced into the solution.

Additional 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 may have the same or a different configuration compared to the mixing device 102, for example the same or different number of subchambers, same or different hole diameter, same or different subchamber size, etc. In any case, like the mixing device 102, the mixing device 103 is configured to produce a swirling fluid flow in each sub-chamber.

The solution enters the mixing device 103 through the inlet in the device, which is hermetically connected to the pipe 101b. The solution enters the mixing chamber, with turbulent mixing occurring in each sub-chamber of the mixing device as the solution passes through the elements in the chamber due to the holes in each element. After mixing in the last sub-chamber, the water leaves the chamber through an outlet in the mixing device, which is hermetically connected to the pipe 101c.

At this point, the reaction is complete and HOCl is formed. The preparation is controlled by measuring pH and electrical conductivity. The pH value is used in conjunction with the electrical conductivity based on a pre-calibrated relationship between the electrical conductivity and the HOCl concentration measured by spectrophotometry. The measured electrical conductivity is a measure of the solvent's ability to conduct electrical current. By comparing the resulting matrix with various known concentrations of HOCl and OCl ^- , a calibration curve was generated (FIG. 8) and used in conjunction with a pH meter to adjust the titration and control the process.

Conduit 101c may be connected to a multi-way valve 112 which switches flow between discharge line 113 and product collection line 114. Shown in FIG. 5 and 6. Valve 112 includes a pH meter and a conductivity meter. These devices measure the concentration (ppm), purity, and pH of the HOCl produced and provide feedback to change such properties of the HOCl produced. If the HOCl produced in pipe 101c meets the required concentration, purity, and pH, valve 112 switches flow from waste line 113 to product collection line 114 to collect the desired product.

The airless HOCl is collected and bottled in an airless environment. Placing liquids in a vial in an airless environment is known in the art. An exemplary method includes placing an inflatable container (such as a balloon) into the vial. The balloon is connected directly to collection line 114 and the HOCl is pumped directly into the balloon in a vial without any exposure to air. Another method involves filling the vials under vacuum. Another airless filling method involves filling the vials in an inert gas atmosphere that does not react with HOCl, such as argon.

The resulting hypochlorous acid does not contain air and will have a pH of about 4.5 to about 7.5. However, the pH of the resulting HOCl can be adjusted after the production process by adding any acid (eg HCl) or alkali (eg NaOCl) to the resulting hypochlorous acid. For example, a pH of about 4.5 to about 7 is particularly suitable for use in the re-sterilization of heat sensitive medical instruments. Other applications, such as non-medical use such as poultry and fish processing, as well as general agricultural and petrochemical applications, bacterial biofilm removal and water treatment, may require different pH levels.

The process can be performed manually or automatically. The fluid systems described herein may be operatively connected to a computer that controls a manufacturing process. The computer may be a PCL logic controller system. The computer opens and closes valves for water inlet, waste water outlet and product outlet according to feedback received from sensors in the system (eg conductivity, pH and product concentration (ppm)). The computer can also store water pressure and water quantity values and can adjust them according to feedback received from the sensors regarding the properties of the HOCl produced. The computer can also control infusion pumps that inject reagents into the water for the receiving process.

The process can be performed multiple times, for example pipe 101c can be connected to a second fluid system and the resulting HOCl flows through the second system where the process described above is repeated with HOCl instead of water as the feed solution. In this way, an increased yield of HOCl is obtained. Any number of fluid systems may be connected according to the methods of the invention.

Fig. 7 is a diagram showing another example of a system 200 for producing hypochlorous acid according to the methods of the invention. The system 200 is configured to adjust the pH of the incoming water and introduce a buffer for stability. In system 200, water is supplied to pipe 201a. Connected to pipe 201a is a pH meter 208. The pH meter 208 measures the pH of the incoming water. The pH meter 208 is connected to the inlet 202. The inlet 202 allows at least one buffer substance to be introduced into the incoming water. The buffering agent is supplied by an infusion pump which is hermetically connected to port 202. In this method, the flow rate and thus the amount of buffering agent injected into the water at any given time is controlled. The infusion pump can be controlled automatically or manually. The rate at which the buffer is added to the water depends on the quality of the incoming water (conductivity and pH), the composition of the buffer, and the pressure and flow of the incoming water. Administration can be by continuous infusion or intermittently. Because the water flows through the pipe 201a in a turbulent manner, there is an initial mixing of the buffer substance with the water when the buffer is introduced into the water. This initial mixing may be sufficient to properly control the performance of the incoming water.

In some embodiments, additional mixing of water and buffer is performed prior to the HOCl production process. In such embodiments, additional mixing occurs when the buffered water 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 may have the same or a different configuration compared to the mixing device 102 , for example the same or different number of subchambers, the same or different hole diameters, the same or different sizes of subchambers, etc. In any case, like the mixing device 102, the mixing device 203 is configured to produce a swirling fluid flow in each sub-chamber.

The solution enters the mixing device 203 through an inlet in the device, which is hermetically connected to the pipe 201a. The solution enters the mixing chamber, with turbulent mixing occurring in each sub-chamber of the mixing device as the solution passes through the elements in the chamber, due to the holes in each element. After mixing in the last sub-chamber, water exits the chamber through a fluid outlet in the mixing device, which is hermetically connected to pipe 202b. The water has a pH of at least about 8.0, preferably 8.4, and a buffer capacity of pH 5.5-9.0.

