EP0287074A2 | 1988-10-19 | |||
USRE41279E | 2010-04-27 | |||
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US20100227004A1 | 2010-09-09 | |||
US20050214386A1 | 2005-09-29 | |||
US20210352905A1 | 2021-11-18 | |||
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CA2616008A1 | 2007-01-25 |
EDWARDS, ADVANCES IN WOUND CARE, vol. 5, 2016, pages 11 - 19
SOUZA, PHOTODIAGNOSIS PHOTODYN. THER., vol. 23, 2018, pages 347 - 352
AMERICAN ASSOCIATION FOR LABORATORY ANIMAL SCIENCE POLICY, 1996
Claims 1. A antimicrobial formulation, comprising: a solid oxidized chlorine salt according to the formula: M n+ [Cl (O) x ]n n- where M is one of an alkali metal, alkaline earth metal, and transition metal ion, n is 1 or 2, x is 1, 2, 3, or 4; an activator according to the formula: R1XOn(R2,)m where R1 comprises from 1 to 10 hydrogenated carbon atoms, optionally substituted with amino, amido, carboxylic, sulfonic or hydroxy groups, X is one of a carbon, phosphorous and sulfur, n and m are each 2 or 3, and R2 is one of H, an alkali metal, an alkaline earth metal, a transition metal ion salt, or an ammonium salt; and a pharmaceutically-acceptable diluent, adjuvant, or carrier. 2. The formulation of claim 1, wherein said oxidized chlorine salt is an alkali metal or alkaline earth metal salt of hypochlorous acid. 3. The formulation of claim 2, wherein said activator is acetic acid. 4. The formulation of claim 1, wherein said oxidized chlorine salt is an alkali metal or alkaline earth metal salt of chlorous acid. 5. The formulation of claim 4, wherein said activator is acetic acid. 6. The formulation of claim 1, wherein said activator is acetic acid. 7. The formulation of claim 1 having an osmolality in the range of about 0.1 mOsm to about 500 mOsm. 8. The formulation of claim 6 having a pH between about 4 and about 8. 9. The formulation of claim 1, further comprising a viscosity-enhancing agent. 10. The formulation of claim 9, wherein the viscosity-enhancing agent is resistant to oxidation by the oxidized chlorine salt. 11. The formulation of claim 9, wherein the viscosity-enhancing agent comprises a water-soluble gelling agent. 12. The formulation of claim 11, wherein the water-soluble gelling agent is selected from the group consisting of poly acrylic acid, polyethylene glycol, poly(acrylic acid)- acrylamidoalkylpropane sulfonic acid co-polymer, phosphino polycarboxylic acid, apoly(acrylic acid)-acrylamidoalkylpropane and sulfonic acid-sulfonated styrene terpolymers. 13. The formulation of claim 1, further comprising a colorimetric dye. 14. The formulation of claim 13, wherein the dye is a reduction-oxidation dye. 15. The formulation of claim 14, wherein color and intensity of color of the dye is dependent on an oxidation state of the oxidized chlorine compound. 16. The formulation of claim 1 formulated in an aqueous solution, gel, cream, ointment, or oil. 17. The formulation of claim 1 produced and stored in a multi-compartment container. 18. The formulation of claim 17, wherein fluid and solid components are contained within separate respective compartments prior to combination of said fluid and solid components to produce a composition. 19. An inhalation formulation, comprising between about 25 ppm and about 100 ppm of hypochlorous acid and about 0.25% acetic acid at about pH of 5.5. 20. The formulation of claim 19, wherein the formulation is isotonic with respect to blood. |
The active ingredient in preferred compositions of the invention is hypochlorous acid (HOCl). This active ingredient is derived from sodium hypochlorite, which is produced as an aqueous solution from the reaction of gaseous Cl2 with water at alkaline pH. A 3% NaOCl is produced and added to the final IS to reach a maximum of 200 ppm (0.01% w/w) HOCl. The other ingredients of the composition include the following: Sodium Hydroxide, Ph.Eur./USP-NF grade, 0.1M solution added to required pH (5.5); pH stabilizer Acetic Acid, Ph.Eur./USP-NF grade glacier, 0.25%; Osmolarity adjuster Sodium Chloride, Ph. Eur./USP-NF grade, added to reach isotonic formulation (303mOsm); and Purified Water, water purified through Reverse Osmosis and deionized by Ion Exchange process or according to Ph.Eur./USP-NF monograph. A preferred clinical dosage for the composition is 5 mL of 25 - 100 ppm hypochlorous acid. The final product also contains 0.25% acetic acid buffer. As such, the solution contains more than 99.1% HOCl and less than 0.9% OCl-. HOCl is the active substance in IS and has been found to be 80 times more effective as a sanitizing agent compared to an equivalent concentration of OCl-. Therefore, HOCl serves the dual effect in IS of being the API and acting as an antimicrobial agent to inhibit the growth of microorganisms in the final product. The composition may be presented in plastic PET vials/bottles. Before administration to the patient, the composition is transferred to a nebulizer/inhalation device reservoir. This transfer is done in the clinic. After transfer to the nebulizer, the solution is administered immediately (within 1-2 h) to the patient through liquid aerosol delivery. The patient should receive 5 mL of nebulized composition. Compositions for viral administration are typically single-dose administration and are delivered to the respiratory tract by nebulization, using, for example, PARI BOY. The nebulizer PARI BOY Classic Inhalation System, containing PARI BOY Classic Compressor, PARI LC SPRINT nebulizer. To obtain relevant deposition of the test solution in the lower and upper airways, the nebulizer will be equipped with a PARI SMARTMASK. It should be noted that other nebulizers and inhalers may be used. HOCl is produced by the body’s own immune cells, i.e., neutrophils and monocytes/macrophages. It is a powerful oxidizing agent that chlorinates and oxidizes molecular structures, especially those with thiol, thiol-ether, and amino groups (e.g., proteins, fatty acids), leading to denaturation and loss of normal function of a wide array of microbes. HOCl is considered by the FDA to be “the form of free available chlorine that has the highest bactericidal activity against a broad range of microorganisms.” HOCl is a strong oxidizing agent, however, in low concentrations (≤0.1%), it is very well tolerated and safe in wound care applications. Incorporation by Reference Any and all references and citations to other documents, such as patents, patent applications, patent publications, journals, books, papers, web contents, that have been made throughout this disclosure are hereby incorporated herein by reference in their entirety for all purposes. Equivalents The invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. The foregoing embodiments are therefore to be considered in all respects illustrative rather than limiting on the invention described herein. EXAMPLES Example 1: General procedure for preparation of dry, air free solid mixtures of API-P and NaCl for loading into a multi-compartment device. calcium 1a. Production of dry powder comprising 50 ppm sodium hypochlorite in sodium chloride In 8.95 g of dry NaCl (mw: 58.44 g/mol), 50 mg of dry sodium hypochlorite (mw: 74.44 g/mol) was blended to a homogenous mixed powder and stored under air free and dry conditions in containers shielded from light. An aliquot of 90 mg of the powder is loaded into compartment 1 of the multi-compartment device. 1b. Production of dry powder comprising 100 ppm sodium hypochlorite in sodium chloride In 8.90 g of dry NaCl (mw: 58.44 g/mol), 100 mg of dry sodium hypochlorite (mw: 74.44 g/mol) was blended to a homogenous mixed powder and stored under air free and dry conditions in containers shielded from light. An aliquot of 90 mg of the powder is loaded into compartment 1 of the multi-compartment device. 1c. Production of dry powder comprising 200 ppm sodium hypochlorite in sodium chloride In 8.8 g of dry NaCl (mw: 58.44 g/mol), 200 mg of dry sodium hypochlorite (mw: 74.44 g/mol) was blended to a homogenous mixed powder and stored under air free and dry conditions in containers shielded from light. An aliquot of 90 mg of the powder is loaded into compartment 1 of the multi-compartment device. 1d. Production of dry powder comprising 500 ppm sodium hypochlorite in sodium chloride In 8.5 g of dry NaCl (mw: 58.44 g/mol), 500 mg of dry sodium hypochlorite (mw: 74.44 g/mol) was blended to a homogenous mixed powder and stored under air free and dry conditions in containers shielded from light. An aliquot of 90 mg of the powder is loaded into compartment 1 of the multi-compartment device. 1e. Production of dry powder comprising 25 ppm calcium dihypochlorite in sodium chloride In 8.975 g of dry NaCl (mw: 58.44 g/mol), 25 mg of dry calium hypochlorite (mw: 142.98 g/mol) was blended to a homogenous mixed powder and stored under air free and dry conditions in containers shielded from light. An aliquot of 90 mg of the powder is loaded into compartment 1 of the multi-compartment device. 1f. Production of dry powder comprising 50 ppm calcium dihypochlorite in sodium chloride In 8.975 g of dry NaCl (mw: 58.44 g/mol), 50 mg of dry calium hypochlorite (mw: 142.98 g/mol) was blended to a homogenous mixed powder and stored under air free and dry conditions in containers shielded from light. An aliquot of 90 mg of the powder is loaded into compartment 1 of the multi-compartment device. 1g. Production of dry powder comprising 100 ppm calcium dihypochlorite in sodium chloride In 8.9 g of dry NaCl (mw: 58.44 g/mol), 100 mg of dry calium hypochlorite (mw: 142.98 g/mol) was blended to a homogenous mixed powder and stored under air free and dry conditions in containers shielded from light. An aliquot of 90 mg of the powder is loaded into compartment 1 of the multi-compartment device. 1h. Production of dry powder comprising 100 ppm calcium dihypochlorite in sodium chloride In 8.9 g of dry NaCl (mw: 58.44 g/mol), 100 mg of dry calium hypochlorite (mw: 142.98 g/mol) was blended to a homogenous mixed powder and stored under air free and dry conditions in containers shielded from light. An aliquot of 90 mg of the powder is loaded into compartment 1 of the multi-compartment device. 