Multi-compartment device and method of preparing a hypochlorous acid based inhalation formulation

Abstract

The present invention provides multi-compartment devices and methods for preparing hypochlorous acid-based inhalation formulations. The multi-compartment devices for preparing hypochlorous acid-based broad-spectrum antiviral and / or antibacterial inhalation formulations provided by the present invention comprise: (a) a first compartment comprising calcium hypochlorite or another pharmaceutically acceptable hypochlorous acid precursor, and (b) a second compartment comprising aqueous acetic acid and water or saline, wherein mixing the contents of the compartments produces an inhalation formulation comprising hypochlorous acid. The hypochlorous acid-based inhalation formulations prepared by the present invention provide a safe and effective approach to treating and preventing respiratory infections, including viral and bacterial infections.

Description

[0001] This application is a divisional application of the invention patent application with application number 202180058393.X, application date July 7, 2021, and invention title "Compositions and methods for disinfection, treatment and prevention of microbial infections". Related application citation

[0002] This application claims priority and benefit to U.S. Provisional Application No. 63 / 048,815, filed July 7, 2020, the contents of which are incorporated herein by reference in their entirety. Technical Field

[0003] The present invention generally relates to a multi-compartment device for preparing a hypochlorous acid-based broad-spectrum antiviral and / or antibacterial inhalation formulation, and a method for preparing a hypochlorous acid-based inhalation formulation. Background Technology

[0004] Infectious diseases are a leading cause of death worldwide, causing more than 13 million deaths annually, including nearly two-thirds of all child deaths. Furthermore, antibiotic resistance is increasing and is raising the prevalence of many human diseases, including pneumonia, tuberculosis, and cholera. Of particular concern is that many human pathogens have developed resistance to common antibiotics. The introduction of new, more effective derivatives of existing antibiotics provides only temporary solutions, as existing resistance mechanisms rapidly adapt to new derivatives. While drug-resistant Gram-positive bacteria pose a significant threat, common Gram-negative pathogens (such as Escherichia coli) also pose a threat. (Escherichia coli) The emergence of multidrug-resistant (MDR) strains of Pseudomonas aeruginosa is particularly worrying. Furthermore, the presence of MDR strains of Pseudomonas aeruginosa is also concerning. (Pseudomonas aeruginosa) Acinetobacter baumannii (Acinetobacter baumannii) and Enterobacteriaceae bacteria (Enterobacteriaceae) The isolates have been shown to be resistant to almost all antibiotics.

[0005] Viruses are also a significant issue in infectious disease epidemiology. Severe viral outbreaks are becoming increasingly common, many of which originate from zoonotic diseases. For example, the SARS (Severe Acute Respiratory Syndrome) and MERS (Middle East Respiratory Syndrome) outbreaks of the early to mid-2000s, the H1N1 outbreak of 2009, and the subsequent SARS-CoV-2 outbreak of 2020 have all focused attention on the treatment and prevention of the spread of these viral pathogens.

[0006] Many respiratory viruses are transmitted through droplet infection. In this case, virus-containing respiratory droplets are expelled by an infected person and can be contaminated by others who come into direct contact with the droplets or with surfaces where the droplets have landed. Typically, infection occurs when the virus enters the nose, eyes, ears, or mouth and binds to receptors on mucous membranes or epithelial cells. Additionally, some viruses are transmitted through virus-containing aerosol particles or through the air. In either case, the virus can survive for hours to days after being expressed by an infected individual.

[0007] Conventional compositions and methods for disinfecting surfaces or contaminated epithelial cells are insufficient to inactivate all infectious agents. Current forms of conventional disinfectant compositions may require long and impractical exposure times, or may use harmful or corrosive solutions or vapors that cannot be used on sensitive instruments or living tissues, thus failing to provide a practical solution to the growing health risks posed by drug-resistant pathogens.

[0008] Chloride oxides, or chlorine oxidants (also referred to as "OC" in this document), comprise a large class of chemicals and are frequently found in nature and in mammalian biological systems. Chloride oxides can also exist as neutral compounds or ions, namely, so-called oxyanions. Several oxyanions of chlorine exist, one of which can be in oxidation states of +1, +3, +5, or +7. The corresponding anion is hypochlorite (ClO₂). - ), chlorite (ClO2) - ), chlorate (ClO3) - ) or perchlorate (ClO4) - Under low pH conditions, the standard reduction potential of hypochlorous acid (HOCl) is +1.63, and that of chlorogenic acid (HClO2) is 1.64. Under alkaline pH conditions, the standard reduction potentials are +0.89 and +0.78, respectively. At pH values ​​between 5 and 7, the reduction potential is higher than +1.

[0009] Therefore, hypochlorite and chlorite ions are generally the most useful oxidation states with the potential to kill microorganisms and parasites. In particular, chloride ions (Cl-) - In its most stable oxidation state, it is non-reactive and not an effective disinfectant. Chlorate and perchlorate ions in their +5 and +7 oxidation states are more reactive and may be more difficult to handle than those in their lower oxidation states.

[0010] Hypochlorite ions have the chemical formula ClO -Chlorine (Cl) is in the +1 oxidation state, a potentially unstable oxidation state because the lowest oxidation state of Cl is -1. Both hypochlorite and chlorite ions combine with many cations to form hypochlorites and chlorites, which are salts of these chlorine oxides. Common examples include sodium hypochlorite (household bleach) and calcium hypochlorite, which is the main active ingredient in commercial products including bleaching powder, chlorine powder, or chlorolime, commonly used in water treatment (e.g., swimming pools). Chlorite and hypochlorite ions are also referred to herein as "major chlorine oxides" and are useful in a variety of situations. Sodium chlorite and sodium hypochlorite are strong oxidizing agents and have been used in water purification, disinfection, and bleaching and deodorizing of animal products.

[0011] Because sodium hypochlorite produces highly toxic chlorine gas under acidic conditions, commercially available household aqueous solutions are strongly alkaline solutions, and their pH value is adjusted using sodium hydroxide.

[0012] Hypochlorous acid is a weak acid known to rapidly inactivate bacteria, algae, fungi, and other organic matter, making it an effective agent against a wide range of microorganisms. Furthermore, hypochlorous acid is generally non-toxic to humans because it is a weak acid, and certain compounds are naturally produced that allow humans to tolerate it. Due to the combination of its biocidal and safe properties, hypochlorous acid has been found to have many beneficial uses in various industries, such as medical, food service, food retail, agriculture, wound care, laboratory, hospitality, dental, and floriculture.

[0013] Hypochlorous acid is formed when chlorine dissolves in water. Specifically, it is produced by the acidification of hypochlorite, in which the chlorine atom is in the +1 oxidation state. Hypochlorous acid exists in equilibrium with chlorine gas, which can escape from the solution. This equilibrium depends on pH, as shown in Equation 1: Please refer to the above formula (Formula 1). A high pH value drives the reaction to the right, promoting the disproportionation of chlorine into chlorate and hypochlorite, while a low pH value drives the reaction to the left, promoting the release of potentially toxic chlorine gas (Cl2).

[0014] A major challenge facing the current medical applications of chlorine solutions (especially those with oxidation states above -1) is their stability, as these chemicals are in a high energy state and tend to revert to chloride ions (Cl-) at ambient temperatures. - It also decomposes in solution. This hinders the shelf-life stability required for pharmaceutical preparations and medical devices containing chlorine oxides under environmental conditions. Therefore, it is difficult to achieve the appropriate shelf life required for medical devices and pharmaceuticals with chlorine oxide solutions. This inherent limitation of all chlorine oxides restricts transportation and storage, especially in high-temperature regions with fluctuating temperatures, light, humidity, and atmospheric gases.

[0015] Therefore, while formulations containing chlorine oxides can be effective antimicrobial agents, conventional formulations have significant drawbacks. For example, the weak acid HOCl is unstable and impure when produced under conventional conditions. Therefore, a more controlled and direct preparation process for chlorine oxides that can provide stability in the field for the intended short-term use is needed. Overall, the medical need for novel therapies to treat drug-resistant microorganisms and viruses remains unmet. Summary of the Invention

[0016] This invention provides a disinfectant composition comprising a precursor of oxidized chlorine dissolved in a pharmaceutically acceptable diluent, adjuvant, or carrier and bound to an activator. The resulting composition provides a modified antimicrobial agent for in vivo and surface disinfection. In a preferred formulation, the composition of this invention comprises an acetic acid activator bound to a hypochlorite form. Optionally, the formulation of this invention may be bound to a thickener and / or dye. For example, a thickener may be used to adjust the viscosity of the formulation of this invention to form a gel. The formulation of this invention is preferably mixed in a container comprising a separate chamber as part of a multi-compartment device prior to use. The composition of this invention can be formulated for oral, intravenous, transdermal, or inhalation administration. Furthermore, the formulation of this invention can be prepared for inhalation via a nebulizer or similar device for rapid introduction into the patient's respiratory system. Therefore, the composition of this invention is a useful disinfectant for treating various bacterial and / or viral pathogens in vivo and on surfaces.

[0017] In one particular aspect, the present invention relates to an antimicrobial agent that provides a safe and effective route for treating and preventing respiratory infections, including viral and bacterial infections. Preferred compositions comprise a broad-spectrum antiviral and / or antibacterial inhalation solution based on hypochlorous acid. The solution of the present invention is preferably nebulized for delivery by inhalation. More specifically, preferred formulations contain hypochlorous acid (HOCl) (approximately 25 ppm to approximately 200 ppm) stabilized with acetic acid (approximately 0.25%), thereby producing a sustainable HOCl concentration with significant antimicrobial effects. The addition of acetic acid improves the stability of HOCl, thus enabling the development of therapeutic agents with extended shelf life. Furthermore, the composition is preferably formulated at pH 5.5 and is physiologically isotonic, thereby improving airway tolerability.

[0018] The compositions of the present invention possess unique antipathogenic properties. In one aspect, the compositions of the present invention act on enveloped viruses and provide excellent antiviral effects against coronaviruses. Therefore, such compositions are particularly suitable for treating and preventing the spread of SARS infection (e.g., COVID-19). More specifically, SARS-CoV-2 and many other viruses have surface proteins (i.e., spike proteins), which are entry points into cells of the respiratory system. These spike proteins contain -SH groups that are easily oxidized by HOCl. Even low concentrations of HOCl oxidize extracellular -SH groups (e.g., on viral spike proteins) without harming normal tissues and intracellular enzymes. Therefore, the antiviral action of the compositions of the present invention disrupts viral particles in the respiratory tract upon initial exposure, during infection, and when the virions are located intracellularly and subsequently released by cells in the respiratory tract.

[0019] Therefore, the unique antiviral properties of the compositions of the present invention (especially against enveloped viruses) make them a powerful tool in ongoing efforts to prevent the spread of coronaviruses. These compositions have shortened the duration of illness and reduced the severity of symptoms in a wide range of patient populations.

[0020] In another aspect, the present invention provides a disinfectant composition comprising a salt of a solid chlorine oxide, an activator (e.g., acetic acid), and a pharmaceutically acceptable diluent, adjuvant, or carrier. The salt of the solid chlorine oxide is based on the chemical formula M. n+ [Cl (O) x ] n n- Where M is an alkali metal ion, alkaline earth metal ion, or transition metal ion, n is 1 or 2, and x is an integer between 1 and 4, inclusive. The activator is based on the chemical formula R1XO. n (R2,) m The R1 group contains 1 to 10 hydride carbon atoms, optionally substituted with amino, amide, carboxyl, sulfonic acid or hydroxyl groups, wherein the group X is selected from carbon, phosphorus and sulfur; n and m are independently 2 or 3, and R2 is selected from H, alkali metal ion salts, alkaline earth metal ion salts, transition metal ion salts and ammonium salts.

[0021] In a preferred embodiment, the chlorine oxide salt comprises an alkali metal or alkaline earth metal salt of hypochlorous acid (HOCl). In such an embodiment, the activator is acetic acid. In other embodiments, the chlorine oxide salt comprises an alkali metal or alkaline earth metal salt of chlorite (HOClO). Again, in such an embodiment, the activator is acetic acid.

[0022] In some embodiments, the composition comprises a molar osmolality in the range of about 0.1 mOsm to about 500 mOsm.

[0023] In some embodiments, a certain amount of a salt of chlorine oxide, acetic acid, or its metal salt or ammonium salt is produced at a pH between 4 and 8.

[0024] In some embodiments, the composition further comprises a tackifier. In some aspects, the tackifier cannot be oxidized by chlorine-oxidizing substances.

[0025] In some embodiments, the tackifier comprises a water-soluble gelling agent. The water-soluble gelling agent may include, but is not limited to, polyacrylic acid, polyethylene glycol, poly(acrylic acid)-acrylamide alkyl propane sulfonate copolymer, phosphino polycarboxylic acid, poly(acrylic acid)-acrylamide alkyl propane, and sulfonated styrene terpolymer.

[0026] In some embodiments, the composition comprises a dye. The dye preferably produces a colorimetric indicator indicating the presence of a chlorine oxide compound in the formulation. The dye may be a vat-oxidation dye. In a preferred embodiment, the color and intensity of the dye depend on the oxidation state of the chlorine oxide compound.

[0027] The formulations of the present invention can be formulated as aqueous solutions, gels, creams, ointments, or oils. The formulations of the present invention can be produced and stored in multi-compartment containers. In some aspects, each individual compartment before assembly contains both aqueous and solid components.

[0028] The formulations of the present invention can be used as surface antibacterial agents and for the treatment of diseases. Therefore, such formulations of the present invention can be used as inhalation products, for example, in conjunction with nebulizers, inhalers, vaporizers, or other suitable delivery devices. Furthermore, the compositions of the present invention can be formulated for application in animal or agricultural breeding for the treatment of skin, wounds, mastitis, or any other infectious diseases; and for antiviral applications. Attached Figure Description

[0029] Figure 1 This is a schematic diagram of an exemplary multi-compartment or multi-chamber container for producing, storing and dispensing disinfectant compositions according to an embodiment of the present invention.

[0030] Figure 2 The results obtained using the sample solution according to the invention are shown.

[0031] Figure 3 The results obtained using the sample solution according to the invention are shown. Detailed Implementation

[0032] This invention generally relates to a composition comprising a solid and liquid precursor of chlorine in its oxidized state, an activator (e.g., acetic acid or a salt thereof), and one or more additional components. Such a composition can be used as a disinfectant to treat a wide variety of bacterial and / or viral pathogens on various biological and non-biological surfaces and in the environment.

[0033] Some preferred formulations of the present invention are multi-component (i.e., two-component, three-component, four-component, etc.) formulations that instantaneously produce compositions with long-term stability. This reduces the shelf-life-related limitations typically observed in the prior art when using conventional hypochlorous acid or chlorine dioxide solutions. More specifically, ready-to-use formulations that directly produce chlorine oxides from solid precursors (API-P) can be used at the point of use in a multi-compartment device or container. Such multi-compartment devices or containers are used for the preparation, dispensing, and long-term stable storage of prepared compositions as described herein. Specifically, such multi-compartment containers described herein may have multiple compartments or chambers, each containing the components required to produce the compositions of the present invention. In one embodiment, the formulation comprises a solid precursor of oxidized chlorine and acetic acid or a salt thereof, a thickener, and a dye, and is subsequently combined at the desired time and place of use to prepare an antimicrobial composition.

[0034] Another chlorine oxide that can be used as an API in antimicrobial agents is chlorine dioxide, in which the chlorine atom is in the +3 oxidation state. The main reaction of sodium chlorite is to produce chlorine dioxide, as shown in Equation (2): Please refer to the above formula (Formula 2). HOR is usually an inorganic acid, such as hydrochloric acid or citric acid, because a proton source is needed to first convert sodium chlorite into chlorite, and then into chlorine dioxide, which is a highly water-soluble gas at room temperature.

[0035] One advantage of chlorine dioxide is that it does not produce chlorine gas (Cl2), which is known to react with chlorinated hydrocarbons such as trihalomethanes, and chlorinated hydrocarbons are toxic environmental pollutants. Another advantage of chlorine dioxide is that its activity as a disinfectant or the stability of its aqueous solutions is not dependent on pH.

[0036] This invention addresses the challenges associated with prior art compositions using chlorine oxides. In particular, it provides a composition comprising a solid precursor of oxidized chlorine (OC) and an activator providing a proton source. A preferred example of the activator is acetic acid or a salt thereof, wherein the disinfectant composition of this invention is formed instantaneously at the point of use through a controlled and direct process, exhibiting stability that allows for the intended short-term use. Such compositions are useful disinfectants for treating a wide variety of microorganisms. In particular, when the active pharmaceutical ingredient is generated at the point of use from a stable solid precursor of chlorine oxide (hereinafter referred to as "API-P"), for example by including acetic acid as an activator and simultaneously buffering the solution or gel to a biocompatible pH, the stability problems of the prior art are no longer present.

