阅读说明：本技术 用于处理生物膜而不诱导抗微生物抗性的组合物和方法 (Compositions and methods for treating biofilms without inducing antimicrobial resistance ) 是由 G·H·阿勒马斯 于 2019-11-01 设计创作，主要内容包括：含有次氯酸和乙酸的消毒组合物可用于处理组织中或组织上的生物膜,包括与伤口或其他皮肤创伤有关的生物膜。所述组合物可用于处理织表面上和组织表面下的各种类型的组织。提供了用于处理生物膜而不诱导抗微生物抗性的组合物。(Disinfecting compositions containing hypochlorous acid and acetic acid are useful for treating biofilms in or on tissue, including biofilms associated with wounds or other skin wounds. The compositions are useful for treating various types of tissue on and below the surface of the tissue. Compositions for treating biofilms without inducing antimicrobial resistance are provided.)

1. A composition comprising acetic acid and hypochlorous acid sufficient to treat a bacterial biofilm and not induce antimicrobial resistance.

2. The composition of claim 1, wherein the acetic acid concentration is from 0.05% to 5.0%.

3. The composition of claim 1, wherein the acetic acid concentration is greater than about 0.25%.

4. The composition of claim 1, wherein the hypochlorous acid concentration is from 5ppm to 2500 ppm.

5. The composition of claim 1, wherein the hypochlorous acid concentration is about 25 ppm.

6. The composition of claim 1, wherein the acetic acid is present in a concentration sufficient to penetrate tissue.

7. The composition of claim 1, wherein the hypochlorous acid is present in a concentration sufficient to modulate the toxicity of the acetic acid.

8. The composition of claim 1, wherein the composition is formulated as a gel, cream, ointment, or oil.

9. The composition of claim 1, wherein the acetic acid is encapsulated in nanoparticles.

10. A method for treating a bacterial infection in a tissue, the method comprising providing to the tissue a composition comprising acetic acid and hypochlorous acid in an amount effective to modulate the toxicity of the acetic acid without inducing antimicrobial resistance.

11. The method of claim 10, wherein the concentration of acetic acid is sufficient to penetrate skin.

12. The method of claim 11, wherein the acetic acid is present in an amount of about 0.05% to about 1.0% and the hypochlorous acid is present at a concentration of about 5ppm to about 100 ppm.

13. The method of claim 11, wherein the tissue is skin, and wherein the hypochlorous acid concentration is about 25ppm, and wherein the acetic acid concentration is about 0.0625% to about 0.25%.

Technical Field

The present invention relates generally to compositions for treating biofilms, in particular acetic acid and hypochlorous acid which do not induce antimicrobial resistance.

Background

Biofilm-producing microbial infections can cause serious health problems. Scientists estimate that up to 80% of all infections affecting mammals are biofilm infections. Biofilm-growing bacteria cause chronic infections characterized by persistent inflammation and tissue damage. Chronic infection refers to the following infections: despite antibiotic therapy and the innate and adaptive immune and inflammatory responses of the host, it persists and, in contrast to colonization, is characterized by an immune response and a persisting pathology. (N,Bjarnsholt Antibiotic resistance of bacterial biofilms (Antibiotic resistance of bacterial biologicals) Int J Antibiotic Agents [ Internet ] T, Givskov M, Molin S, Ciofu O]2010 month 4 [ references from 2014 month 7 and 14 days]；35(4):322–32)。

Bacteria that adhere to the surface and grow in the form of a biofilm are protected from killing by antibiotics. The reduced antibiotic susceptibility contributes to the persistence of biofilm infections. For example, the persistence of infection with staphylococcus aureus (s. aureus) and pseudomonas aeruginosa (p. aeruginosa) is attributed to the formation of biofilms. (Int J Med Microbiol.2002, 7 months; 292(2):107-13.Stewart PS 1).

Antibiotic resistance and antimicrobial resistance of bacteria are increasingly problematic. The widespread use of antibiotics has exacerbated the accelerated development of antibiotic-resistant bacteria (Science, 264: 360-. Antibiotic resistance can rapidly spread to other bacteria, including different species of bacteria. Some bacterial strains are sensitive to only one antibiotic, and it may only be a matter of time that some bacteria are no longer eradicated by any antibiotic. (The Role of The Water David ringer, Di Hu, Marek Basler. Type VI Secretion System Effectors in Target Cell Lysis and Subsequent Horizontal Gene Transfer. Cell Reports, 2017; 21(13):3927DOI:10.1016/j. cell.2017.12.020).

This public Health epidemic is also attributed to the emergence of pathogens that are resistant to several antibiotics at the same time, thereby reducing the possibility of effective treatment (World Health Organization has held back the World's World Global Strategy for Resistance to Antimicrobial Resistance, Geneva, World Health Organization,2001, WHO/CDS/CSR/DRS/2001.2). Without new antimicrobial compositions, treatment of biofilm infections is becoming increasingly difficult due to the natural resistance and antimicrobial resistance of biofilms.

The compositions of the prior art have various disadvantages. Some prior art compositions promote antimicrobial resistance while attempting to treat biofilm infections, making it increasingly difficult to treat biofilms. Other prior art compositions fail to eradicate biofilms without prolonged exposure to affected problems, which induces antimicrobial resistance. While other prior art compositions do not treat mature biofilms.

Disclosure of Invention

Antimicrobial compositions comprising hypochlorous acid and an organic acid (such as acetic acid) as described herein can be used to treat biofilms in or on tissue without inducing antimicrobial resistance, ranging from simple surface disinfectants to wounds or other skin wounds. For example, wounds are often susceptible to microbial infection, including antimicrobial resistant biofilms formed on and below the surface of the wound, which can prevent healing and may lead to chronic conditions or persistent infections. Compositions comprising hypochlorous acid and acetic acid can be used to treat biofilms on tissues and do not induce antimicrobial resistance. The concentrations of HOCl and HAc are balanced to achieve a synergistic effect such that the antimicrobial capacity of the composition is greater than would be expected based on the antimicrobial properties of each component itself, while not inducing antimicrobial resistance. The combination of HOCl and HAc is also effective against antimicrobial resistance. Acetic acid provides an important buffering capacity for hypochlorous acid to perform optimally, especially in environments that tend to drive the pH to steady-state levels. For example, in the oral cavity, the natural pH is about 7.4, while acetic acid provides a buffering capacity in this environment, giving HOCl optimal activity. In addition, hypochlorous acid modulates the toxicity of acetic acid and provides an analgesic effect, allowing for a stronger composition to be applied to the skin or other tissue without adverse side effects, patient discomfort, or antimicrobial resistance. Finally, acetic acid is particularly effective against anaerobic bacteria such as pseudomonas (pseudomonas). In addition to acetic acid, other organic acids such as ascorbic acid, lactic acid, formic acid, malic acid, citric acid, uric acid, and other carboxylic or sulfonic acids. In addition, the combination of hypochlorous acid and acetic acid enhances the antimicrobial properties of other microbial treatments.

Acetic acid concentrations of about 0.1% to about 5% are useful. Hypochlorous acid concentrations greater than 5ppm can be used to treat biofilm infections. Some compositions have a hypochlorous acid concentration of about 5ppm, greater than 25ppm, greater than 50ppm up to about 2500 ppm. The compositions of the present invention are particularly effective in biofilm reduction due to the synergistic balance between hypochlorous acid and acetic acid which gives the composition a dual role of surface treatment of the biofilm, treatment just below the surface and deeper subsurface treatment. Generally, hypochlorous acid is able to act rapidly at or near the surface; while acetic acid takes longer to act and therefore can act below the tissue surface. The synergistic effect of the inventive composition of hypochlorous acid and acetic acid on biofilms also reduces the potential for antimicrobial resistance and cross-resistance.

Application of the disclosed compositions to the affected area helps to treat bacterial infections, including biofilms, without developing resistance to antimicrobial agents and antibiotics.

The compositions of the present invention are effective against immature, young and mature biofilms. Application of the composition to the affected area facilitates treatment of biofilms classified as immature, young and mature without inducing microbial resistance.

The compositions of the present invention may be provided as a gel or cream that allows for longer contact times with tissues without inducing antimicrobial resistance and cross-resistance. For wound treatment, gel and cream compositions also help to keep the wound hydrated, promoting healing. In addition, one or both components of the compositions of the present invention may be encapsulated in nanoparticles for controlled or delayed release.

The compositions described herein may be combined with various excipients and carriers to facilitate topical administration. The hypochlorous acid (and organic acid) product can be in the form of a gel, cream, lotion, spray, liquid, foam, powder, and other delivery formulations known in the art. Alternatively, the composition may be incorporated into a cloth or fibrous wipe or wound dressing.

In certain aspects, the present invention includes compositions consisting of acetic acid and hypochlorous acid. Acetic acid is present at a concentration greater than about 0.05% and preferably less than about 5.0%. Preferred hypochlorous acid concentrations are from about 5ppm to about 2500 ppm. In some embodiments, the specific concentrations of acetic acid and hypochlorous acid depend on the expected exposure time of the composition to the treatment area.

In some embodiments, the acetic acid is present in a concentration sufficient to penetrate below the surface of the tissue. In some embodiments, the acetic acid concentration is greater than about 0.5%, and preferably greater than about 0.25%, and in some embodiments, it is about 0.50% or higher. Acetic acid may be encapsulated in nanoparticles for controlled or delayed release. In some embodiments, the hypochlorous acid is present in a concentration sufficient to treat biofilm on and directly below the wound surface. The composition may further comprise a gel, cream, ointment or oil.