Next, the process is carried out as described above to obtain HOCl. The compound that forms the proton is then injected into the water that flows through the pipe 201b through the injection hole 204. The compound that forms the proton is injected using an infusion pump that is hermetically connected to the hole 204. In this method, the flow rate is controlled and, thus, the amount of a compound that forms a proton introduced into water at any given time. The infusion pump can be controlled automatically or manually. The rate at which a compound that forms a proton is introduced into water depends on the properties of the water (electrical conductivity and pH), the composition of the buffer, and the pressure and flow of the water. In some embodiments, the implementation of the pump allows you to enter from about 6.5 l/h to about 12 l/h of the compound that forms a proton in the water. Administration may be by continuous infusion or intermittently. Since the water flows through the pipes in a turbulent mode, there is an initial mixing of the compound that forms the proton with the water when hydrochloric acid is introduced into the water.

Additional mixing occurs as the solution enters the second mixing device 205. The mixing device 205 includes all of the features discussed above with respect to

- 9 041951 mixing device 102. Mixing device 205 may have the same or different configuration compared to the mixing device 203, for example, the same or different number of subchambers, the same or different hole diameters, the same or different sizes of subchambers, etc. In any case, like the mixing device 203, the mixing device 205 is configured to produce a swirling fluid flow in each sub-chamber.

The solution enters the mixing device 205 through an inlet in the device, which is hermetically connected to the pipe 201b. The solution enters the mixing chamber, with turbulent mixing occurring in each sub-chamber of the mixing device as the solution passes through the elements in the chamber, due to the holes in each element. After mixing in the last sub-chamber, water exits the chamber through a fluid outlet in the mixing device, which is hermetically connected to the pipe 201c.

The compound that forms the hypochlorite anion is then injected into the solution that flows through the pipe 201c through the injection hole 206. The compound that forms the hypochlorite anion is injected using an infusion pump that is hermetically connected to the hole 206. In this method, the the flow rate, and thus the amount of compound that forms the hypochlorite anion, introduced into the water at any given time. The infusion pump can be controlled automatically or manually. The rate of introduction of the compound that forms the hypochlorite anion into water depends on the properties of the solution (electrical conductivity and pH), as well as the pressure and flow of the solution. In some embodiments, the pump allows about 6.5-12 L/hr of the compound that forms the hypochlorite anion to be injected into the solution. The amount to be injected depends on the desired HOCl concentration (ppm) and the flow of water through the pipes. Administration may be by continuous infusion or intermittently. As the solution flows through the pipes in a turbulent manner, there is an initial mixing of the compound that forms the hypochlorite anion with the solution as the compound that forms the hypochlorite anion is introduced into the solution.

Additionally, 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 may have the same or different configuration compared to the mixing devices 205 or 203, for example, the same or a different number of subchambers, the same or different hole diameters, the same or different sizes of subchambers, etc. In any case, like the mixing devices 205 and 203, the mixing device 207 is configured to produce a swirling fluid flow in each sub-chamber.

The solution enters the mixing device 207 through an inlet in the device, which is hermetically connected to the pipe 201c. The solution enters the mixing chamber, with turbulent mixing occurring in each sub-chamber of the mixing device as the solution passes through the elements in the chamber, due to the holes in each element. After mixing in the last sub-chamber, the water leaves the chamber through the fluid inlet in the mixing device, which is hermetically connected to the pipe 201d.

At this point, the reaction is complete and HOCl is obtained. The resulting HOCl can be measured and collected as described above. Pipe 201d may be connected to a multi-way valve that switches flow between a discharge line and a product collection line. The valve includes a pH meter and an electrical conductivity meter. These devices measure the concentration, purity, and pH of the HOCl produced and provide feedback to change these properties of the HOCl produced. If the HOCl produced in pipe 201d meets the desired concentration, purity, and pH, the valve switches the flow from the waste line to the product collection line to collect the desired product.

In another embodiment, the deionizer is located in line with the incoming water. The deionizer deionizes the water, and then a buffer substance is introduced into the deionized water. The production process is then then carried out as described for system 200 embodiments to produce water having a pH of at least about 8, such as 8.4, and a buffer capacity of pH 6-8.

The HOCl produced by the above process can be used in a number of different applications, such as medicine, catering, food retail, agriculture, wound care, laboratories, hospitality, dentistry, delignification, or the flower industry.

Treatment of wounds.

In some embodiments, the compositions of the invention are used to treat a wound. Wound care includes the treatment of injured or diseased skin, including abrasions, lacerations, tears, punctures, or burns. Particular wound treatments include biofilm treatment. Biofilms can form when free-swimming microorganisms such as bacteria and fungi attach to surfaces. Biofilms are known to impair skin wound healing and reduce antibacterial efficacy in healing or treating infected wounds. Other common pathologies associated with biofilms include urinary tract infections, middle ear infections, chronic wounds, and plaque formation. Cystic fibrosis, intrinsic valve endocarditis, otitis media, periodontitis, and chronic prostatitis also include

- 10 041951 microorganisms that form biofilms. Microorganisms commonly associated with biofilms include Candida albicans, coagulase-negative staphylococci, Enterococcus, Klebsiella pneumoniae,

Pseudomonas aeruginosa, Staphylococcus aureus and others.

Biofilms are often resistant to traditional antimicrobials and therefore pose a serious health threat. The persistence of biofilms makes traditional antibiotics and antimicrobials ineffective. Because biofilms can significantly reduce susceptibility to antibiotics and disinfectants, treatments are required that can destroy biofilms without being too toxic to the patient.

Methods are provided for administering the composition to a patient in need of treatment for a biofilm-associated infection. The methods of the invention include the prevention, therapy, or treatment of a biofilm-associated infection. The methods include administering one or more unit doses of the composition in a therapeutically or prophylactically effective amount to treat an existing biofilm infection or to prevent a patient from developing a biofilm infection. In some embodiments, the spread of the biofilm-associated infection to another site in the patient is inhibited. In various embodiments, the implementation of the composition can be used parenterally, orally, topically or topically. The compositions can be administered by intravenous, intramuscular or subcutaneous injection. In the methods of the invention, the compositions may be administered in a pharmaceutically acceptable carrier, examples of which are discussed below.

