Nitric oxide foam for use in treating skin wounds

Abstract

Disclosed herein is a method for treating skin wounds in a patient in need of such treatment, comprising applying a foamed composition comprising nitric oxide to the area of skin requiring such treatment. The wound may be selected from the group consisting of skin ulcers, including diabetic foot ulcers (DFUs), venous leg ulcers (VLUs), arterial ulcers, decubitus (pressure) ulcers or other pressure injuries, deep tissue injuries (DTIs), burns, abrasions, skin tears, or bruises.

Description

Attorney Docket No.: 274507 / NOXY24-001WO / 564918 Method of Treating Skin Wounds Using Nitric Oxide Foam Cross Reference to Related Application

[0001] The present application claims priority under 35 U.S.C. § 119(e) to U.S. ProvisionalApplication 63 / 568,730, filed on March 22, 2024, the entire contents of which is incorporated herein by reference. Background

[0002] A wound is a disruption of the normal structure and function of the skin and soft tissuearchitecture. Depending on the physiology and healing processes, wounds can be either acute or chronic. Chronic wound bioburden plays a significant role in development of infection-related complications and impaired healing (Armstrong DG, 2020). Diabetes is associated with an increased risk for atherogenesis and vascular inflammation, caused by hyperglycemia, excess free fatty acids, insulin resistance, and other factors. Diabetic foot ulcers (DFUs) are a major cause of morbidity (Armstrong, 2017), and account for at least two-thirds of all nontraumatic amputations performed in the US (Ramsey, 1999; Gregg, 2004).

[0003] Nitric oxide (NO) is an important cellular signaling, naturally occurring, and potentantimicrobial, gaseous molecule found in all mammalians (Schairer, 2012). It is an FDA approved vasodilator that selectively dilates the pulmonary vasculature when administered by inhalation. Additionally, it has been demonstrated that NO is essential in the wound healing process (Malone-Povolny, 2019). Nitric oxide has been employed to treat skin wounds. For example, in 2004, Chris Miller reported the use of NO gas to treat leg ulcers. According to the report, gaseous NO was applied to the leg of a patient using a “single patient use” plastic boot. The boot was equipped with a delivery system that administered 200 ppm NO gas for an average of 8.1 hours. After 14 consecutive nights of treatment, the ulcer was significantly reduced in size. Miller, J Cutan Med Surg, 2004 Jul-Aug;8(4):233-8. doi: 10.1007 / s10227-004-0106-8. In US Patent Publication 2005 / 0191372, Chris Miller further discloses NO gas bathing units, including an inflatable bag-like bathing unit in the shape of a boot or mitten or glove that can be placed over a patient’s foot or hand, respectively. Other reports relating to the use of NO to treat skin conditions abound.

[0004] Miller illustrates the challenges of using NO gas for wound treatment. NO gas isreported to be toxic at higher concentration (greater than exposures of 2000 ppm). In Miller’s 1 59870042.1Attorney Docket No.: 274507 / NOXY24-001WO / 564918 bathing units, it is impossible to control the directionality of NO gas diffusion to just the site on the skin requiring treatment. Treatment effects are thus due to serendipitous absorption of NO. As a result, Miller’s method and device requires long treatment times and high concentrations of NO gas, increasing the risk of toxic exposure.

[0005] To avoid the issues posed by NO gas treatment, therapeutic media such as creams andointments have been developed that generate NO in situ from the reaction of nitrite with acid. These creams and ointments are two-part formulations. The first formulation contains the nitrite, and the second formulation contains the acid. When the two formulations are mixed, for instance on skin that requires treatment, the acid and nitrite react to form NO gas, which then diffuses to the skin. Zhu H, Ka B, Murad F, World J. Surg.2007, 31, 624. Zhu H, Wei X, Bian K, Murad F, J. Burn Care Res.2008, 29, 804. Researchers have also utilized foams for NO delivery to skin requiring treatment. In US Patent No.10,751,365 Miller, Hill and Bell disclose a two-part liquid formulation that provides NO to the skin. The first formulation contains nitrite and a surfactant, and the second formulation contains acid and a surfactant. The two formulations can be agitated to produce foams. When the two foamed formulations are mixed, the acid and nitrite react to form NO which is present as NO-containing bubbles in the foam, which then can be placed on skin requiring treatment.

[0006] However, challenges remain with using NO-containing foam as a delivery medium fortreating and reducing the size and severity of skin wounds, including skin ulcers, pressure injuries, deep tissue injuries, burns, abrasions, skin tears, bruises, and the like, and by extension, disrupting biofilms on the skin, eradicating bacterial skin infections, and reducing skin inflammation. Summary of the Invention

[0007] These and other needs are met by the present invention which is directed to a method oftreating a wound in a patient in need thereof, wherein the method comprises applying a foamed composition comprising nitric oxide to the wound. The wound may be selected from the group consisting of diabetic foot ulcers (DFUs), venous leg ulcers (VLUs), arterial ulcers, decubitus (pressure) ulcers or other pressure injuries, deep tissue injuries (DTIs), burns, abrasions, skin tears, and bruises (hereinafter the wounds disclosed herein). Brief Description of the Figures 2 59870042.1Attorney Docket No.: 274507 / NOXY24-001WO / 564918

[0008] FIG. 1 depicts the log reduction in bacteria concentration after NO foam treatment.

[0009] FIG. 2 depicts the mechanism of growth factor regulated skin wound healing, disruptionof growth factor regulated skin wound healing, and the destruction of the extracellular skin matrix associated with skin wounds and skin healing.

[0010] FIG. 3 depicts the reduction of proteolytic activity when exposed to nitric oxide foam.

[0011] FIG. 4A depicts the ability of nitric oxide foam to inhibit MMP-1.

[0012] FIG. 4B depicts the ability of nitric oxide foam to inhibit MMP-2.

[0013] FIG. 4C depicts the ability of nitric oxide foam to inhibit MMP-8.

[0014] FIG. 4D depicts the ability of nitric oxide foam to inhibit MMP-9.

[0015] FIG. 4E depicts the ability of nitric oxide foam to inhibit 4197 bacterial collagenase.

[0016] FIG 4F depicts the ability of nitric oxide foam to inhibit human neutrophil elastase.

[0017] FIG. 5 summarizes data demonstrating the ability of nitric oxide foam to heal skinwounds in patients.

[0018] FIG. 6 depicts the process by which a combination of nitric oxide foam andbenzalkonium chloride heal skin wounds.

[0019] FIG. 7 summarizes data from an additional study demonstrating the ability of nitric oxidefoam to heal skin wounds in patients.

[0020] FIG. 8 summarizes data from an additional study demonstrating the ability of nitric oxidefoam to heal skin wounds in patients.

[0021] FIG. 9 summarizes data from an additional study demonstrating the ability of nitric oxidefoam to heal skin wounds in diabetes patients. Detailed Description Definitions

[0022] The following table of abbreviations is provided.Abbreviation Term3 59870042.1Attorney Docket No.: 274507 / NOXY24-001WO / 564918 Abbreviation Term AST Aspartate Aminotransferase

[0023] A “skin ulcer” is a sore on the skin or a mucous membrane, accompanied by thedisintegration of tissue. Skin ulcers can result in complete loss of the epidermis and often 4 59870042.1Attorney Docket No.: 274507 / NOXY24-001WO / 564918 portions of the dermis and even subcutaneous fat and are most common on the skin of the lower extremities. An ulcer that appears on the skin is often visible as an inflamed tissue with an area of reddened skin. A skin ulcer is often visible in the event of exposure to heat or cold, irritation, or a problem with blood circulation. Skin ulcers can be characterized as decubitus (pressure) ulcers, venous skin ulcers, arterial (ischemic) skin ulcers, diabetic ulcers, and neuropathic skin ulcers. Skin ulcers also include buruli ulcers (caused by Mycobacterium ulcerans) and stasis dermatitis.