In a related aspect, the invention relates to a method for preventing antimicrobial resistance. The method comprises applying to the tissue a composition comprising acetic acid and hypochlorous acid in amounts sufficient to prevent antimicrobial resistance.

In a related aspect, the invention relates to a method for treating a biofilm in or on a tissue. The method comprises applying to the tissue a composition comprising acetic acid at a concentration sufficient to penetrate the skin and hypochlorous acid in an amount sufficient to remove biofilm on and directly below the surface of the tissue and not induce antimicrobial resistance.

Acetic acid may be present in an amount sufficient to remove biofilm beneath the skin surface without causing antimicrobial resistance. Acetic acid may be present in an amount of about 0.5% to about 5.0%, and hypochlorous acid may be present at a concentration of about 5ppm to about 2500 ppm.

In a related aspect, a method for treating biofilm in tissue involves applying nanoparticles comprising acetic acid to a tissue site to protect against antimicrobial resistance. The nanoparticles may be lipid soluble.

Drawings

Figure 1 is a schematic diagram showing an exemplary system for producing hypochlorous acid according to the method of the present invention.

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

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

Fig. 4 is a schematic diagram showing a front view of the means for dividing the mixing chamber into a plurality of sub-chambers. This view shows the aperture in the member.

Fig. 5 is a schematic diagram showing a valve configured with a measurement sensor for switching from a waste line to a product collection line.

Figure 6 is a schematic diagram showing a valve in series with a waste line and a product collection line.

Figure 7 is a schematic diagram showing another exemplary system for producing hypochlorous acid according to the methods of the present invention. The system is configured for automated use with buffered deionized water. The buffer may be contained in the influent water or may be introduced through an injection port. Buffers can also be mixed during the mixing process by using NaOH in NaOCl or NaOH and acetic acid injected separately or other similar acids or bases.

Fig. 8 is a calibration graph showing indirectly calculated HOCl concentration (ppm) versus conductivity.

Fig. 9 is a graph showing spectrophotometric analysis of the generated HOCl. Gases typically produced during HOCl production are ClO2, Cl2O, and Cl2, all of which are detectable in the visible range as yellow or yellowish red. The figure shows that there is no absorption from the colored gas in the generated HOCl.

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

Figure 11 is a graph showing how the pH of the HOCl product changes over time.

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

Figure 13 shows an antimicrobial composition comprising an aqueous hypochlorous acid solution encapsulated in nanoparticles.

Figure 14 is a schematic representation of a method of making an antimicrobial composition comprising an aqueous hypochlorous acid solution encapsulated in nanoparticles.

Figures 15-21 provide data on the reduction of various biofilms when exposed to different concentrations of a composition of acetic acid and hypochlorous acid compared to a commercial biofilm treatment.

Fig. 22 is a graph showing the killing effect of different concentrations of HOCl with 1% or 4% acetic acid.

FIG. 23 is a graph showing the killing effect of 200ppm and 500ppm HOCl with 1%, 2% or 4% acetic acid.

Detailed Description

Treatment of bacterial infections and biofilms is achieved using synergistic compositions comprising an organic acid (such as acetic acid) and hypochlorous acid. The acetic acid component is particularly effective for penetration into tissue, while hypochlorous acid is particularly effective for treating biofilms on the outer surface of tissue. Acetic acid can penetrate up to 2mm or more below the wound surface to treat biofilm that is otherwise difficult to reach.

Preferred compositions of the invention comprise hypochlorous acid in synergistic combination with acetic acid. It has been found that an equilibrium composition of acetic acid (or equivalent organic acid) in a biocidal amount optimized with hypochlorous acid, whether applied to a surface, or in a manner designed to penetrate the skin, achieves maximum therapeutic effect. For example, for surface applications, the relative amounts of acetic acid and hypochlorous acid are lower than for osmotic applications. According to the present invention, surface bacterial contamination or biofilm is adequately treated by hypochlorous acid with lower amounts of acetic acid; but for applications requiring deep penetration (e.g. wounds) the amount of acetic acid must be increased. In that case, hypochlorous acid is used to mitigate the toxicity of acetic acid to surrounding tissues, while allowing the acetic acid to attack the biofilm. In addition, it has been found that a synergistic combination of acetic acid and hypochlorous acid selectively kills harmful biofilm while retaining beneficial biofilm. The compositions of the present invention comprise acetic acid and hypochlorous acid in balanced concentrations to selectively kill harmful biofilms. The concentrations are also balanced in view of the application for which they are intended (e.g. surface treatment of the teeth or skin versus deep tissue treatment of wounds or roots). The present application provides guidance regarding the synergistic effect of various combinations of acetic acid and hypochlorous acid. One skilled in the art can determine the relative amounts of acetic acid and hypochlorous acid required to treat any bacterial infection or biofilm formation based on the information provided in the specification.

Treating both superficial level infections and sub-dermal infections provide a dual action treatment that is particularly desirable for wound care. Chronic wounds and eczema are plagued by biofilms affecting the subsurface of the wound. These wounds often have staphylococcus aureus infections, which are usually present on or near the surface and prevent wound closure and healing. In addition, pseudomonas aeruginosa infections often occur below the surface of the wound, often deeper. When there is a deep infection, it is important to keep the wound open and hydrated while healing from the inside out to prevent the wound from closing before the deeper infection heals. Treating only superficial level infections can cause wound closure and entrap deeper biofilms within the tissue, which can lead to sepsis and other complications.

Features of the invention are also found in the regulation of hypochlorous acid and acetic acid. For example, increasing the concentration of HOCl or acetic acid while keeping the other concentration constant may improve the combined bacterial kill. In addition, increasing acetic acid concentration increases the killing effect of "dirty" samples (i.e., samples containing organic matter such as blood, sputum, and other organic compounds). Therefore, in the dirty products, it is more important to adjust the concentration of acetic acid. Fig. 22 shows the killing effect of different concentrations of HOCl with 1% or 4% acetic acid. Figure 23 shows that the maximum jump in killing occurred between 1% and 2% acetic acid. Both fig. 22 and fig. 23 are the results of applying different concentrations of HOCl and acetic acid to a wound simulation model of Pseudomonas aeruginosa (Pseudomonas aeruginosa) infection.

The present invention has many applications for the prevention and/or treatment of biofilms. For example, a common problem with diabetic foot ulcers is that they close at the surface, forming an open cavity underneath. The cavity will contain pus as part of the immune system response, which is composed of debris, bacteria and white blood cells. Pus in the closed compartment, particularly in foot ulcers, helps to spread the infection and may cause sepsis. However, in an open wound that is properly packed, pus will drain into the wound bed and dressing. Bacteria that survive in the pus environment within the closed wound can spread the infection. Thus, removal of staphylococcus aureus infection without first or simultaneously removing pseudomonas aeruginosa infection may not completely eradicate biofilm infection, leading to premature closure and possibly sepsis.

Antimicrobial solutions, such as the compositions provided herein, reduce infection in the deep regions of the wound bed and allow the wound to heal from inside to outside, such that the surface does not heal faster than the internal wound. Acetic acid is present in an amount sufficient to disinfect biofilm below the wound bed, and hypochlorous acid is present in an amount sufficient to disinfect the wound surface. Thus, the composition allows for complete disinfection of the wound to prevent premature wound closure and entrapment of biofilm below the closed wound surface.

The disclosed compositions are particularly effective because balancing the concentrations of hypochlorous acid and acetic acid allows for treatment of surface level biofilms as well as subsurface biofilms. The exact balance depends on the treatment site and the amount of surface penetration desired. Hypochlorous acid may be present from about 10ppm up to about 500ppm or more. Different uses and types of tissue may require higher or lower concentrations. Acetic acid may be present from about 0.25% up to about 2.0% or more and preferably about 1.0%. By balancing the two components, the composition may have a dual role of treating both the surface and subsurface of the tissue or wound.

Compositions consistent with the present disclosure may be produced for a variety of uses. For example, compositions of relatively low acetic acid (as low as about 0.05%) and about 5-60ppm hypochlorous acid are useful compositions as mouthwashes for combating infection of dental tissue. Lower concentrations of acetic acid are sufficient in mouthwash compositions because microbial infections tend not to penetrate deep into the tissue.

On the other hand, for wound treatment, the composition may comprise 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 biofilms deep in the tissue.

Other uses may require more or less of each component. For example, compositions having a hypochlorous acid concentration of about 80-250ppm can be used to irrigate the bladder, a treatment often required by patients with urinary catheters to prevent infection or blockage. Compositions having hypochlorous acid concentrations of about 15-60ppm are sufficient to treat infected lungs.

In certain embodiments, the composition is in the form of a gel, which allows for longer contact time with the wound. Rinsing with solution may not be sufficient because the contact time of the preservative will be very short. In many cases, the composition should be in contact with the biofilm for an extended period of time, ranging from seconds to minutes to an hour or more, in order to completely remove the biofilm. The composition may be provided against an immediate evaporating or dispersing gel or cream. Gels, creams, ointments, oils and other similar carriers for topical application are known in the art.

Furthermore, for wound treatment, compositions in the form of a gel have the benefit of maintaining moisture at the wound site. It is important to maintain wound hydration during and after treatment with the compositions of the present invention. The disclosed compositions are primarily water (typically 95% or more), allowing the wound to remain hydrated while the antiseptic component of the composition counteracts infection in the wound and prevents new infections from occurring. Maintaining hydration also prevents premature wound closure and entrapment of biofilm within the tissue. Acetic acid is easily formulated into a gel because acetic acid does not over-react. Other organic acids may also be used, and less reactive organic acids are desirable.