1i. Production of dry powder comprising 100 ppm calcium dihypochlorite in sodium chloride In 8.9 g of dry NaCl (mw: 58.44 g/mol), 100 mg of dry calcium hypochlorite (mw: 142.98 g/mol) was blended to a homogenous mixed powder and stored under air free and dry conditions in containers shielded from light. An aliquot of 90 mg of the powder is loaded into compartment 1 of the multi-compartment device. 1j. Production of dry powder comprising 1 ppm sodium chlorite in sodium chloride In 89.99 g of dry NaCl (mw: 58.44 g/mol), 10 mg of dry sodium chlorite (mw: 90.44 g/mol) was blended to a homogenous mixed powder and stored under air free and dry conditions in containers shielded from light. An aliquot of 90 mg of the powder is loaded into compartment 1 of the multi-compartment device. 1k. Production of dry powder comprising 5 ppm sodium chlorite in sodium chloride In 89.99 g of dry NaCl (mw: 58.44 g/mol), 50 mg of dry sodium chlorite (mw: 90.44 g/mol) was blended to a homogenous mixed powder and stored under air free and dry conditions in containers shielded from light. An aliquot of 90 mg of the powder is loaded into compartment 1 of the multi-compartment device. 1l. Production of dry powder comprising 10 ppm calcium chlorite in sodium chloride In 89.99 g of dry NaCl (mw: 58.44 g/mol), 100 mg of dry calcium chlorite (mw: 157.89 g/mol) was blended to a homogenous mixed powder and stored under air free and dry conditions in containers shielded from light. An aliquot of 90 mg of the powder is loaded into compartment 1 of the multi-compartment device. Example 2: General procedure for preparation of 1 L stock solutions of activator for low- volume aliquot loading into a multiple compartment device 2a. Acetic acid activator stock solution (0.125 %, pH 2.95) In 998.75 mL of sterile water, 1.25 mL of acetic acid (mw: 60.05 g/mol) was dissolved. 2b. Acetic acid activator stock solution (0.125 %, pH 4.3) In 998.75 mL of sterile water, 1.25 mL of acetic acid (mw: 60.05 g/mol) was dissolved. The pH was adjusted to 4.3 using 10 N NaOH. 2c. Acetic acid activator stock solution (0.25 %, pH 4.3) In 998.75 mL of sterile water, 2.5 mL of acetic acid (mw: 60.05 g/mol) was dissolved. The pH was adjusted to 4.3 using 10 N NaOH. 2d. Acetic acid activator stock solution (0.25 %, pH 5.0) In 998.75 mL of sterile water, 2.5 mL of acetic acid (mw: 60.05 g/mol) was dissolved. The pH was adjusted to 5.0 using 10 N NaOH. 2e. Acetic acid activator stock solution (1 %, pH 4.3) In 998.75 mL of sterile water, 10 mL of acetic acid (mw: 60.05 g/mol) was dissolved. The pH was adjusted to 4.3 using 10 N NaOH. 2f. Acetic acid activator stock solution (2 %, pH 4.3) In 998.75 mL of sterile water, 20 mL of acetic acid (mw: 60.05 g/mol) was dissolved. The pH was adjusted to 4.3 using 10 N NaOH. 2g. Acetic acid/sodium acetate activator stock solution (0.1 M, pH 5.0) In 800 mL of distilled water, 5.772 g of sodium acetate (mW: 82 g/mol), 1.778 g of acetic acid (mw: 60.05 g/mol) was added to the solution. The pH was adjusted to 5.0 using 10N HCl or 10 N NaOH, and distilled water was added until the volume was 1 L. 2h. Isotonic acetic acid activator stock solution (0.125 %, pH 2.95) In 998.75 mL of sterile water, 1.25 mL of acetic acid (mw: 60.05 g/mol) and 8.4 g NaCl (mw: 58.44 g/mol) was added. 2i. Isotonic acetic acid activator stock solution (0.125 %, pH 4.3) In 998.75 mL of sterile water, 1.25 mL of acetic acid (mw: 60.05 g/mol) and 8.4 g NaCl (mw: 58.44 g/mol) was added. The pH was adjusted to 4.3 using 10 N NaOH. 2j. Isotonic acetic acid activator stock solution (0.25 %, pH 4.3) In 998.75 mL of sterile water, 2.5 mL of acetic acid (mw: 60.05 g/mol) and 8.4 g NaCl (mw: 58.44 g/mol) was added. The pH was adjusted to 4.3 using 10 N NaOH. 2k. Isotonic acetic acid activator stock solution (0.125 %, pH 5.0) In 998.75 mL of sterile water, 1.25 mL of acetic acid (mw: 60.05 g/mol) and 8.4 g NaCl (mw: 58.44 g/mol) was added. The pH was adjusted to 5.0 using 10 N NaOH. 2l. Isotonic acetic acid activator stock solution (0.25 %, pH 5.0) In 998.75 mL of sterile water, 2.5 mL of acetic acid (mw: 60.05 g/mol) and 8.4 g NaCl (mw: 58.44 g/mol) was added. The pH was adjusted to 5.0 using 10 N NaOH 2m. Isotonic acetic acid/sodium acetate activator stock solution (0.1 M, pH 5.0) In 800 mL of distilled water, 5.772 g of sodium acetate (mW: 82 g/mol), 1.778 g of acetic acid (mw: 60.05 g/mol) and 8.4 g NaCl (mw: 58.44 g/mol) was added to the solution. The pH was adjusted to 5.0 using 10N HCl or 10 N NaOH, and distilled water was added until the volume was 1 L. 2n. Acetate buffer (0.1 M, pH 5.0) In 800 mL of sterile water, 5.772 g of sodium acetate (mW: 82 g/mol) and 1.778 g of acetic acid (mw: 60.05 g/mol) was added to the solution. The pH was adjusted to 5.0 using 10N HCl, and distilled water was added until the volume was 1 L. 2o. ACES buffer (0.1 M, pH 6.7) In 800 mL of sterile water, 18.22 g of N-(2-acetamido)-2-aminoethanesulfonic acid (mW: 182.2 g/mol) was added to the solution. The pH was adjusted to 6.7 using pH using 10N NaOH, and distilled water was added until the volume was 1 L. 2p. Citric acid solution (0.1 M, pH 2.2) An amount of 19.2 g of citric acid (mw: 192.1 g/mol) was dissolved in 1L of sterile water. 2q. Citrate buffer (0.1 M, pH 6.0) In 800 mL of sterile water, 12.044 g of sodium citrate (mW: 294.1 g/mol) and 11.341 g of citric acid (mw: 192.1 g/mol) was added to the solution. The pH was adjusted to 6.0 using 0.1 N NaOH, and distilled water was added until the volume was 1 L. 2r. ADA buffer (0.1 M, pH 6.6) In 800 mL of sterile water, 95.11 g of 2-[(2-amino-2-oxoethyl)-(carboxymethyl)amino]acetic acid (ADA, mW: 190.22 g/mol) was added to the solution. ADA dissolved when the pH was adjusted to 6.6 using pH using 10N NaOH, and distilled water was added until the volume was 1 L. 2s. EBBS buffer including the dye Phenol Red (pH 7.0) In 800 mL of sterile water, 200 mg of CaCl2 (mW: 110.98 g/mol), 200 mg of MgSO4-7H2O (mW: 246.47g/mol), 400 mg of KCl (mW: 75 g/mol), 2.2 g of NaHCO 3 (mW: 84.01 g/mol), 6.8 g of NaCl (mw: 58.44 g/mol), 140 mg NaH 2 PO 4 H 2 O (mw: 138 g/mol), 1 g D-Glucose (Dextrose) (mw: 180.16 g/mol) and 10 mg phenol red Phenol Red (mw: 354.38 g/mol) was added to the solution. The pH of the solution was adjusted to 7.0 or another desired pH using HCl or NaOH. 2t. Sterile isotonic oxygenated water A stock volume of 1 L of sterile water saturated with oxygen is added 9 g of NaCl and stored at room temperature in a sealed bottle shielded from light. Example 3: Instant preparation of ready to use disinfectant formulations from solid salts of oxidized chlorine combined with solutions from Example 1. Example 3.1: Non-limiting steps of a general procedure 1. An aliquot of 90 mg of any of the powders from example 1 is loaded into compartment 4 of the multi-compartment device. 2. An aliquot of 10 mL of any of the activator solutions from Example 2 is loaded into compartment 4 of the multi-compartment device. 3. To generate the main product according to the invention, the seal, barrier or port 3 according to FIG.1 between the screw cap and compartment 4 is broken or opened to mix the contents in compartment 1 with the solution in compartment 4, followed by gently squeezing or shaking to generate the disinfectant solution. The resulting solution can be taken out through the opening after removing the screw cap on the multi-compartment device, and are now ready to use. The isotonic solutions have a pH in the interval 4 to 9, preferably between 5 and 6, is generally used for antimicrobial purposes. 4. Optionally, depending on the intended use, a water-soluble dye in solid form with a color that varies with the oxidation state of the API (ROD), in a precalculated amount to generate a concentration of the dye in the concentration range 0.01 - 1000 ppm, is optionally loaded into compartment 9 of the multi-compartment device, and the procedure in 3 is repeated including mixture of compartments 1, 4 and 9. 5. Optionally, depending on the intended use, a precalculated amount an amino acid as a stabilizer of the API, preferably taurine in the same concentration as the API, is optionally loaded into compartment 5 of the multi-compartment device. and the procedure in 3. is repeated including mixture of compartments 1, 4 and 5. 6. Optionally, depending on the intended use, e.g. for skin or wound applications, an amount of a water-soluble viscosity enhancer (VE) that cannot be oxidized by the API, precalculated to gain a concentration of VE in the final solution in the concentration range 0.01 - 25%, is loaded into compartment 5 of the multi-compartment device. A VE concentration of 0.01 - 0.1 % generates a viscous but fluid solution, while 0.3 - 1% produce a gel. The dispersion of the VE in the solution from step 3, 4 and/or 5 is converted to a viscous solution or a gel using a Silverson Mixer or an Ystral Mixer, and used on site for skin or wound applications. The viscous solution or a gel have increased stability because of slower motions of molecules and may be packed into soft bags, bottles protecting the solution or gel from air and light for later use. Example 3.2: General procedure for preparation of reconstitutable hypochlorous acid- based composition 1. An aliquot of reconstitutable agent(s) is mixed with a diluent to thereby form a ready to use disinfectant formulation in accordance with the present invention, including, but not limited to, the hypochlorous acid-based broad-spectrum antimicrobial solution described herein. The reconstitutable agent(s) and diluent are mixed in a multi-compartment device, similar to the device illustrated in FIG.1 and described herein. 