[0024] “Pressure sores” are pressure injuries that occur on areas of the skin that are underpressure and can also be referred to as bed sores, pressure sores, or decubitus ulcers.

[0025] “Deep tissue injuries,” which may lead to pressure ulcers, occur from damage or shear tounderlying soft tissue of the skin. Embodiments Skin Wounds In one aspect, disclosed herein is a method of treating a wound in a patient in need thereof. The method comprises applying a foamed composition comprising nitric oxide to the wound. The wound may be selected from the group consisting of a skin ulcer, which may be a diabetic foot ulcer (DFU), venous leg ulcer (VLU), or arterial ulcer. In another embodiment, the wound may be a pressure injury, burn, deep tissue injury (DTI), abrasion, skin tear, or bruise. In another embodiment, the wound is selected from the group consisting of diabetic foot ulcers (DFUs), veinous leg ulcers (VLUs), pressure injuries, burns, traumatic wounds, deep tissue injuries (DTIs), abrasions, lacerations, skin tears, bruises, and arterial ulcers.

[0026] In one embodiment, the skin ulcer is a pressure ulcer. In another embodiment, the skinulcer is a diabetic ulcer. In another embodiment, the skin ulcer is a venous skin ulcer. In another embodiment, the skin ulcer is an arterial (ischemic) skin ulcer. In a further embodiment, the skin ulcer is a neuropathic skin ulcer. In a further embodiment, the skin ulcer is a Buruli ulcer caused by Mycobacterium ulceran). In another embodiment, a skin ulcer may be caused by stasis dermatitis. Foamed Composition

[0027] The foamed composition used to treat the wounds as disclosed herein employs a nitricoxide-laden, micro-bubble foam to create a substantially air-tight barrier that effectively isolates 5 59870042.1Attorney Docket No.: 274507 / NOXY24-001WO / 564918 the skin to be treated and that minimizes escape of NO to the surrounding environment. Overall, the specified nitric oxide foam may be considered as a temporary dilute dispersion of bubbles, distorted to form polyhedral cells with relatively thin, flat walls, wherein the polyhedral cells are almost regular dodecahedra with Plateau borders. The foamed composition, comprised of a plethora of thin-filmed, micro (to nano) sized bubbles, form a transitional and substantially impermeable bubble system, held together by electro-static and surface tensioning forces.

[0028] In one embodiment, the foamed composition may be a liquid foam, for example, anassociation of cellular materials, for example, gas bubbles, surrounded by a continuous liquid film.

[0029] In one embodiment, the topical foam is comprised of volumes of acidic and nitrite ionsolutions that, when combined, form a foam containing nitric oxide gas bubbles created at a pre- determined reaction rate between the acidic and nitrite solutes. The nitric oxide gas bubbles are internally isolated within the foam. The foam clusters around the nitric oxide gas bubbles creates a barrier that slows diffusion of nitric oxide gas from the foam to the surrounding environment. The foam thus provides a therapeutic covering for, and isolation of, an application site on the skin, ensuring it is free from ambient interference and environmental contaminants such as air- or waterborne pathogens. In effect, the foam is an airtight cover that efficiently protects and allows for the efficient administration of NO to skin in need of treatment.

[0030] Once applied to the skin, the foam forms a barrier overlay of the injury site that traps NObubbles and prevents them from escaping to the surrounding environment, and that naturally compels the film of the bubbles containing NO that are next to the skin surface to rupture due to shock, vibration, temperature gradients, skin texture, or the presence of natural surface tension modifiers, thus liberating nitric oxide gas at sites requiring treatment. Ultimately the foam provides a medium for short or prolonged moistening and administering of nitric oxide to the skin, thereby preparing the application site for sterile bandaging.

[0031] Nitric oxide bubble clusters contained within the foam are optimally suited to coverirregular surfaces that are typical of the wounds disclosed herein. The result is that sites on the skin requiring treatment can be completely covered with a layer of therapeutic nitric oxide bubbles which in turn delivers NO gas to substantially every facet of wound geography. Each bubble is of a predetermined and uniform density. When taken together, the bubbles may 6 59870042.1Attorney Docket No.: 274507 / NOXY24-001WO / 564918 provide an air-tight network over and above the application site, which, apart from the nitric oxide gas itself, may limit exogenous microscopic to nanoscopic elements from either entering, leaving, or disrupting the barrier. These bubbles are designed to be relatively durable to house the nitric oxide gas that is generated in situ, protecting the gas from premature dispersion, as well as carrying, transporting, and offloading individual nitric oxide gas payloads on cue to sites on the skin that require treatment.

[0032] These isolating steps are preparatory to a systematic flooding of a site on the skinrequiring treatment with nitric oxide, thereby effectively and efficiently promoting in situ orthogonal diffusion of the gas into skin tissue. This may result in disrupting biofilms on the skin and treating bacterial skin infections and inflammatory skin conditions, as well as to aide in wound healing. Moreover, since nitric oxide is a signaling molecule and a known vasodilator, increasing the likelihood of nitric oxide absorption through the skin can be harnessed to treat other pathologies in the body, in addition to those associated with the outer layers of the skin.

[0033] The nitric oxide containing foams used in the treatment method can be prepared as hereindescribed. The components include an acid reactant, one or more surfactants, and a source of nitric oxide (a nitrite reactant).

[0034] The acid is typically a protic acid having a pH of 2 to a pH of 6. The acid can be selectedfrom the group consisting of citric acid, lactic acid, salicylic acid, phosphoric acid, ascorbic acid, hydrochloric acid, acetic acid, hyaluronic acid, and hypochlorous acid and combinations thereof.

[0035] The surfactant is a pharmaceutically acceptable cationic surfactant that can be usedtopically on the skin. The surfactants may be a cationic surfactant and may be selected from cetyl trimethyl ammonium bromide, cetrimonium bromide, dodecylbenzenesulfonic acid, cetylpyridinium chloride, stearalkonium chloride, polyquaternium-7, cocamidopropyl betaine, coco betaine, lauryl dimethyl ammonium chloride, polyquaternium-10, behentrimonium chloride, and cetrimonium chloride. In one embodiment, the surfactant is coco betaine.

[0036] The nitrite reactant is a nitrite salt selected from the group consisting of sodium nitrite,calcium nitrite, potassium nitrite, and ammonium nitrite. The nitric oxide source is typically sodium nitrite or potassium nitrite.

[0037] Solutions employed to make the nitric oxide topical foam composition comprise twosolutions, both of which are batch scalable. A first solution may comprise approximately 100 7 59870042.1Attorney Docket No.: 274507 / NOXY24-001WO / 564918 mL of water, 6 g of citric acid, and 3 g of cationic surfactant. A second solution may comprise approximately 100 mL of water, 10 g of sodium nitrite, and 1 g of cationic surfactant.

[0038] Alternatively, a first solution may comprise approximately 100 mL of water, 5 g of lacticacid, 8 g of citric acid, and 3 g of cationic surfactant. A second solution may comprise approximately 100 mL of water, 10 g of sodium nitrite, 2 g of sodium bicarbonate, and 1 g of cationic surfactant.

[0039] Alternatively, the first solution may comprise approximately 100 mL of water, 5 g oflactic acid, 6 g of citric acid, and 6 g of cationic surfactant. A second solution may comprise approximately 100 mL of water, 10 g of sodium nitrite, and 2 g of cationic surfactant.

[0040] Alternatively, a first solution may comprise approximately 100 mL of water, 5 g of lacticacid, 8 g of citric acid, and 6 g of cationic surfactant. A second solution may comprise approximately 100 mL of water, 10 g of sodium nitrite, 2 g of sodium bicarbonate, and 1 g of cationic surfactant.

[0041] Alternatively, a first solution may comprise approximately 50-99 g of water, 0.1 to 50 gof a cationic surfactant, and 9 to 15 g of citric acid. A second solution may comprise approximately 50 to 99 g of water, 0.1 to 50 g of cationic surfactant, and 17 to 23 g of sodium nitrite.