Sustained release compositions may also be used. In some compositions, the acetic acid may be encapsulated in lipid soluble nanoparticles, which allows the acetic acid to be carried below the wound surface before being released from the nanoparticles. Application with nanoparticles of different properties allows the acetic acid to be released slowly over time, preventing diffusion, and providing other benefits to the application. Acetic acid is free diffusing, water soluble, and has a high vapor pressure. These properties increase the difficulty of controlling acetic acid direction. The nanoparticle-encapsulated acetic acid allows for more precise control of the composition. The nanoparticles are described in more detail below and shown in fig. 13 and 14.

In some embodiments, the composition comprises some acetic acid that is not encapsulated within the nanoparticles and some acetic acid that is encapsulated within the nanoparticles. Alternatively, the sustained release formulation may be used alone or in combination with other fast acting formulations. For example, a wound may be treated with a composition of acetic acid and hypochlorous acid, and then treated with a composition of primarily acetic acid encapsulated in nanoparticles to provide a sustained release of acetic acid deep into the wound after initial treatment.

Production of hypochlorous acid compositions

The basis of the compositions and methods of the present invention is the protonation of hypochlorite ion (OCl-). Protonation is achieved by introducing an acid (e.g., HCl) into the solution, exemplified by HCl or acetic acid (HAc) and NaOCl, which will cause the following reactions to occur:

or

The hypochlorous acid in the aqueous solution partially dissociates into anionic hypochlorite (OCl)^-) Thus hypochlorous acid and anions (OCl) in aqueous solution^-) There is always a balance between. The equilibrium is pH dependent and at higher pH the anion predominates. In aqueous solution, hypochlorous acid is also mixed with other chlorine species, especially chlorine gas Cl₂And various oxychlorides. At acidic pH values, chlorine gas becomes more and more dominant, while at neutral pH values, the different equilibria result in solutions dominated by hypochlorous acid. Therefore, it is important to control the exposure to air and the pH in the production of hypochlorous acid.

In addition, the concentration of protons (H +) also affects the stability of the product. The present invention recognizes that proton concentration can be controlled by using an acid that has less ability to donate protons (i.e., the acid can provide buffering capacity) at a given pH. For example, when the desired pH of the final solution is about the pKa of acetic acid, it is optimal to perform the process with acetic acid rather than hydrochloric acid. This can be achieved by mixing in water at a ratio of 250X or more, meaning 1 part proton donor (e.g., HCl or acetic acid) at 100% concentration with 250 parts water.

In certain embodiments, the method of making HOCl involves mixing in water in an airless environment to generate protons (H) in the water⁺) Of (2) to (b)The product and the formation of hypochlorite anion (OCl) in water^-) Thereby producing air-free hypochlorous acid. The water may be tap water or purified water, such as water available from water purification companies, such as Millipore (Billerica, MA). Typically, the pH of the water is maintained from about 4.5 to about 9 during the process, however, the pH may be above or below this range during the production process. Performing the process of the present invention in an airless environment prevents the accumulation of chlorine during the production process. Furthermore, performing the process of the present invention in an airless environment further stabilizes the generated HOCl.

Hypochlorite anion (OCl) can be generated in water^-) Any compound of (1). Exemplary compounds include NaOCl and Ca (OCl)₂. In a particular embodiment, the compound is NaOCl. Production of protons (H) in water⁺) Any of the compounds of (a) can be used with the methods of the present invention. Exemplary compounds are acids such as acetic acid, HCl, and H2SO 4. In a particular embodiment, the compound is hydrochloric acid. In a preferred embodiment, the compound is acetic acid, as it is a weaker acid with a preferred pKa relative to HCl, which means that it donates fewer protons than HCl during the reaction, and is better able to maintain the preferred pH level.

Mixing can be performed in any type of container, chamber, or fluid system. In certain embodiments, a fluidic system 100 as shown in fig. 1 is used to perform the methods of the present invention. The system 100 includes a series of interconnected conduits 101a-c, the conduits 101a-c having a plurality of mixing devices 102 and 103 in series with the plurality of conduits 101 a-c. The piping and mixing apparatus may be interconnected using seals so that all air may be purged from the system, allowing the process of the present invention to be carried out in an airless environment. In certain embodiments, the process of the invention is also carried out under pressure. The preparation of HOCl in an air-free environment and under pressure allows for the generation of HOCl that does not interact with gases in the air (e.g., oxygen), which may destabilize the generated HOCl.

The conduits 101a-c typically have an inner diameter in the range of about 5mm to about 50mm, more preferably about 17mm to about 21 mm. In a particular embodiment, the conduits 101a-c have an inner diameter of about 21 mm. The conduits 101a-c typically have a length of about 10cm to about 400cm, more preferably about 15cm to about 350 cm. In certain embodiments, the conduits 101a-c are of the same length. In other embodiments, the conduits 101a-c have different lengths. In a particular embodiment, the conduit 101a has a length of about 105cm, the conduit 101b has a length of about 40cm, and the conduit 101c has a length of about 200 cm.

The conduits and mixers may be made of any inert material such that the material from the conduits and mixers does not participate in the reactions occurring within the fluid system. Exemplary materials include PVC-U. The tubing is commercially available from Georg Ficher AB. The conduit and the mixer may be configured to have a linear arrangement such that the conduit and the mixer are arranged in a straight line. Alternatively, the piping and mixer may have a non-linear arrangement such that the water must flow through bends and curves throughout the process. The system 100 shows a non-linear configuration of the conduits 101a-c and the mixers 102 and 103.

The conduit 101a is an inlet conduit that receives water to be flowed through the system. Typically, the water in the conduits 101a-c is at a pressure of at least about 0.1 bar (such as, for example, 0.2 bar or greater, 0.3 bar or greater, 0.4 bar or greater, 0.5 bar or greater, 0.7 bar or greater, 0.9 bar or greater, 1.0 bar or greater, 1.2 bar or greater, 1.3 bar or greater, or 1.5 bar or greater). At such pressures, a turbulent water flow is created, thus introducing the reagent into the highly turbulent water flow, which facilitates initial mixing of the reagent with the water prior to further mixing in the mixing devices 102 and 103.

In order to control the pH value during the production process, the feed water should have a buffering capacity in the range of pH 3.5-9.0, more preferably 6.0-8.0, in order to facilitate the addition of the proton-generating compound and the hypochlorite anion-generating compound. Dissolved salts and other molecules found in most tap water impart a buffering capacity to tap water in the pH range of 5.5-9.0, and tap water is therefore a suitable water for use with the method of the invention.

In certain embodiments, deionized water is used with the addition of known buffers to produce water having a buffering capacity in the range of pH 3.5-9.0. An example of a buffer within this particular range is a phosphate buffer. For better process control and consistency, the use of formulated deionized water may be preferred over tap water, as tap water may vary from location to location and over time. In addition, the use of deionized water with known additives also ensures a stable pH of the influent water stream. This process will be discussed in more detail below.

In particular embodiments, the initial pH of the water prior to addition of the proton-generating compound or hypochlorite anion-generating compound is at least about 8.0, including 8.1 or greater, 8.2 or greater, 8.3 or greater, 8.4 or greater, 8.5 or greater, 8.6 or greater, 8.7 or greater, 8.8 or greater, 8.9 or greater, 9.0 or greater, 9.5 or greater, 10.0 or greater, 10.5 or greater, or 10.8 or greater. In a particular embodiment, the pH of the water is 8.4 prior to the addition of the proton-generating compound or hypochlorite anion-generating compound.

The method of making HOCl includes introducing the proton-generating compound and the hypochlorite anion-generating compound into water in any order (e.g., simultaneously or sequentially) and in any manner (aqueous form, solid form, etc.). For example, the proton-generating compound and the hypochlorite anion-generating compound are both aqueous solutions and are introduced into the water sequentially, e.g., the proton-generating compound may be introduced first into the water and the hypochlorite anion-generating compound may be introduced second into the water.

The system 100 is configured for sequential introduction of reagents into a water stream, and the following methods are described herein: wherein the proton generating compound is first introduced into the water and the hypochlorite anion generating compound is second introduced into the water. In certain embodiments, the proton-generating compound and the hypochlorite anion-generating compound are introduced into the water in small aliquots (e.g., from about 0.1mL to about 0.6 mL). Although an acid (proton-generating compound) and a base (hypochlorite anion-generating compound) are added, repetition and microtitre make it possible to control the pH. In certain embodiments, an amount of no more than about 0.6mL of the proton-generating compound is introduced into the water at a single time point. In other embodiments, the hypochlorite anion generating compound is introduced into the water at a single time point in an amount of no more than about 0.6 mL.

To introduce reagents into the water, tubing 101a includes an injection port 104 and tubing 101b includes an injection port 105. Injection ports 104 and 105 allow introduction of reagents into the water stream. In this embodiment, the proton-generating aqueous compound is introduced into the water in conduit 101a via injection port 104. The proton generating compound is introduced by an infusion pump sealably connected to port 104. In this way, the flow rate, and hence the amount, of the proton-generating compound introduced into the water at any given time is controlled. The infusion pump may be controlled automatically or manually. The rate of introduction of the proton-generating compound into the water is based on the feed water quality (conductivity and pH level) and the pressure and flow rate of the feed water. In certain embodiments, the pump is configured to introduce about 6.5 liters of hydrochloric acid per hour into the water. The introduction may be continuous infusion or carried out in a batch mode. Since water flows through the pipe in a turbulent manner, there is an initial mixing of the proton-forming compound with the water as the hydrochloric acid is introduced into the water.