2. The reconstitutable agents may include, but are not limited to, dry powdered agents, including any of the powders from Example 1, as well as any of the activator solutions from Example 2 that have been prepared into dry powder form. For example, in one embodiment, one of the reconstitutable agents may include calcium hypochlorite in dry powder form (provided in a first compartment of a multi-compartment device) and a second reconstitutable agent may include sodium acetate in dry powder form (provided in a second compartment of the multi-compartment device). A diluent, such as water (or other aqueous medium), is provided in a third compartment. 3. Each of the reconstitutable agents (the powdered calcium hypochlorite and powdered sodium acetate) and the diluent are maintained in separate respective compartments of the mixing device until a user is ready to combine and mix the agents and diluent to form an antimicrobial solution as described herein. For example, the device may include one or more breakable seals separating one or more of the compartments from one another, such that a user need only break the seal to thereby initiate mixing of the reconstitutable agents and diluent together. Once the agents and diluent are mixed, a user need only shake the device to agitate and adequately combine the agents and diluent. The device may include one or more filters for filtering the diluent prior to mixing with the reconstitutable agents and/or filtering the resulting antimicrobial solution prior to use. For example, the device may come preloaded with the reconstitutable agents but without the diluent (i.e., water). Accordingly, in the field and at the site of use, a user need only provide water (i.e., from a water source) to the device, at which point a filter may filter out any impurities in the water to provide an adequate diluent. Furthermore, upon producing the resulting antimicrobial solution, the solution may pass through a one way filter when a user applies the solution to the site of intended use (i.e., wound irrigation, hand disinfectant, inhalation solution, countermeasures towards biological and chemical weapons, and the like. Accordingly, the filter prevents contaminated solution from reentering the device during use. 4. Optionally, depending on the intended use, a colorimetric indicator may be added to the composition to provide a visual indication of the level of antimicrobial effects in the solution. The colorimetric indicator may include a dye, for example, such as a reduction-oxidation dye (ROD), such that the color and intensity of color of the dye is dependent on an oxidation state of the oxidized chlorine compound. For example, if the standard half-cell potential of the ROD has a lower positive value than oxidized chlorine (OC), the color of the formulation will be maintained as long as the OC is active. Thereby, the color provides a visual clue in the region wherein the formulation has been applied and where there is active OC. Further, employment of the opposite type of indicator, where the color appears when the oxidizing power of the OC is vanishing, is also useful. The mixing device itself may also include a color scale of sorts that provides various shades of color indicating levels of concentration and appropriate uses/applications for such levels of concentration. 5. Various techniques to prepare dry powders are known and practiced. Such techniques include lyophilization, spray-drying, spray-freeze drying, bulk crystallization, vacuum drying, and foam drying. Lyophilization (freeze-drying) is often a preferred method used to prepare dry powders (lyophilizates) containing proteins. Various methods of lyophilization are well known to those skilled in the art. The lyophilization apparatus and process applies a vacuum that converts liquid portions of a composition into a solid which is subject to a sub-atmospheric pressure to create a vapor. The vapor is drawn from the lyophilization chamber through vapor passages and exhausted to regions external of the lyophilizing apparatus. The lyophilizing process reduces the liquid composition to a dried powdery or granular form. In particular, freeze drying, or lyophilization, is a dehydration technique. It takes place while a product is in a frozen state (ice sublimation under a vacuum) and under a vacuum (drying by gentle heating). These conditions stabilize the product, and minimize oxidation and other degradative processes. The conditions of freeze drying permit running the process at low temperatures, therefore, thermally labile products can be preserved. Freeze drying has become an accepted method of processing heat sensitive products that require long term storage at temperatures above freezing. 6. Steps in freeze drying include pretreatment, freezing, primary drying and secondary drying. Pretreatment includes any method of treating the product prior to freezing. This may include concentrating the product, formulation revision (i.e., addition of components to increase stability and/or improve processing), decreasing a high vapor pressure solvent or increasing the surface area. Methods of pretreatment include: freeze concentration, solution phase concentration, and formulating specifically to preserve product appearance or to provide lyoprotection for reactive products. 7. Accordingly, by providing the reconstitutable agents and diluent(s) within a mixing device, as described herein, the resulting antimicrobial solution can be produced at the desired time and at the desired site of use without facing storage degradation. As previously described herein, certain antimicrobial solutions may typically have a relatively short shelf-life, as they may contain compounds that degrade rapidly and lose their effectiveness. As such, some formulations may require refrigeration and special packaging, or require immediate use upon being produced. Such special treatment, however, adds to operating costs and complicates storage, particularly in areas where such storage is not available. Accordingly, the present invention allows for antimicrobial solutions to be produced by a user at a point of use (e.g., in the field) where access to such solutions is critical, such as in military situations or the like. Example 4: In vitro anti-biofilm effect of example 3 test solutions of HOCl and acetic acid. Three different test solutions were generated form the multi-compartment device. All three test solutions are generated as described in example 3.1 from the multicompartment device, loaded with 90 mg of dry powder comprising 200 ppm sodium hypochlorite in sodium chloride (example 1c) in compartment 1. Three aliquots of 10 mL of acetic acid solutions from (0.125 %, pH 4,3, example 2b) in compartment 4 in three different multicompartment-devices. 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). Experimental setup Test organisms: Pseudomonas aeruginosa or Staphylococcus aureus wild-type strains Biofilm type: 48 hours- or 24 hours-old biofilms grown on semipermeable membranes placed on solidified medium supplemented with 0.5% glucose. In the case of 48h-old biofilms, the membranes with biofilms were transferred onto fresh plates after 24h. Initial viable cell amount: 5 x 10 9 colony forming units (CFUs) Treatment method: Membranes with biofilms were transferred to new plates. Eight-10 layers of sterile gauze were placed on the second membrane, and 1 ml of antimicrobial solution was pipetted on the gauze layers. The treatment was carried out at room temperature for 2-to-3h, or 4- to-6h. In the case of the 4-to-6h treatments, the gauze layers were replaced with fresh gauze layers with 1ml sample solution 2 or 3h after the treatments had been initiated. Evaluation method: The gauze layers were discarded, and each membrane with biofilms was transferred into a 15ml tube containing 5ml 0.9% NaCl, vortexed for 10 sec., sonicated in an ultrasound bath for 10 min, and vortexed again for 10 sec. Ten-fold serial dilutions were made, and 10ul of each dilution was spot-plated on LB plates for viable CFU counting. Results and conclusions FIG. 2 shows the results obtained using the sample solutions. Increasing the HAc concentrations from 0.25% to 1% and 2% in a 200ppm HOCl solution gradually increased the killing of S. aureus biofilms. The effect of 1 % acetic acid alone had only minor effect on the biofilm. The three test solutions were compared to 4 different competing wound healing products on the market which all showed only minor effects on the S. aureus biofilms. An even stronger effect was shown for biofilms from P. Aeruginosa It is concluded that hypochlorous acid and acetic acid at pH 4.3 acts synergistically and efficiently at concentrations that have shown to be safe in other studies. Example 5: In vivo toxicity studies Example 5.1: 7 day inhalation toxicity study in rats. A 7 day inhalation toxicity study in rats is performed as described by Kogel et al in 2913 in https://www.pmiscience.com/resources/docs/default-source/def ault-document- library/2013_ukogel_ict_poster.pdf?sfvrsn=d6a9f606_0. The rat inhalation study is performed according to the Organization for Economic Cooperation and Development (OECD). The test solution is generated as described in example 3.1 from the multicompartment device, loaded with 90 mg of dry powder comprising 100 ppm sodium hypochlorite in sodium chloride (example 1 b) in compartment 1 and an aliquot of 10 mL of acetic acid solution (0.125 %, pH 4.3, example 2b) in compartment 4. Test Guideline 412, Sprague-Dawley rats is exposed to filtered fresh air (sham) as a reference, or the test solution. Care and use of the animals is in accordance with the American Association for Laboratory Animal Science Policy (1996). All animal experiments are approved by the Institutional Animal Care and Use Committee (IACUC). The histopathological evaluation is performed at defined anatomical sites of the nose and of the left lung according to a defined grading system. Free lung cells are determined in bronchoalveolar lavage fluid by flow cytometry, and inflammatory mediators are measured by multi-analytes profiling (MAP). For the Systems Toxicology approach, RNA