[0042] Alternatively, a first solution may comprise approximately 80 to 99 g of water, 1 to 20 gof a cationic surfactant, and 10 to 14 g of citric acid. A second solution may comprise approximately 80 to 99 g of water, 1 to 20 g of cationic surfactant, and 18 to 22 g of sodium nitrite. In this and other embodiments, the cationic surfactant is coco betaine.

[0043] Alternatively, a first solution may comprise approximately 96 to 100 g of water, 8 to 12 gof a cationic surfactant, and 11 to 13 g of citric acid. A second solution may comprise approximately 96 to 100 g of water, 2 to 5 g of cationic surfactant, and 19 to 21 g of sodium nitrite.

[0044] Alternatively, a first solution may comprise approximately 99 to 100 g of water, 10.5 to11.5 g of a cationic surfactant, and 11.5 to 12.5 g of citric acid. A second solution may comprise approximately 99 to 100 g of water, 3 to 4 g of cationic surfactant, and 19 to 20 g of sodium nitrite.

[0045] As noted above, in these solutions, increasing the surfactant concentration can lead to8 59870042.1Attorney Docket No.: 274507 / NOXY24-001WO / 564918 more robust foams that enhance nitric oxide exposure times. Thus, in one embodiment, the total surfactant concentration (w / w%) of the combined first solution and second solutions may be about 0.1 to 50.0 percent. In another embodiment, the total surfactant concentration (w / w%) of the combined first solution and second solutions may be increased from about 1.0 to 2.0 percent to about 2.1 to 50.0 percent. In another embodiment, the total surfactant concentration (w / w%) of the combined first solution and second solutions may be increased from about 1.0 to 2.0 percent to about 2.1 to 30.0 percent. In another embodiment, the total surfactant concentration (w / w%) of the combined first solution and second solutions may be increased from about 1.0 to 2.0 percent to about 2.1 to 10.0 percent. In another embodiment, the total surfactant concentration (w / w%) of the combined first solution and second solutions may be increased from about 1.0 to 2.0 percent to about 2.1 to 6.0 percent.

[0046] In a further embodiment, the foamed composition comprises:a first solution comprising water, at least one nitrite reactant, and a cationic surfactant; a second solution comprising water, at least one acidic reactant, and the cationic surfactant; and a foam which is the foamed composition, obtained by mixing the first solution and the second solution.

[0047] In this and other embodiments, the nitrite reactant is sodium nitrite, the acidic reactant iscitric acid, and the surfactant is coco betaine.

[0048] In a further embodiment, the foamed composition comprises:a first solution comprising water, at least one nitrite reactant, and a cationic surfactant; a second solution comprising water, at least one acidic reactant, and the cationic surfactant; a first foam obtained by aerating the first solution; a second foam obtained by aerating the second solution; and a foam which is the foamed composition, obtained by mixing the first foam and the second foam.

[0049] In a further embodiment, the effective amount of the foamed composition per cm2comprises 0.01 to 0.99 g of an acidic solution comprising a cationic surfactant and 0.01 to 0.99 g of a sodium nitrite solution comprising a cationic surfactant. 9 59870042.1Attorney Docket No.: 274507 / NOXY24-001WO / 564918

[0050] In a further embodiment, the effective amount of the foamed composition per cm2comprises 0.05 to 0.9 g of an acidic solution comprising a cationic surfactant and 0.05 to 0.9 g of a sodium nitrite solution comprising a cationic surfactant.

[0051] In a further embodiment, the effective amount of the foamed composition per cm2comprises 0.08 to 0.8 g of an acidic solution comprising a cationic surfactant and 0.08 to 0.8 g of a sodium nitrite solution comprising a cationic surfactant.

[0052] In a further embodiment, the effective amount of the foamed composition per cm2comprises 0.09 to 0.9 g of an acidic solution comprising a cationic surfactant and 0.09 to 0.9 g of a sodium nitrite solution comprising a cationic surfactant.

[0053] In a further embodiment, the effective amount of the foamed composition per cm2comprises 0.1 to 0.8 g of an acidic solution comprising a cationic surfactant and 0.1 to 0.8 g of a sodium nitrite solution comprising a cationic surfactant.

[0054] In a further embodiment, the effective amount of the foamed composition per cm2comprises 0.1 to 0.7 g of an acidic solution comprising a cationic surfactant and 0.1 to 0.7 g of a sodium nitrite solution comprising a cationic surfactant.

[0055] In a further embodiment, the effective amount of the foamed composition per cm2comprises 0.1 to 0.6 g of an acidic solution comprising a cationic surfactant and 0.1 to 0.6 g of a sodium nitrite solution comprising a cationic surfactant.

[0056] In a further embodiment, the effective amount of the foamed composition per cm2comprises 0.1 to 0.5 g of an acidic solution comprising a cationic surfactant and 0.1 to 0.5 g of a sodium nitrite solution comprising a cationic surfactant.

[0057] In a further embodiment, the effective amount of the foamed composition per cm2comprises 0.2 to 0.4 g of an acidic solution comprising a cationic surfactant and 0.2 to 0.4 g of a sodium nitrite solution comprising a cationic surfactant.

[0058] In a further embodiment, the effective amount of the foamed composition per cm2comprises 0.25 to 0.35 g of an acidic solution comprising a cationic surfactant and 0.25 to 0.35 g of a sodium nitrite solution comprising a cationic surfactant.

[0059] In a further embodiment, the effective amount of the foamed composition comprises 0.01to 0.99 g of an acidic solution comprising a cationic surfactant and 0.01 to 0.99 g of a sodium nitrite solution comprising a cationic surfactant. 10 59870042.1Attorney Docket No.: 274507 / NOXY24-001WO / 564918

[0060] In a further embodiment, the effective amount of the foamed composition comprises 0.05to 0.9 g of an acidic solution comprising a cationic surfactant and 0.05 to 0.9 g of a sodium nitrite solution comprising a cationic surfactant.

[0061] In a further embodiment, the effective amount of the foamed composition comprises 0.08to 0.8 g of an acidic solution comprising a cationic surfactant and 0.08 to 0.8 g of a sodium nitrite solution comprising a cationic surfactant.

[0062] In a further embodiment, the effective amount of the foamed composition comprises 0.09to 0.9 g of an acidic solution comprising a cationic surfactant and 0.09 to 0.9 g of a sodium nitrite solution comprising a cationic surfactant.

[0063] In a further embodiment, the effective amount of the foamed composition comprises 0.1to 0.8 g of an acidic solution comprising a cationic surfactant and 0.1 to 0.8 g of a sodium nitrite solution comprising a cationic surfactant.

[0064] In a further embodiment, the effective amount of the foamed composition comprises 0.1to 0.7 g of an acidic solution comprising a cationic surfactant and 0.1 to 0.7 g of a sodium nitrite solution comprising a cationic surfactant.

[0065] In a further embodiment, the effective amount of the foamed composition comprises 0.1to 0.6 g of an acidic solution comprising a cationic surfactant and 0.1 to 0.6 g of a sodium nitrite solution comprising a cationic surfactant.

[0066] In a further embodiment, the effective amount of the foamed composition comprises 0.1to 0.5 g of an acidic solution comprising a cationic surfactant and 0.1 to 0.5 g of a sodium nitrite solution comprising a cationic surfactant.

[0067] In a further embodiment, the effective amount of the foamed composition comprises 0.2to 0.4 g of an acidic solution comprising a cationic surfactant and 0.2 to 0.4 g of a sodium nitrite solution comprising a cationic surfactant.

[0068] In a further embodiment, the effective amount of the foamed composition comprises 0.25to 0.35 g of an acidic solution comprising a cationic surfactant and 0.25 to 0.35 g of a sodium nitrite solution comprising a cationic surfactant.