[0037] As mentioned above, existing technologies fail to address how to ensure the ionic strength or molar osmotic pressure weight concentration of the final antimicrobial solution that is biocompatible with the biological fluid. Furthermore, existing technologies do not demonstrate how to adjust and increase the contact time and persistence of the API in the target treatment area (e.g., by adjusting rheology and flowability). Moreover, existing technologies fail to provide a simple yet effective method to monitor the oxidation state of the API during the mixing of disinfectant compositions and to visually indicate the location where the API is applied.

[0038] Furthermore, in some embodiments, the compositions of the present invention may also contain a combination of a thickener (also referred to herein as "VE") and / or a solid precursor containing oxidized chlorine and an activator (e.g., acetic acid or a salt thereof).

[0039] Another embodiment of the invention includes a dye in the formulation, which is preferably a dye whose color changes with the oxidation state of chlorine atoms, such as a redox-sensitive dye.

[0040] In particular, some preferred compositions of the present invention are multi-component (i.e., two-component, three-component, four-component, etc.) formulations in solid form that immediately produce compositions with long-term stability, the components being separated by fragile walls or barriers. This eliminates any shelf-life-related problems found in the prior art when using hypochlorous acid or chlorine dioxide solutions.

[0041] More specifically, ready-to-use formulations that directly generate chlorine oxides from solid precursors (API-P) can be produced at the point of use in a multi-compartment device or container. Such a multi-compartment device or container can be used to prepare, dispense, and stably store prepared compositions as described herein. Specifically, such a multi-compartment container described herein can have multiple compartments or chambers, each containing the components required to produce the compositions of the invention. For example, a solid precursor of oxidized chlorine and an activator (e.g., acetic acid or a salt thereof), a thickener, and a dye are mixed, and the composition then produces the desired disinfectant formulation at the desired time and location of use.

[0042] Acetic acid is an abundant natural compound found in various mammalian tissues. It is also a byproduct of bacterial fermentation of carbohydrates.

[0043] Sodium acetate is non-toxic and permitted for use in pharmaceutical preparations for oral and parenteral administration. The bactericidal properties of acetic acid are well-known. It is effective against bacteria such as Proteus vulgaris (…). P. vulgaris ), Pseudomonas aeruginosa ( P. aeruginosa ) and Acinetobacter baumannii ( A. Baumannii Acetic acid has a proven effectiveness against problematic Gram-negative bacteria such as [list of bacteria]. Acetic acid has a broad microbial spectrum, even when tested at low concentrations of 0.5–3%. Acetic acid concentrations effective in eradicating pre-formed biofilms range from 0.10% to 2.5%. Therefore, acetic acid and its metal salts are very attractive compounds for use in antimicrobial agents because they can act as buffers to stabilize pH, together with their metal salts.

[0044] In addition to its antimicrobial properties, acetic acid is attractive due to its inability to be further oxidized by oxidants such as OC and its endogenous presence in high concentrations in living tissues.

[0045] Therefore, multi-compartment containers realize the practical application of mixing the required components to generate an active solution of API on the spot at the point of use. It should be noted that, in order to ensure the ionic strength or molar osmolality of the final antimicrobial solution is adapted to the molar osmolality in the area of ​​use in medical applications, a pre-calculated amount of sodium chloride may be included in the multi-compartment device, depending on the intended use.

[0046] A preferred embodiment of the present invention is an inhaled formulation for respiratory administration. Therefore, nebulizers or inhalers that convert liquids into aerosols, commonly used to treat cystic fibrosis, asthma, COPD, and other respiratory diseases or conditions, are useful in this invention. The device for inhalation administration can use compressed air or ultrasonic energy to nebulize the formulation of the present invention. Any type of metered-dose inhaler (pMDI), dry powder inhaler (DPI), or slow mist inhaler (SMI) can also be used. Any electrostatic or non-electrostatic inhaler (e.g., VORTEX, Pari, or Sympotec) can also be used to implement the present invention.

[0047] The pre-loaded multi-compartment containers described herein produce stable, broad-spectrum antimicrobial solutions when the components are mixed, leaving only biocompatible, inactive chemicals in nature.

[0048] As described above, the activation of the API of the present invention is achieved using an activator such as acetic acid, which works synergistically with chlorine oxide to combat microorganisms and also maintains the acidity within a pH range of 4-8. The method and formulation of the present invention avoid the inherent drawback of insufficient long-term stability of chlorine oxide OC in solution because it eliminates the need to store the disinfectant composition as an aqueous solution.

[0049] Another advantage of this invention is the option to add other compounds that facilitate application. For example, in wound healing applications, it is necessary to increase the viscosity (µ) of the product on the skin to prolong contact time. This invention solves this problem by using a water-soluble or soluble thickener (VE) that is chemically non-oxidizable by the API, thereby improving the regulation of the API's contact time and persistence in the target treatment area. VE ensures that the rheology and flowability are suitable for the corresponding sterilization method and area to produce a solution or gel with full flowability. VE may, for example, contain water-soluble gelling agents such as polyacrylic acid, polyethylene glycol, or any other oligomers or polymers that cannot be oxidized by the API.

[0050] Furthermore, the compositions of the present invention may comprise one or more dyes, preferably selected from the group consisting of reduction-oxidation dyes (also referred to herein as "RODs"), wherein the color and intensity depend on the oxidation state of chlorine oxide. It should be noted that, in addition to providing a visual indication of the oxidation state of the chlorine atom (i.e., provided by color), the ROD also provides its own antimicrobial effect. This enhances the synergistic effect between the various components in the formulation in a novel way. The ROD is able to maintain its color for a sufficiently long time to monitor the oxidative activity of the API (chlorine oxide) and also provides a visual indication of the area where the formulation has been applied.

[0051] Other advantages and features of the invention will become apparent from the description of the invention provided below.

[0052] In a preferred embodiment, the chlorine oxide (OC) has the following general formula: Where M is any alkali metal, alkaline earth metal, or transition metal ion, n is an integer from 1 to 5, and x is an integer from 1 to 4.

[0053] If M = Na, n = 1, x = 1, then API-P is solid NaOCl. If M = Ca, n = 2, x = 1, then API-P is solid Ca(OCl)2. If M = Na, n = 1, x = 2, then API-P is solid NaClO2. If M = Ca, n = 2, x = 2, then API-P is solid Ca(ClO2)2. In the case of x = 3 or 4, API-P produces more reactive chlorate and perchlorate substances.

[0054] A non-restrictive example is Figure 1 Hypochlorous acid is generated instantaneously from sodium hypochlorite or calcium hypochlorite in cap 2, and a sodium acetate buffer solution in compartment 4 provides a ready-to-use solution of API hypochlorous acid with a pH between 5 and 6 in compartment 9, optionally with color and thickener.

[0055] Another non-limiting example is calcium dichlorate (Ca(OCl)2), a stable, water-soluble API-P of HOCl. It dissolves instantaneously in water, leaving only naturally occurring calcium hydroxide and producing HOCl, one of the two active ingredients of this invention, which degrades into Cl. - And biocompatible substances containing hydrogen and oxygen.

[0056] Another preferred embodiment of the invention provides a solid precursor of chlorine oxide, which is tetrachlorodecoxide (TCDO), CAS number 92047-76-2, a stable solution referred to as WF10 or OXO-K993, prepared according to the method described by Meuer et al. in CA2616008, which is incorporated herein by reference. It can be prepared by reacting chlorite ions (ClO2) in water... - It is prepared by combining alkali metal or alkaline earth metal salts with excess oxygen.

[0057] Therefore, one advantage of the present invention is that the precursor API-P in dry and anhydrous solid form does not have drug stability issues, thus the present invention solves one of the main technical problems in the prior art.

[0058] One aspect of the invention is a combination of API-P with a molecule comprising a carboxylic acid functional group -COOH, a sulfonic acid functional group -SO3H, a phosphate functional group -PO3H, or a boric acid functional group -B(OH)2, each of these components acting as an activator of API-P in the formulation. Typically, the activator has the general formula R1XO. n (R2,) m In this formula, group R1 can be a group containing about 1 to about 10 hydride carbon atoms, optionally substituted with an amino, amide, carboxyl, or hydroxyl group. Group X can be a carbon, phosphorus, or sulfur atom, n and m are 2 or 3, and R2 is a proton (H) or any alkali metal, alkaline earth metal, or transition metal ion. The nature of the substituents varies depending on the application and the type of chlorine, and can be any compound containing an amino group (e.g., ammonia), an amino acid (e.g., taurine), or a therapeutic agent that enhances the synergistic potential of the formulation. The activator can be of the general formula R1XO. n R2 defines any combination or mixture of two or more compounds.

[0059] A preferred, non-limiting example is carboxylic acid R3COOH, wherein R3 is H or a straight-chain or branched saturated or unsaturated hydrocarbon chain having about 1 to about 24 carbon atoms, optionally substituted with a hydroxyl group. Non-limiting examples of the activator may be acetic acid, citric acid, tartaric acid, lactic acid, hippuric acid, maleic acid, boric acid, sulfuric acid, phosphoric acid, boric acid, 3-(N-morpholino)propanesulfonic acid (MOPS), 2-(carbamoylmethylamino)ethanesulfonic acid (ACES), 2-(carbamoylmethylamino)ethanesulfonic acid (ADA), 2-(carbamoylmethylamino)ethanesulfonic acid (diglycine), piperazine-N,N'-bis(2-ethanesulfonic acid) (PIPES), or any amino acid.

[0060] Taurine is particularly preferred because it is an endogenous amino acid that usually modulates the function of OC in the body, and it can combine with OC to form endogenous N-chloro-amino acids, such as ClNH-CH2CH2-SO3H, which itself has antibacterial properties.

[0061] Acetic acid is preferred because it is endogenous in the human body, has antibacterial properties, has very low toxicity, and forms a buffer when mixed with its metal salt, and is used as a non-limiting example in further description of the invention.

[0062] One advantage of this invention is that the solid multicomponent product of this invention is not affected by stability issues in pharmaceutical or medical device environments, regardless of temperature, air, humidity, light, oxygen or other environmental conditions, because API-P is a solid and is readily available from the market.

[0063] The API-P disclosed herein can be water-soluble and reaches physiological pH and ionic strength almost instantly in the final solution in combination with acetic acid and / or its salts.

[0064] The ability to generate antimicrobial APIs in situ instantly enhances the product's ease of use and versatility. Furthermore, the components can be packaged separately and combined as needed, further impacting storage stability and field use.

[0065] This invention considers small, stable, single-use two- or three-part devices ideal for travel, disaster response, military personnel, or microbial outbreaks. Furthermore, larger-scale (e.g., tank) designs incorporating active antimicrobial precursors (sometimes referred to herein as API-P) are suitable for agricultural environments, aquaculture, or military operations, and are well-suited for disinfecting larger areas.

[0066] Thickening agents used in the preparation of viscous solutions and gels In some embodiments of the invention, components other than the API may be included. For example, a thickener is preferred for wound healing or skin disinfection. A preferred thickener is a water-soluble gelling agent that does not oxidize the API. The gelling agent provides extended API durability in the area of ​​interest, such as the skin.

[0067] Examples of the gelling agents of the present invention include, but are not limited to, polyacrylic acid (CARBOMER), polyethylene glycol or any other oligomers, polymers or block copolymers thereof. Furthermore, the tackifier may be selected from poly(acrylic acid)-acrylamide alkyl propane sulfonate copolymers, phospho-based polycarboxylic acids, and poly(acrylic acid)-acrylamide alkyl propane and sulfonate-sulfonated styrene terpolymers.

[0068] Polymers such as acrylate copolymers perform well in the formulations of the present invention at concentrations ranging from about 0.01% to about 5%. Acrylate copolymers are homopolymers and copolymers crosslinked with acrylic acid and polyalkenyl polyether. Acrylate copolymers exist at various grafting densities. An exemplary crosslinking agent is pentaerythritol, which is very stable. Polyacrylic acid (PAA) polymers known to stabilize H2O2 formulations can be used in the present invention.

[0069] According to the present invention, polymer-stabilized OC solutions can be applied to a wide range of applications, such as wound treatment, aseptic packaging, electronics manufacturing, and pulp and paper bleaching. APIs can be formulated into gels or viscous fluids that can be applied to inanimate target surfaces or target surfaces representing infected epithelial mucosa or skin to ensure prolonged close contact with the necessary levels of API. Non-viscous API formulations can also be dispersed as a mist into the air of confined spaces for environmental disinfection or for inhalation purposes to treat respiratory illnesses. For example, polyacrylic acid carbomer exhibits increased viscosity at concentrations of 0.01-0.1%. If desired, it forms a conventional gel in the concentration range of 0.1-1%.

[0070] Antimicrobial redox-sensitive antimicrobial dyes as indicators Another additive in the formulation of the present invention is a reduction-oxidation dye (hereinafter referred to as ROD), wherein the color and intensity of the dye depend on the oxidation state of the OC. More advantageously, the ROD itself has antimicrobial effects, thereby enhancing the antimicrobial synergy between the components of the formulation described herein. If the standard half-cell potential of the ROD has a lower positive value than that of the OC, then the color of the formulation will be maintained as long as the OC is active. Thus, the color provides a visual indication in areas where the formulation has been applied and where active OC is present. This is particularly advantageous, for example, when the formulation of the present invention is used to treat mastitis and in cases where a large number of cattle require treatment for mastitis; the coloring formulation of the present invention visually indicates which animals have been treated. Furthermore, it is also useful to use an indicator of the opposite type, in which case the color appears when the oxidizing power of the OC disappears.

[0071] Non-limiting examples of suitable dyes that can be used in this invention are pH-independent dyes that are visible in the presence of OC. Preferred examples are N-phenyl-o-aminobenzoic acid (purple-red), N-ethoxychrysoidine (cyan), o-anisidine (red), sodium diphenylamine sulfonate (red-purple), diphenylbenzidine (purple), diphenylamine (purple), and viologen, which is colorless in the presence of OC but deep blue in the absence of OC.

[0072] Examples of pH-dependent dyes that are deep blue in the presence of reactive OC but colorless in the absence of OC include sodium 2,6-dibromophenol-indophenol or sodium 2,6-dichlorophenol-indophenol, sodium o-cresol-indophenol, thione (also known as Laureth violet), methylene blue, gentian violet, indigo tetrasulfonic acid, indigo carmine (also known as indigo disulfonic acid), and indigo monosulfonic acid. Examples of dyes that are red or reddish-purple in the presence of OC include phenol saffron, saffron T, neutral red, and dialkyl-p-phenylenediamine (SPD, reddish-purple).

[0073] Many of these dyes have antibacterial effects on their own (i.e., methylene blue (MB) and gentian violet (GV)), and combinations of them have been used with polymers such as polyvinyl alcohol or polyurethane as antibacterial dyes in wound dressing foams, for example, as described by Edwards in Wound Care (2016), 5, pp 11-19.

[0074] One particularly useful class of dyes that can be used in this invention are microbial phenazines, which are coloring, redox-active nitrogen-containing aromatic compounds with metabolic, ecological and evolutionary significance.

[0075] Another class of phenazines includes bis-N-oxide phenazines, which have stronger antimicrobial properties than their parent phenazines. Most of these compounds are naturally occurring compounds produced by bacteria and are heteroaromatic N-oxide compounds, referred to below as HANOX. In addition to being redox dyes (RODs), HANOX compounds are also useful in this invention because their color depends on the oxidation state of the OC.

[0076] In addition, there are certain phenazine derivatives. In particular, they exhibit high activity against a variety of bacteria, yeasts, and fungi, such as *Streptococcus agalactiae* (…). Streptococcus agalactiae Staphylococcus aureus Staphylococcus aureus ), Escherichia coli ( Escherichia coli ), Corynebacterium pyogenes ( Corynebacterium pyogenes ), Moraxella tamariscina ( Moraxella bovis ), Pseudomonas aeruginosa ( Pseudomonas aeruginosa Candida albicans and Microsporum canis Microsporum canis Therefore, phenazine derivatives are particularly useful in agriculture for treating animal diseases caused by microorganisms.

[0077] A surprising finding when using these derivatives is that they do not cause damage to tissues under the conditions of use, which makes them particularly suitable for topical application, preferably in amounts of 0.05% to 1.0% of the composition by weight.