Further mixing occurs as the water enters the first mixing device 102. Fig. 2 shows an enlarged view of the mixing device 102 shown in fig. 1. In the embodiment shown, the mixing device comprises a length of about 5.5cm and a diameter of about 5 cm. Those skilled in the art will recognize that these are exemplary dimensions and that the method of the present invention may be performed with mixing devices having dimensions different from the illustrated dimensions. The mixing device 102 comprises a fluid inlet 106 sealably coupled to the pipe 101a and a fluid outlet 107 sealably coupled to the pipe 101 b. In this manner, water may enter the mixing chamber 108 of the device 102 from the conduit 101a and exit the mixing chamber 108 of the device 102 through the conduit 101 b.

The mixing device 102 is configured to generate a plurality of fluid vortices within the device. An exemplary device constructed in this manner is shown in fig. 3, which provides an interior view of the chamber 108 of the device 102. The chamber 108 includes a plurality of members 109 spaced perpendicular to the inlet and outlet and fixed within the chamber 108 to form a plurality of sub-chambers 110. Each member 109 includes at least one aperture 111 that allows fluid to flow therethrough. Fig. 4 shows a front view of the member 109 such that the aperture 111 can be seen. The size of the orifice will depend on the water flow and pressure in the system.

Any number of components 109 may be secured in the chamber 108, and the number of components 109 secured in the chamber 108 will depend on the amount of mixing desired. Fig. 4 shows four members 109a-d secured in a chamber to create four sub-chambers 110 a-d. The members 109 may be spaced apart at uniform distances within the chamber 108, resulting in sub-chambers 110 of uniform size. Alternatively, the members 109 may be spaced apart at different distances within the chamber 108, thereby creating sub-chambers 110 of different sizes. The members 109 are sized so that they can be secured to the inner wall within the chamber 108. In this way, water cannot flow around the members and can only pass through the apertures 111 in each member 109 to move through the mixing device 102. Typically, the member will have a diameter of about 1cm to about 10 cm. In a specific embodiment, the member has a diameter of about 3.5 cm.

A vortex of fluid is created within each sub-chamber 110 a-d. The vortex is created by the water flowing through the apertures 111 in each member 109. The method of the present invention allows for any arrangement of apertures 111 around each member 109. Fig. 4 shows a non-limiting example of a different arrangement of apertures 111 in the member 109. The orifice may be of any shape. Fig. 4 shows a circular orifice 111. In certain embodiments, all of the apertures 111 are located within the same location of the member 109. In other embodiments, the apertures 111 are located in different locations of the member 109. Within a single member 109, all of the apertures 111 may have the same diameter. Alternatively, at least two apertures 111 have different sizes within a single member 110. In other embodiments, all apertures 111 within a single member 110 are of different sizes.

In certain embodiments, the apertures 111 in the members 110 have a first size and the apertures 111 in different members 110 have a second, different size. In other embodiments, the apertures 111 in at least two different members 110 are the same size. The size of the orifice will depend on the water flow and pressure in the system. An exemplary orifice diameter is about 1mm to about 1 cm. In a specific embodiment, the orifice has a diameter of about 6 mm.

The solution enters the mixing device 102 through an inlet 106 sealably engaged with the conduit 101 a. The solution enters chamber 108 and turbulent mixing occurs in each sub-chamber 110a-d as the solution passes through members 109a-d via orifices 111 in each member 109 a-d. After mixing in the final sub-chamber 110d, the water exits the chamber 108 via the fluid outlet 107 sealably fitted to the pipe 101 b.

Next, the compound that generates hypochlorite anions is introduced into the solution flowing through the pipe 101b via the injection port 105. The hypochlorite anion generating compound is introduced by an infusion pump sealably connected to port 105. In this way, the flow rate, and therefore the amount, of the hypochlorite anion generating compound introduced into the water at any given time is controlled. The infusion pump may be controlled automatically or manually. The rate of introduction of the hypochlorite anion generating compound into the water is based on the nature of the solution (conductivity and pH level) and the pressure and flow rate of the solution. In certain embodiments, the pump is configured to introduce about 6.5 liters of hypochlorite anion generating compound into the solution per hour. The introduction may be continuous infusion or carried out in a batch mode. Since the solution flows through the pipe in a turbulent manner, there is initial mixing of the hypochlorite anion generating compound with the solution as it is introduced into the solution.

Further mixing occurs as 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 be configured the same as or different from the mixing device 102, e.g., the same or different number of sub-chambers, orifices of the same or different diameters, sub-chambers of the same or different sizes, etc. However, similar to the mixing device 102, the mixing device 103 is configured to generate a vortex of fluid within each sub-chamber.

The solution enters the mixing device 103 through an inlet in the device which sealingly engages the conduit 101 b. The solution enters the mixing chamber and turbulent mixing occurs in each sub-chamber of the mixing device as the solution passes through the members in the chamber via the apertures in each member. After mixing in the final sub-chamber, the water exits the chamber via a fluid outlet in the mixing device sealably fitted to the conduit 101 c.

At this point, the reaction is complete and HOCl has been formed. Production was controlled in series by measuring pH and conductivity. The pH value was used in combination with the conductivity based on a pre-calibrated relationship between conductivity and HOCl concentration measured using spectrophotometry. The measured conductivity is a measure of the solvent's ability to conduct current. The same substrate was compared to different known concentrations of HOCl and OCl-and a calibration curve was established (fig. 8) which was used in combination with a pH meter to regulate titration and control the process.

The conduit 101c may be connected to a switching valve 112 that switches between a waste line 113 and a product collection line 114. As shown in fig. 5 and 6. Valve 112 includes a pH meter and a conductivity measurement device. These devices measure the concentration (ppm), purity and pH of the generated HOCl and provide feedback for changing the properties of the generated HOCl. Once the HOCl produced in conduit 101c meets the required concentration, purity and pH, valve 112 switches from waste line 113 to product collection line 114 to collect the desired product.

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

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

The process may be performed manually or in an automated manner. The fluid systems described herein may be operably connected to a computer that controls the production process. The computer may be a PCL logic controller system. The computer opens and closes the valves for the water inlet, wastewater outlet, and product outlet based on feedback received from sensors in the system (e.g., conductivity, pH, and concentration of product produced (ppm)). The computer may also store values for water pressure and water quantity, and may adjust these values based on feedback received from the sensors regarding the nature of the HOCl produced. The computer may also control an infusion pump that injects the reagent into the water for the manufacturing process.

This process can be repeated as the pipe 101c can be attached to a second fluid system and the produced HOCl then flowed through the second system where the process is repeated with the starting solution being HOCl instead of water. In this way, increased HOCl yields result. Any number of fluid systems may be interconnected with the method of the present invention.

Figure 7 is a schematic diagram showing another exemplary system 200 for producing hypochlorous acid according to the methods of the present invention. System 200 is configured to adjust the pH of the influent water and inject a buffer for stability. In system 200, water is introduced into conduit 201 a. A pH meter 208 is connected to the pipe 201 a. The pH meter 208 measures the pH of the influent water. A pH meter 208 is connected to injection port 202.

Injection port 202 allows for the introduction of at least one buffer into the water. The buffer is introduced by an infusion pump sealably connected to port 202. In this way, the flow rate, and hence the amount, of buffer introduced into the water at any given time is controlled. The infusion pump may be controlled automatically or manually. The rate at which the buffer is introduced into the water is based on the influent water quality (conductivity and pH level), the buffer composition, and the pressure and flow rate of the influent water. The introduction may be continuous infusion or carried out in a batch mode. Since the water flows through the conduit 201a in a turbulent manner, there is an initial mixing of the buffer with the water as it is introduced into the water. This initial mixing may be sufficient to properly adjust the properties of the influent water.

In certain embodiments, further mixing of water with buffer is performed prior to performing the process for producing HOCl. In those embodiments, further mixing occurs when the water with the buffer 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 be configured the same as or different from the mixing device 102, e.g., the same or different number of sub-chambers, orifices of the same or different diameters, sub-chambers of the same or different sizes, etc. However, similar to the mixing device 102, the mixing device 203 is configured to generate a vortex of fluid within each sub-chamber.

The solution enters the mixing device 203 through an inlet in the device which sealingly engages the conduit 201 a. The solution enters the mixing chamber and turbulent mixing occurs in each sub-chamber of the mixing device as the solution passes through the members in the chamber via the apertures in each member. After mixing in the final sub-chamber, the water exits the chamber via a fluid outlet in the mixing device sealably fitted to the conduit 202 b. The water has a pH of at least about 8.0, preferably 8.4, and a buffering capacity of pH 5.5-9.0.

The process is now carried out as described above to produce HOCl. Next, the proton generating compound is introduced into the water flowing through the pipe 201b via the injection port 204. The proton generating compound is introduced by an infusion pump sealably connected to port 204. In this way, the flow rate, and hence the amount, of the proton-generating compound introduced into the water at any given time is controlled. The infusion pump may be controlled automatically or manually. The rate of introduction of the proton-generating compound into the water depends on the properties of the water (conductivity and pH level), the buffer composition, and the pressure and flow rate of the water.

In certain embodiments, the pump is configured to introduce from about 6.5 liters per hour to about 12 liters per hour of the proton-generating compound into the water. The introduction may be continuous infusion or carried out in a batch mode. Since water flows through the pipe in a turbulent manner, there is an initial mixing of the proton-forming compound with the water as the hydrochloric acid is introduced into the water.