[0069] The foamed composition disclosed herein can be formed in a number of ways known tothe skilled person. For example, the first and second solutions can be agitated, which can include agitation by whipping, pumping, shaking, or the like, to each form a foam, and then these 11 59870042.1Attorney Docket No.: 274507 / NOXY24-001WO / 564918 resultant foams can be mixed together using a stirring rod, spatula, or other common device. For example, the first and second solutions are available in first and second plastic bottles that can be shaken by hand to produce foams. The two foams are mixed, typically again by hand.

[0070] When a portion of the first and second solutions are mixed, a resultant foam is producedthat contains nitric oxide which can be readily applied to the skin. The resultant foam is a carrier for the generation of nitric oxide due to the reaction of sodium nitrite with acid, according to the reaction sequence shown below.

[0071] Themayeffectively provide a barrier that helps to keep the nitric oxide contained near the skin, to promote topical treatment and healing of wounds as described herein and also including cuts and scrapes, as well as to disrupting biofilms on the skin, as well as treating bacterial skin infections and inflammatory skin conditions, and promoting the absorption of nitric oxide through the skin. Although not bound by theory, a bubble containing nitric oxide gas may burst near the skin due to shock vibrations, irregularities in the surface of the skin, temperature, or another perturbation, allowing the nitric oxide to be absorbed transdermally. The foam may be placed on the skin and then allowed to collapse as opposed to being rubbed in.

[0072] The foam carrier may provide a relatively easy process for metering the amount of nitricoxide generated and put in contact with the skin as needed to treat wounds as disclosed herein. The foam carrier may also provide a method that uses less media as compared to other media. The resulting foams can be evaluated using a number of protocols and can be tailored to meet various therapeutic needs.

[0073] In a further embodiment, the foamed composition can prolong the protection and othereffects of nitric oxide, by creating an impermeable barrier that covers the area needed to treat the wounds disclosed herein. Other Composition Components

[0074] A further aspect of the invention relates to a treatment combination according to any oneof the previous embodiments of the nitric oxide topical foam formulation, comprising a second 12 59870042.1Attorney Docket No.: 274507 / NOXY24-001WO / 564918 active ingredient which is a topical dermatological agent. The second active ingredient which is a topical dermatological agent can be incorporated in the nitric oxide topical foam formulation or may be comprised in a separate formulation.

[0075] In case the nitric oxide topical foam formulation and the second active ingredient arecomprised in separated formulations, they can be administered simultaneously, sequentially or separately; preferably the separated formulations are administered simultaneously or sequentially, especially sequentially. In case the nitric oxide topical foam formulation is for example administered once daily and the second active ingredient once daily, then the separated compositions are preferably administered one time per day simultaneously or sequentially, especially sequentially. If administered sequentially or separately, the separated compositions may be administered in one or the other order. The number of administrations per day may be the same or different for the separated pharmaceutical compositions. For instance, one composition may be administered twice daily, and the other composition may be administered once or twice daily. Preferably the nitric oxide topical foam formulation is administered once daily and the composition comprising the second active ingredient is administered twice daily. Further, the separated formulations are administered by topical application to the skin.

[0076] In an additional aspect, the nitric oxide topical foam formulation of the invention can beused with one or more additional topical dermatological agents, wherein the one or more additional topical dermatologic agents is incorporated in the nitric oxide topical foam formulation or is in a separate formulation as described above.

[0077] The one or more additional topical dermatological agents does one or more of modifyingthe treatment profile of the foam, modifying the physical properties of the foam, modifying the chemical properties of the foam, or modifying the surface properties of the application site. Since the nitric oxide topical foam formulations disclosed herein have prolonged exposure times, they can also prolong the exposure times for other topical dermatological agents.

[0078] “Topical dermatological agent” means a therapeutic agent that can be applied directly onthe skin to treat skin conditions. Types of dermatological agents include topical agents for miscellaneous use, topical acne agents, topical anesthetics, topical anti-infectives, topical anti- rosacea agents, topical antibiotics, topical antifungals, topical antihistamines, topical antineoplastics, topical antipsoriatics, topical antivirals, topical astringents, topical debriding 13 59870042.1Attorney Docket No.: 274507 / NOXY24-001WO / 564918 agents, topical, depigmenting agents, topical emollients, topical keratolytics, topical non- steroidal anti-inflammatories, topical photochemotherapeutics, topical rubefacients, topical steroids, topical steroids with anti-infectives, and topical antiseptics.

[0079] Miscellaneous dermatologic agents are used to treat a variety of skin conditions. Non-limiting examples of miscellaneous topical dermatologic agents used to treat skin conditions include aluminum chloride hexahydrate, crisaborole, eflornithine, tacrolimus, pimecrolimus, minoxidil, glycopyrronium, menthol, zinc oxide, coal tar, capsaicin, selenium sulfide, bimatoprost, diphenhydramine, hydrocortisone, sodium hyaluronate, salicylic acid, Vitamin E, bexarotene, mequinol / tretinoin, becaplermin, dexpanthenol, alitretinoin, and calamine.

[0080] Topical acne agents include antiseptic washes that contain ingredients to gently cleansethe skin; and creams, lotions, or gels that exfoliate the skin, inhibit bacterial growth, speed up skin cell renewal or decrease the formation of comedones. Non-limiting examples of topical acne agents include adapalene, benzoyl peroxide, clindamycin, dapsone, tretinoin, azelaic acid, tazarotene, salicylic acid, clascoterone, erythromycin, and resorcinol.

[0081] Topical anesthetics are medicines that numb and reduce the sensation of pain in the areato which they are applied. Non-limiting examples of topical anesthetics include lidocaine, pramoxine, phenol, benzocaine, dibucaine, xylocaine, tetracaine, prilocaine, and dyclonine.

[0082] Topical anti-infective agents act by either killing or inhibiting the spread of infectiousagents. They include antibiotics, antibacterial, antifungal and antiviral agents. Non-limiting examples of topical anti-infectives include docosanol, ivermectin, imiquimod, hydrogen peroxide, crotamiton, spinosad, cadexomer iodine, silver, malathion, piperonyl butoxide, pyrethrins, permethrin, sinecatechins, abametapir, acetic acid, iodoquinol, nitrofurazone, and chloroxine.

[0083] Topical anti-rosacea agents are used for the treatment of inflammatory papules, pustulesand erythema of rosacea. Non-limiting examples of antirosacea agents include ivermectin, brimonidine, oxymetazoline, and azelaic acid.

[0084] Antibiotics are medicines that destroy or inhibit the growth of susceptible bacteria. Non-limiting examples of topical antibiotics include mupirocin, sulfacetamide sodium / sulfur, retapamulin, silver sulfadiazine, ozenoxacin, erythromycin, polymyxin b, pramoxine, mafenide, gentamicin, and mupirocin. 14 59870042.1Attorney Docket No.: 274507 / NOXY24-001WO / 564918

[0085] Topical antifungals are products that treat fungal infections caused by dermatophytes,yeasts, or mold. Non-limiting examples of topical antifungals include efinaconazole, tavaborole, ketoconazole, terbinafine, undecylenic acid, ciclopirox, nystatin, econazole, ciclopirox, naftifine, oxiconazole, terbinafine, clotrimazole, sertaconazole, tolnaftate, butenafine, luliconazole, and sulconazole.

[0086] Topical antihistamines are products that have been manufactured for use on the skin, inthe nose, or in the eye. They contain antihistamines which are medicines that block histamine release from histamine-1 receptors and are used to treat the symptoms of an allergic reaction such as edema (swelling), itch, inflammation (redness), sneezing, or a runny nose or watery eye. Non-limiting examples of topical antihistamines include doxepin and diphenhydramine.

[0087] Topical antineoplastics work by different mechanisms to prevent the development andspread of neoplastic cells that characterize cancers such as melanoma. Non-limiting examples of topical antineoplastics include imiquimod, fluorouracil, ingenol, ruxolitinib, tirbanibulin, and mechlorethamine.