[0078] They have particular value in localized applications, such as in solid or gel formulations (including finely divided powder and particulate materials) and in liquid formulations (including solutions, suspensions, concentrates, tinctures, slurries and aerosols, creams, gels, gels, ointments and pastes).

[0079] Methylene blue is another particularly preferred dye for use in this invention because the FDA has approved it as an excipient in pharmaceutical formulations, and it has antibacterial properties, and its efficacy as a therapeutic agent can be enhanced using photodynamic therapy.

[0080] Multi-compartment device for the present invention Figure 1 This is a schematic diagram of an exemplary multi-compartment device for instant formulation generation according to the present invention.

[0081] The design of the device (including the number of compartments) can be adapted to usage specifications. Device 8 includes a screw cap 1 associated with a main compartment containing a solid precursor of API in dry form, designated API-P (2). By rotating the screw cap 1 in one direction, the screw cap 1 can open a seal or port 3, allowing API-P to enter a second compartment 4, which contains an aqueous solution of an activator and a pre-calculated amount of sodium chloride to make the final molar osmotic weight concentration of the solution is isotonic with body fluids. To obtain the desired final pH, optionally, an aqueous solution of the activator and optionally a pre-calculated amount of its metal or amino acid salt can be pre-filled into compartments 4, 5, or 10. Smaller particles in compartment 4 indicate that API-P dissolves rapidly in the activator solution, thereby generating API. A third compartment 5 is optional and is used to contain a solution of a redox dye (ROD), depending on the technical application of the respective device. Compartments 4 and 5 are separated by a wall 6. Compartments 4 and 5 are also separated from compartment 10 by a fragile septum or wall 7. Optionally, a fourth compartment at the same height as compartments 4 and 5 may contain amino acids, such as essential or non-essential amino acids for API stabilization or taurine. For simplicity, in this illustration, the fourth compartment 10 may optionally be pure water or an activator solution. This ready-to-use disinfectant solution is released from the device when the screw cap 2 is turned in the opposite direction and can be applied to the area of ​​concern for disinfection. One aspect of the invention is the solid precursor API-P in the screw cap 2, which is a chlorine oxidizing substance. The solution obtained from the multi-compartment device can ultimately be used in aqueous solution to produce a thickener (VE) solution. Any multi-compartment device that allows mixing of precursor components and additives can be used in this invention, including bottles, bags, syringes, inhalers, hand sanitizers, spray bottles, flasks, or canisters. As described above, devices that can be easily activated at the bedside or in the field without complex mixing procedures and can be stored at ambient temperature are preferred.

[0082] The multi-compartment device of the present invention is a closed system and can be designed to eliminate mixing errors to avoid undue contact with patients and staff, and complies with the Joint Commission and USO 797 ​​guidelines.

[0083] Non-limiting examples of designs that can be used in this invention include B Braun's Duplex Container, CredenceCompanion safety syringe system, Dual-Mix multi-compartment bag, or Easyrec kit including a screw cap that releases a solid or solid mixture to mix into one or more fluid phases, thereby producing a ready-to-use formulation of an API.

[0084] The use of this invention for antibacterial purposes in photodynamic therapy. It has been demonstrated that the use of antimicrobial photodynamic therapy (aPDT) to eliminate bacteria is an alternative treatment method in the treatment of peri-implantitis. Therefore, another preferred embodiment of the formulation of the present invention, comprising OC, acetic acid or a salt thereof, and optionally a thickener, is to include ROD (e.g., methylene blue) for use in photodynamic therapy, such as for improving wound healing or bacterial infection in mammals. In this case, light having a wavelength suitable for producing the photodynamic effect of the dye can be used to irradiate the application site of the product of the present invention.

[0085] In Photodiagnosis Photodyn. Ther. (2018), 23, pp 347-352, Souza et al. demonstrated the antimicrobial activity of hypochlorite solution and reciprocating instruments combined with photodynamic therapy on root canals infected with Enterococcus faecalis using photodynamic therapy. However, the test solution did not contain an antimicrobial dye. These techniques are incorporated herein by reference.

[0086] Step-by-step method for API generation This invention provides a composition of a solid precursor API-P of a chloride substance combined with an activator (e.g., acetic acid or a salt thereof) and a method of using the same. An exemplary method includes the following six steps: 1. Obtain a pre-calculated quantity API-P, which has the following M... n+ [Cl (O) x ] n n- The general formula is represented, where M can be any alkali metal, alkaline earth metal, or transition metal ion, where n is an integer from 1 to 5, x is an integer from 1 to 4, and y is an integer from 1 to 2. Solid (API-P) is produced in the final solution in the form of OC at a concentration in the range of 0.01-1000 ppm, preferably in the range of 0.1-100 ppm. The resulting solution is then loaded into compartment 1 of a multi-compartment apparatus. API-P is mixed with a pre-calculated amount of sodium chloride to produce a final molar osmotic pressure weight concentration in the range of 0.1-500 mOsm, and optionally any other stable solid.

[0087] 2. Obtain a pre-calculated amount of activator having the general formula R1XO. n (R2,) m The activator is preferably acetic acid, optionally a mixture thereof with its metal salt or ammonium salt. The activator is dissolved in a pharmaceutically acceptable diluent, adjuvant, or carrier to produce an activator concentration in the range of 0.05-10%, preferably 0.08-0.5%, more preferably 0.10-0.2%. If API-P is not premixed with sodium chloride, the solution in step 2 may contain a certain amount of sodium chloride, and in either case, produce a final molar osmotic pressure weight concentration in the range of 0.1-500 mOsm, preferably about 300 mOsm, which corresponds to 150 mM sodium chloride. One portion of the solution is loaded into the second compartment of the multi-compartment device.

[0088] 3. To produce the main product of the invention, compartments 1 and 2 are mixed by opening the port or breaking the sealing diaphragm between the first and second compartments to mix the contents of the compartments, followed by environmental squeezing or shaking to produce a disinfectant solution. The resulting solution can be removed through a cap on the multi-compartment device before use. This solution is isotonic, has a pH value in the range of 4 to 9, preferably in the range of 5 to 6, and is generally used for antimicrobial purposes, such as for inhalation therapy using devices such as asthma inhalers or nebulizers to combat viral infections in the upper respiratory tract of mammals.

[0089] 4. For applications where the use of a color indicator in step 4 can provide information or indicate the oxidative activity of the API during treatment (e.g., in the treatment of mastitis), the dye, whose color changes with the oxidation state of the API (ROD), is optionally loaded into an optional compartment of the multi-compartment device in a pre-calculated amount capable of producing a dye concentration in the range of 0.01-1000 ppm.

[0090] 5. Optionally, according to the intended use, an amino acid (preferably taurine at the same concentration as the API) as a stabilizer is loaded into an optional compartment of the multi-compartment device in a pre-calculated amount. Step 4 is then performed to reduce oxidative stress on the biological surface.

[0091] 6. Depending on the intended use, a certain amount of a water-soluble thickener (VE) that cannot be oxidized by API, in the concentration range of 0.01-25%, preferably in the range of 0.1-10%, and even more preferably in the range of 0.2-1%, is mixed with the solution produced in a selected order of steps 1-3 (optionally combined with any combination of steps 4-5). A VE concentration of 0.01-0.1% produces a viscous but fluid solution, while a VE concentration of 0.3-1% produces a gel. For skin or wound applications, a third compartment containing VE may be included in step 3 during the mixing process to produce a viscous or gel-like product.

[0092] Applications of this invention in agriculture In agriculture, particularly on animal farms, a variety of infectious diseases caused by bacteria, viruses, and fungi affect the daily operations of farms and impact facility operating costs. In these situations, the formulations designed in this invention have therapeutic or preventative effects, and are particularly suitable for skin infections.

[0093] A significant example is bovine mastitis, which costs the U.S. dairy industry approximately $1.7-2 billion annually. Effective and environmentally friendly treatment for mastitis has proven difficult because milk from cows treated with long-term antibiotics cannot be sold until residual drugs have left their bodies. There is no effective vaccine because the infection in the udder and teats is far from the animal's main bloodstream. To identify treated cows, dairy plant workers use tape to provide warnings and label the treated cows.

[0094] Therefore, a preferred aspect of the invention is the treatment of mastitis using a gel or viscous solution comprising OC, acetic acid or a salt thereof, a thickener VE, and ROD (e.g., methylene blue). The colored gel is held in the area of ​​the breast and nipple, allowing the acetic acid to penetrate the skin of the nipple, and the color eliminates the need for adhesive strips. Furthermore, the applied gel can be irradiated with light of an appropriate wavelength to enhance its therapeutic effect. In this case, steps 1-4 and step 6 are performed to produce a formulation ready for immediate use.

[0095] Applications of this invention in aquaculture Water quality is a prerequisite for the successful aquaculture of aquatic animals such as fish, oysters, shrimp, and prawns. Open water systems often introduce organisms such as viruses, bacteria, lice, protozoa, fungal pathogens, algae, and parasites. Common viral infections that lead to high mortality rates in aquatic species impacting food production include koi herpesvirus disease, pancreatic disease (PD), and infectious salmon anemia (ISA). Often, inadequate water quality or a sufficient quantity of purified water is lacking. Current aquaculture facilities typically lack the means to prevent these infectious species from approaching and affecting farmed species. Furthermore, once infected, there are no effective cures available for these diseases.

[0096] A preferred embodiment of the chlorine dioxide OC of the present invention is an effective treatment for all these infected and harmful organisms and cells. Immediate formulations of OC are highly effective in controlling these waterborne pathogens. For example, chlorine dioxide is a broad-spectrum biocide that effectively addresses identified problems in the prior art. The formulations of the present invention can even be used in dedicated tanks for repeated treatment of farmed salmon, etc., without harming the gills or any other part of the farmed species, while simultaneously having a destructive effect on disease-causing microorganisms. In these applications, the preparation sequence used is to load API-P (NaOClO2 or Ca(OClO2)2) into compartment 1 and mix it with a pre-calculated amount of acetic acid in steps 1-3.

[0097] Antiviral applications of the present invention The methods disclosed herein are used to treat contaminated surfaces, equipment (e.g., medical devices), furniture surfaces, door handles, devices, clothing, or people. The formulations of this invention can be applied in the form of gels, aqueous solutions, or by atomizing or evaporating the API onto surfaces or confined spaces. The on-demand formulation feature allows for the treatment of areas with highly effective antimicrobial agents without concerns about storage degradation.

[0098] The method of this invention considers dispersing active agents into crevices and microenvironments, and even onto individuals suspected of being contaminated with infectious tissues or bodily fluids. The evaporation of these formulations can produce beneficial therapeutic or preventative effects against drug-resistant viral, bacterial, or fungal infections.

[0099] The formulations of the present invention pose no substantial toxic risk upon administration. A preferred embodiment is the treatment of viral infections of the upper respiratory tract. Therefore, the systems and methods of the present invention provide chlorine oxide OC as a means of treating respiratory viral infections. The compositions of the present invention can be used to treat SARS, MERS, and other infections, including but not limited to SARS CoV-2 infection. This treatment is now made easier for the first time by combining an immediate precursor of the API with the multi-compartment device of the present invention, as it eliminates the need to assess whether the solution stored under ambient conditions is insufficiently active.

[0100] In particular, inhalable hypochlorous acid formulations of OC, activators (e.g., acetic acid), excipients that adjust the rheology of the final solution, and osmotic pressure regulators (e.g., sodium chloride) can be used. This immediate formulation can now be prepared on-site and delivered using a nebulizer (e.g., a soft mist inhaler, a jet nebulizer, an ultrasonic nebulizer, and a vibrating mesh nebulizer). In use, the inhaler and nebulizer atomize the composition of the present invention for delivery by inhalation.

[0101] Formulations for nebulization can be provided in dry powder, solution, or suspension form. Fine droplets, sprays, and aerosols can be delivered via intranasal or intrapulmonary pump dispensers or squeeze bottles. The composition can also be inhaled via inhalers (e.g., metered-dose inhalers or dry powder inhalers). The composition can also be inhaled via nebulizers (e.g., ultrasonic nebulizers), thereby delivering the composition of OC and acetic acid directly to the respiratory tract via an inhalable formulation. This enables the prevention and treatment of respiratory infections caused by viruses and other microorganisms. According to the present invention, the formulations described herein are safe and effective for the prevention and treatment of viral infections.

[0102] The compositions of the present invention may also contain a pharmaceutically acceptable carrier (e.g., a diluent) to facilitate delivery to the respiratory 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 non-toxic and pharmaceutically acceptable carriers are known to those skilled in the art. Various carriers may be particularly suitable for different formulations of the composition, for example, whether used as drops or as a spray, suspension, or other form for pulmonary delivery.

[0103] Inhalation formulations can be provided in dry powder, solution, or suspension form. The composition can be delivered using various devices known in the art for administering drops, microdroplets, and sprays. The composition can be delivered using a dropper, pipette, or dispenser. Microdroplets, sprays, and aerosols can be delivered using intranasal or intrapulmonary pump dispensers or squeeze bottles.

[0104] Intranasal administration can be delivered via a nasal spray device. Therefore, the formulation of the present invention can be designed as a nasal spray. This nasal spray is sprayed into the nose and delivered to the respiratory tract.

[0105] Soft mist inhalers use mechanical energy stored in a spring, driven by the user, to pressurize a liquid container, causing the liquid to be expelled from the nozzle and inhaled as a soft mist. Soft mist inhalers do not rely on gaseous propellants or electricity to operate. The average droplet size in a soft mist inhaler is approximately 5.8 micrometers.

[0106] Jet nebulizers are the most common type and can be referred to simply as nebulizers. Jet nebulizers use compressed gas (such as air or oxygen) to atomize liquid medication as it is released at high speed from it. The resulting atomized droplets of treatment solution or suspension are then inhaled by the user for treatment. The compressed gas can be pre-compressed in a storage container or, as needed, by a compressor within the nebulizer.

[0107] Ultrasonic nebulizers rely on an electronic oscillator to generate high-frequency ultrasound waves, which atomize the medication as they are guided through a reservoir of a therapeutic solution suspension for inhalation.

[0108] Vibrating mesh nebulizers utilize the vibration of a membrane with thousands of holes at the top of a reservoir to atomize fine droplets for inhalation. Vibrating mesh nebulizers avoid some of the drawbacks of ultrasonic nebulizers, thus providing more efficient nebulization, reducing treatment time, and minimizing the need for heating the atomized liquid.

[0109] Treatment of viral infections is achieved using a synergistic combination of acetic acid and hypochlorous acid. The acetic acid component is particularly effective at penetrating tissues, while hypochlorous acid is particularly effective at treating infections on the outer surfaces of tissues. As described above, these combinations are effective for treating and preventing respiratory infections.

[0110] The disclosed composition is particularly effective because balancing the concentrations of hypochlorous acid and acetic acid with sodium chloride allows for safe treatment of viruses. Precise balancing depends on the formulation, the treatment site, and even the desired surface penetration. Hypochlorous acid can be present in amounts from about 5 ppm to about 1000 ppm or higher. Different applications, different delivery methods, and tissue types may require higher or lower concentrations. Acetic acid can be present in concentrations from about 0.1% to about 5.0% or higher, preferably about 1.0%. By balancing these two components, the composition can have a dual effect of treating both the surface and subsurface of the tissue to which it is applied.

[0111] When the OC is hypochlorous acid (HOCl), an immediate-use composition with an OC concentration of approximately 15-60 ppm is usually sufficient to treat infected lungs. When the OC is chlorine dioxide (OCl2), a concentration of 0.1-5 ppm is usually adequate.

[0112] In some cases, to completely destroy the virus or prevent it from entering the respiratory tract, the composition should be in contact with the virus for a longer period of time, from seconds to minutes, to an hour or longer. Therefore, in some embodiments, the composition is in gel form, which allows for longer contact with the site of infection.

[0113] Combining the composition with known antiviral treatments can enhance its efficacy. In some embodiments, the method of the present invention further includes (simultaneously or sequentially with the composition of the present invention) administering one or more doses of an antiviral substance. These antiviral substances may include, but are not limited to, acyclovir, adefovir, amantadine, boprevir, brivudine, cidofovir, emtricitabine, entecavir, famciclovir, fomivir, phosphonoformate (sodium), ganciclovir, lamivudine, penciclovir, telbivudine, tenofovir, valacyclovir, valganciclovir, vidarabine, M2 inhibitors, neuraminidase inhibitors, interferon, ribavirin, nucleoside reverse transcriptase inhibitors, non-nucleoside reverse transcriptase inhibitors, non-structural protein 5a (ns5a) inhibitors, chemokine receptor antagonists, integrase strand transfer inhibitors, protease inhibitors, and purine nucleosides.