samples from specific sites in the respiratory tract are obtained, i.e., respiratory nasal epithelium (RNE) and lung. For lung RNA isolation, respiratory epithelium of main bronchus and lung parenchyma is separated by Laser Capture Microdissection (LCM) and further processed, and analyzed on whole genome Affymetrix microarrays (GeneChip® Rat Genome 2302.0 Array).No major perturbations are found related to inflammation, cell stress, cell proliferation in bronchi or lung parenchyma. Example 6: Treatment of mastitis For applications where a color indicator in step 4 can add information in the therapeutic procedure, e.g. in or for indication of the oxidative activity of the API, the compartment comprising the ROD is included in the procedure. Example 7: Clinical antiviral therapy The medicine cup of Gima Aerosol Corsia Nebulizer is loaded with 5 mL of the test solution generated as described in example 3.1 from the multicompartment device, loaded with 90 mg of dry powder comprising 1 ppm sodium chlorite in sodium chloride (example 1j) in compartment 1 and an aliquot of 10 mL of citric acid solution (0.1 M, pH 2,2, example 2p) in compartment 4. The mouth of a patient with a coronavirus lung infection is attached to the hose and the face mask attached to the nebulizer, which is started. After 10-15 minutes of breathing, the fluid is used up, and the nebulizer is turned off. The patient is monitored for several hours to secure that no side effects of the treatment is taking place. The mucosa and cilia of the patient is investigated for potential side effects. Example 8: Pharmacology of inhalation solution (IS) The virus-inactivating properties of the inhalation solution (IS) of the composition against modified vaccinia virus Ankara (MVA) have been investigated. The IS products at 50, 100, and 200 ppm HOCl (pH 5.5) (and diluted 50% solutions) showed virus inactivation properties suggesting that the lowest concentration of the IS product showing virus inactivation was at 25 ppm HOCl. Further dilutions were tested and diluted solutions with concentrations of 5, 10 and 20 ppm did not show any virus inactivation and effect suggesting that the non-active lower range was demonstrated. IS products with 50, 100 and 200 ppm, HOCl (pH 5.5) demonstrated antiviral activity against the enveloped DNA vaccinia virus for all tested HOCl concentrations. Products that have antiviral activity against the vaccinia virus are considered active against all enveloped viruses, including SARS-CoV-2. In a separate study, IS has been shown to inactivate SARS- CoV-2 between 10 and 200 ppm HOCl. As for antibacterial activity, overnight cultures of S. aureus and P. aeruginosa were grown for 2 and 24 hours, respectively, to test IS against both planktonic and biofilm growing bacteria. Full effect was seen for 50 ppm HOCl IS for P. aeruginosa and S. aureus (though 100 ppm HOCl IS for S. aureus biofilm). In summary, the IS products with HOCl concentrations between 50 and 200 ppm show virus inactivation in two different MVA in vitro tests. After dilutions of the test products, the lowest concentration showing antiviral activity was at 25 ppm HOCl and the lowest diluted concentrations tested showing no antiviral activity were 5, 10 and 20 ppm HOCl. From these experiments the antiviral effective concentration range was between 25 and 200 ppm HOCl. IS has been shown to inactivate SARS-CoV-2 in various concentrations. Example 8.1: Antiviral efficacy Antiviral effectiveness of HOCl against vaccinia virus Antiviral assays were performed to evaluate the virucidal activity of HOCl against modified vaccinia virus Ankara (MVA). The product used was IS containing 50, 100, and 200 ppm HOCl at the following concentrations: · Undiluted (80.0%) · Diluted with aqua bidest. (50.0%) · Diluted with aqua bidest. (10.0%) · Diluted with aqua bidest. (1.0%) – 200 ppm HOCl only The test methods involved exposing the test products (50, 100, & 200 ppm HOCl) at dilutions between 1-80% to BHK21-cells infected with MVA, as confirmed via infectivity assay. The product was in contact with MVA infected cells for either 1 or 2 minutes then an inactivation assay was performed to determine virucidal activity. Determination of cytotoxicity was also performed following product contact. Method To prepare the test virus suspension, BHK 21-cells were cultivated with MEM and 10% or 2% fetal calf serum. Cells were infected with a multiplicity of infection of 0.1. The test product was tested undiluted. Due to the addition of interfering substance and test virus suspension an 80.0% solution resulted. Infectivity was determined as endpoint titration according to EN 5.5 transferring 0.1 mL of each dilution into eight wells of a microtiter plate to 0.1 mL of freshly splitted cells (10-15 x 103 cells per well), beginning with the highest dilution. Microtiter plates were incubated at 37 °C in a 5% CO2-atmosphere. The cytopathic effect was read by using an inverted microscope. Calculation of the infective dose TCID50/mL was calculated with the method of Spearman and Kärber. The virucidal activity of the test disinfectant was evaluated by calculating the decrease in titer in comparison to the control titration without disinfectant. The difference is given as reduction factor (RF). According to the EN 14476, a disinfectant or a disinfectant solution at a particular ·oncentration has virus-inactivating efficacy if the titer is reduced at least by 4 log10 steps within the recommended exposure period. This corresponds to an inactivation of ≥99.99%. Determination of virucidal activity has been carried out according to EN 5.5. Inactivation tests were carried out in sealed test tubes in a water bath at 20 °C ± 1.0 °C. Aliquots were retained after appropriate exposure times and residual infectivity was determined. Determination of cytotoxicity was performed according to EN 5.5.4.1. As reference for test validation a 0.7% formaldehyde solution according to EN 5.5.6 was included. Contact times were 5, 15, 30 and 60 minutes. In addition, cytotoxicity of formaldehyde test solution was determined according to EN 5.5.6.2 with dilutions up to 10 -5 . Results All undiluted test products (i.e., 50, 100, 200 ppm HOCl) in an 80.0% assay were able to inactivate MVA after 1 minute of exposure time. The reduction factors were the following: · 50 ppm HOCl: ≥5.25±0.33 · 100 ppm HOCl: ≥5.13±0.25 · 200 ppm HOCl: ≥5.25±0.33 These corresponded to an inactivation of ≥99.999%. The 50.0% solutions were also able to inactivate MVA after 1 minute of exposure time. The reduction factors were the following: · 50 ppm HOCl: ≥4.25±0.33 · 100 ppm HOCl: ≥4.13±0.25 · 200 ppm HOCl: ≥4.25±0.33 These corresponded to an inactivation of ≥99.99%. The 10.0% solutions were not able to inactivate MVA within 1 minute of exposure time. The 1.0% solution (200 ppm HOCl) was also not able to inactivate MVA within 1 minute. In conclusion, the products for inhalation IS at 50, 100, and 200 ppm HOCl tested undiluted demonstrated activity against MVA after an exposure time of 1 minute (0.3 g/L BSA). Example 8.2: Antibacterial and anti-biofilm efficacy Example 8.2.1. Antibacterial and Anti-Biofilm Efficacy of IS An antibacterial assay was performed to evaluate the bactericidal activity of IS against P. aeruginosa and S. aureus grown for either 2 or 24 hours to represent planktonic and biofilm bacteria, respectively. The product used was the IS (i.e., with 0.25% acetic acid, pH 5.5, isotonic) at the following concentrations: · 10 ppm HOCl · 50 ppm HOCl · 100 ppm HOCl · 200 ppm HOCl · 500 ppm HOCl The product was in contact with either P. aeruginosa or S. aureus for 1 hour then an aliquot was plated and left to incubate overnight. The next day the plates were evaluated for growth and log reductions were quantified in the case of partial growth. Method MH340 (P. Aeruginosa PAO1) was grown in 5 mL LB and NCTC-8325-4 (S. aureus) in 5 mL TSB in culture tubes overnight (17 hours) at 37°C, shaking at 180 rpm. Overnight cultures were thereafter diluted 50 times and 200 µL of the diluted bacterial suspension were deposited per well in 96 rounded well microtiter plates (8 technical replicates). One microtiter plate per condition and treatment per bacteria. Two hours growth (“planktonic” bacteria) + 1 hour treatment and 24 hours growth (“biofilm” bacteria) + 1 hour treatment. The bacteria were incubated at 37°C for 2 and 24 hours, respectively. Thereafter bacteria were treated with 0.9% NaCl (control), 10 ppm HOCl, 50 ppm HOCl, 100 ppm HOCl, 200 ppm HOCl, and 500 ppm HOCl IS at 37°C for 1 hour. After the treatment period (one hour) 20 µL per well was spotted on LB plates and cultured at 37°C overnight. The day after the plates were checked for growth (saline is control) or no growth. Results As seen in Table 2 below, P. aeruginosa planktonic bacteria and biofilms were eradicated at lower product concentrations than S. aureus. There is full antibacterial effect of the final IS product (100 ppm HOCl) across the board in representative planktonic and biofilm S. aureus and P. aeruginosa. In conclusion, IS at concentrations of 10 or 50 ppm HOCl kill the common planktonic bacterial pathogens P. aeruginosa and S. aureus, respectively. IS with 50 or 100 ppm HOCl kill biofilm forms of P. aeruginosa and S. aureus, respectively. Example 8.2.2. Antibacterial and Anti-Biofilm Efficacy of IS and Acetic Acid Another antibacterial assay was performed to evaluate the bactericidal activity of IS and acetic acid against P. aeruginosa and S. aureus grown for 2 hours to represent planktonic bacteria. The product used was the IS (i.e., with 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 The product was in contact with either P. aeruginosa or S. aureus for 1 hour then an aliquot was plated and left to incubate overnight. The next day the plates were evaluated for growth and log reductions were quantified in the case of partial growth. Method Diluted overnight cultures (OD of 0.5, ~10 7 for S. aureus and ~10 8 for