[0088] Topical antipsoriatics are agents, which are applied on the skin surface to treat psoriasis.Non-limiting examples of topical antipsoriatics include betamethasone, calcipotriene, tazarotene, and halobetasol.

[0089] Topical antiviral agents are applied locally to treat viral infections. Non-limitingexamples of topical antivirals include penciclovir and acyclovir.

[0090] Topical astringents are agents that cause skin cells or mucus membranes to contract orshrink, by precipitating proteins from their surface. When applied topically they dry, harden and protect the skin. A non-limiting example of a topical astringent is witch hazel.

[0091] Topical debriding agents are chemicals that are used locally to clean an open wound byremoving foreign material and dead tissue, so that the wound heals without increased risk of infection. This accelerates healing. Non-limiting examples of topical debriding agents include collagenase, balsam peru combined with castor oil and trypsin, and anacaulase.

[0092] Topical depigmenting agents work in different ways to inhibit melanogenesis (thepigmentation pathway by which cells produce melanin). Some agents cause reversible depigmentation and some cause irreversible depigmentation. These agents are applied on the skin, on the affected area to treat hyperpigmentation. Non-limiting examples of topical 15 59870042.1Attorney Docket No.: 274507 / NOXY24-001WO / 564918 depigmenting agents include fluocinolone combined with hydroquinone and tretinoin, and monobenzoin.

[0093] Topical emollients, or moisturizers, contain ingredients that soothe and soften the skin. .Non-limiting examples of topical emollients include salicylic acid / urea, ammonium lactate, urea, vitamin a, d, and e, petrolatum, lanolin, and aloe vera.

[0094] Topical keratolytics are agents that are applied on the skin to soften the keratin. Theyloosen and assist exfoliation of the skin cells. Keratolytics also help the skin to bind moisture and is useful in treating dry skin conditions. They are used to treat psoriasis, acne, warts, corns and other forms of keratosis. Non-limiting examples of topical keratolytincs include podofilox, salicylic acid, and trichloroacetic acid.

[0095] Topical non-steroidal anti-inflammatories (often abbreviated to NSAIDs) are creams,gels, rubs, solutions or sprays that contain a nonsteroidal anti-inflammatory agent and are designed to be applied directly to the skin overlying a painful joint or area of bone. They are used to relieve pain and to treat symptoms of arthritis such as inflammation, swelling, and stiffness. Topical NSAIDs may also be used in the treatment of actinic keratosis (a precancerous patch of thick, scaly or crusted skin). Non-limiting examples of topical NSAIDs include diclofenac, capsaicin / diclofenac, and diclofenac / lidocaine.

[0096] Topical photochemotherapeutics make skin more sensitive to light. They work by causinga reaction with light that can destroy certain types of diseased skin cells. They may be used in the treatment of vitiligo or actinic keratosis in combination with light treatment. Non-limiting examples of topical photochemotherapeutics include aminolevulinic acid, methoxsalen, methylaminolevulinate, and aminolevulinic acid.

[0097] Topical rubefacients cause irritation and reddening of the skin, due to increased bloodflow. They are used in the treatment of pain in various musculoskeletal conditions. Non-limiting examples of topical rubefacients include methyl salicylate, camphor, menthol, phenol, trolamine salicylate, and capsaicin / menthol.

[0098] Topical steroids contain corticosteroids (often abbreviated to steroids) which are designedto be applied externally to the scalp or the skin, depending on the condition being treated. Corticosteroids control inflammation by mimicking naturally occurring corticosteroid hormones produced by our adrenal glands, which are two small glands that sit on top of our kidneys. In 16 59870042.1Attorney Docket No.: 274507 / NOXY24-001WO / 564918 addition to reducing inflammation (redness and swelling) in the area that they are applied, topical corticosteroids also suppress the immune response, reduce cell turnover, and constrict (narrow) blood vessels. Non-limiting examples of topical steroids include mometasone, clobetasol, triamcinolone, fluocinonide, flurandrenolide, clocortolone, halobetasol, desoximetasone, desonide, hydrocortisone, betamethasone, halcinonide, fluocinolone, prednicarbate, diflorasone, fluocinolone, triamcinolone, flurandrenolide, diflorasone, fluticasone, and alclometasone.

[0099] Topical antiseptics are used to reduce the microbial count and reduce the risk ofinfections on the skin. Non-limiting examples of topical antiseptics include alcohol (ethanol and isopropanol), benzethonium chloride, benzalkonium chloride (BAC), camphorated metacresol, eucalyptol 0.091%, hexylresorcinol hydrogen peroxide topical solution, iodine tincture, iodine topical solution, menthol, methylbenzethonium chloride, methyl salicylate, phenol, povidone- iodine, and thymol. BAC represents a mixture of N,N-dimethyl alkyl amines, which conform generally to the formula: .

[0100] All amounts are as percents on the total weight (that is, 100weight percent) of a given solution. Dosing and Administration

[0101] In one embodiment, the nitric oxide topical foam formulation as disclosed hereinis administered as needed to treat the wounds disclosed herein.

[0102] In one embodiment, “as needed” use of the foamed compositing nitric oxidemeans that the composition can applied to the wound at least once a day, or twice a day, or three times a day, or four times a day, or five times a day, or 6 times a day, or 12 times a day or 24 times a day.

[0103] In another embodiment, “as needed” means that the composition can be appliedto the wound at least once an hour, or every two hours, or every three hours, or every four hours, or every 6 hours, or every eight hours or every 12 hours.

[0104] In another embodiment, “as needed” means that the composition can be applied tothe wound daily, every other day, once a week twice a week, or once a week for a week or 17 59870042.1Attorney Docket No.: 274507 / NOXY24-001WO / 564918 multiple weeks. In another embodiment, the foamed composition is applied to the wound each day for from 2 to 12 weeks, or from 2 to 10 weeks, or from 2 to 8 weeks, or from 2 to 6 weeks, or from 2 to 4 weeks, or from 4 to 12 weeks, or from 4 to 10 weeks, or from 4 to 8 weeks, or from 4 to 6 weeks, or from 6 to 12 weeks, or from 6 to 10 weeks, or from 6 to 8 weeks, or from 8 to 12 weeks or from 10 to 12 weeks.

[0105] In another embodiment, the foamed composition is applied to the wound each dayfor from 1 day to 12 weeks, 1 week to 12 weeks, 2 to 12 weeks, or from 2 to 10 weeks, or from 2 to 8 weeks, or from 2 to 6 weeks, or from 2 to 4 weeks, or from 4 to 12 weeks, or from 4 to 10 weeks, or from 4 to 8 weeks, or from 4 to 6 weeks, or from 6 to 12 weeks, or from 6 to 10 weeks, or from 6 to 8 weeks, or from 8 to 12 weeks or from 10 to 12 weeks.

[0106] In one embodiment, the foamed composition is sprayed onto the skin, producing afoam. The duration of application can be from one minute, to sixty minutes, or from 1 minute to 24 hours. In a further embodiment, upon application, the foamed composition overlays the wound to be treated without completely collapsing, from more than 1 minute to up to an hour. Treatment

[0107] The use of the nitric oxide topical foam formulation leads to the reduction in sizeand severity of wounds disclosed herein. In one embodiment, the wounds, which include skin ulcers, including skin ulcers, pressure injuries, deep tissue injuries, burns, abrasions, skin tears, and bruises, upon treatment with the foamed composition are reduced in area and / or volume by from 50% to 100%, compared to before treatment, after 4 weeks of treatment, or after 5 weeks, or after 6 weeks, or after 7 weeks, or after 8 weeks, or after 9 weeks, or from 10 weeks, or after 11 weeks, or after 12 weeks. In a further embodiment, the volume or the area, or both the volume and the area, of the wound decreases over time in from less than a week to 4 months.