[0114] The compositions of the present invention can also be used in combination with known antimicrobial treatments. In some embodiments, the method of the present invention further includes (simultaneously or sequentially with the compositions of the present invention) administering one or more doses of an antibiotic substance, including but not limited to ciprofloxacin, β-lactam antibiotics (e.g., ampicillin or carbapenem), azithromycin, cephalosporins, doxycycline, fusidic acid, gentamicin, linezolid, levofloxacin, norfloxacin, ofloxacin, rifampin, tetracycline, tobramycin, vancomycin, amikacin, ceftazidime, cefepime, trimethoprim / sulfamethoxazole. Azoxystrobin, piperacillin / tazobactam, aztreanam, meropenem, colistin, or chloramphenicol.

[0115] In some embodiments, the method of the present invention further includes administering one or more doses of an antibiotic substance derived from antibiotic classes, including but not limited to aminoglycosides, carbapenems, first-generation cephalosporins, second-generation cephalosporins, third-generation cephalosporins, fourth-generation cephalosporins, glycopeptides, macrolides, monobactams, penicillins, polypeptides, quinolones, sulfonamides, tetracyclines, lincosamides, and... Azoxyl ketones. In some embodiments, the method of the present invention includes the administration of non-antibiotic antimicrobial substances, including but not limited to sertraline, racemic and stereoisomer forms of thiopyridazine, benzoyl peroxide, taurolidine, and hexitidine.

[0116] The administration regimen of the composition may include the amount of composition, frequency, and duration of exposure. The administration regimen may depend on the severity of the infection or on a protocol developed for the treatment or prevention of a viral infection.

[0117] The composition can be administered in a single daily dose or in multiple daily doses (e.g., 2, 3, 4 or more daily). A person receiving the composition may be exposed to it for hours or minutes. The duration of exposure may depend on the frequency, amount, and even severity of the infection.

[0118] Depending on the nature of the OC, the total daily dose of API formed from the solid precursor in an immediate solution can range from 0.01 to 1000 mg. Actual dosage can vary depending on the specific delivery composition, method of administration, and other factors known in the art.

[0119] The composition can be applied to any location in the respiratory tract, such as the respiratory epithelium, nasal cavity, nasal epithelium, pharynx, esophagus, larynx, epiglottis, trachea, carina, bronchi, bronchioles, or lungs. Using this composition for respiratory treatment can prevent any virally transmitted disease or disorder.

[0120] In some other embodiments, for example, the compositions of the present invention can be used to sterilize entire rooms, facilities, medical devices, and surgical instruments. Medical devices are typically initially sterile when provided, but may require additional or subsequent cleaning and disinfection or sterilization. In particular, it is especially important to sterilize or disinfect reusable medical devices using any known technique before reuse. The compositions can be applied to medical devices. For example, the compositions can be applied by wiping or spreading the composition onto the surface of the device, by spraying the composition onto the device in the form of an aerosol or mist, by immersing the device in a container containing a volume of the composition, or by placing the device into a flow of the composition (e.g., from a valve). Alternatively, medical devices and surgical instruments can be stored immersed in the composition and removed when needed.

[0121] Some disclosed compositions contain 2% or more acetic acid and, when combined with OC (acetic acid compound), have been shown to be safe and effective for treating skin and other tissues. It has been found that the OC in these compositions modulates the acetic acid content. This allows the compositions to utilize the disinfecting properties of acetic acid without causing damage to tissues.

[0122] Use of this invention in the treatment of COVID-19 and other respiratory infectious diseases. As mentioned above, in one aspect, the present invention relates to a disinfectant composition for providing a safe and effective method for treating and preventing the spread of respiratory infections, including SARS-CoV-2.

[0123] Compositions for treating SARS infection include a hypochlorous acid-based nebulized broad-spectrum antiviral and antibacterial inhalation solution. More specifically, the formulation contains hypochlorous acid (HOCl) (25 ppm to 200 ppm) stabilized with acetic acid (approximately 0.25%), resulting in a sustained HOCl concentration with positive antimicrobial effects. The addition of acetic acid improves the stability of HOCl, thus enabling the development of therapeutic agents with extended shelf life. Furthermore, the composition is formulated with an increased pH (5.5) and isotonicity, thereby improving airway tolerance.

[0124] The compositions of the present invention possess unique antiviral properties (especially against enveloped viruses) and provide excellent antiviral activity. Therefore, for example, such compositions are particularly suitable for the treatment and prevention of COVID-19. More specifically, SARS-CoV-2 and many other viruses have surface proteins (i.e., spike proteins), which are known as “gatekeepers” of human cells in the respiratory system. These spike proteins contain -SH groups that are easily oxidized by HOCl. Even low concentrations of HOCl oxidize extracellular -SH groups (e.g., viral spike proteins) without harming normal tissues and intracellular enzymes. Therefore, the antiviral action of the compositions of the present invention can destroy viral particles in the respiratory tract at first exposure, during infection, and when the virion is located inside cells and subsequently released by human respiratory cells. Thus, the unique antiviral properties of the compositions of the present invention (especially against enveloped viruses) make them a powerful potential tool for ongoing efforts to prevent the spread of coronaviruses. Considering the pathogenicity of coronaviruses worldwide, such compositions could shorten the duration of illness and reduce the severity of symptoms in a broad population of patients infected with COVID-19, especially when there is an unprecedented need.

[0125] Table 1 (below) provides a list of the components of the composition of the present invention consisting of 25 ppm - 200 ppm HOCl + 0.25% acetic acid. Inert gas: Argon (100%) The sodium hypochlorite solution can be replaced with a product from another GMP manufacturer.

[0126] The active ingredient in the preferred composition of this invention is hypochlorous acid (HOCl). This active ingredient is derived from sodium hypochlorite, which is an aqueous solution produced by the reaction of gaseous Cl2 with water under alkaline pH conditions. 3% NaOCl is generated and added to the final IS to achieve a maximum HOCl concentration of 200 ppm (0.01% w / w). Other components of the composition include: sodium hydroxide, Ph. Eur. / USP-NF grade, 0.1M solution, added to the desired pH (5.5); acetic acid, Ph. Eur. / USP-NF grade, glacial acetic acid, 0.25%; sodium chloride, Ph. Eur. / USP-NF grade, added to obtain an isotonic formulation (303 mOsm); and purified water, purified by reverse osmosis and deionized by ion exchange or according to the Ph. Eur. / USP-NF monograph.

[0127] The preferred clinical dose of the composition is 5 mL of 25-100 ppm hypochlorous acid. The final product also contains 0.25% acetate buffer. Therefore, the solution contains more than 99.1% HOCl and less than 0.9% OCl. - HOCl is the active substance in IS, and it has been found that it acts as a disinfectant more effectively than an equivalent concentration of OCl. - It is 80 times more effective. Therefore, HOCl has a dual role in IS, acting as both an API and an antimicrobial agent to inhibit the growth of microorganisms in the final product. The composition can be stored in plastic PET vials / bottles. Before administration to the patient, the composition is transferred to the reservoir of the nebulizer / inhalation device. This transfer is performed in the clinic. After transfer to the nebulizer, the solution is given to the patient immediately (within 1-2 hours) via liquid aerosol delivery. The patient should receive 5 ml of the nebulized composition.

[0128] Compositions used for viral administration are typically administered as a single dose and delivered to the respiratory tract via nebulization, for example, using PARI BOY. The PARI BOY Classic nebulizer inhalation system comprises a PARI BOY Classic compressor and a PARILC SPRINT nebulizer. To obtain relevant deposition of the test solution in the upper and lower airways, the nebulizer is equipped with PARISMARTMASK. It should be noted that other nebulizers and inhalers can be used.

[0129] HOCl is produced by the body's own immune cells, namely neutrophils and monocytes / macrophages. It is a strong oxidizing agent capable of chlorinating and oxidizing molecular structures, especially those containing thiols, thiol ethers, and amino groups (such as proteins and fatty acids), leading to denaturation and loss of normal function in a variety of microorganisms. HOCl is considered by the FDA to be "the free available chlorine form with the highest bactericidal activity against a wide range of microorganisms." While HOCl is a strong oxidizing agent, it exhibits good tolerability and safety in wound care applications at low concentrations (≤0.1%).

[0130] By incorporating via reference Any and all references and citations in this disclosure to other documents such as patents, patent applications, patent announcements, journals, books, papers, online content, etc., are incorporated herein by reference in their entirety for all purposes.

[0131] Equivalent form This invention may be practiced in other specific forms without departing from its spirit or essential characteristics. Therefore, the foregoing embodiments are to be considered exemplary in all respects and not as limitations on the invention described herein.

[0132] Example Example 1: General procedure for preparing a dry, air-free mixture of API-P and sodium chloride solids for loading into a multi-compartment device.

[0133] 1a. A dry powder containing 50 ppm sodium hypochlorite in sodium chloride. Mix 50 mg of dry sodium hypochlorite (molecular weight: 74.44 g / mol) with 8.95 g of dry sodium chloride (molecular weight: 58.44 g / mol) to form a homogeneous powder mixture, and store the powder mixture in a light-proof container under airless and dry conditions. Pack one 90 mg portion of the powder into compartment 1 of the multi-compartment apparatus.

[0134] 1b. The production of dry powder containing 100 ppm sodium hypochlorite in sodium chloride. Mix 100 mg of dry sodium hypochlorite (molecular weight: 74.44 g / mol) with 8.90 g of dry sodium chloride (molecular weight: 58.44 g / mol) to form a homogeneous powder mixture, and store the powder mixture in a light-proof container under airless and dry conditions. Pack one 90 mg portion of the powder into compartment 1 of the multi-compartment apparatus.

[0135] 1c. The production of dry powder containing 200 ppm sodium hypochlorite in sodium chloride. Mix 200 mg of dry sodium hypochlorite (molecular weight: 74.44 g / mol) with 8.8 g of dry sodium chloride (molecular weight: 58.44 g / mol) to form a homogeneous powder mixture, and store the powder mixture in a light-proof container under airless and dry conditions. Pack one 90 mg portion of the powder into compartment 1 of the multi-compartment apparatus.

[0136] 1d. A dry powder containing 500 ppm sodium hypochlorite was produced in sodium chloride. Mix 500 mg of dry sodium hypochlorite (molecular weight: 74.44 g / mol) with 8.5 g of dry sodium chloride (molecular weight: 58.44 g / mol) to form a homogeneous powder mixture, and store the powder mixture in a light-proof container under airless and dry conditions. Pack one 90 mg portion of the powder into compartment 1 of the multi-compartment apparatus.

[0137] 1e. A dry powder containing 25 ppm calcium dichlorate in sodium chloride. Mix 25 mg of dry calcium hypochlorite (molecular weight: 142.98 g / mol) with 8.975 g of dry sodium chloride (molecular weight: 58.44 g / mol) to form a homogeneous powder mixture, and store the powder mixture in a light-proof container under air-free and dry conditions. A 90 mg portion of the powder is then packed into compartment 1 of a multi-compartment apparatus.

[0138] 1f. A dry powder containing 50 ppm calcium dichlorate in sodium chloride is produced. Mix 50 mg of dry calcium hypochlorite (molecular weight: 142.98 g / mol) with 8.975 g of dry sodium chloride (molecular weight: 58.44 g / mol) to form a homogeneous powder mixture, and store the powder mixture in a light-proof container under air-free and dry conditions. A 90 mg portion of the powder is then placed into compartment 1 of a multi-compartment apparatus.

[0139] 1g. A dry powder containing 100 ppm calcium dichlorate in sodium chloride. Mix 100 mg of dry calcium hypochlorite (molecular weight: 142.98 g / mol) with 8.9 g of dry sodium chloride (molecular weight: 58.44 g / mol) to form a homogeneous powder mixture, and store the powder mixture in a light-proof container under airless and dry conditions. Pack one 90 mg portion of the powder into compartment 1 of the multi-compartment apparatus.

[0140] 1 hour. A dry powder containing 100 ppm calcium dichlorate in sodium chloride is produced. Mix 100 mg of dry calcium hypochlorite (molecular weight: 142.98 g / mol) with 8.9 g of dry sodium chloride (molecular weight: 58.44 g / mol) to form a homogeneous powder mixture, and store the powder mixture in a light-proof container under airless and dry conditions. Pack one 90 mg portion of the powder into compartment 1 of the multi-compartment apparatus.

[0141] 1i. A dry powder containing 100 ppm calcium dichlorate in sodium chloride. Mix 100 mg of dry calcium hypochlorite (molecular weight: 142.98 g / mol) with 8.9 g of dry sodium chloride (molecular weight: 58.44 g / mol) to form a homogeneous powder mixture, and store the powder mixture in a light-proof container under airless and dry conditions. Pack one 90 mg portion of the powder into compartment 1 of the multi-compartment apparatus.

[0142] 1j. A dry powder produced containing 1 ppm sodium chlorite in sodium chloride. Mix 10 mg of dry sodium chlorite (molecular weight: 90.44 g / mol) with 89.99 g of dry sodium chloride (molecular weight: 58.44 g / mol) to form a homogeneous powder mixture, and store the powder mixture in a light-proof container under airless and dry conditions. Pack a 90 mg portion of the powder into compartment 1 of the multi-compartment apparatus.

[0143] 1k. A dry powder produced containing 5 ppm sodium chlorite in sodium chloride. Mix 50 mg of dry sodium chlorite (molecular weight: 90.44 g / mol) with 89.99 g of dry sodium chloride (molecular weight: 58.44 g / mol) to form a homogeneous powder mixture, and store the powder mixture in a light-proof container under airless and dry conditions. A 90 mg portion of the powder is then placed into compartment 1 of a multi-compartment apparatus.

[0144] 1. A dry powder containing 10 ppm calcium chlorite in sodium chloride. Mix 100 mg of dry calcium chlorite (molecular weight: 157.89 g / mol) with 89.99 g of dry sodium chloride (molecular weight: 58.44 g / mol) to form a homogeneous powder mixture, and store the powder mixture in a light-proof container under airless and dry conditions. A 90 mg portion of the powder is then placed into compartment 1 of a multi-compartment apparatus.

[0145] Example 2: General steps for preparing a 1-liter activator stock solution for loading small-volume samples into a multi-compartment device. 2a. Acetic acid activator stock solution (0.125%, pH 2.95) Dissolve 1.25 mL of acetic acid (molecular weight (mw): 60.05 g / mol) in 998.75 mL of sterile water.

[0146] 2b. Acetic acid activator stock solution (0.125%, pH 4.3) Dissolve 1.25 mL of acetic acid (molecular weight: 60.05 g / mol) in 998.75 mL of sterile water. Adjust the pH to 4.3 using 10N sodium hydroxide.

[0147] 2c. Acetic acid activator stock solution (0.25%, pH 4.3) Dissolve 2.5 mL of acetic acid (molecular weight: 60.05 g / mol) in 998.75 mL of sterile water. Adjust the pH to 4.3 using 10N sodium hydroxide.

[0148] 2d. Acetic acid activator stock solution (0.25%, pH 5.0) Dissolve 2.5 mL of acetic acid (molecular weight: 60.05 g / mol) in 998.75 mL of sterile water. Adjust the pH to 5.0 using 10N sodium hydroxide.

[0149] 2e. Acetic acid activator stock solution (1%, pH 4.3) Dissolve 10 mL of acetic acid (molecular weight: 60.05 g / mol) in 998.75 mL of sterile water. Adjust the pH to 4.3 using 10N sodium hydroxide.

[0150] 2f. Acetic acid activator stock solution (2%, pH 4.3) Dissolve 20 mL of acetic acid (molecular weight: 60.05 g / mol) in 998.75 mL of sterile water. Adjust the pH to 4.3 using 10N sodium hydroxide.

[0151] 2g. Acetic acid / sodium acetate activator stock solution (0.1 M, pH 5.0) Add 5.772 g of sodium acetate (molecular weight: 82 g / mol) and 1.778 g of acetic acid (molecular weight: 60.05 g / mol) to 800 ml of distilled water. Adjust the pH to 5.0 using 10N hydrochloric acid or 10N sodium hydroxide, and add distilled water until the volume is 1 liter.

[0152] 2h. Isotonic acetic acid activator stock solution (0.125%, pH 2.95) Add 1.25 mL of acetic acid (molecular weight: 60.05 g / mol) and 8.4 g of sodium chloride (molecular weight: 58.44 g / mol) to 998.75 mL of sterile water.

[0153] 2i. Stock solution of isotonic acetic acid activator (0.125%, pH 4.3) Add 1.25 mL of acetic acid (molecular weight: 60.05 g / mol) and 8.4 g of sodium chloride (molecular weight: 58.44 g / mol) to 998.75 mL of sterile water. Adjust the pH to 4.3 using 10N sodium hydroxide.