Further 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 the mixing device 102. The mixing device 205 may be configured the same as or different from the mixing device 203, e.g., the same or different number of sub-chambers, orifices of the same or different diameters, sub-chambers of the same or different sizes, etc. However, similar to the mixing device 203, the mixing device 205 is configured to generate a vortex of fluid within each sub-chamber.

The solution enters the mixing device 205 through an inlet in the device which sealingly engages the conduit 201 b. The solution enters the mixing chamber and turbulent mixing occurs in each sub-chamber of the mixing device as the solution passes through the members in the chamber via the apertures in each member. After mixing in the final sub-chamber, the water exits the chamber via a fluid outlet in the mixing device sealably fitted to the conduit 201 c.

Next, the hypochlorite anion generating compound is introduced into the solution flowing through the pipe 201c via the injection port 206. The hypochlorite anion generating compound is introduced by an infusion pump sealably connected to port 206. In this way, the flow rate, and therefore the amount, of the hypochlorite anion generating compound introduced into the water at any given time is controlled. The infusion pump may be controlled automatically or manually. The rate of introduction of the hypochlorite anion generating compound into the water is based on the nature of the solution (conductivity and pH level) and the pressure and flow rate of the solution. In certain embodiments, the pump is configured to introduce about 6.5-12 liters of hypochlorite anion generating compound into the solution per hour. The amount introduced depends on the desired HOCl concentration (ppm) and the water flow through the pipe. The introduction may be continuous infusion or carried out in a batch mode. Since the solution flows through the pipe in a turbulent manner, there is initial mixing of the hypochlorite anion generating compound with the solution as it is introduced into the solution.

Further mixing occurs as 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 be configured the same as or different from the mixing device 205 or 203, e.g., the same or different number of sub-chambers, the same or different diameter orifices, the same or different sized sub-chambers, etc. However, similar to mixing devices 205 and 203, mixing device 207 is configured to create a vortex of fluid within each subchamber.

The solution enters the mixing device 207 through an inlet in the device which sealingly engages the conduit 201 c. The solution enters the mixing chamber and turbulent mixing occurs in each sub-chamber of the mixing device as the solution passes through the members in the chamber via the apertures in each member. After mixing in the final sub-chamber, the water exits the chamber via a fluid outlet in the mixing device sealably fitted to the conduit 201 d.

At this point, the reaction is complete and HOCl has been formed. The generated HOCl can be measured and collected as described above. The conduit 201d may be connected to a switching valve that switches between a waste line and a product collection line. The valve includes a pH meter and a conductivity measuring device. These devices measure the concentration, purity and pH of the generated HOCl and provide feedback for these property changes of the generated HOCl. Once the HOCl produced in conduit 201d meets the required concentration, purity and pH, the valve switches from the waste line to the product collection line to collect the desired product.

In another embodiment, a deionizer is placed in series with the feed water. The deionizer deionizes water and then adds a buffer to the deionized water. The production process is then carried out as described for the embodiment of system 200 to produce water having a pH of at least about 8 (e.g., 8.4) and a buffering capacity of pH 6-8.

HOCl produced by the above process can be used in many different applications, such as medical, food service, food retail, agriculture, wound care, laboratory, hotel, dental, delignification or floral industries.

Wound care

In certain embodiments, the compositions of the present invention are used for wound care. Wound care involves the treatment of damaged or broken skin, including abrasions, lacerations, ruptures, perforations, or burns. Certain wound care treatments involve the treatment of biofilms. Biofilms can form when free-floating microorganisms, such as bacteria and fungi, attach themselves to surfaces. Biofilms are known to impair skin wound healing and reduce the surface antibacterial efficacy in healing or treating infected wounds. Other common health conditions associated with biofilms include urinary tract infections, middle ear infections, chronic wounds, and plaque formation. Cystic fibrosis, natural valve endocarditis, otitis media, periodontitis and chronic prostatitis also involve biofilm-producing microorganisms. Microorganisms commonly associated with biofilms include Candida albicans (Candida albicans), coagulase negative Staphylococcus (Staphyloccci), Enterococcus (Enterococcus), Klebsiella pneumoniae (Klebsiella pneumoniae), Pseudomonas aeruginosa, Staphylococcus aureus, and the like.

Biofilms are often resistant to traditional antimicrobial treatments and are therefore a serious health risk. Biofilm resistance has rendered traditional antibiotic and antimicrobial therapies ineffective. Because biofilms can greatly reduce sensitivity to antibiotics and disinfectants, there is a need for therapeutic approaches that can break down biofilms but are less toxic to patients.

Methods of administering the compositions to an individual in need of treatment for a biofilm-associated infection are provided. The methods of the invention include the prevention, treatment, or cure of biofilm-associated infections. The methods comprise administering a therapeutically or prophylactically effective amount of one or more unit doses of the compositions for treating an existing biofilm-associated infection or preventing the establishment of a biofilm-associated infection in an individual. In some embodiments, the spread of the biofilm-associated infection to another site in the individual is inhibited. In various embodiments, the composition may be administered parenterally, orally, topically, or topically. The composition may 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.

Treatments include killing microorganisms that inhabit biofilms or removing biofilms, inhibiting biofilm formation, and disrupting existing biofilms. The compositions disclosed herein are particularly effective for treating microbial biofilms in or on wounds. The composition may be in the form of a topically applicable wound treatment composition comprising hypochlorous acid and an acetic acid compound. The composition may be combined with additional antimicrobial agents.

The compositions of the present invention may be topically administered to a subject, for example, by applying or painting the composition directly to the epidermis or epithelial tissue of the subject. The composition may be formulated as a liquid, powder, lotion, cream, gel, oil, ointment, gel, solid, semi-solid formulation, or aerosol spray. Such formulations may be produced in conventional manner using suitable carriers well known to those skilled in the art.

Suitable carriers for topical application preferably remain in place on the skin as a continuous film and resist 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 provided within or on a wound contacting surface of the wound dressing. The wound dressing may be intended to be applied to a wound to be treated and it comprises a substrate containing the composition of the invention. Such a dressing is particularly convenient as it delivers the composition of the invention to the wound to be treated and at the same time provides a dressing for that wound. The wound dressing may be, for example, a fiber, a foam, a hydrocolloid, a collagen, a film, a sheet-like hydrogel, or a combination thereof. The wound dressing may be in the form of a layered dressing, wherein one or more layers of the dressing are formed from at least part or one or both of the following: natural fibers, alginates, chitosan derivatives, cellulose, carboxymethyl cellulose, cotton, rayon, nylon, acrylics, polyesters, polyurethane foams, hydrogels, hydrocolloids, polyvinyl alcohol, starch films, collagen, hyaluronic acid and its derivatives, biodegradable materials, and other materials known in the art. The methods of the present invention may further comprise negative pressure wound therapy as known in the art. Such therapies include applying negative pressure to the wound, such as with a vacuum dressing.

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

The use of the compound in combination with another known antimicrobial therapy may increase the efficacy of the antimicrobial agent. In some embodiments, the methods of the invention further comprise administering (simultaneously or sequentially with the compositions of the invention) one or more doses of an antibiotic substance, including, but not limited to, ciprofloxacin (ciprofloxacin), ampicillin (ampicilin), azithromycin (azithromycin), cephalosporin, doxycycline, fusidic acid (fusidic acid), gentamycin (gentamycin), linezolid (linezolid), levofloxacin (levofloxacin), norfloxacin (norfloxacin), ofloxacin (ofloxacin), rifampin (rifampin), tetracycline (tetracycline), tobramycin (tobramycin), vancomycin (vancomycin), amikacin (amikacin), defuzidime, cefepime (cefepime), trimethoprim (trimethoprim)/sulfamethoxazole (sulfamethoxazole)Oxazole (sulfamethoxazole), piperacillin (piperacillin)/tazobactam (tazobactam), aztreonam (aztreonam), meropenem (meropenem), colistin, or chloramphenicol. In some embodiments, the methods of the invention further comprise administering one or more doses of an antibiotic substance from the class of antibiotics, including but not limited to aminoglycosides, carbacephems, antibiotics, or antibiotics,Carbapenems, first generation cephalosporins, second generation cephalosporins, third generation cephalosporins, fourth generation cephalosporins, glycopeptides, macrolides, monobactams, penicillins, polypeptides, quinolones, sulfonamides, tetracyclines, lincosamides, andoxazolidinones. In some embodiments, the methods of the present invention comprise administering non-antibiotic antimicrobial substances, including, but not limited to, sertraline (sertraline), racemic and stereoisomeric forms of thioridazine (thioridazine), benzoyl peroxide, taurolidine (taurolidine), and aminocyclopyrimidine (hexitidine).

Treating biofilms on other tissues

The compositions of the present invention are useful for treating biofilms affecting parts of the body or adhering to various surfaces. In some embodiments, the methods of the invention comprise administering to an individual in need thereof a therapeutically effective composition for treating a biofilm-associated infection in the bladder, kidney, heart, middle ear, sinus, skin, lung, joint, subcutaneous tissue, soft tissue, vascular tissue, and/or eye. In other embodiments, a therapeutically effective amount of the composition is administered to an individual in need thereof for treating one or more of the following conditions associated with a biofilm: urinary tract infection; chronic bacterial vaginosis; prostatitis; bacterial infections derived from diabetes, such as diabetic skin ulcers; pressure ulcers; venous catheter related ulcers; or a surgical wound (e.g., a surgical site infection). In some embodiments, the biofilm is on the skin of the individual. In some embodiments, the biofilm is associated with a wound, including an abrasion, laceration, fracture, puncture, burn, and chronic wound. In some embodiments, the biofilm is beneath the surface of the skin, in subcutaneous tissue, such as a deep tissue wound or surgical site.