P. aeruginosa) of S. aureus (NCTC-8325-4) and P. aeruginosa PAO1 (MH340) were grown in 96-well microtiter plates for 2 hours, to test antibacterial properties against planktonic gram-positive and gram- negative bacteria. Wells were thereafter treated with IS at varying concentrations of HOCl (25, 50, 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) for one hour before harvest. After one hour, Dey-Engley neutralizing broth (Sigma Aldrich, D3435) was added to all wells to inactivate IS and the content of the wells were diluted in 10-fold series and plated on relevant agar plates (down to 10 -8 ). The plates were grown aerobically for 18 hours at 37°C. The CFU counts were calculated from the number of colonies in the countable dilutions to calculate log reductions. Test was run with three technical replicates of each bacterium. Results As seen in Table 3 below, 25, 50 and 100 ppm HOCl IS eradicated both gram-positive (S. aureus) and gram-negative (P. aeruginosa) bacteria. Isotonic 0.25% acetic acid (pH 5.5) did not eradicate the bacteria. In conclusion, IS efficiently eradicates planktonic gram-positive (S. aureus) and gram-negative bacteria (P. aeruginosa) at HOCl concentrations of 25 ppm and 10 ppm, respectively. Acetic acid does not eradicate gram-positive (S. aureus) bacteria and shows minimal reduction in gram- negative bacteria (P. aeruginosa). Example 8.3: Anti-SARS-CoV-2 efficacy Viral inactivation and cytotoxicity assays were performed to evaluate the virucidal activity of IS against SARS-CoV-2 infected Vero E6 cells. The product used was IS at the following concentrations: · 10 ppm HOCl · 50 ppm HOCl · 100 ppm HOCl · 200 ppm HOCl The test method involved exposing the test product at concentrations between 10-200 ppm HOCl to Vero E6 cells infected with SARS-Cov-2 for 48 hours. The cells were then stained, and the number of virus antigen positive cells were enumerated. A cell proliferation assay was performed to evaluate cytotoxicity. Method Vero E6 cells/well were seeded in 96-well plates, the virus (multiplicity of infection 0.002) was added and incubated for 1 h at 37°C or media only for non-treated controls and for cytotoxicity assays. The virus was removed and IS 10 ppm HOCl, 50, 100, or 200 ppm HOCl, either undiluted or diluted by half was added for 15 min, thereafter the assay was incubated for 48 h. The incubated cells were fixed and stained with primary antibody SARS-CoV-2 spike chimeric monoclonal antibody and with secondary antibody F(ab')2-Goat anti-Human IgG Fc Cross- Adsorbed Secondary Antibody, HRP. Single infected cells were visualized with DAB substrate and counted automatically by an ImmunoSpot series 5 UV analyzer. Cytotoxicity assays were performed using the Cell Titer AQueous One Solution Cell Proliferation Assay. Results In this study, the antiviral effect of the test compound was evaluated by amount of VERO cells free of virus compared to the control. Based on the results IS lowered the amount of virus positive VERO cells, thus IS inactivated SARS-CoV-2 in concentration from 10 ppm to 100 ppm HOCl without killing the VERO cells. VERO cells have been reported to be extremely fragile and not well suited to study IS thus even better antiviral activity might have been obtained with more robust cells. However, it has not been possible to run these experiments in other cell types due to the classification of SARS-CoV-2 as a Class 3 microorganism. The viral inactivation and cytotoxicity results are presented in FIG.3. Referring to FIG.3, each bar represents the mean with standard error of the mean (error bars). Left axis shows the number of virus antigen positive cells normalized to non-treated controls (in percentage). Right axis shows the cell viability (absorbance) normalized to non-treated controls (in percentage). MOI = Multiplicity of infection. Note, undiluted experiments at 50, 100, and 200 ppm killed the VERO cells due to an unknown mechanism and therefore are not reported in the figure above. However, 10 ppm undiluted and 50:50 dilution of 50, 100 and 200 ppm did not kill the VERO cells and SARS-Cov-2 inactivation was observed. In conclusion, at various concentrations, IS inactivates SARS-CoV-2. Example 9: Toxicology of inhalation solution (IS) Several in vivo studies have been performed and are on-going to characterize the toxicology of IS. Non-GLP in vivo inhalation toxicity studies in Göttingen minipigs have been performed at Ellegaard Göttingen Minipigs in Denmark. These studies include a 5-day repeated dosing study in minipigs by intubation with nebulized IS, including a recovery period of 2 or 4 weeks for selected animals. In addition, a small pilot study by intubation was performed to aid selection of dose levels for the subsequent studies. Intubation was selected as the dose route in these studies to maximize the amount of the IS that reached the lungs. Following these studies, a further study of 5 days duration was also performed at Ellegaard Göttingen minipigs with dosing nebulized IS by mask to mimic the intended human exposure to be studied in the proposed clinical trials. A further non-GLP Maximum Tolerated Dosage study in minipigs was performed as a preliminary study to a 14-day repeat dose GLP inhalation toxicity study in minipigs. Both studies (preliminary and main study) have dosing via mask, again to mimic as closely as possible the human administration. Due to animal welfare restrictions, the minipigs may only be dosed once per day and therefore are exposed to nebulized IS for 60 minutes to deliver the full day dosing intended for the clinical studies (i.e., 18 mL at 100 ppm) as opposed to 5 mL dosing multiple times per day. Example 9.1: Repeat-dose toxicity The initial toxicity studies (non-GLP) were conducted at Ellegaard Göttingen Minipigs in Denmark. An additional preliminary non-GLP study was performed at Covance in England and a GLP study is on-going at Covance in England. All completed and planned repeat-dose toxicity studies are summarized in the following subsections. Example 9.1.1: In vivo inhalation study - intubated Forty-two healthy young-adult Göttingen minipigs, 21 males and 21 females, 6-8 months of age, were used in this experiment. The minipigs weighed approximately 12 kg. The minipigs were bred and housed at Ellegaard Göttingen Minipigs in AAALAC International approved barrier facility housing and according to the facilities’ standard for local environment, feeding, and care. The experimental protocol was approved by the Danish Animal Experiments Inspectorate (license no.2020-15-0201-00530), and all procedures were carried out according to the Danish Animal Testing Act. The study was not performed according to GLP, however data were recorded and reported according to the documented Study Plan and to local Standard Operating Procedures. The study was performed in two separate phases. In the first phase, 32 animals (4 males and 4 females per group) were treated for 5 days and terminated. In the second phase, a further 10 animals (5 males and 5 females) were treated at the highest dose; 1 male and 1 female were killed on Day 5 following the last treatment, and 2 males and 2 females were killed respectively after a 14 or 28-day recovery period. Both phases are summarized and reported here as a single study for convenience. The animals were allocated to the dosing groups as follows: Main phase · 0.9% NaCl as 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 (1 male and 1 female killed following the final dose) · 200 ppm HOCl + 0.25% HAc, pH 5.5, isotonic (2 males and 2 females killed after 2 weeks recovery) · 200 ppm HOCl + 0.25% HAc, pH 5.5, isotonic (2 males and 2 females killed after 4 weeks recovery) Additionally, four minipigs were used in a pilot study, where three were dosed with a 500 ppm + 0.25% HAc, pH 5.5, isotonic IS, whilst one received saline and acted as a control. All minipigs were anaesthetized (with propofol potentiated by butorphanol by intravenous catheter) daily for five days to receive 5 mL nebulized product (saline for the control group) through an endotracheal tube. The minipigs were ventilated using a GE anesthesia machine at volume-controlled ventilation with a total flow of 2 L/min (50% oxygen) and a tidal volume of 10 mL/kg. Spirometry, including P peak (our major outcome parameter, to assess potential bronchoconstriction), was recorded every two minutes as well as capnometry, non-invasive blood pressure, heart rate (ECG), and temperature. The animals were allowed at least 10 min of stabilization at the ventilator system before observations, including P peak , were recorded. The animals were monitored for 10 min as baseline measurements; thereafter the nebulization of 5 mL product was started (Aerogen Solo nebulizer, Timik Aps, Kolding, Denmark). Nebulization lasted 11-20 min (as according to manufacturer, 2- 5 min/mL). After all product was nebulized, the animals were monitored for another 15 min (post-inhalation) before they could regain consciousness. Every morning before and every afternoon after the anesthesia/inhalation, all animals were scored to assess general condition, appetite, behavior, coughing, lung function, and mobility. Blood samples were taken before the first dose and again after the last dose and evaluated for clinical pathology parameters. For recovery animals, blood was also evaluated for clinical pathology during the off-dose period. All animals were killed on Day 5 after completion of dosing except for the recovery group animals which were killed after 2 or 4 weeks off-dose. Routine necropsy with special attention to the respiratory system was performed following euthanasia by an experienced veterinary pathologist to observe potential macroscopic signs of toxicity in situ. Lungs and mediastinal lymph nodes were weighed. Samples for histopathology were collected proximally (including the main bronchus) and distally from all seven lung lobes, from the trachea, carina, mediastinal lymph nodes, heart (right and left ventricular