[0154] 2j. Stock solution of isotonic acetic acid activator (0.25%, pH 4.3) Add 2.5 mL of acetic acid (molecular weight: 60.05 g / mol) and 8.4 g of sodium chloride (molecular weight: 58.44 g / mol) to 998.75 mL of sterile water. Adjust the pH to 4.3 using 10N sodium hydroxide.

[0155] 2k. Isotonic acetic acid activator stock solution (0.125%, pH 5.0) Add 1.25 mL of acetic acid (molecular weight: 60.05 g / mol) and 8.4 g of sodium chloride (molecular weight: 58.44 g / mol) to 998.75 mL of sterile water. Adjust the pH to 5.0 using 10N sodium hydroxide.

[0156] 2l. Stock solution of isotonic acetic acid activator (0.25%, pH 5.0) Add 2.5 mL of acetic acid (molecular weight: 60.05 g / mol) and 8.4 g of sodium chloride (molecular weight: 58.44 g / mol) to 998.75 mL of sterile water. Adjust the pH to 5.0 using 10N sodium hydroxide.

[0157] 2m. Isotonic acetic acid / sodium acetate activator stock solution (0.1 M, pH 5.0) Add 5.772 g sodium acetate (molecular weight: 82 g / mol), 1.778 g acetic acid (molecular weight: 60.05 g / mol), and 8.4 g sodium chloride (molecular weight: 58.44 g / mol) to 800 mL of distilled water. Adjust the pH to 5.0 using 10N hydrochloric acid or 10N sodium hydroxide, and add distilled water until the volume is 1 liter.

[0158] 2n. Acetate buffer (0.1 M, pH 5.0) Add 5.772 g sodium acetate (molecular weight: 82 g / mol) and 1.778 g acetic acid (molecular weight: 60.05 g / mol) to 800 mL of sterile water. Adjust the pH to 5.0 using 10N hydrochloric acid and add distilled water until the volume is 1 liter.

[0159] 2o. ACES buffer (0.1 M, pH 6.7) Add 18.22 g of N-(2-acetamido)-2-aminoethanesulfonic acid (molecular weight: 182.2 g / mol) to 800 mL of sterile water. Adjust the pH to 6.7 using 10N sodium hydroxide and add distilled water until the volume is 1 liter.

[0160] 2p. Citric acid solution (0.1 M, pH 2.2) Dissolve 19.2 grams of citric acid (molecular weight: 192.1 g / mol) in 1 liter of sterile water.

[0161] 2q. Citrate buffer (0.1 M, pH 6.0) Add 12.044 g of sodium citrate (molecular weight: 294.1 g / mol) and 11.341 g of citric acid (molecular weight: 192.1 g / mol) to 800 mL of sterile water. Adjust the pH to 6.0 using 0.1 N sodium hydroxide and add distilled water until the volume is 1 liter.

[0162] 2r. ADA buffer (0.1 M, pH 6.6) Add 95.11 g of 2-[(2-amino-2-oxoethyl)-(carboxymethyl)amino]acetic acid (ADA, molecular weight: 190.22 g / mol) to 800 mL of sterile water. After adjusting the pH to 6.6 using 10 N sodium hydroxide, the ADA dissolves, and distilled water is added until the volume is 1 liter.

[0163] 2s. EBBS buffer containing phenol red dye (pH 7.0) Add the following to 800 mL of sterile water: 200 mg calcium chloride (molecular weight: 110.98 g / mol), 200 mg magnesium sulfate heptahydrate (molecular weight: 246.47 g / mol), 400 mg potassium chloride (molecular weight: 75 g / mol), 2.2 g sodium bicarbonate (molecular weight: 84.01 g / mol), 6.8 g sodium chloride (molecular weight: 58.44 g / mol), 140 mg NaH₂PO₄H₂O (molecular weight: 138 g / mol), 1 g dextrorotatory glucose (molecular weight: 180.16 g / mol), and 10 mg phenol red (molecular weight: 354.38 g / mol). Adjust the pH of the solution to 7.0 or another desired pH using hydrochloric acid or sodium hydroxide.

[0164] 2 tons of sterile isotonic oxygenated water Add 9 grams of sodium chloride to 1 liter of sterile water stock solution containing saturated oxygen and store in a light-protected, sealed bottle at room temperature.

[0165] Example 3: Preparation of a ready-to-use disinfectant formulation from a solid salt of chlorine oxide combined with the solution of Example 1.

[0166] Example 3.1: Non-limiting steps of a general procedure 1. A 90 mg sample of any powder obtained from Example 1 is packed into compartment 4 of the multi-compartment device.

[0167] 2. Fill a 10 ml portion of any activator solution obtained from Example 2 into compartment 4 of the multi-compartment device.

[0168] 3. In order to produce the main product of this invention, breaking or opening such as Figure 1 The seal, barrier, or port 3 shown, located between the screw cap and compartment 4, mixes the contents of compartment 1 with the solution in compartment 4, followed by gentle squeezing or shaking to produce a disinfectant solution. After removing the screw cap from the multi-compartment device, the resulting solution can be removed through the opening and is immediately usable. This isotonic solution has a pH value in the range of 4 to 9, preferably between 5 and 6, and is typically used for antibacterial purposes.

[0169] 4. Optionally, depending on the intended use, a water-soluble dye in solid form, the color of which varies with the oxidation state of API (ROD), is loaded into compartment 9 of the multi-compartment device in a pre-calculated amount capable of producing a dye concentration in the range of 0.01-1000 ppm, and the procedure in step 3, including the mixing of compartments 1, 4 and 9, is repeated.

[0170] 5. Optionally, depending on the intended use, an amino acid (preferably taurine with the same concentration as the API) acting as a stabilizer is loaded into compartment 5 of the multi-compartment device in a pre-calculated amount. The procedure in step 3, including the mixing of compartments 1, 4, and 5, is repeated.

[0171] 6. Optionally, depending on the intended use (e.g., for skin or wound application), a water-soluble thickener (VE) that cannot be oxidized by the API is loaded into compartment 5 of the multi-compartment device in a pre-calculated amount, in which a concentration of VE in the final solution within the range of 0.01-25%, is obtained. A VE concentration of 0.01-0.1% produces a viscous but fluid solution, while a VE concentration of 0.3-1% produces a gel. The dispersion of VE in solution obtained from steps 3, 4, and / or 5 is converted into a viscous solution or gel using a Silverson mixer or a Ystral mixer and applied to the skin or wound site. The stability of this viscous solution or gel is improved due to the slower molecular motion, and it can be packaged into a soft bag or bottle to protect the solution or gel from air and light for subsequent use.

[0172] Example 4: In vitro anti-biofilm effect of HOCl and acetic acid test solutions from Example 3.

[0173] Three different test solutions were generated from the multi-compartment apparatus. All three test solutions were generated from the multi-compartment apparatus as described in Example 3.1, with compartment 1 containing 90 mg of dry powder containing 200 ppm sodium hypochlorite in sodium chloride (Example 1c). Three 10 mL aliquots of acetic acid solution (0.125%, pH 4.3, Example 2b) were contained in compartment 4 of the three different multi-compartment apparatuses: Solution 1: (0.25%, pH 4.3, Example 2c), Solution 2: (1%, pH 4.3, Example 2e), Solution 3: (2%, pH 4.3, Example 2f).

[0174] Experimental setup Test organisms: Pseudomonas aeruginosa or Staphylococcus aureus (Staphylococcus aureus) wild type strain Biofilm type: 48-hour or 24-hour biofilms grown on a semi-permeable membrane placed on a solidified medium supplemented with 0.5% glucose. In the case of 48-hour biofilms, the membrane with biofilm was transferred to a new plate after 24 hours.

[0175] Initial live cell count: 5 × 10 9 One colony forming unit (CFU).

[0176] Processing method: Transfer the membrane with biofilm to a new plate. Place 8-10 layers of sterile gauze on the second membrane layer and pipette 1 mL of antimicrobial solution onto the gauze layer. Perform this treatment at room temperature for 2-3 hours, or 4-6 hours. In the case of a 4-6 hour treatment, replace the gauze layer with a fresh gauze layer containing 1 mL of sample solution 2 or 3 hours after the start of the treatment.

[0177] Evaluation method: Discard the gauze layer, transfer each membrane with biofilm to a 15 mL tube containing 5 mL of 0.9% sodium chloride, vortex for 10 seconds, sonicate in an ultrasonic bath for 10 minutes, and vortex again for 10 seconds. Perform 10-fold serial dilutions, spotting 10 μL of each dilution onto an LB agar plate for live CFU counting.

[0178] Results and Conclusions Figure 2Results obtained using the sample solutions are shown. In a 200 ppm HOCl solution, increasing the HAc concentration from 0.25% to 1% and 2% gradually enhanced the bactericidal effect against Staphylococcus aureus biofilm. 1% acetic acid alone had minimal effect on biofilm. Comparison of these three test solutions with four different competing wound healing products on the market showed that all of them had only a slight effect on Staphylococcus aureus biofilm. The effect on Pseudomonas aeruginosa biofilm was stronger. The conclusion is that hypochlorous acid and acetic acid at pH 4.3 work synergistically and efficiently at concentrations that have been shown to be safe in other studies.

[0179] Example 5: In vivo toxicity study Example 5.1: 7-day inhalation toxicity study in rats.

[0180] A 7-day inhalation toxicity study in rats was conducted according to the instructions on page 2913 of Kogel et al. at https: / / www.pmiscience.com / resources / docs / default-source / default-document-library / 2013_ukogel_ict_poster.pdf?sfvrsn=d6a9f606_0. This rat inhalation study was conducted in accordance with OECD guidelines. The test solution was generated from a multi-compartment apparatus according to the instructions in Example 3.1, with compartment 1 containing 90 mg of dry powder containing 100 ppm sodium hypochlorite in sodium chloride (Example 1b), and compartment 4 containing one 10 mL aliquot of acetic acid solution (0.125%, pH 4.3, Example 2b). Sprague-Dawley rats were exposed to filtered fresh air (sham) as a control, or to the test solution, according to Test Guideline 412. Animal management and use complied with the policies of the American Society for Laboratory Animal Science (1996). All animal experiments were approved by the Institutional Animal Management and Use Committee (IACUC). Histopathological evaluation was performed at prescribed anatomical locations in the nose and left lung, following the prescribed grading system. Free lung cells in bronchoalveolar lavage fluid were determined by flow cytometry, and inflammatory mediators were determined by multiple analyte analysis (MAP). For systemic toxicology methods, RNA samples were obtained from specific locations in the respiratory tract (i.e., the nasal respiratory epithelium (RNE)) and the lungs. For lung RNA isolation, respiratory epithelium from the main bronchus and lung parenchyma was isolated using laser capture microdissection (LCM) and further processed and analyzed on a whole-genome Affymetrix microarray (GeneChip® rat genome 230 2.0 array). No serious perturbations associated with inflammation, cellular stress, or cell proliferation in the bronchi or lung parenchyma were found.

[0181] Example 6: Treatment of mastitis For applications where the use of color indicators in step 4 can add information during treatment (e.g., indication of the oxidative activity of the API), the process includes a compartment containing ROD.

[0182] Example 7: Clinical antiviral treatment 5 ml of test solution, as described in Example 3.1, was added to the drug cup of the Gima Aerosol Corsia nebulizer. 90 mg of dry powder containing 1 ppm sodium chlorite in sodium chloride was added to compartment 1 (Example 1j). One 10 ml portion of citric acid solution (0.1 M, pH 2.2, Example 2p) was added to compartment 4. The mouth of a patient with coronavirus lung infection was placed over the tubing, the mask was attached to the nebulizer, and the nebulizer was activated. After 10–15 minutes of breathing, when the liquid was used up, the nebulizer was turned off. The patient was monitored for several hours to ensure no side effects occurred. The patient's mucous membranes and cilia were studied to examine for potential side effects.

[0183] Example 8: Pharmacological (IS) Study of Inhalation Solution The inhalation solution (IS) of the composition was investigated for its viral inactivation properties against modified Ankara vaccinia virus (MVA). IS products at concentrations of 50 ppm, 100 ppm, and 200 ppm HOCl (pH 5.5) (as well as a diluted 50% solution) exhibited viral inactivation properties, with the lowest concentration of IS product demonstrating viral inactivation capability being 25 ppm HOCl. Further dilutions were tested, and diluted solutions at concentrations of 5, 10, and 20 ppm showed no viral inactivation capability or effect, indicating a lower range of inactivity. For all tested HOCl concentrations, IS products at 50 ppm, 100 ppm, and 200 ppm HOCl (pH 5.5) demonstrated antiviral activity against enveloped DNA vaccinia virus. Products exhibiting antiviral activity against vaccinia virus are considered effective against all enveloped viruses, including SARS-CoV-2. Another independent study showed that IS with 10–200 ppm HOCl was able to inactivate SARS-CoV-2.

[0184] For antibacterial activity, overnight cultures of Staphylococcus aureus and Pseudomonas aeruginosa were grown for 2 hours and 24 hours, respectively, to test the inhibitory effect of IS on planktonic bacteria and biofilm-growing bacteria. IS at 50 ppm HOCl showed complete efficacy against Pseudomonas aeruginosa and Staphylococcus aureus (although IS at 100 ppm HOCl was used against Staphylococcus aureus biofilm).

[0185] In summary, IS products with HOCl concentrations of 50–200 ppm demonstrated virus inactivation capabilities in two different MVA in vitro assays. After dilution of the test products, the lowest concentration exhibiting antiviral activity was 25 ppm HOCl, while the lowest dilutions showing no antiviral activity were 5 ppm, 10 ppm, and 20 ppm HOCl. Based on these experiments, the effective antiviral concentration range is between 25 ppm and 200 ppm HOCl. It has been demonstrated that IS can inactivate SARS-CoV-2 at various concentrations.

[0186] Example 8.1: Antiviral efficacy Antiviral effect of HOCl against vaccinia virus Antiviral tests were conducted to evaluate the antiviral activity of HOCl against modified Ankara vaccinia virus (MVA). The products used were IS solutions containing 50 ppm, 100 ppm, and 200 ppm HOCl at the following concentrations: • Undiluted (80.0%) • Use double-distilled water (aqua bidest.) Dilute (50.0%) • Dilute with double-distilled water (10.0%) • Dilute with double-distilled water (1.0%) - only for 200 ppm HOCl.

[0187] The assay involved exposing the test product (50 ppm, 100 ppm, and 200 ppm HOCl) at dilutions of 1-80% to MVA-infected BHK21 cells, as confirmed by infectivity assays. The product was exposed to the MVA-infected cells for 1 or 2 minutes, followed by an inactivation assay to determine antiviral activity. Cytotoxicity assays were also performed after product exposure.

[0188] method To prepare the test virus suspension, BHK 21 cells were cultured in MEM and with 10% or 2% fetal bovine serum. The multiplicity of infection (MOI) for cell infection was 0.1. The test product was undiluted at the time of the experiment. Due to the addition of interfering substances and the test virus suspension, the solution reached 80.0%.

[0189] Starting with the highest dilution, transfer 0.1 mL of each dilution to eight wells of a microtiter plate, following EN 5.5, to transfer 0.1 mL of each solution to newly dividing cells (10⁻¹⁵ × 10⁻¹⁰). 3Infectivity was determined by endpoint titration (cells / well). Microtiter plates were incubated at 37°C in a 5% CO2 atmosphere. Cytopathic effects were read using an inverted microscope. The infectious dose (TCID50 / mL) was calculated using the Spearman-Kärber method.

[0190] The virucidal activity of the test disinfectant is assessed by calculating the decrease in titer compared to a control titer without disinfectant. This difference is given as the reduction factor (RF). According to EN 14476, a reduction in titer of at least 4 logs over the recommended exposure period is considered acceptable. 10 If a disinfectant or disinfectant solution of a specific concentration is effective in inactivating viruses, then it corresponds to an inactivation rate of ≥99.99%.

[0191] Viricidal activity was determined according to EN 5.5. Inactivation tests were performed in sealed test tubes in a water bath at 20°C ± 1.0°C. Samples were retained after appropriate exposure times, and residual infectivity was determined. Cytotoxicity assays were performed according to EN 5.5.4.1. A 0.7% formaldehyde solution as specified in EN 5.5.6 was used as a reference for test validation. Exposure times were 5, 15, 30, and 60 minutes. Furthermore, the cytotoxicity of the formaldehyde test solution was determined by dilution to 10... -5 It was subsequently measured in accordance with EN5.5.6.2.