Treating biofilms on non-tissue surfaces

Other applications for treating biofilms are also contemplated. For example, the compositions of the present invention may be applied to treat microbial biofilms on surfaces, such as surfaces in hospitals (such as operating rooms or patient care rooms) and other surfaces (e.g., home work surfaces). The present invention also encompasses treating biofilms formed on implanted medical devices and prostheses.

As is known in the art, implanted medical devices are prone to biofilm formation, including fungal and bacterial biofilms. The methods and compositions of the present invention may also be used to treat biofilms formed on the surfaces of implanted medical devices, such as catheters and prostheses. The compositions of the present invention may be applied to a pre-implant medical device. Alternatively, the medical device may comprise a reservoir containing the composition such that the composition may be released in a controlled manner following implantation. Methods for treating implanted medical devices can be found in U.S. patents 5,902,283 and 6,589,591 and U.S. patent publication 2005/0267543, each of which is incorporated herein by reference in its entirety.

Dental treatment

In another embodiment of the present invention, a method for treating an oral cavity-associated biofilm (such as dental plaque) is provided. The present invention provides methods for oral plaque prevention, treating oral plaque infection, treating dental hypersensitivity, sterilizing root canals, or treating dental disease.

The methods of the present invention comprise contacting an oral surface (such as teeth, gums, or tongue) with a therapeutically effective amount of the composition. Some methods of the invention comprise preventing an oral-related biofilm by administering to an individual a prophylactically effective amount of a composition. The compositions can be formulated as dentifrices, such as toothpastes, 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 formulated as a mouthwash. In some embodiments, the composition is formulated as a coating (paint), foam, gel, or paint (varnish), for example in a fluorochemical composition. In one embodiment, the composition is in the form of a gel or foam in a mouth guard that is worn by the patient for several minutes for fluoride treatment. In other embodiments, the composition is contacted with an adhesive strip that can be applied to the teeth or other oral surfaces. The composition may comprise a liquid polymer formulation which is preferably a composition for topical application to a surface such as teeth, skin, mucous membranes, and which dries to a film that adheres to the surface, which resists removal under normal conditions, such as eating or brushing for application to teeth and oral mucosa, or normal washing and abrasion when applied to skin. Alternatively, the composition may be applied to bandages, dressings, gauze, brushes, implants, and the like, and allowed to dry into a film prior to application to a patient.

Mastitis treatment

In another embodiment of the present invention, compositions and methods for treating mastitis are provided. Mastitis is an inflammation of a 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 (Staphylococcus epidermidis), Streptococcus agalactiae (Streptococcus agalactiae), Streptococcus uberis (Streptococcus uberis), and others. Some bacteria known to cause mastitis also form biofilms, but not all mastitis cases are caused by biofilm formation.

Mastitis can occur in any mammal, such as humans, cows (cows) and other animals. Mastitis is a particular problem in dairy cattle. In cattle, the condition occurs when leukocytes are released into the mammary gland, often as a response to bacteria in the teat canal. Cows that are often repeatedly infected must be slaughtered to prevent widespread infection in the herd. Loss of milk from infected cows, as well as loss of cows and entire herds due to infection, results in significant economic loss to the dairy industry worldwide. For example, in the united states, mastitis is estimated to incur losses of up to 20 billion dollars each year in the dairy industry.

Methods of administering compositions to a mammal in need of treatment for mastitis are provided. The methods of the present invention include the prevention, treatment, or cure of mastitis. In some embodiments, the spread of mastitis to another stable or to another animal is inhibited.

The formulations, dosages, and routes of administration discussed above are suitable for use in these embodiments of the invention. For example, in various embodiments, the composition may be administered parenterally, orally, topically, or topically. The composition may be administered by intravenous, intramuscular or subcutaneous injection. As known in the art, the composition may be applied by infusion through the teat canal. In the methods of the present invention, the compositions may be administered in a pharmaceutically acceptable carrier, which may include emollients, emulsifiers, thickeners, solvents and the like.

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

The composition may be applied topically to the udder of a cow by applying or smearing the composition directly onto the udder or nipple of the cow. The composition may be formulated as a liquid, powder, lotion, cream, gel, oil, ointment, gel, solid, semi-solid formulation, or aerosol spray. The method of the invention may further comprise dipping the teat into the composition. Teat dipping may be used to treat an already infected udder or to prophylactically prevent the development of mastitis. The composition may be applied immediately prior to milking, immediately after milking, or both. Methods of teat dipping are known in the art and are described in more detail in U.S. patent 4,113,854 and U.S. patent publications 2003/0235560 and 2003/0113384, each of which is incorporated herein by reference in its entirety. The method may further comprise using a teat sealant to create a physical barrier for the teat orifice after administration of the composition.

In other embodiments, the composition may be provided via intramammary infusion. Intramammary infusion involves forcing the antibiotic up through the teat canal into the udder. Infusion fluids may comprise a composition disclosed herein in combination with a pharmaceutically acceptable carrier, such as canola oil. Prior to infusion, the teats are cleaned, for example with an alcohol swab. The antibiotic infusion device may include a cannula sized and shaped to fit into the teat canal. The cannula may be inserted fully or partially through the teat canal (streamk canal). Methods for infusion are known in the art and are described, for example, in U.S. patents 4,983,634 and 5,797,872, the entire contents of each of which are incorporated herein by reference.

The methods of the invention may further comprise administering an antibiotic in combination with a composition of the invention, or in a continuous dose either before or after administration of the composition.

Wound and surgical use

The composition may also be used for the prevention and treatment of biofilm on other types of living tissue. Tissues include, for example, skin, mucosa, wound, or ostomy site (ostomy). As described above, the composition may be used for wound treatment. Wounds include pressure sores, chronic wounds, burns, pressure wounds, diabetic wounds and other forms of skin trauma. Wounds are often prone to biofilm formation, which hinders healing and may lead to chronic conditions. The composition of hypochlorous acid and acetic acid can be used for debridement and cleaning of damaged tissues.

The compositions may also be used in a surgical environment for treating the skin before or after surgery. The composition prevents infections that would lead to biofilm formation. Sometimes, areas requiring surgery, such as traumatic wounds, may already be at risk of biofilm having formed. The hypochlorous acid composition can be used to disinfect the area prior to surgical incision, which not only helps to treat the biofilm, but also reduces the likelihood that it will diffuse to other tissues during surgery. The composition may be used to disinfect any surface within the surgical field.

Other medical uses

In addition to wound care, the HOCl compositions of the present invention may also be used for non-traumatic tissue treatment. They are useful for bladder irrigation, for the prevention or treatment of bladder infections or catheter-related urinary tract infections, and the like. They may similarly be used to treat infections in the upper respiratory digestive tract, such as sinus and lung infections, or infections in the mouth, pharynx, paranasal sinuses, nasal sinuses (sinonasal tract), larynx, piriformis or oesophagus. They are useful in combating the growth of microorganisms that cause infections and reducing allergens that cause adverse immune responses. The compositions may also be administered to the gastrointestinal tract, including the stomach, intestine and colon, to combat microbial infections, such as gastroenteritis, Clostridium difficile (Clostridium difficile) infections and Small Intestine Bacterial Overgrowth (SIBO).

In various other embodiments, the compositions may be administered in the form of eye drops to combat ocular 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 mouth rinse or mouthwash to combat the accumulation of biofilm in the oral cavity, or it may be used to clean or store dentures.

In addition to being used as a preservative to treat or prevent biofilm on living tissue, the composition may also be used as a disinfectant on other surfaces, such as for health care facilities, food preparations, cooking utensils, and the like. The composition can be used for disinfecting a work table, a hospital bed or a food preparation surface.

The compositions are useful, for example, for disinfecting medical devices and surgical instruments. Medical devices are often initially supplied in sterile form, but may require additional or subsequent cleaning and sterilization or sterilization. Particularly reusable medical devices, must be sterilized or disinfected before reuse. The composition may be applied to the medical device using any known technique. For example, the composition may be applied by wiping or painting the composition onto the surface of the device, by spraying an aerosol or mist form of the composition onto the device, by dipping the device into a container containing a volume of the composition, or by placing the device in a stream of the composition (such as from a faucet). Additionally or alternatively, medical devices and surgical instruments may also be stored submerged in the composition and removed at the time of use.

Treatment with the hypochlorous acid compositions disclosed herein may be performed in addition to other known techniques, such as autoclaving. Alternatively, the composition may be applied instead of autoclaving. Since heat sterilization is not useful for all devices (e.g., some devices contain delicate parts or electronics that cannot withstand high temperatures), hypochlorous acid compositions are a useful alternative, providing an effective means of sterilizing or disinfecting such devices.

The composition may also be used to disinfect implants and prostheses prior to their introduction into the body. Such devices include orthopedic implants, wires, screws, rods, artificial intervertebral discs, prosthetic joints, soft tissue fillers, pacemakers, intrauterine devices, coronary stents, ear tubes, intraocular lenses, dental implants, and many others known in the art.

As will be understood in the art, the various embodiments and uses described above relate to various methods of administration.

Hypochlorous acid compositions are particularly effective for transdermal therapy due to the small size of the HOCl molecules. Hypochlorous acid is able to penetrate the epithelium and wound surface, thus generally reaching deeper tissue layers without injection. This is particularly useful for biofilm infections that form under the top layer of the skin. Unlike many other antimicrobial treatments, acetic acid and similar organic acids penetrate deeper into the skin without the need for invasive delivery mechanisms.