Treatment includes killing the microorganisms inhabiting the biofilm or removing the biofilm, inhibiting the formation of the biofilm, and destroying the 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 topical wound treatment composition which contains a compound of hypochlorous acid and acetic acid. The composition may be combined with an additional antimicrobial agent.

Compositions of the invention may be administered topically to a subject, for example by direct application or distribution of the composition to the subject's epidermal or epithelial tissue. The composition may be in the form of a liquid, powder, lotion, cream, gel, oil, ointment, gel, solid, semi-solid, or aerosol spray. Such dosage forms can be manufactured in a standard way using suitable carriers, which are well known to the person skilled in the art.

Suitable topical carriers preferably remain in place on the skin as a continuous film and are resistant to removal by perspiration or immersion in water. The carrier may include pharmaceutically acceptable emollients, emulsifiers, thickeners, solvents, and the like.

The composition may be provided as part of a wound dressing, wherein the composition is present within the wound dressing or on its wound contact surface. The wound dressing may be intended to be applied to the wound being treated and includes a substrate containing compositions in accordance with the invention. Such a dressing is particularly convenient because it delivers the composition according to the invention to the treated wound and at the same time provides its dressing. The wound dressing may be, for example, fibrous, foam, hydrocolloid, collagen, film, sheet, hydrogel, or a combination thereof. The wound dressing may be in the form of a multi-layered dressing in which one or more layers are formed, at least in part, of or are one of or consist of the following: natural fibers, alginate, chitosan, chitosan derivatives, cellulose, carboxymethyl cellulose, cotton, viscose, nylon , acrylic, polyester, polyurethane foam, hydrogels, hydrocolloids, polyvinyl alcohol, starch, starch film, collagen, hyaluronic acid and its derivatives, biodegradable materials and other materials known in the art. The methods of the invention may further include negative pressure wound therapy, as is known in the art. Such therapies include applying negative pressure to the wound, for example with a vacuum dressing.

The composition may be administered in a single daily dose or in multiple doses, eg 2, 3, 4 or more doses per day. The total daily amount of the composition may be about 0.01, 0.1, 1, 2 mg, etc., up to about 1000 mg. In some embodiments, the total daily amount administered is about 0.01 to about 1 mg, about 1 to about 10 mg, about 10 to about 100 mg, about 100 to about 500 mg, or about 500 to about 1000 mg . The actual dose may vary depending on the particular composition administered, the route of administration, the type or location of the biofilm being treated, and other factors known in the art. In some embodiments, the implementation of the dose can also be adjusted to provide a given amount of composition per kilogram of patient weight.

The use of a compound in combination with another known antimicrobial treatment may increase the effectiveness of the antimicrobial agent. In some embodiments, the methods

- 11 041951 according to the invention further include the administration (simultaneously or sequentially with compositions according to the invention) of one or more doses of an antibiotic substance, including, without limitation, ciprofloxacin, ampicillin, azithromycin, cephalosporin, doxycycline, fusidic acid, gentamicin, linezolid, levofloxacin, norfloxacin , ofloxacin, rifampicin, tetracycline, tobramycin, vancomycin, amikacin, deftazidime, cefepime, trimethoprim/sulfamethoxazole, piperacillin/tazobactam, aztreonam, meropenem, colistin, or chloramphenicol. 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 include administering a non-antibiotic antimicrobial agent including, but not limited to, sertraline, racemic and stereoisomeric forms of thioridazine, benzoyl peroxide, taurolidine, and hexetidine.

Treatment of biofilm on other tissues.

Compositions according to the invention can be used to treat biofilms affecting various parts of the body or attached to various surfaces. In some embodiments, the methods of the invention comprise administering a therapeutically effective composition to a patient in need thereof for treating a biofilm-associated infection in the bladder, kidney, heart, middle ear, sinuses, skin, lung, joint, subcutaneous tissue, soft tissue, vascular tissue, and / or in the eye. In other embodiments, a therapeutically effective amount of the composition is administered to a patient in need thereof for the treatment of one or more of the following biofilm-related conditions: urinary tract infection; chronic bacterial vaginosis; prostatitis; a bacterial infection associated with diabetes, such as a diabetic skin ulcer; pressure ulcer; an ulcer associated with a venous catheter; or a surgical wound (eg, infection of the surgical field). In some embodiments, the implementation of the biofilm is on the skin of a person. In some embodiments, the biofilm is associated with a wound, including scratches, lacerations, tears, punctures, burns, and chronic wounds. In some embodiments, the implementation of the biofilm is located under the surface of the skin, in subcutaneous tissue, such as a deep wound or infection of the surgical field.

Biofilm treatment on non-tissue surfaces.

Other applications for processing biofilms are also contemplated. For example, the composition according to the invention can be used to treat microbial biofilms on surfaces, such as surfaces in hospitals (such as operating rooms or patient care rooms), as well as other surfaces (eg, work surfaces in the home). The invention also covers the treatment of biofilms that form on implanted medical devices and prostheses.

As is known in the art, implantable medical devices are susceptible to the formation of biofilms, including fungal biofilms and bacterial biofilms. The methods and compositions of the invention can also be used to treat biofilms that form on the surfaces of implantable medical devices such as catheters and prostheses. The compositions of the invention may be applied to a medical device prior to implantation. Alternatively, the medical device may comprise a reservoir containing the composition, wherein the composition may be released in a controlled manner after implantation. Methods for processing implantable medical devices can be found in US Pat.

Dental treatment.

In another embodiment, the invention provides a method for treating an oral associated biofilm such as dental plaque. The invention provides methods for preventing plaque formation, treating plaque-associated infection, treating tooth hypersensitivity, sterilizing a root canal, or treating a dental disease.