muscles), kidney and liver of all animals (plus 2 sentinel, untreated animals from the animal facility). In the pilot study with 500 ppm HOCl, there was moderate ciliary loss in the respiratory epithelium, mainly in the proximal lung samples. Based on this finding, it was decided that 200 ppm HOCl would be a suitable high dose level for the main study. In the main study, all minipigs were found to be normal at the twice daily clinical evaluations. Hematological and biochemical parameters were unremarkable for all groups at baseline (Day 1 before inhalation) and at the end of the experiment (Day 5 after inhalation). There were no findings indicative of an effect of treatment. In the spirometry measurements, the major outcome parameter, Ppeak, did not differ between the different treatment groups or control in relation to and after inhalation. Further, the largest difference in P peak seen per minipig per experiment was 1 cm H 2 O, which is within the limits of detection of the machine and of no clinical significance; however, for two pigs (one in the control group and one in the 200 ppm HOCl group) the differences in P peak was 2 cm H2O. This clearly underlines that the inhalation of the nebulized products did not induce bronco- constriction. All other parameters were unaffected by treatment. At necropsy, no apparent macroscopic signs of reaction to treatment were observed. In the first part of the study (treatment of 4 + 4 animals per group at 0, 50, 100, or 200 ppm, plus the pilot group of three animals dosed at 500 ppm HOCl) pathological findings related to drug exposure were local lymph node hyperplasia, loss of epithelial ciliation in the carina area (tracheal bifurcation) and main bronchi. For the main bronchi, loss of ciliation was primarily present proximally in the lung lobes, and all lobes were equally affected. Neutrophilic granulocyte infiltration was seen in mucosa and submucosa of trachea, carina and main bronchi, and the incidence followed the pattern of ciliation loss. The pathological findings related to drug exposure were present when 500 and 200 ppm HOCl were administered. However, 500 ppm resulted in the most pronounced findings. Only minimal ciliation loss was observed in single animals which received 100 ppm HOCl. All the other minor macroscopic and microscopic findings are considered either related to the daily anesthetic procedure or being incidental findings. No differences were observed between male and female animals. In the second part of the study (animals dosed at 200 ppm and killed immediately after the last dose (1 + 1), or after 2 weeks recovery (2 + 2) or after 4 weeks recovery (2 + 2)), the histopathological findings in the 200 ppm HOCl group without recovery were found to be comparable to the 200ppm group in the main study. Thus, it was confirmed that daily inhalation of 200ppm HOCl for five days results in loss of cilia in trachea, carina area, and main bronchi. Recovery of ciliation loss was found after both 2 and 4 weeks of recovery. Hyperplasia was seen in the bronchial and bronchiolar epithelium in the recovery groups which represents signs of cellular regeneration. Neutrophilic granulocyte infiltration in mucosa and submucosa of trachea, carina, and main bronchi was not seen after recovery. The recovery groups showed an increased amount of intra-alveolar macrophages. However, a large variation was seen which, together with the low number of animals in each group, make it difficult to clearly relate the finding to the tested drug. In addition, the estimated number of intra-alveolar macrophages in the 200 ppm HOCl group in the first study was much higher than in the second. Furthermore, focal infiltration of alveolar macrophages, sometimes associated with mineralization, are reported as common findings in Göttingen minipigs. No differences were observed between female and male animals. SIS at 100 ppm HOCl (5.0 mL) was considered to be the NOAEL following dosing of minipigs by intubation. Example 9.1.2: In vivo inhalation study - masked In this study, healthy minipigs were treated daily for five days with 10 mL (10 mL is added to the nebulizer, but the expected delivery was 8.8 mL, as residual volume is 1.2 mL) of the nebulized IS or nebulized saline solution (0.9% w/v NaCl, as a control) by mask covering the snout. The previously found NOAEL of 100 ppm was tested as well as 50 ppm and compared to saline control. Twelve healthy young-adult Göttingen minipigs (6-8 months of age) were used in this experiment (31355). The minipigs (6 males and 6 females) weighed approximately 12 kg. The minipigs were bred and housed at Ellegaard Göttingen Minipigs in AAALAC International approved barrier facility housing and according to the facilities’ standard for local environment, feeding, and care. The experimental protocol was approved by the Danish Animal Experiments Inspectorate (license no.2020-15-0201-00530), and all procedures were carried out according to the Danish Animal Testing Act. The animals were randomly divided into the following dosing groups with 4 animals (2 males and 2 females): · 0.9% NaCl as 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) The minipigs were trained to accept the sling confinement on two occasions during the week before the study. During the study, two minipigs at a time were placed in slings in a calm and light-dimmed procedure room. The animals were lightly sedated with low-dose midazolam (0.3- 0.7 mg/kg – increased during the five days as necessary to keep each animal calm) and their eyes were covered to keep them calm. Thereafter a mask was placed over the snout and the mask was connected to a Pari Boy® classic nebulizer. The nebulizer chamber was initially filled with 4 mL IS or saline, and was continuously refilled (three times @ 2 mL) until 10 mL was administered after approximately 30 min. According to the manufacturer, the residual volume is approximately 1.2 mL, therefore the administered dose was ~8.8 mL. A pulse oximeter was connected to the tail of each animal to measure pulse and oxygen saturation; measurements, including counting of respiratory frequency, were noted after 5, 10, 15, and 20 minutes of inhalation. After the procedure, the animals were placed in a recovery box and observed until full recovery and thereafter guided back to their stall. The procedure was repeated daily for five days. On day five, the animals were euthanized after the last inhalation. Every morning before and afternoon after the procedure, all animals were scored to assess general condition, appetite, behavior, coughing, lung function, and mobility. Blood samples were drawn the first day before inhalation (baseline) and on the last day of inhalation after the inhalation. Standard biochemistry and hematology, including differential count, were performed. Routine necropsy with special attention to the respiratory system was performed following euthanasia by an experienced veterinary pathologist to observe potential macroscopic signs of toxicity in situ. Lungs and mediastinal lymph nodes were weighed. Samples for histopathology were collected proximally (including the main bronchus) and distally from the right cranial and the left caudal lung lobes, from the trachea, carina, mediastinal lymph nodes, heart (right and left ventricular muscles), kidney, and liver. The nasal passages were collected for histopathology by using a standardized approach to investigate three nasal levels. Lungs (including trachea, carina, bronchi, and bronchioles), lymph nodes (sub carinal), nasal passages (the squamous, transitional, respiratory, and olfactory epithelium covering the nasal opening, nasoturbinate, maxiloturbinate, vomernasal organ, ethmoturbinates and nasopharynx), liver, kidney, and heart were examined histologically. On a few occasions, a few coughs or a sneeze were heard in relation to inhalation or after removal of the mask from the snout; this was noted for one animal in the control group, two animals in the 50ppm group, and one animal in the 100ppm group. This could likely be a reaction to the humid local environment the mask creates around the snout. Since the incidence was similar between the groups, it is not considered to be attributable to IS. Respiratory rate, pulse, and oxygen saturation were similar between groups. Animals recovered from the mild sedation in maximum 10 minutes following dosing. No clinical signs were seen at the regular daily checks. Hematological and biochemical parameters’ development from baseline (Day 1 before inhalation) to after the experiment (Day 5 after inhalation) was unremarkable for all groups. Creatine kinase elevations which were seen in all groups are considered most likely due to struggling in relation to handling and blood collection. At necropsy, no apparent macroscopic signs of toxicity were observed. Pathological findings related to drug exposure were not observed in trachea, carina-area, and lungs. All minor macroscopic and microscopic findings seen in trachea, carina-area, and lungs were considered either related to the daily sedation procedure, non-successful attempts to sample blood from the jugular vein, euthanasia, or were incidental findings. For example, focal infiltration of alveolar macrophages, sometimes associated with mineralization were seen in some animals of all groups and is reported as a common finding in Göttingen minipigs. It has also been reported that euthanasia by pentobarbital can induce lung tissue damage including congestion, oedema, hemorrhage, emphysema, and necrosis based on studies in rats, mice, rabbits, guinea pigs, sheep, non-human primates, dogs, and cats, and consistency appears across species. Within the nose and nasopharynx, hyperemia, epithelial desquamation, loss of cilia, and lymphoid hyperplasia were seen within all three groups. The changes were most often seen in focal areas. For the nasopharynx more animals were registered with changes within both the 100 ppm and 50 ppm HOCl groups when compared to