However, in some embodiments, it may be desirable to prevent the penetration of HOCl into the skin, and thus the composition may be combined with excipients, carriers, emulsifiers, polymers, or other ingredients, examples of which are discussed in U.S. patent application 2016/0271171, which is incorporated herein by reference in its entirety.

In addition to topical applications, wherein the composition may be sprayed, wiped or rubbed onto the skin, in other embodiments, the composition may be injected into a specific tissue in need of treatment. The composition may be ingested in the form of a capsule for administration to the gastrointestinal tract. They may be provided in sustained release or delayed release capsules. The compositions may be provided as suppositories for insertion into the rectum or vagina.

In other embodiments, the composition may be provided as a nasal spray for the treatment of the upper aerodigestive tract, which treatment may include the treatment of allergies, sinus infections, and the like. Nasal sprays can be in the form of droplets, aerosols, gels, or powders. The buffered hypochlorous acid and acetic acid composition may be combined with one or more of a decongestant or an anti-inflammatory or antihistamine agent as desired. The composition may be atomized with a nasal spray dispenser as known in the art.

Nasal sprays can also contain pharmaceutically acceptable carriers, such as diluents, 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 tears. Suitable pharmaceutically acceptable non-toxic carriers are known to those skilled in the art. Various carriers may be particularly suitable for different formulations of the composition, for example whether it is intended for use as a drop, or a spray, nasal suspension, nasal ointment, nasal gel or another nasal form. Other additives, excipients, emulsifiers, dispersants, buffers, preservatives, wetting agents, consistency aids and gelling agents may also be included. Preferably, additives should be selected that impart the desired characteristics without reducing the stability of hypochlorous acid. The additive may help to apply the composition evenly to the mucosa, or help to reduce or delay the rate of absorption of the composition.

The composition may be delivered by various means known in the art for administering drops, droplets and sprays. The nasal spray composition may be delivered by dropper, pipette or dispensing. Fine droplets, sprays and aerosols can be delivered by an intranasal pump dispenser or squeeze bottle. The compositions may also be inhaled via a metered dose inhaler, such as a dry powder inhaler or a nebulizer.

Having nanoparticlesControlled release of encapsulation

A stable aqueous solution of hypochlorous acid and/or acetic acid may be encapsulated in nanoparticles that allow for controlled release of the acid from the nanoparticles. The controlled release allows for a long lasting antimicrobial protection.

Fig. 13 shows an antimicrobial composition 1301 comprising an aqueous solution 1303 of hypochlorous acid encapsulated in nanoparticles 1305. An aqueous solution 1303 of hypochlorous acid is made by the methods described herein to produce a solution in which the acid is stable. The stabilized hypochlorous acid solution 1303 is then encapsulated in nanoparticles 1305. The nanoparticles allow gradual release of hypochlorous acid. Although not depicted, acetic acid may also be encapsulated in the nanoparticles for controlled release.

The nanoparticles may be any type of nanoparticles that provide for the controlled release of acid from the nanoparticles. The nanoparticles may comprise a polymer, such as an organic polymer. Examples of polymers suitable for controlled release of nanoparticles include acrylic acid, carrageenan, cellulosic polymers (e.g., ethyl cellulose or hydroxypropyl cellulose), chitosan, cyclodextrin, gelatin, guar gum (guar gum), high amylase starch, hyaluronic acid, locust bean gum, pectin, polyacrylamide, poly (D, L-lactide-co-glycolide acid), poly (lactic acid), poly (xylitol adipate salicylate), polyanhydride, poly (ethylene oxide), poly (ethylene imine), polyglycerol esters of fatty acids, polysaccharides, polyvinyl alcohol, povidone, sodium alginate, and xanthan gum. For details on the use of polymers to form 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. pharmaceuticals 12: 3479-; gao et al, The Journal of Antibiotics 64: 625-; lee et al, International Journal of Nanomedicine 11: 285-; and U.S. patent No. 8,449,916 (incorporated by reference). The nanoparticles may contain aluminosilicates (such as zeolites, e.g., analcime, chabazite, clinoptilolite, heulandite, leucite, montmorillonite, natrolite, phillipsite, or stilbite), calcium ammonium nitrate, hydroxylapatites (e.g., urea-modified hydroxylapatites), metal hydroxides, metal oxides, polyphosphates, or silicon compounds (e.g., silica). The nanoparticle may contain a lipid, i.e. it may be a lipid nanoparticle. The nanoparticles may comprise liposomes. For details on the formation of controlled release nanoparticles using liposomes, see Weiniger et al, Anaesthesia 67:906-916 (2012). Liposomes can be multilamellar. The nanoparticles may contain gels, sol-gels, emulsions, colloids, or hydrogels. For details on the formation of controlled release nanoparticles using hydrogels, see Grijalvo et al, Biomate.Sci.4: 555 (2016). The nanoparticles may contain combinations of forms, such as hydrogels encapsulated within liposomes. The nanoparticles may have a core-shell structure. The nanoparticles may be biodegradable. The compositions of the present invention may comprise an antimetabolite agent. The antimetabolite agent may be a metal ion. For example, the antimetabolite may be zinc, copper or silver.

Nanoparticles that allow for the controlled release of hypochlorous acid or acetic acid allow for the diffusion of acid to proceed more slowly than the diffusion of acid from an equal volume of the same aqueous solution of acid not encapsulated in the nanoparticles. The controlled release of hypochlorous acid or acetic acid can be attributed to the permeability characteristics of the nanoparticles (e.g., partially or poorly permeable to acid nanoparticles). The controlled release nanoparticles may be nanoparticles that release acid in a time-dependent manner due to degradation of the nanoparticles or impairment of their structural integrity. The release of acid from the nanoparticles may be triggered by environmental conditions such as: pH, temperature, light, pressure, redox conditions, or the presence of specific chemicals.

Figure 14 is a schematic representation of a method 1401 of making an antimicrobial composition comprising an aqueous solution 1403 of hypochlorous acid encapsulated in nanoparticles 1405. The method entails mixing 1411 a compound 1415 that generates protons (H +) in water and hypochlorite anions (OCl) in water in a chamber 1413 that has been purged of air^-) Compound 1417. Mixing 1411 produces an air-free aqueous solution 1403 of hypochlorous acid. The solution 1403 is then encapsulated 1421 in the nanoparticle 1405. Encapsulation can be performed in an airless environment to produce a substantially airless composition.

Acetic acid for biofilm treatmentAnd hypochlorous acid

The disclosed formulations of acetic acid and hypochlorous acid are excellent for treating biofilms on surfaces, including skin or other tissues. The compositions use a balanced formulation in which the combination of acetic acid and hypochlorous acid provides better disinfecting qualities than either material alone. Indeed, the present invention recognizes that the particular combinations disclosed provide greater disinfection capacity than would be expected by the addition of acetic acid and hypochlorous acid. In other words, the compositions have been found to be greater than the sum of their parts. These benefits are shown in the accompanying data in fig. 15-21, which demonstrates how a balanced composition of acetic acid and hypochlorous acid provides enhanced disinfection capacity against biofilms and performs better than all other products on the market. The performance difference is shown over a wide range of concentrations.

In addition, since acetic acid is toxic at high concentrations, the prior art has taught not to use it on skin or other tissues except in trace amounts. Some of the disclosed compositions contain 2% or greater of acetic acid and, when combined with HOCl, have proven safe and effective for treating skin and other tissues. HOCl in these compositions has been found to have a modulating effect on acetic acid. This allows the composition to take advantage of the antimicrobial properties of acetic acid without causing damage to the tissue. In addition, HOCl has an analgesic function, so it also allows the use of higher concentrations of HAc on the skin or other tissue without causing excessive pain or discomfort to the patient.

For example, fig. 15 shows various concentrations of HOCl and acetic acid compared to other commercially available antimicrobial compositions. Eight different treatments were tested, as listed along the x-axis. Each composition was exposed to a 24 hour filter-grown staphylococcus aureus biofilm, and biofilm reduction was measured in colony forming units per milliliter (cfu/ml) and reported on a logarithmic scale along the y-axis. Measurements of biofilm reduction were recorded at 3 hours and 6 hours. Thus, each column has two bars and shows the effect of each composition on the biofilm over time.

The first three columns show the results for 200ppm HOCl with three different concentrations of acetic acid (0.25%, 1.0%, and 2.0%, respectively). The fourth column shows 1% acetic acid alone. The next four columns show commercial antimicrobial products: prontosan; octenilin; pyrisept; and Microdacyn, which is a hypochlorous acid composition.

The results show that all three combinations of acetic acid and hypochlorous acid are more effective on biofilm than any other combination. At 3 hours, the performance of test composition a (200ppm HOCl and 0.25% HAc) was roughly comparable to that of Prontosan (market leader of current biofilm treatment). It also performs well over 1% of HAc or other commercial products. However, after 6 hours, composition a showed much greater efficacy than even Protosan.

At the same time, test composition B (200ppm HOCl and 1.0% HAc) was even more effective at treating biofilms. Comparing composition B with 1% HAc (in column four) shows the unexpected benefit of adding HOCl. Despite having the same concentration of acetic acid, composition B performed far better than 1% HAc alone at both 3 and 6 hours.

Of the tested compositions, composition C (200ppm HOCl and 2.0% HAc) showed the greatest biofilm reduction. At 3 hours and 6 hours, the effect is several orders of magnitude higher than the commercial product.

These data show that in addition to being more effective in reducing biofilm than any commercially available product, compositions containing both acetic acid and hypochlorous acid are more effective than either acetic acid alone (1% HAc) or hypochlorous acid alone (microdacyn), and those excellent results cannot be explained by the additive effect of the two components alone. Without being bound by 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 otherwise be predicted based on the efficacy of each component alone.