Methods of the invention include contacting an oral surface such as teeth, gums or tongue with a therapeutically effective amount of the composition. Some methods of the invention include preventing the formation of an oral associated biofilm by administering a prophylactically effective amount of the composition to a patient. The composition may be formulated as a dentifrice, such as toothpaste, for the treatment or prevention of dental plaque. In other embodiments, the biofilm may be located on the tongue, oral mucosa, or gums. In some embodiments, the composition is in the form of a mouthwash. In some embodiments, the composition is in the form of a paint, foam, gel, or varnish, such as a fluoride composition. In one embodiment, the composition is in the form of a gel or foam in a cap that the patient wears for several minutes for fluoride treatment. In other embodiments, the composition is in contact with an adhesive

- 12 041951 strips that can be applied to teeth or other surfaces in the oral cavity. The composition may contain a liquid polymeric composition which is a composition which is preferably applied to a surface such as a tooth, skin, mucous membrane and which dries as a film adhered to that surface which is resistant to removal under normal conditions such as eating or brushing, for application to the teeth and oral mucosa, or normal washing and brushing when applied to the skin. Alternatively, the composition may be applied to dressings, dressings, gauze, brushes, implants, and the like. and leave to dry to form a film prior to administration to the patient.

Mastitis treatment.

In another embodiment, the invention provides compositions and methods for the treatment of mastitis. Mastitis is inflammation of tissue in the mammary gland or udder of a mammal. It is often associated with bacterial infections such as Pseudomonas aeruginosa, Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus agalactiae, Streptococus uberis and others. Some bacteria known to cause mastitis also form biofilms, however mastitis does not always result from biofilm formation.

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, this condition occurs when white blood cells infiltrate the mammary gland, often in response to bacteria in the teat canal. Cows that are constantly infected often need to be culled to prevent widespread infection in the herd. The loss of milk from infected cows and the loss of cows and entire herds due to infection causes great economic losses in the dairy industry worldwide. In the United States, for example, mastitis is estimated to cost the dairy industry $2 billion annually.

Methods are provided for administering the composition to a mammal in need of mastitis treatment. The methods of the invention include the prevention, therapy or treatment of mastitis. In some embodiments, the spread of mastitis to another quarter of the udder or to another animal is prevented.

Dosage forms, doses and routes of administration discussed above may apply to these embodiments of the invention. For example, in various embodiments, the composition may be administered parenterally, orally, topically, or topically. The compositions may be administered by intravenous, intramuscular or subcutaneous injection. The compositions may be administered by infusion through the teat canal, as is known in the art. In the methods of the invention, the compositions may be administered in a pharmaceutically acceptable carrier, which may include emollients, emulsifiers, thickeners, solvents, and the like.

The composition may be administered in a single daily dose or in multiple doses, eg 2, 3, 4 or more doses per day. The total daily amount of the composition may be about 0.01, 0.1, 1, 2 mg, etc., up to about 1000 mg. In some embodiments, the total daily amount administered is about 0.01 to about 1 mg, about 1 to about 10 mg, about 10 to about 100 mg, about 100 to about 500 mg, or about 500 to about 1000 mg . The actual dose may vary depending on the particular composition administered, route of administration, and other factors known in the art. The composition may be administered in combination with another known antimicrobial treatment such as an antibiotic.

The compositions can be applied topically to the udder of a cow by direct application or distribution of the composition to the udder or teat. The composition may be in the form of a liquid, powder, lotion, cream, gel, oil, ointment, gel, solid, semi-solid, or aerosol. The methods of the invention may further include dipping the teat into the composition. Teat dip can be used to treat an already infected udder or as a prophylactic to prevent the development of mastitis. The composition can be applied immediately before milking and/or immediately after milking. Teat dipping techniques are known in the art and are described in more detail in US Pat. No. 4,113,854 and in US Patent Publications 2003/0235560 and 2003/0113384, which are incorporated herein by reference in their entirety. The methods may further include the use of a nipple sealant to provide a physical barrier to seal the opening of the nipple after application of the composition.

In other embodiments, the compositions may be provided by intramammary infusion. Intramammary infusion involves the administration of an antibiotic through the teat canal into the udder. The infusion fluid may contain the composition disclosed herein in combination with a pharmaceutically acceptable carrier such as canola oil. Before infusion, the nipple is cleaned, for example, with an alcohol swab. The antibiotic infusion device may include a cannula sized and shaped to fit the teat canal. The cannula may be inserted wholly or partially through the teat canal. Infusion methods are known in the art and are described, for example, in US Pat. Nos. 4,983,634 and 5,797,872, each of which is incorporated herein by reference in its entirety.

The methods of the invention may further include administering the antibiotics in combination with the compositions of the invention, or in sequential doses, before or after the administration of the compositions.

Wounds and surgical applications.

The compositions can be used to prevent and treat biofilm on other types of living tissue. Tissue includes, for example, skin, mucous membranes, wounds, or stomas. As described above, the composition can be used to treat wounds. Wounds include bedsores, chronic wounds, burns, decubitus wounds, diabetic wounds, and other forms of skin injury. Wounds are often prone to biofilm formation, which prevents healing and can lead to chronic disease. Compositions of hypochlorous and acetic acids can be used to remove and clean damaged tissues.

The compositions may also be used in a surgical setting to treat the skin before or after surgery. The composition prevents infection, which can lead to the formation of a biofilm. Sometimes an area requiring surgery, such as a traumatic injury, may already be at risk for developing a biofilm. The hypochlorous acid composition can be used to disinfect the area before the surgical incision, which will not only help eliminate the biofilm, but also reduce the likelihood of it spreading to other tissues during surgery. The compositions can be used to disinfect any surface within the surgical field.