[0192] result In the 80.0% test, all undiluted test products (i.e., 50 ppm, 100 ppm, and 200 ppm HOCl) were able to inactivate MVA after a 1-minute exposure time. The reduction factors are as follows: • 50 ppm HOCl: ≥5.25±0.33 •100 ppm HOCl: ≥5.13±0.25 •200 ppm HOCl: ≥5.25±0.33 This corresponds to an inactivation rate of ≥99.999%.

[0193] A 50.0% solution can also inactivate MVA after 1 minute of exposure. The reduction factors are as follows: • 50 ppm HOCl: ≥4.25±0.33 •100 ppm HOCl: ≥4.13±0.25 •200 ppm HOCl: ≥4.25±0.33 This corresponds to an inactivation rate of ≥99.99%.

[0194] A 10.0% solution cannot inactivate MVA within a 1-minute exposure time. A 1.0% solution (200 ppm HOCl) also cannot inactivate MVA within 1 minute.

[0195] In summary, the inhaled IS products at undiluted concentrations of 50 ppm, 100 ppm, and 200 ppm HOCl exhibited anti-MVA activity after 1 minute of exposure (0.3 g / L BSA) during testing.

[0196] Example 8.2: Antibacterial and anti-biofilm effects Example 8.2.1: Antibacterial and antibiofilm effects of IS Antimicrobial tests were conducted to evaluate the bactericidal activity of IS against *Pseudomonas aeruginosa* and *Staphylococcus aureus*, representing planktonic and biofilm bacteria, respectively, after 2 or 24 hours of growth. The product used was IS at the following concentrations (i.e., containing 0.25% acetic acid, pH 5.5, isotonic): • 10 ppm HOCl • 50 ppm HOCl •100 ppm HOCl •200 ppm HOCl • 500 ppm HOCl.

[0197] Contact the product with *Pseudomonas aeruginosa* or *Staphylococcus aureus* for 1 hour, then place equal aliquots on plates and incubate overnight. The next day, assess growth on the plates; in cases of partial growth, quantify log reductions.

[0198] method MH340 (Pseudomonas aeruginosa PAO1) was grown in 5 mL LB, and NCTC-8325-4 (Staphylococcus aureus) was grown in 5 mL TSB. The culture tubes were stored at 37°C overnight (17 hours) and shaken at 180 rpm.

[0199] The overnight culture was then diluted 50-fold, and 200 mL of the diluted bacterial suspension was deposited into each well of a 96-well microtiter plate (8 technical replicas). One microtiter plate was used for each condition and each bacterial treatment. Two-hour growth (“planktonic” bacteria) + 1-hour treatment and 24-hour growth (“biofilm” bacteria) + 1-hour treatment were employed. The bacteria were incubated at 37°C for 2 hours and 24 hours, respectively.

[0200] Subsequently, the bacteria were treated at 37°C for 1 hour with 0.9% sodium chloride (control), 10 ppm HOCl, 50 ppm HOCl, 100 ppm HOCl, 200 ppm HOCl and 500 ppm HOCl.

[0201] After the treatment period (1 hour), apply 20 μL per well to LB plates and incubate overnight at 37°C. The next day, check the plates for growth (with saline as a control) or no growth.

[0202] result As shown in Table 2 below, Pseudomonas aeruginosa planktonic bacteria and biofilms were eradicated at product concentrations lower than those for Staphylococcus aureus. The final IS product (100 ppm HOCl) demonstrated comprehensive antimicrobial activity against representative planktonic and biofilm-bearing Staphylococcus aureus and Pseudomonas aeruginosa. In summary, HOCl at concentrations of 10 ppm or 50 ppm can kill common planktonic bacterial pathogens such as Pseudomonas aeruginosa and Staphylococcus aureus. IS with HOCl concentrations of 50 ppm or 100 ppm can kill Pseudomonas aeruginosa and Staphylococcus aureus in biofilm form, respectively.

[0203] Example 8.2.2. Antibacterial and antibiofilm effects of IS and acetic acid Another antimicrobial test was conducted to evaluate the bactericidal activity of IS and acetic acid against *Pseudomonas aeruginosa* and *Staphylococcus aureus*, which had grown for 2 hours to represent planktonic bacteria. The products used were either IS (i.e., containing 0.25% acetic acid, pH 5.5, isotonic) or acetic acid alone at the following concentrations: •25 ppm HOCl • 50 ppm HOCl •100 ppm HOCl • 0.25% acetic acid, pH 5.5, isotonic.

[0204] Contact the product with *Pseudomonas aeruginosa* or *Staphylococcus aureus* for 1 hour, then place equal aliquots on plates and incubate overnight. The next day, assess growth on the plates; in cases of partial growth, quantify the logarithmic decrease.

[0205] method Overnight cultures of Staphylococcus aureus (NCTC-8325-4) and Pseudomonas aeruginosa PAO1 (MH340) diluted (OD 0.5, Staphylococcus aureus ~10). 7 Pseudomonas aeruginosa is ~10 8The bacteria were grown in 96-well microtiter plates for 2 hours to test their antimicrobial properties against planktonic Gram-positive and Gram-negative bacteria. Subsequently, before harvest, the wells were treated for 1 hour with different concentrations of HOCl (25 ppm, 50 ppm, and 100 ppm HOCl, 0.25% acetic acid, pH 5.5, isotonic), isotonic 0.25% acetic acid (pH 5.5), and 0.9% saline (control).

[0206] One hour later, Dai-Engelli neutralizing broth (Sigma Aldrich, D3435) was added to all wells to inactivate IS, and the contents of the wells were diluted 10-fold serially and placed on the relevant agar plates (down to 10). -8 Incubate plates aerobically at 37°C for 18 hours. Calculate CFU counts from the colony counts in countable dilutions to determine the logarithmic reduction. Perform experiments using three technical backups for each bacterium.

[0207] result As shown in Table 3 below, IS concentrations of HOCl at 25 ppm, 50 ppm, and 100 ppm eradicated both Gram-positive (Staphylococcus aureus) and Gram-negative (Pseudomonas aeruginosa) bacteria. Isotonic 0.25% acetic acid (pH 5.5) did not eradicate bacteria. In summary, IS at HOCl concentrations of 25 ppm and 10 ppm effectively eradicated planktonic Gram-positive bacteria (Staphylococcus aureus) and Gram-negative bacteria (Pseudomonas aeruginosa). Acetic acid did not eradicate Gram-positive bacteria (Staphylococcus aureus) and showed minimal reduction in Gram-negative bacteria (Pseudomonas aeruginosa).

[0208] Example 8.3: Anti-SARS-CoV-2 efficacy Virus inactivation and cytotoxicity assays were performed to evaluate the antiviral activity of IS against Vero E6 cells infected with SARS-CoV-2. The product used was IS at the following concentrations: • 10 ppm HOCl • 50 ppm HOCl •100 ppm HOCl •200 ppm HOCl.

[0209] The assay involved exposing Vero E6 cells infected with SARS-CoV-2 to the test product at concentrations of 10 ppm–200 ppm HOCl for 48 hours. The cells were then stained, and the number of virus antigen-positive cells was counted. Cell proliferation assays were performed to assess cytotoxicity.

[0210] method Vero E6 cells / well were seeded in 96-well plates, virus (multiplicity of infection 0.002) was added and incubated at 37°C for 1 hour, or incubated in culture medium alone as an untreated control for cytotoxicity assay. After removing the virus, 10 ppm HOCl, 50 ppm, 100 ppm, or 200 ppm HOCl (undiluted or half-diluted) was added to IS solution and incubated for 15 minutes, followed by incubation for 48 hours.

[0211] Incubated cells were fixed and stained with HRP using a primary anti-SARS-CoV-2 spike chimeric monoclonal antibody and a secondary antibody F(ab')2-goat anti-human IgG Fc cross-adsorption secondary antibody. Infected cells were visualized using DAB substrate and automatically counted using an ImmunoSpot 5 series UV analyzer. Cytotoxicity assays were performed using the Cell Titer AQueous One Solution Cell Proliferation Assay.

[0212] result In this study, the antiviral efficacy of the test compound was assessed by the number of virus-free VERO cells compared to the control. Based on the results, IS reduced the number of virus-positive VERO cells, thus IS inactivated SARS-CoV-2 at concentrations ranging from 10 ppm to 100 ppm HOCl without killing VERO cells. VERO cells have been reported to be extremely fragile and not well-suited for IS studies; therefore, using more robust cells might yield better antiviral activity. However, because SARS-CoV-2 is classified as a Group 3 microorganism, it is not possible to conduct these experiments in other cell types. Virus inactivation and cytotoxicity results are as follows: Figure 3 As shown.

[0213] Please refer to Figure 3 Each bar represents the mean, accompanied by the standard error of the mean (error bars). The left axis shows the number of virus antigen-positive cells normalized relative to the untreated control (expressed as a percentage). The right axis shows the cell viability (absorbance) normalized relative to the untreated control (expressed as a percentage). MOI = Multiplicity of Infection.

[0214] Note that undiluted experiments at concentrations of 50 ppm, 100 ppm, and 200 ppm killed VERO cells due to an unknown mechanism and are therefore not reported in the above figure. However, undiluted 10 ppm and 50:50 diluted 50 ppm, 100 ppm, and 200 ppm did not kill VERO cells, and SARS-CoV-2 inactivation was observed. In conclusion, IS inactivates SARS-CoV-2 at various concentrations.

[0215] Example 9: Toxicological Study of Inhalation Solution (IS) Several in vivo studies have been conducted and are ongoing to characterize the toxicological properties of IS.

[0216] Non-GLP in vivo inhalation toxicity studies were conducted in Göttingen miniature pigs at Ellegaard Göttingen Minipigs in Denmark. These studies included repeated 5-day dosing studies of nebulized IS via cannulation in miniature pigs, including a 2- or 4-week recovery period for the selected animals. Additionally, a small pilot study via cannulation was conducted to help select the dose level for subsequent studies. In these studies, cannulation was chosen as the route of administration to maximize the amount of IS reaching the lungs.

[0217] Following these studies, a further 5-day study was conducted at Ellegaard Göttingen Minipigs via mask nebulization of IS to simulate the intended human exposure to be studied in the proposed clinical trial.

[0218] Further non-GLP maximum tolerated dose studies were conducted in miniature pigs as a preliminary study of 14-day repeated-dose GLP inhalation toxicity in miniature pigs. Both studies (the preliminary and the primary studies) were administered via face mask, which was also intended to mimic human administration as closely as possible. Due to animal protection restrictions, miniature pigs could only be administered once daily; therefore, they were exposed to nebulized IS for 60 minutes to provide the full-day dose used in the clinical studies (i.e., 18 ml, 100 ppm), rather than multiple daily doses of 5 ml.

[0219] Example 9.1: Repeated-dose toxicity A preliminary toxicity study (non-GLP) was conducted at Ellegaard Göttingen Minipigs in Denmark. Another preliminary non-GLP study was conducted at Covance in the UK, and a GLP study is currently underway at Covance in the UK. All completed and planned repeated-dose toxicity studies are summarized in the following subsections.

[0220] Example 9.1.1: In vivo inhalation study - intubation method Forty-two healthy, young adult Göttingen miniature pigs, aged 6-8 months, were used in this experiment (21 males and 21 females). The pigs weighed approximately 12 kg. These pigs were bred and raised in a barrier facility housed by Ellegaard Göttingen Minipigs, approved by AAALAC International, according to the facility's local environmental, feeding, and care standards. The experimental protocol was approved by the Danish Animal Testing Authority (License No.: 2020-15-0201-00530), and all procedures were performed in accordance with Danish animal testing laws. This study was not conducted in accordance with GLP (Good Laboratory Practice), but the data were recorded and reported according to the documented research plan and local standard operating procedures.

[0221] This study was conducted in two phases. In the first phase, 32 animals (4 males and 4 females per group) were treated for 5 days and then the treatment was terminated. In the second phase, another 10 animals (5 males and 5 females) were treated with the highest dose; after the final treatment, one male and one female were sacrificed on day 5, and two males and two females were sacrificed after a 14-day or 28-day recovery period, respectively. For convenience, both phases are summarized and reported as separate studies.

[0222] Animals were assigned to the following dosage groups: Main stages • 0.9% sodium chloride was used as a control (4 males and 4 females). • 50 ppm HOCl + 0.25% HAc, pH 5.5, isotonic (4 males and 4 females) • 100 ppm HOCl + 0.25% HAc, pH 5.5, isotonic (4 males and 4 females) • 200 ppm HOCl + 0.25% HAc, pH 5.5, isotonic (4 males and 4 females) Recovery phase • 200 ppm HOCl + 0.25% HAc, pH 5.5, isotonic (one male and one female were euthanized after the last application). • 200 ppm HOCl + 0.25% HAc, pH 5.5, isotonic (2 males and 2 females were euthanized 2 weeks after recovery). • 200 ppm HOCl + 0.25% HAc, pH 5.5, isotonic (2 males and 4 females were euthanized 2 weeks after recovery). In addition, four miniature pigs were used in the preliminary study. Three of them were given 500 ppm + 0.25% HAc, pH 5.5, isotonic IS, while the other received saline as a control.

[0223] All miniature pigs were anesthetized daily (using butorphanol-enhanced propofol via intravenous catheter) for five days to receive 5 ml of nebulized product via endotracheal tube (saline was used in the control group). The pigs were ventilated using a GE anesthesia machine in volumetric ventilation mode at a total flow rate of 2 L / min (50% oxygen) and a tidal volume of 10 ml / kg. Vital capacity measurements, including P… peak (The main outcome parameters we used to evaluate potential bronchoconstriction) and recorded blood gas monitoring values, non-invasive blood pressure, heart rate (ECG), and body temperature.

[0224] Recording observations (including P) peak Before inhalation, stabilize the animal on the ventilator system for at least 10 minutes. Monitor the animal for 10 minutes as a baseline measurement; then begin nebulization of 5 ml of product (using an Aerogen Solo nebulizer manufactured by TimikAps in Köling, Denmark). Nebulization should continue for 11–20 minutes (following the manufacturer’s instructions, 2–5 minutes / ml). After all product has been nebulized, monitor the animal for an additional 15 minutes (post-inhalation) until the animal regains consciousness.

[0225] All animals were scored every morning before anesthesia / inhalation and every afternoon after anesthesia / inhalation to assess general condition, appetite, behavior, cough, lung function, and activity. Blood samples were collected before the first dose and after the last dose for evaluation of clinicopathological parameters. For animals in the recovery phase, clinicopathological evaluation of blood was also performed during the withdrawal period.

[0226] All animals were euthanized on day 5 after administration, except for the recovery group, which was euthanized 2 or 4 weeks after drug withdrawal. Following euthanasia, routine necropsy was performed by an experienced veterinary pathologist, with particular attention to the respiratory system, to observe for potential macroscopic toxicities in situ. Lungs and mediastinal lymph nodes were weighed. Samples for histopathological analysis were collected from all animals (plus 2 tagged, untreated animals from the animal facility) from the proximal (including the main bronchus) and distal lobes of all seven lungs, trachea, carina, mediastinal lymph nodes, heart (right and left ventricular myocardium), kidneys, and liver.

[0227] In a pilot study using 500 ppm HOCl, moderate ciliary loss was observed in respiratory epithelial cells, primarily in proximal lung samples. Based on this finding, 200 ppm HOCl was determined to be an appropriate high-dose level for the primary study.

[0228] In the primary study, all miniature pigs were found to be normal during twice-daily clinical assessments.

[0229] At baseline (1 day before inhalation) and at the end of the experiment (5 days after inhalation), hematological and biochemical parameters were not significant in all groups. No indication of therapeutic effect was found.

[0230] In vital capacity measurement, the primary outcome parameter P peak There were no differences before and after inhalation between different treatment groups or control groups. Furthermore, P was observed for each miniature pig in each experiment. peak The largest difference was 1 cm of water column, which is within the detection limits of the machine and has no clinical significance; however, for two miniature pigs (one in the control group and the other in the 200 ppm HOCl group), P peak The difference was 2 cm of water column. This clearly demonstrates that inhalation of the nebulized product did not cause bronchoconstriction. All other parameters were unaffected by the treatment.

[0231] No obvious macroscopic signs of response to treatment were observed during the autopsy.