[0108] In a further embodiment, the wounds disclosed herein are improved upontreatment with the nitric oxide topical foam formulation. In one embodiment, the improvement is measured according to the University of Texas Staging System for Diabetic Foot Ulcers, the Wagner–Meggitt classification system, the Saint Elian Wound Score System (SEWSS), the SINBAD Classification System and Score, and / or the Society for Vascular Surgery Lower Extremity Threatened Limb Classification System (WIfI).

[0109] In a further embodiment the area and / or the volume of the wound to be treated18 59870042.1Attorney Docket No.: 274507 / NOXY24-001WO / 564918 decreases to complete wound closure upon treatment with the collapsible foam. “Complete wound closure” is defined as substantially complete re-epithelialization without drainage ordressing requirements confirmed at two consecutive study visits two weeks apart. “Complete re-epithelialization” means 100% re-epithelialization.

[0110] In a further embodiment, the patient’s level of methemoglobin is + / - 1% of thepatient’s pre-treatment level of methemoglobin during treatment with the collapsible foam. Methemoglobin is a form of hemoglobin wherein its heme has been oxidized from Fe+2to Fe+3. Unlike normal hemoglobin, methemoglobin does not bind oxygen and as a result cannot deliver oxygen to the tissues. Systemic nitric oxide can oxidize hemoglobin to methemoglobin, reducing the oxygen carrying capacity of the blood to tissue.

[0111] In a further embodiment, the foamed composition can be combined with othertreatments, including treatments that are considered “standard of care.” “Standard of care” can include one or more of moisture management, debridement, dressing changes, wound off- loading, revascularization, infection control, glycemic control, and multidisciplinary care.

[0112] In a further embodiment, the nitric oxide topical foam formulation contains nitricoxide, such that the concentration of ambient nitric oxide gas produced from the foamed composition within 1 foot of the target site is at or below 25 ppm, and / or the maximum levels for humans as defined by OSHA. In further embodiments, the concentration of ambient nitric oxide gas within 1 foot of the target ulcer site is at or below 20 ppm, 15 ppm, 10 ppm, or 5 ppm over an eight-hour period. In a further embodiment, the gas produced from the foamed composition comprises nitrogen dioxide (NO2) within the OSHA exposure limits.

[0113] In a further embodiment, use of the nitric oxide topical foam formulation canreduce pathogen concentration at the site of the wound to be treated.

[0114] In a further embodiment, use of the nitric oxide topical foam formulation reducesthe need for debridement at the wound to be treated.

[0115] The use of the nitric oxide topical foam formulation as disclosed herein is safeand tolerable to the subject. Safety and tolerability are determined by the absence of a serious treatment-related adverse event which is caused by the treatment, generally as determined by a physician. The event can be a treatment-emergent adverse events (TEAE), including serious adverse events and adverse events as described herein and may include cardiac arrest, death, or 19 59870042.1Attorney Docket No.: 274507 / NOXY24-001WO / 564918 another serious event.

[0116] The invention will now be demonstrated by the following non-limiting examples.Example 1

[0117] A topical, foam-based wound care product (the nitric oxide topical foamformulation disclosed herein) indicated for the treatment of chronic Diabetic Foot Ulcers (DFUs) in adult patients as an adjunct to standard of care (SoC) is disclosed.

[0118] The topical, foam-based wound care product is produced by mixing the foamgenerated from each component of the foam composition described herein, identified as Solution A and Solution B. The mixing of these foams causes a chemical reaction resulting in a single foam made up of thousands of NO-filled microbubbles. The topical, foam-based wound care product may be similar in size and consistency to commonly used foam soaps and hand- sanitizers. The topical, foam-based wound care product is intended to be applied directly on the wound using a sanitary applicator such as cotton swabs, tongue depressors, sterile gloves, syringe, and the like.

[0119] The application of the topical, foam-based wound care product is performed byapplying a sufficient amount of foam on the wound so that the wound is adequately covered. The recommended dosing is one pump of Solution A and one pump of Solution B thoroughly mixed together with the resulting combined foam then applied to the wound and surrounding area. The foam can be applied per squared centimeter area of wound. For example, for a wound with an area of 3 cm2, three pumps of Solution A mixed with three pumps of Solution B would create the resulting combined foam which is then applied to the wound and surrounding area. Alternatively, the foam can be applied per centimeter of wound length, rounded up to the nearest centimeter. For example, for a wound with dimensions of 2cm by 3.2cm, four pumps of Solution A would be mixed with four pumps of Solution B with the resulting combined foam then applied to the wound and surrounding area. When the topical, foam-based wound care product is applied on the wound, it forms a substantially air-tight barrier, for example, dome, trapping the produced NO against the wound tissue. The formation of this air-tight dome assures that the bubbles closest to the tissue burst first and release NO orthogonally into the wound tissue.

[0120] This application medium also prevents the NO gas from escaping to theatmosphere as it is released. Additionally, the foam prevents outside air from oxidizing the NO 20 59870042.1Attorney Docket No.: 274507 / NOXY24-001WO / 564918 before it diffuses into the tissue. NO diffusion into the tissue in a time-controlled and targeted manner is also assured. As each successive layer of bubbles reaches the skin surface, they erupt and release their NO payload. Each treatment takes five minutes. This treatment is anticipated to clear infection, increase local blood flow, and reduce inflammation in the wound—all functions which are critical to wound healing.

[0121] The amount of NO produced over the treatment period after the Solution A andSolution B foams are mixed is primarily related to the concentration of acid, for example, citric acid, and nitrite, for example, sodium nitrite, in the respective Solutions. Previous results from acidified nitrite gel testing provided a starting point. The concentration of each solution for the acidified nitrite foam formulation was selected after many designed experiments were conducted to measure the effect of concentration on the NO release profile, the foam duration, and the mixed foam’s effect on the skin.

[0122] The topical, foam-based wound care product is generated only after the twosolutions A and B are mixed. The output of one pump each of Solution A and B from the foamers, (whenever the plunger is fully (and easily) compressed, it can be partially pumped), generates 0.6 (+ / - 5%) grams of topical, foam-based wound care product foam which is sufficient to cover approximately a 1 square centimeter wound. For larger wounds, a greater volume of foam can be generated by using multiple pumps, in a 1 to 1 ratio, per square centimeter of wound area. For example, a 3 square centimeter wound would require 3 pumps each of Solution A and Solution B that are then thoroughly mixed. Summary of Prior Clinical Studies Antimicrobial Studies

[0123] Biofilms of the various Gram-positive and Gram-negative microbes that arecommonly associated with wound infection were grown using a Drip Flow Reactor (DFR - DFR 110-6, Biosurface Technologies Corp., Bozeman, MT) at the Medical Biofilms Laboratory (MBL) of the Center for Biofilm Engineering (CBE) at Montana State University. The DFR models a low-shear environment and has been approved by American Society for Testing and Materials (ASTM) as standard method for growing P. aeruginosa biofilms (E2647-20). A modified procedure, more relevant to wound biofilms, was utilized. Biofilms were treated with the foamed composition as disclosed herein for five minutes. 21 59870042.1Attorney Docket No.: 274507 / NOXY24-001WO / 564918

[0124] Each biofilm was exposed to a five-minute treatment of foamed composition asdisclosed herein. The results of the foamed composition as disclosed herein treatments demonstrated a significant reduction in Colony Forming Units per square centimeter (CFU / cm2) of the biofilm. To date, Gram-negative Pseudomonas aeruginosa, Acinetobacter baumannii, Proteus mirabilis, gram-positive Staphylococcus aureus, Candida albicans, and Staphylococcus epidermidis biofilms have been tested against a five-minute treatment of foamed composition as disclosed herein. The study concluded that foamed composition as disclosed herein effectively and significantly reduced the CFU / cm2levels of all tested biofilms.