the saline group. However, the description of the lesions was similar across the groups and based on the low number of animals it was concluded that no clear difference can be seen between the three study groups. It should be noted that epithelial desquamation can be seen as an artifact of tissue sampling. It is concluded that mask inhalation of 100 and 50 ppm HOCl could not be associated with an increase in findings of significance compared with the saline control group. In conclusion, findings were seen in all groups, including the saline controls, but there were none in the IS treated animals that were considered to be attributable to IS. Based on this study, the NOAEL for IS was 100 ppm (8.8 mL) for administration by mask. Example 9.1.3: In vivo multi-dose safety study The IS has been tested in an in vivo inhalation model in minipigs to assess the maximum tolerated dose to aid the selection of doses in the subsequent GLP study. Healthy minipigs (n=6; 3 groups of 1 male and 1 female per group) were treated daily for seven days with aerosol concentrations of 1.2, 2.3, or 5.4 μg/L (using 50, 100, or 200 ppm HOCl + 0.25% acetic acid, pH 5.5, isotonic) by a mask covering the snout. Daily treatment duration was 60 minutes and each animal received respectively 19.9, 19.1 or 22.2 mL in the groups dosed with 50, 100 and 200 ppm of IS daily. Animals were euthanized on Day 8, following 7 days of inhalation of IS. During the study, clinical condition, body weight, food consumption, hematology (peripheral blood), blood chemistry, organ weights, macroscopic pathology and histopathology investigations were undertaken. HOCl concentrations, calculated from the achieved aerosol concentrations and nominal hypochlorous concentration of the formulations, were 99%, 92% and 110% of target for Groups 1, 2 and 3, respectively. There were no test item related effects on clinical condition, body weight, food consumption, hematology or blood chemistry parameters, or organ weights and there were no, item-related macroscopic pathology or histopathology findings. It was concluded that IS was well tolerated when administered to Göttingen minipigs via a face mask for 60 minutes per day for 7 consecutive days and that the 50, 100 or 200 ppm concentrations were considered appropriate for 60-minute daily exposures on longer term toxicity studies in Göttingen minipigs. Example 10: Cytotoxicity of wound irrigation solution (WIS) In vitro cytotoxicity of a Wound Irrigation Solution (WIS) at 200 ppm HOCl has been evaluated in two cytotoxicity studies. The objective of the studies was to determine whether WIS is toxic to cultured mammalian L929 cells in vitro. The tests comply with the methods described in ISO 10993-5 and the formulation of the test items were prepared in compliance with ISO 10993-12. The following subsections summarize the in vitro cytotoxicity studies. Example 10.1: In vitro cytotoxicity of WIS (200 ppm HOCl) The test item WIS (SOF 0001/05-01), containing 0.25% acetic acid and 200 ppm HOCl, pH: 4.3, was examined to determine the potential cytotoxic activity on cultured mammalian cells (mouse fibroblasts). The test was performed in accordance with the US Pharmacopeia, Method <87> and the ISO 10993-5 guidelines. A formulation of WIS (SOF 0001/05-01) was prepared with complete cell culture medium (Ham’s F12 medium supplemented with 10% fetal bovine serum and 50 μg/mL gentamycin). A diluent ratio of 0.2 g test item/mL diluent medium was used. This formulation was tested undiluted as well as diluted 1 part formulation + 3 parts fresh cell culture medium. Positive control (sodium lauryl sulphate (SLS), 0.2 mg/mL) and untreated control cultures (served also as negative control, treated with complete cell culture medium) were included in the study. Triplicate cell cultures were treated at each test point for 48 hours. The control treatments produced appropriate responses, demonstrating the correct functioning and sensitivity of the test 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). Under the test conditions of this study (prolonged exposure, 48 hours), undiluted WIS (0.25% acetic acid and 200 ppm hypochlorous acid, pH: 4.3), showed cytotoxic effects on cultured L929 cells. Based on these results, it is concluded that WIS 0.25% acetic acid and 200 ppm hypochlorous acid, pH: 4.3 did not pass the requirements of ISO 10993-5 and USP<87> as the cytotoxicity grade was >2. However, the diluted formulation of WIS (SOF 0001/05-01) showed no toxicity (cytotoxicity grade 0 in all cases). Example 10.2: In vitro cytotoxicity of WIS (200 & 448 ppm HOCl) In this study, the in vitro cytotoxicity of the WIS (200 ppm HOC1, 0.25% acetic acid), SOF 003/53 (448 ppm HOC1, 1% acetic acid) and SOF 003/51 (200 ppm HOC1, 1% acetic acid) formulations were evaluated. The applied in vitro assays measure the release of lactate dehydrogenase (LDH) from ruptured cell membranes and the metabolic activity (MTT) in the cell line NCTC clone 929 (L-929) after exposure towards the formulations for 1, 4, 24, and 48 hours. The assays were performed according to the EUNCL SOP (EUNCL-GTA-03). For all the tested formulations, no significant membrane rupture was measured at the tested concentrations (10-0.005%) and exposure periods (1, 4, 24, and 48 hours). According to the guidelines in the ISO-10993-5 international standard, none of the WIS formulations caused a cytotoxic effect (i.e., more than 30% reduction in cell viability) in the NCTC clone 929 (L-929) cells at the two shortest exposure periods (1 and 4 hours). After 24 and 48 hours of exposure, the WIS did not have a cytotoxic effect on the cells, (i.e., less than 30% reduction in cell viability) whereas the SOF 003/53 and SOF 003/51 formulations induced cytotoxicity at these timepoints (i.e., reduced the viability by 40-45% after 24 hours of exposure and by 55-60% after 48 hours respectively). Example 11: Genotoxicity of inhalation solution (IS) GLP in vitro studies with IS have been performed at Charles River Laboratories, Hungary. Example 11.1: In vitro bacterial reverse mutation assay An inhalation solution of the invention was tested for potential mutagenic activity using the Bacterial Reverse Mutation Assay. The study was performed according to GLP. The experiment was carried out using histidine-requiring auxotroph strains of Salmonella typhimurium (Salmonella typhimurium TA98, TA100, TA1535, and TA1537) and the tryptophan- requiring auxotroph strain of Escherichia coli (Escherichia coli WP2 uvrA) in the presence and absence of a post-mitochondrial supernatant (S9 fraction) prepared from the livers of phenobarbital/β-naphthoflavone-induced rats. The study included a Preliminary Compatibility Test and an Assay 1 (Plate Incorporation Method). The following concentrations were selected and provided by the Sponsor with appropriate documentation as follows: 50 ppm, 100 ppm, 200 ppm and 500 ppm, these are equivalent to 0.05, 0.1, 0.2 and 0.5 mg/mL. At the highest treatment volume (500 µL) these were equivalent to 25, 50, 100 and 250 µg/plate; these concentrations were used in Assay 1. Due to cytotoxicity, additional treatment plate concentrations were also used with lower treatment volumes per plate of the 50ppm test item concentration: 0.3162, 1.0, 3.162 and 10 µg/plate using treatment volumes of the supplied material at 6.3 µL, 20 µL, 63.2 µL and 200 µL, respectively. The maximum test concentration was 250 μg and the minimum was 0.3162 µg test item/plate (a total of eight concentrations). Inhibitory, cytotoxic effect of the test item (absent / slight reduced background lawn development) was observed in all examined bacterial strains without metabolic activation at 250, 100 and 50 µg/plate concentrations, and with metabolic activation at 250 µg/plate concentration. In the assay the number of revertant colonies did not show any biologically relevant increase compared to the solvent controls. There were no reproducible dose-related trends and there was no indication of any treatment-related effect. The reported data of this mutagenicity assay show that under the experimental conditions applied the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used, and therefore in conclusion, IS had no mutagenic activity under the test conditions used in this study. Example 11.2: In vitro mammalian cell micronucleus assay An inhalation solution of the invention was tested in an in vitro micronucleus test using mouse lymphoma L5178Y TK+/- 3.7.2 C cells. The study was performed according to GLP. Two assays were performed (Assay 1 and Assay 2). In both assays, a 3-hour treatment with metabolic activation (in the presence of S9-mix) and a 3-hour and 24-hour treatment without metabolic activation (in the absence of S9-mix) were performed. Sampling was performed 24 hours after the beginning of the treatment. The examined concentrations of the test item in Assay 1 (with and without metabolic activation) were selected and provided by the Sponsor as follows: 50 ppm, 100 ppm, 200 ppm and 500 ppm, these are equivalent to 0.05, 0.1, 0.2 and 0.5 mg/mL considering the treatment value which was 1 mL as determined in OECD No.487 guideline (10% (v/v) in the final treatment medium. In Assay 1, the study was terminated because excessive cytotoxicity of the test item was observed. The selected concentration intervals were not sufficiently refined to evaluate at least three test concentrations to meet the acceptability criteria (within the appropriate cytotoxicity range). Any result with a Relative Increase in Cell Count (RICC) of<~40% was not acceptable for the assay, the aim should be to have a cytotoxicity of approximately 40%-50% achieved in the assay to demonstrate the concentrations used were sufficient to meet the guideline criteria. Therefore, an additional experiment (Assay 2) was performed with modified and more closely spaced concentrations to give further information about the cytotoxic effects and to meet the regulatory acceptability criteria. The examined