Other medical applications.

In addition to wound care, the HOCl compositions of the invention can also be used for non-traumatic tissue healing. They can be used for bladder irrigation, to prevent or treat bladder infections or catheter-associated urinary tract infections, and the like. They may also be used to treat infections in the aerodigestive tract, such as sinus and lung infections, or infections in the mouth, pharynx, paranasal sinuses, sinonasal tract, larynx, piriform sinus, or esophagus. They can be used to control microbial growth that leads to infection and to reduce allergens that cause unwanted immune responses. The compositions may also be administered to the gastrointestinal tract, including the stomach, intestines and colon, to combat microbial infections such as gastroenteritis, Clostridium difficile infection, and Small Intestine Bacterial Overgrowth Syndrome (SIBO).

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

In addition to use as an antiseptic to treat or prevent biofilm formation on living tissue, the compositions can be used as a disinfectant on other surfaces, such as healthcare, food preparation, kitchen utensils, and the like. The compositions can be used to disinfect work surfaces, hospital beds, or food preparation surfaces.

The compositions can be used, for example, for the disinfection of medical devices and surgical instruments. Medical devices are initially supplied sterile, but may require additional or subsequent cleaning, disinfection, or sterilization. Reusable medical devices in particular need to 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 spreading the composition over the surface of the device, spraying an aerosol form of the composition onto the device, immersing the device in a container containing a volume of composition, or by placing the device in a stream of composition, such as from a tap. Additionally or alternatively, medical devices and surgical instruments can also be stored immersed in the composition and removed for the duration of use.

Treatment with the hypochlorous acid composition disclosed herein can be performed in addition to other known methods such as autoclaving. Alternatively, the composition may be used instead of autoclaving. Since thermal sterilization may not be applicable to all devices (for example, some devices contain delicate parts or electronics that cannot withstand high temperatures), hypochlorous acid compositions are a suitable alternative, providing an effective way to sterilize or disinfect such devices.

The compositions can also be used to disinfect implants and prostheses prior to their introduction into the body. Such devices include orthopedic implants, pins, screws, rods,

- 14 041951 artificial discs, joint endoprostheses, soft tissue fillers, pacemakers, intrauterine devices, coronary stents, tympanostomy tubes, artificial lenses, dental implants and many others known in the art.

The stabilized hypochlorous acid and acetic acid compositions described herein can be used to both prevent and remove biofilm on all of the surfaces and tissues discussed herein. Because biofilms make microbes much more resistant to traditional antimicrobials, the microbes that form biofilms are more able to transfer to each other and change their resistance genes and spread through the air and environment. As a result of biofilm development, a simple infection can become chronic, antibiotics and antiseptics no longer work, and new strains of infections appear.

Both acetic acid (or other organic acids) and hypochlorous acid, however, are particularly useful in the treatment and prevention of biofilm formation. The HOCl compositions disclosed herein mimic the immune system's natural disinfectant. Therefore, the compositions are not susceptible to microbial resistance. In addition, they are non-toxic, do not cause burning and relieve itching.

The various embodiments and uses described above include a variety of routes of administration, as will be known in the art.

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 reach deeper tissue layers without the need for injection. This is especially useful in case of a biofilm infection that forms under the top layer of the skin. Unlike many other antimicrobials, acetic acid and similar organic acids can penetrate deeper layers of the skin without requiring an invasive delivery mechanism.

However, in some embodiments, it may be desirable to prevent HOCl from penetrating the skin, and therefore the compositions may be combined with excipients, carriers, emulsifiers, polymers, or other components, examples of which are discussed in US Patent Application 2016/0271171, which is incorporated in its entirety. this document by reference.

In addition to topical use, where the compositions may be sprayed, rubbed, or rubbed into the skin, in other embodiments, the compositions may be administered to the specific tissue in need of treatment. The compositions may be taken orally in the form of capsules for administration to the gastrointestinal tract. They may be supplied in sustained or delayed release capsules. The compositions may be presented in the form of a suppository for insertion into the rectum or vagina.

In other embodiments, the compositions may be in the form of 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 may be in the form of drops, aerosol, gel, or powder. The buffered composition of hypochlorous acid and acetic acid may be combined with one or more of an anticongestant, or an anti-inflammatory or antihistamine, as needed. The composition can be dispensed with a nasal spray dispenser as is known in the art.

The nasal spray may also include a pharmaceutically acceptable carrier, such as a diluent, to facilitate delivery to the nasal mucosa. The carrier may be an aqueous carrier such as sodium chloride solution. The composition may be isotonic, having the same osmotic pressure as blood and lacrimal fluid. Suitable non-toxic pharmaceutically acceptable carriers are known to those skilled in the art. Various carriers may be particularly suitable for different dosage forms of the composition, for example, for use as drops or as a spray, nasal suspension, nasal ointment, nasal gel, or other nasal form. Other additives, adjuvants, emulsifiers, dispersing agents, buffering agents, preservatives, wetting agents, consistency modifiers and gelling agents may also be included. Preferably, additives should be selected that impart the required performance without compromising the stability of the hypochlorous acid. Additives may aid in uniform application of the composition to the mucosa, or to reduce the rate or delay in absorption of the composition.

The composition can be delivered using various devices known in the art for the introduction of droplets, airborne dispersions and aerosols. The nasal spray composition can be delivered using a dropper, pipette or dispenser. Small drops, sprays and aerosols can be delivered using an intranasal dosing pump or soft squeezable vial. The composition can also be inhaled through a metered dose inhaler such as a dry powder inhaler or nebulizer.

Controlled release when encapsulated in nanoparticles.

Stable aqueous solutions of hypochlorous acid and/or acetic acid can be encapsulated in nanoparticles which provide controlled release of the acid from the nanoparticles. Controlled release provides consistent antimicrobial protection.