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

As shown in the figure, the reduction at 2 hours is greater at higher HOCl concentrations, with a particularly pronounced spike at 150 ppm. At 4 hours, spikes also appeared at even lower HOCl concentrations.

FIG. 17 shows the effect of different compositions on Pseudomonas aeruginosa, with the concentration of HOCl maintained at 100ppm and the percentage of acetic acid varied from 25% to 2%. Fig. 18 shows the effect with increasing both HOCl and HAc.

FIGS. 19-21 show different compositions of HOCl and HAc against Staphylococcus aureus and Pseudomonas aeruginosa under various conditions. The figure shows excellent results obtained with a combination of hypochlorous acid and acetic acid, confirming the synergistic effect of the two compounds.

Various formulations are disclosed that are effective in treating biofilm infections in different types of tissues. For example, 200ppm HOCl and 0.25% HAc composition may be used for surface application, such as hand disinfection or mouthwash. As shown in fig. 15, the composition was more effective at treating surface level biofilms than other commercial products. To penetrate deeper into the tissue for treatment, or to clear particularly poor biofilm infection or invasive biofilms that have penetrated below the surface, a higher percentage of HAc may be used, such as a formulation of 200ppm HOCl and 2% HAc. The composition can be used for treating infected wounds, preventing biofilm in wounds, treating eczema, or treating other infections. The formulation has been found to be effective against biofilm that has formed in the tooth root.

Figures 20-21 show additional data supporting the unexpected efficacy of acetic and hypochlorous acid compositions, particularly compared to prior art compositions and commercial compositions, on various biofilms. As the figure clearly shows, various compositions that balance HOCl and HAc concentrations in different ways provide various disinfecting compositions that can target different types of biofilms on different types of tissues.

Minimum inhibitory concentration

The disclosed minimal inhibitory concentrations of hypochlorous acid and acetic acid compositions are useful for biofilm removal on surfaces and tissues as discussed herein. Determination of Minimum Inhibitory Concentrations (MIC) against 5 pathogenic bacteria (Acinetobacter baumannii (carbapenem-resistant), pseudomonas aeruginosa (carbapenem-resistant), Enterococcus faecium (Enterococcus faecium) (vancomycin-resistant), candida species (fluconazole-resistant) and staphylococcus aureus) was performed by broth microdilution (2-fold dilution) in 96-well microtiter plates. After 24 hours incubation in a microtiter dish, optical density will be measured to assess growth. In addition, the next day the suspension was spread on agar and growth was controlled. 5 microorganisms were tested against 10 different concentrations of the antimicrobial compound HOCl and acetic acid, including growth control and sterility control.

The MICs for all tested organisms were surprisingly low (25 ppm and 0.25% for HOCl and acetic acid, respectively) and the Minimum Bactericidal Concentrations (MBC) for HOCl and acetic acid were 50ppm and 0.5%, respectively.

Treating biofilms without inducing antimicrobial resistance

The stabilized hypochlorous and acetic acid compositions described herein are useful for biofilm prevention and biofilm removal on all surfaces and tissues discussed herein. Because biofilms greatly enhance the resistance of microorganisms to traditional antimicrobial agents, biofilm-forming microorganisms are more able to share and modify their resistance genes and transmit into the air and surrounding environment. As a result of biofilm development, simple infections may become chronic, antibiotics and antiseptics cease to function, and new infected strains emerge.

However, both acetic acid (or other organic acids) and hypochlorous acid are particularly useful for treating and preventing biofilms. The composition of HAc and HOCl disclosed herein mimics the natural disinfectant of the immune system. Thus, the composition is not susceptible to microbial resistance.

Tests were conducted to investigate whether P.aeruginosa would develop resistance to the disclosed compositions. Clinical isolates of P.aeruginosa were grown daily and passaged into M ü eller Hinton broth solutions containing increasing concentrations of the disclosed compositions. Isolates were passaged daily (1:100 and 1:1000) to new tubes containing the same concentration or challenged with higher concentrations for 14 days. At the end of the experiment, pseudomonas aeruginosa was isolated from the highest concentration at which growth was observed and tested for sensitivity to 3 different antibiotics (tobramycin, colistin and ciprofloxacin). As a control, the same isolate that had not been exposed to the drug was also tested for antibiotic susceptibility. Surprisingly, the results show that the disclosed compositions do not induce resistance or cross-resistance to the tested antimicrobial substances. The compositions of acetic acid and hypochlorous acid disclosed herein are effective in treating and preventing biofilms without inducing antimicrobial resistance. In addition, they are non-toxic, non-stinging, and relieve itching.

Is incorporated by reference

Any and all references and citations to other documents, such as patents, patent applications, patent publications, periodicals, books, papers, web content, made throughout this disclosure are hereby incorporated herein by reference in their entirety for all purposes.

Equivalent content

The present invention may be embodied in other specific forms without departing from its spirit or essential characteristics. The foregoing embodiments are therefore to be considered in all respects illustrative rather than limiting of the invention described herein.

Examples

Example 1: analysis of the product

When spectrophotometry is extended to also cover the visible range, color can be detected. The gas typically produced during HOCl production is ClO₂、Cl₂O and Cl₂All of these gases are detectable as yellow or yellowish red in the visible range. Tzanavaras et al (Central European J.of Chemistry,2007,5(1) 1-12). The data in FIG. 9 illustrates that HOCl produced by the method of the present invention shows no contribution from colorThe absorption of gas, as indicated by the absence of colored species. HOCl is known to produce a peak at 292nm (Feng et al, 2007, j.environ.eng.sci.6, 277-284).

Example 2:

HOCl produced by the above process was stored under thermal stress at 40 ℃ to accelerate degradation using four different types of aqueous solutions: (1) reagent grade water (deionized water); (2) tap water; (3) reagent grade water with phosphate buffer; and (4) tap water with phosphate buffer. In the initial reaction (T ═ 0); 4 weeks (T ═ 4); 8 weeks (T ═ 8); and the HOCl product was monitored for characteristics after 12 weeks (T ═ 12).

Fig. 10 is a graph showing the amount of HOCl initially produced (parts per million (ppm)) (T ═ 0) and its stability over time. The data show that reagent grade water (deionized water) without phosphate buffer is most stable over 12 weeks, showing minimal product degradation from initial production. Deionized water produces a product that is much more stable than that produced using tap water. Additionally, and surprisingly, the data show that phosphate buffer may negatively impact the amount of HOCl product produced.

Figure 11 is a graph showing how the pH of the HOCl product changes over time. In all cases the pH decreased with time, however, for all cases the pH remained in the range of pH 4 to pH 7 for 12 weeks.

Fig. 12 is a graph showing the oxidation capacity of HOCl product over time. The data show that the product retains oxidative capacity over 12 weeks regardless of the starting water.

Example 3: acetic acid in comparison with hydrochloric acid

Using the above process, HOCl was produced using hydrochloric acid (HCl) and acetic acid and then stored under thermal stress at 40 ℃. The amount of HOCl initially produced (T ═ 0) was recorded, and then the amount of HOCl product remaining after 12 days was recorded. Each yielding three batches. Data for HCl-generated HOCl are shown in table 1. Data for acetic acid-produced HOCl are shown in table 2.

Table 1: HOCl production with HCl

Table 2: HOCl production with acetic acid

The data show that the use of acetic acid provides higher product stability, most likely due to higher pH stability. Without being bound by any particular theory or mechanism of action, it is believed that the different protonation capabilities of acetic acid (i.e., acetic acid supplies fewer protons to the liquid than hydrochloric acid) results in higher stability of HOCl over time as compared to hydrochloric acid.

Example 4: hand disinfectant

The hands of healthy subjects were tested to determine the instant germ killing effect of the disclosed compositions.

In this experiment, 18 subjects washed their hands and their fingertips were immersed in the E.coli solution. After the hands of the subject are dry, an aliquot of the disclosed composition (5mL of 160ppm HOCl, 0.13% Hac) is applied to the dry hands of the subject and rubbed into the skin 30 to stimulate removal of transient bacteria on the skin according to the hand rub procedure (Handrub procedure) PN-EN1500: 2013-07. The same procedure was carried out using 3mL of reference alcohol (60% v/v 2-propanol).

The results show that both the disclosed compositions and the reference alcohol reduce the presence of transient bacteria by 4-log.

In another experiment, an aliquot of the disclosed composition (3mL of 0.03% HOCl, 0.13% Hac) was applied to each hand of 20 subjects. For each subject, one hand was exposed and the other hand was covered with a surgical glove. The composition was rubbed into the hand and forearm for 5 minutes according to the hand rub procedure PN-EN 12791:2005 to maintain exposure to the composition. The same procedure was performed using 3mL of reference alcohol (60% v/v propan-1-ol) for a 3 minute maintenance exposure.

Bacterial kill of escherichia coli, pseudomonas aeruginosa, staphylococcus aureus and Enterococcus hirae (Enterococcus hirae) was measured again immediately after application and 3 hours after application. The results demonstrate that the reference alcohol reduced native bacteria by 2.48log immediately after application and by 2.16log 3 hours after application. The disclosed compositions reduced native bacteria by 0.69log 3 hours after application and by 1.01log immediately after application.

Surprisingly, the disclosed compositions of the present invention are more effective in targeting transient pathogenic bacteria without damaging the natural bacteria than prior art alcohol-based disinfectants. Thus, the disclosed compositions are effective in the targeted treatment of pathogenic biofilm infections without compromising the natural flora of the body that protects the skin.