concentrations of the test item in Assay 2 (with and without metabolic activation) were the same as in Assay 1, however, additional lower treatment concentrations were applied. Therefore, acceptable concentrations of 10, 5 and 2 µg/mL (a total of three) were chosen for evaluation in case of the short treatment with metabolic activation, and concentrations of 6, 2 and 1 µg/mL (a total of three) were chosen for evaluation in case of the short treatment without metabolic activation, and concentrations of 7, 6, 2 and 1 ppm (a total of four) were chosen for evaluation in case of the long treatment without metabolic activation. None of the treatment concentrations caused a biologically or statistically significant increase in the number of micronucleated cells when compared to the appropriate negative (vehicle) control value in the experiments with and without metabolic activation. In conclusion, IS did not cause statistically or biologically significant reproducible increases in the frequency of micronucleated mouse lymphoma L5178Y TK+/- 3.7.2 C cells in the performed experiments with and without metabolic activation. Therefore, IS was considered as not being genotoxic in this test system under the conditions of the study. Example 12: Other toxicity studies Example 12.1: In vitro lung surfactant functionality An Inhalation Solution of the invention was tested in a simulated alveoli model to evaluate its’ effect on lung surfactant function. Lung surfactant reduces lung surface tension, allowing normal expansion and contraction during respiration. Inhalation of aerosols that interfere with the lung surfactant may induce a toxic response. The test method involved exposing a small volume of lung surfactant to nebulized IS during simulated breathing cycles while quantifying lung surfactant surface tension. Change in surface tension was evaluated. Method A previously well-described constant flow through set-up of a constrained drop surfactometer was used to test the product’s effect on lung surfactant function. This method has shown 100% sensitivity in detecting harmful substances when compared to in vivo studies. A drop of lung surfactant (10 µg) was exposed to nebulized 500 ppm HOCl IS (5 mL over five minutes) during simulated breathing cycles of the lung surfactant (to mimic an alveoli). The surface tension was evaluated continuously by axisymmetric drop shake analysis to detect potential critically low surface tension (below 10 mN/m) as would induce atelectasis in vivo. Results No inhibition of the lung surfactant function was measured when lung surfactant was exposed to nebulized inhalation product in the highest concentration (500 ppm HOCl + 0.25 % acetic acid, pH 5.5, isotonic). Similar results were obtained for 0.9% NaCl (control). Example 12.2: Ocular irritation test using the isolated chicken eye method Since the solution will be delivered to the mouth and nose, a study to investigate possible irritant properties to the eye was performed. A GLP study, Test for Ocular Irritation: Isolated Chicken Eye Method with Inhalation Solution (SIS) was performed according to the method described in guideline OECD 438. Four concentrations of IS were provided by the Sponsor with respectively 500, 200, 100 or 50 ppm hypochlorous acid (HOCl). The study was performed over 2 days and each day was referred to as an Experiment (i.e., Experiment 1 and Experiment 2). In each experiment, three eyes were treated with 30 µL of test item (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 in a similar way with 30 µL of 5% (w/v) Benzalkonium chloride solution. The negative control eye was treated with 30 µL of physiological saline (0.9% (w/v) NaCl solution). Corneal thickness, corneal opacity and fluorescein retention were measured and any morphological effects (e.g., pitting or loosening of the epithelium) were evaluated. The results from all eyes used in the study met the quality control standards. The negative control and positive control results were within the historical control data range the in experiment. Thus, the study was considered valid. According to the guideline, the outcome of this study is that the test substance is allocated to one of three categories; either non-irritant or severe irritant or that there is a requirement for further information. Based on this in vitro eye irritation assay in isolated chicken eyes with different concentrations of Inhalation Solution (SIS), the 500 ppm, 200 ppm and 100 ppm test item concentrations were classified as needing further information. An in vivo study is indicated at these concentrations. The 50ppm test item concentration was classified as non-irritant. Example 13: Antibacterial tests with inhalation solution (IS) An inhalation solution of the invention was tested in an antibacterial assay against planktonically-grown gram-positive (Staphylococcus aureus) and gram-negative (Pseudomonas aeruginosa) bacteria, and it showed efficient killing of both bacteria at concentrations of 10-25 ppm HOCl. The tests were performed at Costerton Biofilm Center, University of Copenhagen. The results of such tests are provided in Table 4 below.
Results: As seen in Table 4 antibacterial effect was seen already at 10 ppm HOCl for P. aeruginosa, and neither of the bacteria grew at 25 ppm HOCl. Summary: Inhalation solutions of the invention efficiently eradicates gram-positive (S. aureus) and gram-negative bacteria (P. aeruginosa) at HOCl concentrations of 25 ppm and 10 ppm, respectively, and above. Full effect for all bacteria was seen for IS 25 ppm HOCl. Based on these observations, it is evident that any potential bacterial contamination during the manufacturing of IS, will be eradicated immediately in the product by the broad-spectrum antimicrobial activity of HOCl. Thus, production of any endotoxins is highly unlikely since it takes bacteria to produce endotoxins. Example 14: Antiviral activity according to the standard EN 14476 of IS (starting with 25 ppm HOCl), against vaccinia virus EN 14476 for general virucidal activity is conducted on chemical disinfectants and antiseptics. This is a quantitative suspension test for the evaluation of virucidal activity in the medical area and is performed by an accredited laboratory. Results for IS: The test product of IS, 50 ppm as 50% dilution, 25 ppm HOCl, was able to inactivate the vaccina virus after 1 minute of exposure time under clean conditions (see Table 5).. Therefore, the activity was not measured after 30 or 60 minutes. The reduction factor was ≥4.25±0.33 (1 minute). According to the EN 14476 standard, products that have antiviral activity against the vaccinia virus are considered active against all enveloped viruses. Results for Hand and Surface Disinfectants: EN tests according to the biocidal product regulations were also performed on hand disinfectant and surface disinfectant solutions (HOCl, 200 ± 30 ppm, HAc 0.25%, pH 4.3). The results show antimicrobial efficiency against E. coli, fungi, yeast, and vaccina virus (data not shown). Summary: All EN tests show a fast and effective inactivation of yeast, fungi, bacteria, and viruses from 1 min to 30 sec according to the standard for hand disinfectant and surface disinfectants. The results of IS were not significantly different from the hand disinfectant solutions and indicate similar disinfecting properties, also at 25 ppm HOCl. Example 15: Antimicrobial effectiveness testing Challenge testing may be performed according to USP42-NF372S chapter<51> efficacy or Ph. Eur.5.1.3 antimicrobial preservation. solution batches (pH 4.3, representative HOCl batches to IS were challenged with various microorganisms and below, testing according to USP42-NF37 2S chapter<51> is presented in Table 6 below: Results and Summary: The acceptance criteria for antimicrobial efficacy test as described in USP 42-NF372S chapter <51> were met for all test microorganisms both at day 14 and day 28. In addition, the lowest concentration of IS may be tested according to USP42-NF372S chapter <51> for antimicrobial effectiveness. Example 16: Minimum inhibitory concentration (MIC) tests Determination of minimal inhibitory concentration (MIC) against five pathogenic bacterial strains was carried out by broth microdilution (dilutions of the highest concentration of the test substance, HOCl solution, pH 4.3, 100 ppm HOCl + 1% acetic acid and dilutions), representative to IS. Following incubation for 24 hours after the treatment in the microtiter tray, the optical density was measured to evaluate growth. Furthermore, the suspensions were plated on agar and controlled for growth the following day. All tests were performed at Biofilm Test Facility, University of Copenhagen, Faculty of Health and Medical Sciences, Department of Immunology and Microbiology. Results: For all microorganisms tested the MIC was 25 ppm HOCl + 0.25% acetic acid. The growth was determined by both optical density (plate reader) and by growth on Müeller Hinton agar plates. Conclusion on Microbiological attributes of IS Results of the antimicrobial tests carried out with the inhalation solution of the invention (IS) product, starting at 10 ppm HOCl, as well as other representative HOCl formulations, clearly support that the IS product has excellent antimicrobial activity, and thus we are confident that also IS should be delivered devoid of any microorganisms, as for the other, representative products. This is due to the broad-spectrum antimicrobial activity of HOCl in the products, both at pH 4.3 and pH 5.5 (SIS), the acid form of HOCl in the solution is heavily dominating (≥99.1%). This is also supported by the literature reporting on the antimicrobial activity of HOCl (the acid form) and that HOCl has been used as a preservative to inhibit microbial growth in various health care products (e.g., wound irrigation/rinse solutions) already approved and sold on the market. Therefore, the IS is produced in a non-aseptic, non-sterile facility.
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