[0232] In the first part of the study (4 + 4 animals per group treated with 0 ppm, 50 ppm, 100 ppm, or 200 ppm, plus a pilot group of three animals treated with 500 ppm HOCl), the pathological findings associated with drug exposure were localized lymph node hyperplasia, loss of epithelial cilia in the carina (tracheal bifurcation), and main bronchus. For the main bronchus, ciliary loss occurred primarily proximal to the lobe, with all lobes affected similarly. Neutrophil infiltration was observed in the mucosa and submucosa of the trachea, carina, and main bronchus, with an incidence following the pattern of ciliary loss. Drug-exposed pathological results occurred with administration of 500 ppm and 200 ppm HOCl. However, 500 ppm resulted in the most pronounced results. Only very slight ciliary loss was observed in all animals treated with 100 ppm HOCl. All other minor macroscopic and microscopic results were considered to be related to routine anesthesia procedures or incidental. No differences were observed between male and female animals.

[0233] In the second part of the study (animals were administered at a dose of 200 ppm and sacrificed immediately after the last dose (1+1), or sacrificed after 2 weeks of recovery (2+2), or sacrificed after 4 weeks of recovery (2+2)), the histopathological results of the 200 ppm HOCl group without recovery were found to be comparable to those of the 200 ppm group in the main study. This confirms that inhalation of 200 ppm HOCl daily for five consecutive days results in ciliary loss in the trachea, carina, and main bronchus. Recovery of ciliary loss was observed after 2 and 4 weeks of recovery. Hyperplasia was observed in the bronchial and bronchiolar epithelium of the recovery group, representing a sign of cell regeneration. No neutrophil infiltration was found in the mucosa and submucosa of the trachea, carina, and main bronchus after recovery. The recovery group showed an increased number of alveolar macrophages. However, the large differences observed, and the small number of animals in each group, made it difficult to clearly correlate the findings with the test drug. Furthermore, the estimated number of alveolar macrophages in the 200 ppm HOCl group in the first study was significantly higher than that in the second study. Additionally, localized alveolar macrophage infiltration (sometimes associated with mineralization) has been reported to be common in Göttingen miniature pigs. No differences were observed between females and males.

[0234] The SIS (5.0 mL) of 100 ppm HOCl was considered the no significant adverse effect level (NOAEL) after cannulation administration to miniature pigs.

[0235] Example 9.1.2: In vivo inhalation study - mask method In this study, healthy miniature pigs were treated daily for five days with 10 ml (10 ml was added to the nebulizer, but the expected delivery volume was 8.8 ml, as the residual volume was 1.2 ml) of either IS or nebulized saline solution (0.9% w / v sodium chloride, as a control) via a mask covering the nose and mouth. NOAELs of 100 ppm and 50 ppm, previously identified, were tested and compared to the saline control.

[0236] This experiment used 12 healthy young adult Göttingen miniature pigs (6-8 months old) (31355). The miniature pigs (6 males and 6 females) weighed approximately 12 kg. These pigs were bred and raised in a barrier facility housed by Ellegaard Göttingen Minipigs, approved by AAALAC International, according to the facility's local environmental, feeding, and care standards. The experimental protocol was approved by the Danish Animal Testing Authority (License No.: 2020-15-0201-00530), and all procedures were carried out in accordance with Danish animal testing laws.

[0237] The animals were randomly divided into the following dosage groups, with 4 animals in each group (2 males and 2 females): • 0.9% sodium chloride was used as a control (n=4) • 50 ppm HOCl + 0.25% acetic acid, pH 5.5, isotonic (n=4) • 100 ppm HOCl + 0.25% acetic acid, pH 5.5, isotonic (n=4).

[0238] One week prior to the study, miniature pigs were trained to undergo two suspension confinement sessions. During the study, two miniature pigs were placed in a suspension in a quiet, dimly lit operating room. The animals were mildly sedated with a low dose of midazolam (0.3–0.7 mg / kg – increasing the dose as needed over five days to keep each animal calm), and their eyes were covered to maintain calm. A mask was then placed over the mouth and nose and connected to a Pari Boy® Classic nebulizer. The nebulizer chamber was initially filled with 4 ml of IS or saline and continuously refilled (three times, 2 ml each time) until approximately 10 ml was administered after about 30 minutes. According to the manufacturer's instructions, the residual volume is approximately 1.2 ml, therefore the administered dose is approximately 8.8 ml. A pulse oximeter was attached to the tail of each animal to measure pulse and oxygen saturation; measurements, including respiratory rate counts, were recorded after 5, 10, 15, and 20 minutes of inhalation. After surgery, the animals are placed in a recovery box and observed until fully recovered, then returned to their pens. This procedure is repeated daily for five days. On the fifth day, the animals are euthanized after the final inhalation.

[0239] All animals were scored each morning before the procedure and each afternoon after the procedure to assess their overall condition, appetite, behavior, cough, lung function, and activity.

[0240] Blood samples were drawn on the first day before inhalation (baseline time) and the last day after inhalation. Standard biochemical and hematological tests were performed, including differential counts.

[0241] Following euthanasia, a routine autopsy was performed by an experienced veterinary pathologist, with particular attention to the respiratory system, to observe in situ for potential gross toxicities. The lungs and mediastinal lymph nodes were weighed. Samples for histopathological analysis were collected from the proximal (including the main bronchus) and distal parts of the right and left caudate lobes, the trachea, carina, mediastinal lymph nodes, heart (right and left ventricular myocardium), kidneys, and liver. Nasal samples were collected using standardized methods for histopathological analysis to investigate the three nasal cavity levels.

[0242] Histological examination of the lungs (including the trachea, carina, bronchi, and bronchioles), lymph nodes (subcarina), nasal passages (covering the squamous epithelium, transitional epithelium, respiratory epithelium, and olfactory epithelium of the nostrils, nasal turbinates, maxillonasal turbinate bones, vomeronasal organ, ethmoid turbinate, and nasopharynx), liver, kidneys, and heart.

[0243] In rare cases, or after the mask was removed from the mouth and nose, some inhalation-related coughing or sneezing was heard; this was observed in one animal in the control group, two animals in the 50 ppm group, and one animal in the 100 ppm group. This is likely a reaction to the moist local environment created by the mask around the mouth and nose. Because the incidence was similar between the two groups, it was not considered to be caused by IS. Respiratory rate, pulse, and oxygen saturation were similar between the groups. Animals recovered from mild sedation within a maximum of 10 minutes after administration.

[0244] No clinical symptoms were detected during routine daily examinations. From baseline (1 day before inhalation) to post-experiment (5 days after inhalation), the development of hematological and biochemical parameters was not significant in all groups. The elevated creatine kinase levels observed in all groups were considered most likely to be caused by the struggles associated with handling and collecting blood.

[0245] No obvious macroscopic toxic symptoms were observed during the autopsy.

[0246] No pathological findings related to drug exposure were observed in the trachea, carina, and lungs. All minor macroscopic and microscopic findings in the trachea, carina, and lungs were considered to be related to daily sedation procedures, unsuccessful attempts at jugular venous blood collection, and euthanasia, or to be incidental results. For example, focal infiltration of alveolar macrophages (sometimes associated with mineralization) was found in some animals across all groups; this has been reported to be common in Göttingen miniature pigs. Furthermore, based on studies in rats, mice, rabbits, guinea pigs, sheep, non-human primates, dogs, and cats, it has been reported that euthanasia using pentobarbital may induce lung tissue damage, including congestion, edema, hemorrhage, emphysema, and necrosis, and this showed consistency across species.

[0247] Congestion, epithelial shedding, ciliary loss, and lymphoid tissue hyperplasia were observed in the nose and nasopharynx in all three groups. These changes were most common in the lesion area. For the nasopharynx, more animals showed changes in the 100 ppm and 50 ppm HOCl groups compared to the saline group. However, the descriptions of the damage were similar across groups, and the number of animals was small, so it can be concluded that there were no significant differences between the three study groups. It should be noted that epithelial shedding can be considered an artifact of tissue sampling. The conclusion is that mask inhalation of 100 ppm and 50 ppm HOCl compared to the saline control group may not be associated with a significant improvement in the results.

[0248] In summary, the results were observed in all groups (including the saline control group), but the results in animals treated with IS cannot be attributed to IS. Based on this study, the NOAEL for IS administered via mask was 100 ppm (8.8 ml).

[0249] Example 9.1.3: In vivo multi-dose safety study In vivo inhalation of IS was tested in a miniature pig model to assess the maximum tolerated dose, thus aiding in dose selection for subsequent GLP studies. Healthy miniature pigs (n = 6; 3 groups, 1 male and 1 female per group) were treated daily with aerosol concentrations of 1.2 μg / L, 2.3 μg / L, or 5.4 μg / L (using 50 ppm, 100 ppm, or 200 ppm HOCl + 0.25% acetic acid, pH 5.5, isotonic) via a mask covering their mouth and nose for 7 consecutive days. Treatment lasted 60 minutes daily, with each animal receiving 19.9 mL, 19.1 mL, or 22.2 mL daily in the groups receiving 50 ppm, 100 ppm, and 200 ppm IS, respectively. Animals were euthanized on day 8 after 7 days of IS inhalation. Clinical condition, body weight, food consumption, hematology (peripheral blood), blood chemistry, organ weight, macropathology, and histopathology were studied during the study period. For groups 1, 2, and 3, the target values ​​for HOCl concentrations, calculated based on the achieved aerosol concentration of the formulation and the nominal hypochlorous acid concentration, were 99%, 92%, and 110%, respectively. The study had no relevant effects on clinical condition, body weight, food consumption, hematological or blood chemistry parameters, or organ weight, and no macroscopic or histopathological findings were found related to the study. The conclusion is that in long-term toxicity studies of Göttingen miniature pigs, IS was well tolerated when administered via mask for 60 minutes daily for 7 days, and concentrations of 50 ppm, 100 ppm, or 200 ppm were considered suitable for 60-minute daily exposure.

[0250] Example 10: Cytotoxicity of Wound Irrigation Solution (WIS) The in vitro cytotoxicity of wound irrigation solution (WIS) at 200 ppm HOCl was evaluated in two cytotoxicity studies. The aim of the studies was to determine whether WIS was toxic to cultured mammalian L929 cells in vitro. The assays were performed in accordance with the methods described in ISO 10993-5, and the formulations for the assays were prepared according to ISO 10993-12. The following subsections summarize the in vitro cytotoxicity studies.

[0251] Example 10.1: In vitro cytotoxicity of WIS (200 ppm HOCl) The assay WIS (SOF 0001 / 05-01) containing 0.25% acetic acid and 200 ppm HOCl (pH: 4.3) was investigated to determine its potential cytotoxicity to cultured mammalian cells (mouse fibroblasts). The assay was performed according to the United States Pharmacopeia (USP) method. <87> It was conducted in accordance with the ISO 10993-5 guidelines.

[0252] The WIS formulation (SOF 0001 / 05-01) was prepared using complete cell culture medium (Ham's F12 medium supplemented with 10% fetal bovine serum and 50 μg / mL gentamicin). A dilution ratio of 0.2 g test item / mL dilution medium was used. The undiluted formulation and a formulation diluted at 1 part formulation + 3 parts fresh cell culture medium were tested.

[0253] The study included a positive control (sodium dodecyl sulfate (SLS), 0.2 mg / mL) and an untreated control culture (also used as a negative control, treated with complete cell culture medium). Three cell cultures were treated for 48 hours at each test site. The control treatment produced an appropriate response, indicating proper functioning and sensitivity of the assay system. The diluted formulation showed no toxicity (cytotoxicity grade 0 in all cases), while the undiluted formulation showed cytotoxicity (cytotoxicity grade 4 in all cases).

[0254] Under the test conditions of this study (long-term exposure, 48 hours), undiluted WIS (0.25% acetic acid and 200 ppm hypochlorous acid, pH: 4.3) exhibited cytotoxic effects on cultured L929 cells. Based on these results, it can be concluded that WIS 0.25% acetic acid and 200 ppm hypochlorous acid, pH: 4.3 does not comply with ISO 10993-5 and USP <87> The requirement was met because the cytotoxicity grade was >2. However, the diluted formulation of WIS (SOF 0001 / 05-01) did not exhibit toxicity (cytotoxicity grade 0 in all cases).

[0255] Example 10.2: In vitro cytotoxicity of WIS (200 ppm and 448 ppm HOCl) In this study, the in vitro cytotoxicity of WIS (200 ppm HOCl, 0.25% acetic acid), SOF 003 / 53 (448 ppm HOCl, 1% acetic acid), and SOF 003 / 51 (200 ppm HOCl, 1% acetic acid) formulations was evaluated. The in vitro assays used measured lactate dehydrogenase (LDH) released from ruptured cell membranes and metabolic activity (MTT) in the NCTC clone 929 (L-929) cell line after 1, 4, 24, and 48 hours of exposure to the formulations. These assays were performed according to EUNCL SOP (EUNCL-GTA-03).

[0256] No significant membrane rupture was detected for any of the tested formulations at the tested concentrations (10–0.005%) and exposure times (1, 4, 24, and 48 hours).

[0257] According to the guidance in the ISO-10993-5 international standard, no WIS agent caused cytotoxicity (i.e., a reduction in cell viability of more than 30%) in NCTC clone 929 (L-929) cells within the two shortest exposure times (1 hour and 4 hours).

[0258] After 24 and 48 hours of exposure, WIS had no cytotoxic effect on cells (i.e., cell viability was reduced by less than 30%), while SOF 003 / 53 and SOF 003 / 51 formulations induced cytotoxicity at these time points (i.e., survival was reduced by 40-45% after 24 hours of exposure and by 55-60% after 48 hours of exposure).

[0259] Example 11: Genotoxicity of Inhalation Solution (IS) In vitro studies of GLP using IS were conducted at Charles River Laboratories in Hungary.

[0260] Example 11.1: In vitro bacterial reverse mutation assay The potential mutagenic activity of the inhalation solution of the present invention was tested using a bacterial reverse mutation assay. This study was conducted in accordance with GLP.

[0261] In the presence and absence of mitochondrial-demitochondrial supernatant (S9 fraction) prepared from rat liver induced by phenobarbital / β-naphthylflavonoids, Salmonella typhimurium was used ( Salmonella typhimuriumExperiments were conducted using histidine-dependent auxotrophic strains of *Salmonella typhimurium* (TA98, TA100, TA1535, and TA1537) and tryptophan-dependent auxotrophic strains of *Escherichia coli* (*E. coli* WP2 uvrA). This study included a preliminary compatibility test and Experiment 1 (plate incorporation method). The funder selected and provided the following concentrations and corresponding documentation: 50 ppm, 100 ppm, 200 ppm, and 500 ppm, which are equivalent to 0.05 mg / mL, 0.1 mg / mL, 0.2 mg / mL, and 0.5 mg / mL, respectively. At the highest treatment volume (500 μL), these values ​​are equivalent to 25 μg / plate, 50 μg / plate, 10 μg / plate, and 250 μg / plate; these concentrations were used in Experiment 1. Due to cytotoxicity, other plate concentrations were also used. For a 50 ppm test concentration, lower per-plate treatment amounts were employed: 0.3162 μg / plate, 1.0 μg / plate, 3.162 μg / plate, and 10 μg / plate, providing treatment volumes of 6.3 μL, 20 μL, 63.2 μL, and 200 μL, respectively. The highest test concentration was 250 μg test item / plate, and the lowest was 0.3162 μg test item / plate (a total of 8 concentrations). Inhibitory cytotoxicity of the test items (no or only slight reduction in background development) was observed in all tested bacterial strains. No metabolic activation was observed at concentrations of 250 μg / plate, 100 μg / plate, and 50 μg / plate, while metabolic activation was observed at 250 μg / plate.

[0262] In the study, the number of revertant mutant colonies did not show any biologically relevant increase compared to the solvent control. There was no reproducible dose-related trend, nor any indication of a treatment-related effect.

[0263] The reported data from this mutagenicity test indicate that, under the experimental conditions used, the test items did not induce gene mutations due to base pair changes or frameshifts in the genome of the strain used. Therefore, in conclusion, IS does not have mutagenic activity under the experimental conditions used in this study.

[0264] Example 11.2: In vitro mammalian cell micronucleus assay The inhalation solution of the present invention was tested in an in vitro micronucleus assay using mouse lymphoma L5178Y TK+ / - 3.7.2 C cells. This study was conducted according to GLP. Two assays (Assay 1 and Assay 2) were performed. In both assays, a 3-hour treatment (with metabolic activation) (in the presence of S9-mix) and 3-hour and 24-hour treatments (without metabolic activation) (in the absence of S9-mix) were performed. Samples were taken 24 hours after the start of treatment.