In FIG. 13 shows an antimicrobial composition 1301 comprising an aqueous hypochlorous acid solution 1303 encapsulated in a nanoparticle 1305. An aqueous hypochlorous acid solution 1303 is obtained by the method described herein to obtain a solution in which the acid is stable. The stable hypochlorous acid solution 1303 is then encapsulated in a nanoparticle 1305. The nanoparticle provides a gradual release of hypochlorous acid. Although not shown, acetic acid can also be encapsulated in a controlled release nanoparticle.

The nanoparticle can be any type of nanoparticle that provides controlled release of acid from the nanoparticle. The nanoparticle may include a polymer such as an organic polymer. Examples of polymers suitable for controlled release nanoparticles include acrylic acid, carrageenan, cellulose polymers (e.g. 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-glycolidic acid, polylactic acid, poly-(xylitol salicylate adipate), polyanhydride, polyethylene oxide, polyethyleneimine, glycerol fatty acid polyester, polysaccharides, polyvinyl alcohol, povidone, sodium alginate, and xanthan gum. For more information on the use of polymers to produce controlled release nanoparticles, see Binnebose, et al., PLOS Negl Trop Dis 9:e0004713 (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 Pat. No. 8,449,916 (incorporated by reference). The nanoparticle may contain an aluminosilicate (such as a zeolite, e.g., analcime, chabasite, clinoptilolite, heulandite, leucite, montmorillonite, natrolite, phillipsite, or stilbite), ammonium calcium nitrate, hydroxyapatite (e.g., urea-modified hydroxyapatite), metal hydroxide, metal oxide, polyphosphate or a silicon compound (eg silicon dioxide). The nanoparticle may contain lipids, ie. it may be a lipid nanoparticle. The nanoparticle may include a liposome. For more information on the use of liposomes to produce controlled release nanoparticles, see Weiniger et al., Anaesthesia 67:906-916 (2012). The liposome may be multilayered. The nanoparticle may contain a gel, gel-sol, emulsion, colloid or hydrogel. For more information on the use of hydrogels to produce controlled release nanoparticles, see Grijalvo et al., Biomater. sci. 4:555 (2016). The nanoparticle may contain a combination of formats such as a hydrogel encapsulated in a liposome. A nanoparticle can have a core-shell structure. The nanoparticle may be biodegradable. Compositions according to the invention may include an antimetabolic agent. The antimetabolite agent may be a metal ion. For example, the antimetabolite may be zinc, copper, or silver.

A nanoparticle that provides controlled release of hypochlorous acid or acetic acid allows acid to diffuse more slowly than the acid would diffuse from an equal volume of the same aqueous acid that is not encapsulated in the nanoparticle. The controlled release of hypochlorous acid or acetic acid may be due to the permeability characteristics of the nanoparticles, for example nanoparticles that are partially or poorly acid permeable. A controlled release nanoparticle may be a nanoparticle that releases an acid when the nanoparticle breaks down or its structural integrity is compromised in a time dependent manner. The release of acid from the nanoparticles can be triggered by environmental conditions such as pH, temperature, light, pressure, redox conditions, or the presence of a specific chemical.

Fig. 14 is an illustration of a method 1401 for preparing an antimicrobial composition that includes an aqueous solution 1403 of hypochlorous acid encapsulated in a nanoparticle 1405. The method includes mixing 1411 in water, in an evacuated chamber 1413, compound 1415, which forms a proton (H) ⁺ in water, and compound 1417, which forms hypochlorite anion (OCl) ^- in water. Mixing 1411 gives an air-free aqueous solution 1403 of hypochlorous acid. Solution 1403 is then encapsulated 1421 in nanoparticle 1405. Encapsulation can be performed in an airless environment to produce a composition that is substantially free of air.

Compositions for the treatment of the aerodigestive tract.

The invention provides compositions and methods for treating and/or regulating the aerodigestive tract using compositions comprising hypochlorous acid. According to the invention, hypochlorous acid compositions prepared as described herein are safe and effective for the treatment of aerodigestive infections and/or regulation of the gut biome.

Compositions comprising hypochlorous acid and acetic acid may be presented as a nasal spray for the treatment of the aerodigestive tract. The nasal spray can be shaped

- 16 041951 airborne dispersion, aerosol, gel or powder. The buffered composition of hypochlorous acid and acetic acid may be combined with one or more of an anticongestant, or an anti-inflammatory or antihistamine, as needed. The composition can be dispensed with a nasal spray dispenser as is known in the art.

The nasal spray may also include a pharmaceutically acceptable carrier, such as a diluent, to facilitate delivery to the nasal mucosa. The carrier may be an aqueous carrier such as saline. The composition may be isotonic, having the same osmotic pressure as blood and lacrimal fluid. Suitable non-toxic pharmaceutically acceptable carriers are known to those skilled in the art. Various carriers may be particularly suitable for different dosage forms of the composition, for example, for use as drops or spray, nasal suspension, nasal ointment, nasal gel or other nasal form. Other additives, adjuvants, emulsifiers, dispersing agents, buffering agents, preservatives, wetting agents, consistency modifiers and gelling agents may also be included. Preferably, additives should be selected that give the desired performance without compromising the stability of the hypochlorous acid. Additives may aid in uniform application of the composition to the mucosa or reduce the rate or delay absorption of the composition.