[0125] The topical, foam-based wound care product was found to be an effectivetreatment against biofilms by both antibiotic sensitive and resistant Gram-negative and Gram- positive microbes. Results of this study are provided in the table below. Overall, the use of foamed composition as disclosed herein resulted in biofilm dispersal and reduction and demonstrates a promising potential strategy for the treatment of bioburden and infection in chronic wounds. Microbes Tested and Average Log Reduction of CFU / cm2Method Tested Log Reduction Pseudomonas aeruginosa 58Ex Vivo Pig Explant Study

[0126] To further study the topical, foam-based wound care product’s effect on biofilmsof the various Gram-positive and Gram-negative microbes within dermal tissue, screening studies were conducted of the anti-biofilm properties of wound care formulations using an ex vivo model of mature biofilm. Miller CM et al. Acidified nitrite foam anti-microbial action in an ex vivo porcine dermal model. Journal of Wound Management 2023;24(3):81-85. DOI doi.org / 10.35279 / jowm2024.25.01.02.

[0127] The model has been used extensively to test the anti- biofilm properties ofcommercial and experimental wound treatments within a natural tissue matrix. The model is 22 59870042.1Attorney Docket No.: 274507 / NOXY24-001WO / 564918 suitable for screening solid dressings, gels, and solutions using a variety of testing formats including biofilm prevention, biofilm eradication, barrier testing, debridement, and combination treatments. The study was designed with two arms: testing the topical, foam-based wound care product for preventing biofilm formation, and for testing topical, foam-based wound care product for eradicating existing biofilms in porcine dermal explants using the ex vivo model of mature biofilm.

[0128] The test organisms included the same microbes as used in the AntimicrobialStudy described above. For the prevention studies, the log reduction of colony forming units (CFU) relative to untreated controls ranged from 4 to 9 log CFU / explant for the topical, foam- based wound care product, with the constituents (Solution A and Solution B) also showing the ability to prevent biofilm proliferation. In the eradication studies, 72 hour-old mature biofilms produced in the pig skin explants showed susceptibility to the topical, foam-based wound care product treatment and appeared to be species specific. Namely, biofilms of P. aeruginosa, S. aureus, and C. albicans showed the highest susceptibility at 4-log reduction relative to untreated controls. S. epidermidis exhibited the lowest susceptibility at approximately 2.5-log reduction. This study also assessed the impact of foamed composition as disclosed herein dosing on biofilm eradication, and results indicate that two 5-minute treatments were more effective than a single treatment for all species tested. Together, these results highlight the strong anti-planktonic and anti- biofilm properties of the topical, foam-based wound care product and provide foundational data for further development. STUDY RATIONALE

[0129] A topical, foam-based wound care product is disclosed in US 10,052,348, US10,751,364, and US 11,304,972, incorporated herein by reference and relied upon. The topical, foam-based wound care product produces NO and its microfoam bubble delivery system in order to administer NO topically. The topical, foam-based wound care product is intended for the treatment of chronic DFUs as an adjunct to standard of care (SoC). The topical, foam-based wound care product is proposed to deliver exogenous NO to the tissue of complex and chronic wounds and thereby increase localized blood flow through enhanced vasodilation facilitating wound healing.

[0130] The supplementation of NO to wounded tissues has been demonstrated to enhance23 59870042.1Attorney Docket No.: 274507 / NOXY24-001WO / 564918 the skin’s response to infection and increase wound healing (Adler, 2015; Frank, 2002). Furthermore, NO has also been shown to resolve inflammation and is a powerful pro-healing agent (Barraud, 2015).

[0131] NO that is generated in sufficient concentration by endothelial nitric oxidesynthase (eNOS) and inducible nitric oxide synthase (iNOS) in humans has long been known as a critical and essential agent in the cellular and molecular healing sequence. NO has been demonstrated to be a potent antimicrobial agent (Malone-Povolny, 2019) and vasodilator (Burke, 2009).

[0132] The active ingredient of the topical, foam-based wound care product is gaseousNO that improves localized oxygenation (Castilla, 2012) and perfusion (Reinke, 2012); reduces inflammation (Guzik, 2003), and induces the natural removal of exudate (Waite, 2018) and infectious material (Murray, 2012). The above-mentioned actions of NO also can reduce or eliminate the requirement for: wound debridement (Krzyszczyk, 2018), Hyperbaric Oxygen Therapy (HBOT) or extracorporeal oxygenation (INOmax, FDA Approver Label, NDA 20845 / S20, 2019), antibiotics, and other complex and expensive treatments (Malone-Povolny, 2019). The role and mechanisms of NO-mediating dispersal of bacterial biofilms and its potential for novel therapeutics has been reviewed by Barraud et al (Barraud 2015) and has been identified as potentially being a key treatment for many infectious diseases. STUDY OBJECTIVES AND OUTCOME MEASURES ▪Primary Objective

[0133] To demonstrate the safety and tolerability of the topical, foam-based woundcare product as adjunct to an SOC. ▪Secondary Objective

[0134] The secondary objective of the study is to evaluate the clinical benefit of dailyadministration of the foamed composition as disclosed herein, as an adjunct to a standard of care (SOC), in the treatment of chronic, non-healing diabetic foot ulcers. a. STUDY OUTCOME MEASURES▪ Primary Outcome Measures

[0135] The primary outcome measure in this study is:● Incidence and severity of treatment-emergent adverse events (TEAEs), including serious24 59870042.1Attorney Docket No.: 274507 / NOXY24-001WO / 564918 adverse events and adverse events resulting in permanent discontinuation of protocol- defined therapy ●Number of subjects with a Methemoglobin >5% at any assessment point▪ Secondary Outcome Measures

[0136] The secondary outcome measures in this study are:● Proportion of subjects with complete wound closure during the 12 weeks of theTreatment Phase ●Complete wound closure is defined as 100% re-epithelialization without drainage ordressing requirements confirmed at two consecutive study visits 2 weeks apart. Complete wound closure will be evaluated by the blinded evaluator. ●Wound Area Change (%) during the 12 weeks of the Treatment Phase● Wound area change is defined as the percentage of wound area change as measured bythe Swift Imaging device. ●Wound volume change (%) during the 12 weeks of the Treatment Phase● Wound volume change is defined as the percentage of change based on manualmeasurements of L x W x D in centimeters ●Time to complete wound closure during the 12 weeks of the Treatment Phase● Proportion of subjects who do not develop an infection during the 12 weeks of theTreatment Phase ●Laboratory data changes from baseline to subsequent scheduled visits● Changes in physical examinations from baseline to subsequent scheduled visits● Changes in vital signs from baseline to subsequent scheduled visits● Changes in patient outcomes during the 12 weeks of the Treatment Phase as measured bychanges in the subject response to the Wound-Q Health-Related Quality of Life (Life Impact Psychological, Social) scales at T1 (pre-treatment), T5, and T13 / EOT STUDY DESIGN

[0137] This is a multi-center, randomized, controlled, evaluator-blinded study to assessand measure the safety and clinical benefit of the topical, foam-based wound care product as an adjunct to standard of care (SOC) in the treatment of chronic diabetic foot ulcers. Subjects will be randomized to receive treatment with topical, foam-based wound care product as an adjunct to 25 59870042.1Attorney Docket No.: 274507 / NOXY24-001WO / 564918 SOC or SOC alone. Standard of care will include evaluation to document, offloading, adequate arterial flow, wound cleansing, removal of necrotic, infected and / or nonviable tissue by debridement, maintenance of a moist wound environment, and management of infection.