[0265] The sponsor selected and provided the study concentrations for the test items in Trial 1 (with and without metabolic activation) as follows: 50 ppm, 100 ppm, 200 ppm, and 500 ppm. These concentrations correspond to 0.05 mg / mL, 0.1 mg / mL, 0.2 mg / mL, and 0.5 mg / mL, respectively, based on the treatment value of 1 mL (10% (v / v)) in the final treatment medium as defined in OECD Guideline 487. In Trial 1, the study was terminated due to observed excessive cytotoxicity of the test items. The selected concentration range was not precise enough to assess whether at least three test concentrations met the acceptability criteria (within an appropriate range of cytotoxicity). Any result with a relative increase in cell count (RICC) of <~40% was unacceptable for this trial; the goal should have been to achieve approximately 40%–50% cytotoxicity in the trial to demonstrate that the concentrations used were sufficient to meet the guidance criteria. Therefore, an additional experiment (Trial 2) was conducted using a modified, smaller concentration range to provide more information on cytotoxic effects and to meet the acceptability regulatory criteria.

[0266] The study concentrations (with and without metabolic activation) for the test items in Experiment 2 were the same as in Experiment 1; however, additional lower treatment concentrations were applied. Therefore, in the case of short-term treatment with metabolic activation, acceptable concentrations of 10 μg / mL, 5 μg / mL, and 2 μg / mL (three in total) were selected for evaluation; in the case of short-term treatment without metabolic activation, concentrations of 6 μg / mL, 2 μg / mL, and 1 μg / mL (three in total) were selected for evaluation; and in the case of long-term treatment without metabolic activation, concentrations of 7 ppm, 6 ppm, 2 ppm, and 1 ppm (four in total) were selected for evaluation. In both experiments with and without metabolic activation, none of the treatment concentrations resulted in a biologically or statistically significant increase in micronucleated cell numbers compared to the appropriate negative (carrier) control.

[0267] In summary, in experiments with and without metabolic activation, IS did not induce a statistically or biologically significant reproducible increase in the frequency of micronucleated mouse lymphoma L5178Y TK+ / - 3.7.2 C cells. Therefore, under the conditions of this study, IS was considered to be non-genotoxic in this experimental system.

[0268] Example 12: Other toxicity studies Example 12.1: In vitro pulmonary surfactant function The inhaled solution of this invention was tested in a simulated alveolar model to evaluate its effect on the function of pulmonary surfactant. Pulmonary surfactant reduces lung surface tension, thereby allowing normal expansion and contraction during respiration. Inhalation of aerosols that interfere with pulmonary surfactant may cause toxic reactions.

[0269] The testing method involves exposing a small amount of pulmonary surfactant to a nebulized IS during a simulated respiratory cycle, while quantifying the surface tension of the pulmonary surfactant. Changes in surface tension are then assessed.

[0270] method The effect of the product on pulmonary surfactant function was tested using a constant flow through a confined droplet surface tensiometer device, as described previously. This method demonstrated 100% sensitivity in detecting harmful substances compared to in vivo studies.

[0271] During a simulated respiratory cycle of pulmonary surfactant (used to simulate alveoli), one drop of pulmonary surfactant (10 μg) was exposed to nebulized 500 ppm HOCl IS (5 mL, over 5 min). Surface tension was continuously assessed by axisymmetric droplet analysis to detect potential critically low surface tension (below 10 mN / m) that could induce pulmonary edema in vivo.

[0272] result No inhibition of pulmonary surfactant function was measured when exposed to the highest concentration of the nebulized inhaled product (500 ppm HOCl + 0.25% acetic acid, pH 5.5, isotonic). Similar results were obtained with 0.9% sodium chloride (control).

[0273] Example 12.2: Eye Stimulation Test Using Ex vivo Corn Method Because the solution was to be delivered to the mouth and nose, a study was conducted to investigate its potential eye irritation. The GLP study “Eye Irritation Tests Using Inhalation Solutions (SIS): Ex vivo Corn Method” was performed according to the methodology described in OECD Guideline 438. Four concentrations of SIS were provided by the sponsor: 500 ppm, 200 ppm, 100 ppm, or 50 ppm hypochlorous acid (HOCl). The study lasted two days, with each day designated as an experiment (i.e., Experiment 1 and Experiment 2). In each experiment, three eyes were treated with 30 μL of the test solution (500 ppm or 200 ppm in Experiment 1, and 100 ppm or 50 ppm in Experiment 2). In each experiment, three positive control eyes were treated similarly with 30 μL of 5% (w / v) benzalkonium chloride solution. Negative control eyes were treated with 30 μL of physiological saline (0.9% (w / v) sodium chloride solution). Corneal thickness, corneal opacity, and fluorescein retention were measured, and any morphological effects (such as epithelial pitting or laxity) were assessed.

[0274] Results from all eyes used in the study met quality control standards. Negative and positive control results were within the range of historical control data in the experiment. Therefore, this study is considered valid.

[0275] According to the guidelines, the results of this study resulted in the test substances being categorized into one of three groups: non-irritating, severely irritating, or requiring further information. Based on this in vitro eye irritation test using inhalation solutions (SIS) at different concentrations on isolated corns, test substance concentrations of 500 ppm, 200 ppm, and 100 ppm were classified as requiring further information. This indicates that in vivo studies should be conducted at these concentrations. The test substance concentration of 50 ppm was classified as non-irritating.

[0276] Example 13: Antibacterial test using inhalation solution (IS) The inhalation solution of the present invention was tested in antibacterial tests against planktonic Gram-positive bacteria (Staphylococcus aureus) and Gram-negative bacteria (Pseudomonas aeruginosa), and the inhalation solution showed highly effective bactericidal activity against both bacteria at HOCl concentrations of 10–25 ppm. The tests were conducted at the Costerton Biofilm Centre, University of Copenhagen. The results of these tests are presented in Table 4 below. Results: As shown in Table 4, an antibacterial effect against Pseudomonas aeruginosa was observed at a HOCl concentration of 10 ppm, and neither bacterium grew at a HOCl concentration of 25 ppm.

[0277] In summary, the inhalation solution of this invention effectively eradicates Gram-positive bacteria (Staphylococcus aureus) and Gram-negative bacteria (Pseudomonas aeruginosa) at HOCl concentrations of 25 ppm and 10 ppm and above, respectively. At a HOCl concentration of 25 ppm, IS is effective against all bacteria.

[0278] Based on these observations, it is clear that any potential bacterial contamination during IS production is immediately eradicated from the product by the broad-spectrum antibacterial activity of HOCl. Therefore, the generation of any endotoxins is highly unlikely, as this would require bacteria to produce endotoxins.

[0279] Example 14: Antiviral activity of IS against vaccinia virus tested according to standard EN 14476 (starting from 25 ppm HOCl) The EN 14476 test for general antiviral activity is performed on chemical disinfectants and preservatives. This is a quantitative suspension test conducted by an accredited laboratory to assess antiviral activity in the medical field. Results of IS: After 1 minute of exposure under clean conditions, the IS test product at a 50% dilution of 50 ppm (25 ppm HOCl) was able to inactivate the vaccine virus (see Table 5). Therefore, no activity was measured after 30 or 60 minutes. The reduction factor was ≥4.25 ± 0.33 (1 minute). According to EN 14476, products with antiviral activity against vaccinia virus are considered to be active against all enveloped viruses.

[0280] Results for hand and surface disinfectants: EN tests were also conducted on hand and surface disinfectant solutions (HOCl, 200 ± 30 ppm, HAc 0.25%, pH 4.3) in accordance with biocidal product regulations. Results show antimicrobial efficiencies against Escherichia coli, fungi, yeast, and vaccinia virus (data not shown).

[0281] In summary, based on the standards for hand sanitizers and surface disinfectants, all EN tests demonstrated rapid and effective inactivation of yeast, fungi, bacteria, and viruses within 1 minute to 30 seconds. The results for IS were not significantly different from those for hand sanitizer solutions and exhibited similar disinfection properties, with an HOCl concentration of 25 ppm.

[0282] Example 15: Antibacterial Efficacy Test You can refer to the USP42-NF37 2S section. <51> Perform a challenge test according to Chapter "Efficacy" or Section 5.1.3 "Antibacterial and Antiseptic" of the European Pharmacopoeia (Ph. Eur.). Use various microbial challenge solution batches (pH 4.3, representative HOCl batch of IS), according to USP42-NF37 2S. <51> The chapter was tested, as shown in Table 6 below: Results and Summary: On days 14 and 28, all test microorganisms met the requirements of USP 42-NF37 2S. <51> The acceptance criteria for antimicrobial efficacy testing described in this chapter. Additionally, it can be performed according to USP42-NF37 2S. <51> This chapter tests the antimicrobial efficacy of the lowest concentration of IS.

[0283] Example 16: Minimum Inhibitory Concentration (MIC) Test The minimum inhibitory concentration (MIC) against five pathogenic bacterial strains was determined by microdiluted a representative broth of the IS (a diluent of the highest concentration of the test substance, HOCl solution, pH 4.3, 100 ppm HOCl + 1% acetic acid and diluent). Following treatment, the suspension was incubated in a microtiter dish for 24 hours, and optical density was measured to assess growth. Furthermore, the suspension was plated on agar the following day for growth control. All experiments were conducted at the Biofilm Laboratory of the Department of Immunology and Microbiology, Faculty of Health and Medical Sciences, University of Copenhagen. Results: The MIC was 25 ppm HOCl + 0.25% acetic acid for all tested microorganisms. Growth was determined by optical density (plate reader) and growth on Müeller Hinton agar plates.

[0284] Conclusions regarding the microbial properties of IS The results of antimicrobial tests conducted using the inhalation solution (IS) product of this invention (starting from a concentration of 10 ppm HOCl) and other representative HOCl formulations clearly demonstrate that the IS product possesses excellent antimicrobial activity. Therefore, we are confident that, like other representative products, IS should be delivered under conditions free of any microorganisms. This is because the HOCl in the product exhibits broad-spectrum antimicrobial activity at pH 4.3 and pH 5.5 (SIS), and in solution, the acidic form of HOCl is dominant (≥ 99.1%). This is also supported by literature reporting the antimicrobial activity of HOCl (acidic form), and HOCl has been used as a preservative to inhibit microbial growth in various approved and commercially available healthcare products (e.g., wound irrigation / rinsing solutions). Therefore, IS is manufactured in a non-sterile, non-sterile facility.

[0285] This application may also include the following implementation schemes: 1. An antimicrobial agent comprising: Solid chloride salts according to the following formula: M n+ [Cl (O) x ] n n- Where M is one of the alkali metal ions, alkaline earth metal ions, and transition metal ions, n is 1 or 2, and x is 1, 2, 3, or 4. Activator according to the following formula: R1XO n (R2,) m R1 contains 1-10 hydride carbon atoms, optionally substituted with amino, amide, carboxyl, sulfonic acid, or hydroxyl groups; X is one of carbon, phosphorus, and sulfur; n and m are 2 or 3 respectively; R2 is one of H, an alkali metal ion salt, an alkaline earth metal ion salt, a transition metal ion salt, or an ammonium salt; and Pharmaceutically acceptable diluents, adjuvants, or carriers.

[0286] 2. The formulation according to embodiment 1, wherein the chlorine oxide salt is an alkali metal salt or an alkaline earth metal salt of hypochlorous acid.

[0287] 3. The formulation according to embodiment 2, wherein the activator is acetic acid.

[0288] 4. The formulation according to embodiment 1, wherein the chlorine oxide salt is an alkali metal salt or an alkaline earth metal salt of chlorite.

[0289] 5. The formulation according to embodiment 4, wherein the activator is acetic acid.

[0290] 6. The formulation according to embodiment 1, wherein the activator is acetic acid.

[0291] 7. The formulation according to embodiment 1, wherein the formulation has a molar osmotic pressure weight concentration in the range of about 0.1 mOsm to about 500 mOsm.

[0292] 8. The formulation according to embodiment 6, wherein the formulation has a pH of about 4 to about 8.

[0293] 9. The formulation according to embodiment 1, wherein the formulation further comprises a thickener.

[0294] 10. The formulation according to embodiment 9, wherein the thickener resists oxidation of the chloride salt.

[0295] 11. The formulation according to embodiment 9, wherein the thickener comprises a water-soluble gelling agent.

[0296] 12. The formulation according to embodiment 11, wherein the water-soluble gelling agent is selected from the group consisting of polyacrylic acid, polyethylene glycol, poly(acrylic acid)-acrylamide alkyl propane sulfonate copolymer, phospho-based polycarboxylic acid, poly(acrylic acid)-acrylamide alkyl propane and sulfonated styrene terpolymer.

[0297] 13. The formulation according to embodiment 1, wherein the formulation further comprises a colorimetric dye.

[0298] 14. The formulation according to embodiment 13, wherein the dye is a vat-oxidation dye.

[0299] 15. The formulation according to embodiment 14, wherein the color and color intensity of the dye depend on the oxidation state of the chlorine oxide compound.

[0300] 16. The formulation according to embodiment 1, wherein the formulation is formulated as an aqueous solution, gel, cream, ointment or oil.

[0301] 17. The formulation according to embodiment 1, wherein the formulation is generated and stored in a multi-compartment container.

[0302] 18. The formulation according to embodiment 17, wherein the fluid and solid components are contained in separate respective compartments before the fluid and solid components are mixed to produce the composition.

[0303] 19. An inhalation formulation comprising about 25 ppm to about 100 ppm of hypochlorous acid and about 0.25% of acetic acid, and at a pH of about 5.5.

[0304] 20. The formulation according to embodiment 19, wherein the formulation is isotonic relative to blood.

Claims

1. A multi-compartment device for preparing a hypochlorous acid-based broad-spectrum antiviral and / or antibacterial inhalation formulation, comprising: (a) A first compartment containing calcium hypochlorite or other pharmaceutically acceptable hypochlorite precursor, and (b) A second compartment containing an aqueous solution of acetic acid and water or saline solution. The contents of the compartment are mixed to produce an inhaled formulation containing hypochlorous acid.

2. The apparatus according to claim 1, wherein the hypochlorous acid precursor is calcium hypochlorite.

3. The device according to claim 2, comprising about 0.1 mg to about 200 mg of calcium hypochlorite.

4. The apparatus of claim 1, wherein the first compartment contains calcium hypochlorite and the second compartment contains acetic acid in a saline solution.

5. The apparatus of claim 1, wherein mixing the contents of the compartment produces a formulation comprising about 0.25 wt% acetic acid.

6. The apparatus of claim 1, wherein mixing the contents of the compartment produces an isotonic formulation comprising about 25 ppm to about 200 ppm of hypochlorous acid.

7. The apparatus of claim 1, wherein mixing the contents of the compartment produces an isotonic formulation comprising about 25 ppm to about 200 ppm of hypochlorous acid and about 0.25% acetic acid.

8. The apparatus of claim 1, configured to deliver the formulation to the lungs of a subject.

9. The device of claim 1, configured to deliver the formulation intranasally to the upper respiratory tract of a subject.

10. The apparatus of claim 1, wherein mixing the contents of the compartment produces an isotonic inhalation formulation comprising about 25 ppm to 200 ppm hypochlorous acid and about 0.25% acetic acid.

11. The apparatus of claim 1, wherein mixing the contents of the compartment produces an isotonic formulation for intranasal administration, comprising about 25 ppm to about 200 ppm of hypochlorous acid and about 0.25% acetic acid.

12. The apparatus according to claim 1, wherein the preparation comprises hypochlorous acid, acetic acid, saline solution and water.

13. The apparatus of claim 1, wherein the apparatus is an atomizer, an inhalation device, or a spray device.

14. The apparatus of claim 1, wherein the compartment is separated by a breakable seal.

15. The apparatus of claim 1, wherein the apparatus does not contain any additional excipients or adjuvants.

16. A method for preparing a hypochlorous acid-based inhalation formulation, the method comprising: (a) A hypochlorous acid precursor containing calcium hypochlorite is provided in the first compartment; (b) Provide an acid activator containing acetic acid in the second compartment; (c) Keep the hypochlorous acid precursor and the acid activator separate before use; and (d) Mix the hypochlorous acid precursor and the acid activator before application to generate an inhalation formulation containing hypochlorous acid.

17. The method of claim 16, wherein the inhaled formulation comprises 25 ppm to 500 ppm of hypochlorous acid.

18. The method of claim 17, wherein the inhaled formulation comprises 25 ppm to 200 ppm of hypochlorous acid.

19. The method of claim 18, wherein the inhaled formulation comprises 25 ppm to 100 ppm of hypochlorous acid.

20. The method according to any one of claims 16 to 19, wherein the inhaled formulation comprises about 0.25% acetic acid.

21. The method according to any one of claims 16 to 20, wherein the inhaled formulation is isotonic.

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