Compositions for use according to the invention are prepared as described herein and formulated for delivery to all or part of the aerodigestive tract. For example, hypochlorous acid can be formulated into an aerosol for intranasal delivery as described herein. Intranasal delivery can be a means to target the entire aerodigestive tract, but is mainly used to treat infections in the nose, sinuses, lungs, and upper gastrointestinal tract. In other examples, hypochlorous acid is included in an oral delivery form. Compositions of the invention are formulated in a suitable diluent or adjuvant for pharmaceutical delivery (eg, oral administration) or incorporated into a liquid that is delivered as a beverage. According to the invention, hypochlorous acid compositions can be formulated as a hydrating drink (eg, sports drink), milkshake, electrolyte replenishment drink, smoothie, and the like. Compositions according to the invention can also be added to foods such as yogurt. In addition, the compositions according to the invention can be formulated for delivery directly to the digestive tract, for example in the form of a suppository, enema, or for direct use during surgery.

Compositions according to the invention may be included in the dosage form along with other components. For example, hypochlorous acid compositions can be combined with antibiotics, antivirals, probiotics, and other drugs or nutraceuticals. Thus, the compositions according to the invention can be used both for the regulation of the aero-digestive tract and for the treatment of infections in the aero-digestive tract.

The composition can be delivered using various devices known in the art for the introduction of droplets, airborne dispersions and aerosols. The nasal spray composition can be delivered using a dropper, pipette or dispenser. Small drops, sprays and aerosols can be delivered using an intranasal dosing pump or soft squeezable vial. The composition can also be inhaled through a metered dose inhaler such as a dry powder inhaler or nebulizer.

Compositions of acetic acid and hypochlorous acid for biofilm treatment.

The disclosed dosage forms of acetic acid and hypochlorous acid are excellent for treating biofilms on surfaces, including skin or other tissues. The compositions have a balanced formula in which the combination of acetic acid and hypocholoric acid provides superior disinfectant properties than either substance alone. In fact, in the present invention, the specific disclosed combinations provide a higher disinfectant capacity than would be expected with the addition of acetic acid and hypochlorous acid. In other words, the compositions have been found to exceed the sum of their components. These advantages are shown in the data presented in FIG. 15-21, which demonstrate how balanced compositions of acetic and hypochlorous acids provide improved disinfectant properties against biofilms and outperform all other products on the market. Performance differences are shown over a wide range of concentrations.

In addition, because acetic acid is toxic at high concentrations, there has been no prior art knowledge of its use on skin or other tissues except in trace amounts. Some of the disclosed compositions contain acetic acid in an amount of 2% or more, and in combination with HOCl have proven to be safe and effective for the treatment of skin and other tissues. It was found that HOCl in these compositions has a modulating effect on acetic acid. This allows the use of the antimicrobial properties of acetic acid in the compositions without damaging the tissue. In addition, HOCl has an analgesic function, so this also allows higher concentrations of HAc to be used on the skin or other tissues without causing undue pain or discomfort to the patient.

In FIG. 15, for example, shows a comparison of different concentrations of HOCl and acetic acid with other commercially available antimicrobial compositions. Eight different therapies were tested, listed on the x-axis. Each formulation was subjected to 24-hour contact with filter-grown S. aureus biofilm, and biofilm reduction was measured in colony forming units per ml (cfu/ml) and plotted logarithmically on the y-axis. Measurement of biofilm reduction was recorded after 3 and 6 hours. Therefore, each column contains two columns and demonstrates the effect of each composition on the biofilm over time.

The first three columns show the effect of 200 ppm. HOCl with three different concentrations of acetic acid (0.25, 1.0 and 2.0%, respectively). The fourth column shows only 1% acetic acid. The next four columns show the commercially available antimicrobial products: Prontosan; Octenilin; Pyrisept; as well as Microdacyn, which is a composition of hypochlorous acid.

The results show that all three combinations of acetic acid and hypochlorous acid were more effective against biofilm than any other composition. After 3 hours, test composition A (200 ppm HOCl and 0.25% HAc) showed approximately the same efficacy as Prontosan, the current market leader in biofilm treatment. It was also significantly superior to 1% HAc or other products available on the market. After 6 hours, however, Composition A showed a much higher efficacy even than Protosan.

Meanwhile, test composition B (200 ppm HOCl and 1.0% HAc) was even more effective in treating the biofilm. Comparison of composition B with 1% HAc (in the fourth column) showed an unexpected benefit from the addition of HOCl. Despite the presence of the same concentration of acetic acid, formulation B was significantly superior to 1% HAc at 3 and 6 hours.

Composition C (200 ppm HOCl and 2.0% HAc) showed by far the largest biofilm reduction of the compositions tested. After 3 and 6 hours, it was several orders of magnitude more effective than commercially available products.

These data show that in addition to being more effective at reducing biofilm than any of the commercially available products, formulations containing acetic acid and hypochlorous acid were more effective than acetic acid alone (1% HAc) or one hypochlorous acid (Microdacyn), while such excellent results cannot be explained simply by the additive effect of the two components. Without being limited 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 would be predicted based on the effectiveness of each individual component.

In FIG. 16-19 show the effect of various formulations of HOCl and HAc on P. aeruginosa biofilms. In FIG. 16 shows a comparison of compositions containing 1% acetic acid and various concentrations of HOCl. Five other therapies were tested with HOCl at concentrations of 0, 50, 100, 150 and 200 ppm. Each composition was subjected to 24-hour contact with a filter-grown P. aeruginosa biofilm and biofilm reduction was measured in colony forming units per ml (CFU/mL) and recorded on a logarithmic scale on the y-axis. Biofilm reduction measurements were recorded at 2 and 4 hours.

As shown in the graph, the decrease after 2 hours was greater with increasing HOCl concentration, with a particularly significant peak at 150 ppm. After 4 hours, a peak was observed at even lower HOCl concentrations.

In FIG. 17 shows the effect on P. aeruginosa of various compositions, where the concentration of HOCl was maintained at 100 ppm, and the percentage of acetic acid varied from 0.25 to 2%. In FIG. 18 shows the effects of increasing both HOCl and HAc.

In FIG. 19-21 show different formulations of HOCl and HAc against S. aureus and P. aeruginosa under different conditions. The drawings show excellent results obtained with combinations of hypochlorous acid and acetic acid, which demonstrate the synergistic effect of the two compounds.

The various disclosed compositions may be effective in treating biofilm infections in various tissue types. For example, a composition containing 200 ppm HOCl and 0.25% HAc, can be applied for topical applications such as hand sanitizer or mouthwash. This composition is more effective than other commercially available products in treating biofilms at the surface level, as shown in FIG. 15. For treatment in case of deeper tissue penetration, or to eliminate especially severe biofilm infections or invasive biofilms that have penetrated below the surface, a higher percentage of HAc can be used, for example, a composition of 200 ppm. HOCl with 2% HAc. This composition can be used to treat infected wounds, prevent biofilm formation in wounds, treat eczema, or treat other infections. This composition was found to be effective in combating biofilms that had formed at the root of the teeth.

In FIG. 20 and 21 show additional data supporting the unexpected efficacy of acetic acid and hypochlorous acid compositions on various biofilms, especially

- 18 041951 in comparison with the prior art and commercially available compositions. As can be seen in the drawings, different compositions that contain different concentrations of HOCl and HAc provide a range of disinfectant compositions that can act on different types of biofilms on different types of tissue.

Another advantage of the disclosed HOCl and HAc compositions is that although they are particularly effective in reducing pathogenic biofilms, they have not been found to inhibit the growth of good biofilms and other microbes that live symbiotically on and in tissues. In some studies, good biofilm decreased to a lesser extent than pathogenic biofilm and grew faster than the target biofilm. Especially when compared to alcohol-based disinfectants, the disclosed compositions have proven to be more effective in controlling pathogenic biofilm infection without harming beneficial microbes. Thus, the disclosed compositions are targeted treatments for combating biofilm infections without disturbing the body's natural flora.

Inclusion by referral.

Any possible references to other documents, such as patents, patent applications, patent publications, journals, books, articles, web sources, which have been made in the text of this specification are hereby incorporated by reference in their entirety in all respects.

Equivalents.

The invention may be embodied in certain other forms without departing from its spirit or essential features. Thus, the foregoing embodiments are to be considered in all respects illustrative and not limiting of the invention described herein.

Examples

Example 1 Product Analysis.

If the spectrophotometry is extended to also cover the visible range, colors can be detected. Typically, HOCl production produces gases such as ClO2, Cl2O and Cl ₂ which can be detected in the visible range as yellow or yellow-red (Tzanavaras et al., Central European J. of Chemistry, 2007, 5(1) 1-12 ). The data in FIG. 9 show that HOCl made using the methods of the invention shows no absorption from colored gases, as indicated by the absence of colored matter. HOCl is known to peak at 292 nm (Feng et al., 2007, J. Environ. Eng. Sci. 6, 277-284).

Example 2

The HOCl obtained in the process described above was stored under thermal stress at 40° C. to accelerate decomposition using four different types of aqueous solutions: (1) pure water (deionized water); (2) tap water; (3) Phosphate buffered reagent grade water; and (4) Phosphate buffered tap water. The characteristics of the HOCl product were monitored immediately after the reaction (T=0); after four weeks (T=4); eight weeks (T=8) and twelve weeks (T=12).

Fig. 10 is a graph showing the amount (in parts per million (ppm)) of HOCl produced immediately after the reaction (T=0) and its stability over time. The data show that pure water (deionized water) without phosphate buffer was the most stable over twelve weeks, showing the least level of product degradation relative to its original amount. Deionized water gives a much more stable product than using tap water. In addition, and unexpectedly, the data show that phosphate buffer can adversely affect the amount of HOCl product produced.

Fig. 11 is a graph showing how the pH of the HOCl product changed over time. In all cases, the pH decreased over time, however, in all cases, within twelve weeks, the pH remained in the pH range of 4-7.

Fig. 12 is a graph showing the oxidizing power of the HOCl product over time. The data show that the product retained its oxidative capacity for twelve weeks, regardless of the original water.

Example 3 Acetic acid versus hydrochloric acid.

Using the above process, HOCl was obtained using hydrochloric acid (HCl) and acetic acid, and then stored under heat load at 40°C. The amount of HOCl initially received was recorded (T=0) and then the amount of HOCl product remaining after twelve days was recorded. Three batches of each product were received. Data for HOCl, obtained with HCl, are shown in table. 1. Data for HOCl, obtained with acetic acid, are shown in table. 2.

- 19 041951

Table 1

HOCl made with HCl

Batch number Initial amount (ppm) Initial pH Quantity after 12 days (ppm) pH after 12 days Degree of decomposition The degree of pH change 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 made with acetic acid

Batch number Initial amount (ppm) Initial pH Amount after 12 days (ppm) pH after 12 days Degree of decomposition The degree of pH change 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%

The data show that the use of acetic acid provides higher product stability, most likely due to higher pH stability. Without being limited by any particular theory or mechanism of action, it is believed that the different proton capacities of acetic acid and hydrochloric acid, i.e. the fact that acetic acid donates fewer protons to the liquid than hydrochloric acid results in higher stability of HOCl over time.

Claims (2)

CLAIM 1. An air-free antimicrobial composition for treating a biofilm in or on a tissue surface comprising a solution of acetic acid and hypochlorous acid having a balanced concentration of each of the acids to achieve a synergistic effect such that the composition is sufficient to treat a bacterial biofilm without damaging the environment. tissue, while acetic acid is encapsulated in a nanoparticle and has a concentration of approximately 2 wt.%, and hypochlorous acid has a concentration of approximately 200 ppm. 2. The composition according to claim 1, where the composition is in the form of a gel, cream, ointment or oil.