[0138] A total of 40 subjects will be randomized (25 in the treatment group and 15 in thecontrol group) to receive either topical, foam-based wound care product plus SOC or SOC, alone. a. ELIGIBILITY CRITERIA▪ Inclusion Criteria● Subjects will be eligible for enrollment in the study only if they meet ALL the followingcriteria at time of Screening: ●Male or female subjects aged 18 to 80 years (inclusive) with Type 1 or Type 2 diabetesundergoing therapy for glycemic control. ●Subject has a glycosylated hemoglobin, HbA1c ≤ 12%. Note: Prior documented HbA1cwithin the last 3 months of the Screening Visit is acceptable. ●Presence of at least one diabetic foot ulcer that meets all of the following criteria:● A full-thickness ulcer of University of Texas Wound Classification (UTWCS) Grade I orII ●At least 50% of the ulcer is located below the malleoli● Ulcer size (area) is ≥ 1 cm2 and ≤ 10 cm2 (post-debridement at time of randomization)● Unresponsive to standard ulcer care for ≥ 3 months (at time of screening)● There is a minimum 1 cm margin between the qualifying Target Ulcer and any otherulcers on the specified foot, post-debridement) ●No exposed bone and no tunneling, undermining, or sinus tracts● Ulcer must be non-healing as defined as < 25% reduction in size in response to standardof care during the two-week run-in Screening Period (between the first Screening Visit and Baseline). Note: Criterion 3(g) will be evaluated at the time of randomization. If the subject has more than one qualifying diabetic foot ulcer, the ulcer designated as the Target Ulcer will be at the discretion of the Investigator. ●Subject has adequate circulation to the affected foot as demonstrated by a dorsumtranscutaneous oxygen measurement (TCOM) or a skin perfusion pressure (SPP) 26 59870042.1Attorney Docket No.: 274507 / NOXY24-001WO / 564918 measurement of ≥ 30 mmHg or an Ankle-Brachial Index (ABI) between 0.7 and ≤ 1.3, or a Toe-Brachial Index (TBI) of >0.6 within 3 months of the first Screening Visit. The assessment may also be performed between SV1 and SV2. ●Subject does not smoke or use tobacco products.● Subject, if female of child-bearing potential, has a negative pregnancy test at screening,must not be breastfeeding, and willing to use acceptable methods of contraception (birth control pills, barriers, or abstinence) throughout the study. ●Subject is able and willing to comply with study procedures and applicable dressingchanges. ●Subject demonstrates cognitive and physical ability to administer the treatment asdetermined by the clinician. If a caregiver will administer the treatment, the caregiver must demonstrate cognitive and physical ability. ●A signed and dated informed consent form has been obtained from the subject.▪ Exclusion Criteria● Subjects meeting ANY of the following criteria at time of Screening will be excludedfrom enrollment: ●Ulcers with exposed bone or associated with osteomyelitis. Note: Osteomyelitis shouldbe ruled out by clinical examination (probing of the wound) or X-ray findings, if necessary, by the Investigator. ●Subject has ulcers secondary to a disease other than diabetes, for example, fungalulcerations, malignant ulcerations, and ulcerations due to venous or arterial insufficiency, or due to hematological disorders, in the opinion of the Investigator. ●Ulcer, which in the opinion of the Investigator is suspicious for cancer. Note: Ulcerspresent for > 6 months would require biopsy to be performed to rule out malignancy. ●Subjects with a gangrenous or ischemic toe that may need to be amputated in theopinion of the Investigator. ●Body mass index (BMI) > 40 kg / m2● Methemoglobin > 5% at SV1● Laboratory values at Screening of:o Hemoglobin < 8.5 g / dL27 59870042.1Attorney Docket No.: 274507 / NOXY24-001WO / 564918 oWhite Blood Cells (WBC) < 3.0 X 109 cells / L and > 11 x 109 cells / Lo Liver function studies [Total bilirubin, aspartate aminotransferase (AST)and alanine transaminase (ALT)] > 3x the upper limit of normal oAlbumin < 2.5 g / dLo Renal function studies [Estimated Glomerular Filtration Rate] < 45● Presence of any clinically significant medical condition(s) that, in the opinion ofthe Investigator, could interfere with wound healing, including but not limited to the following: oVasculitis or connective tissue diseaseo Buerger’s disease, Raynaud’s or other peripheral vascular disease.o Clinically significant claudication or peripheral edema on the affected limbo Acute or unstable Charcot footo Aplastic anemia or sickle cell anemiao Current sepsiso Severe heart diseases such as congestive heart failure (NYHA Class III orIV), coronary heart disease with ST segment elevation, myocardial infarction, or coronary artery bypass graft or percutaneous transluminal coronary angioplasty within the last 6 months oSevere liver diseaseo End-stage renal diseaseo Severe malnutritiono Immunosuppressiono Acquired immune deficiency syndrome (AIDS) or HIV positivePast or present malignancy below the knee on the same limb as the Target Ulcer; ●History of radiation at the Target Ulcer site.● Subject is currently receiving (that is, within 30 days of T1 visit) or scheduled toreceive any of following medication or therapies during the course of the study. oImmunosuppressants (including chronic systemic corticosteroids)o Cytotoxic chemotherapyo Cytostatic therapy28 59870042.1Attorney Docket No.: 274507 / NOXY24-001WO / 564918 oLower limb revascularization surgery (for example, angioplasty, arterybypass surgery,) oApplication of bioengineered tissue or skin substituteso Use of any investigational drug(s)● Subjects who have previously received treatment using the foamed composition asdisclosed herein ●Has a known hypersensitivity to any of the investigational drug components● Subject is susceptible to hemorrhaging or has a congenital or acquiredpredisposition to hemorrhaging. ●Any reason that the subjects may need to be admitted to inpatient acute care in theopinion of the Investigator. ●Has any other factor which may, in the opinion of the investigator, compromiseparticipation and / or follow-up in the study. ▪Screening Phase

[0139] The Screening Phase is designed to determine whether subjects are eligible toproceed to the Treatment Phase of the study. This phase consists of a series of screening assessments designed to determine eligibility.

[0140] Screening assessments will commence after obtaining signed informed consent.All subjects who fail to meet eligibility criteria are considered screen failures and are exited from the study without further evaluation. At Screening Visit 1 (SV1), the Investigator will identify the potential Target Ulcer(s). Investigator can identify multiple potential Target Ulcer(s) if a subject has more than one diabetic ulcer present on or below the malleoli that meets the eligibility criteria at the Screening Visit (SV1).

[0141] Investigator will continue to follow and provide SOC treatment to potentialTarget Ulcer during the Screening Phase. To ensure that subjects receive optimal and effective offloading, an offloading boot will be used for all plantar DFUs and offloading device selection for non-plantar DFUs will be determined by the PI, who has expertise in managing the complexities of diabetic foot ulcers. For subjects using an offloading boot, the SOC dressing change frequency will be at least once weekly or more frequently based on the condition of the wound. The patient will be instructed to return to clinic or for second Screening Visit (S2) 7 days 29 59870042.1Attorney Docket No.: 274507 / NOXY24-001WO / 564918 (±3 days) after the first Screening Visit (S1) in order to receive SOC treatment. Standard of care will include evaluation to document, offloading, adequate arterial flow, wound cleansing, removal of necrotic, infected and / or nonviable tissue by debridement, maintenance of a moist wound environment, and management of infection. Treatment Phase

[0142] The treatment phase begins at T1 visit with a series of assessments designed toconfirm subjects’ continued eligibility. The T1 visit will take place 14 ± 3 days after the first Screening Visit (S1). Investigators will cleanse and debride the ulcer, perform hemostasis as required, and use Swift Imaging device to compare the change in ulcer area recorded at SV1 and T1.

[0143] Only one diabetic lower extremity ulcer which continues to meet eligibilitycriteria will be designated as the Target Ulcer at the Baseline / T1 Visit. ●If a potential Target Ulcer has decreased > 25% between the first Screening (SV1)and T1 visit, then this potential Target Ulcer cannot be designated as Target Ulcer. ●If none of the potential Target Ulcer(s) meet eligibility criteria at T1 Visit, subjectwill be considered as screen failure and exit the study without further evaluation. ●If more than one potential Target Ulcer(s) continue to meet eligibility criteria atT1, it is up to the clinical judgment of the Investigator to designate one of them as Target Ulcer. ●Eligible consenting patients will be randomized into one of the following groups:o The foamed composition as disclosed herein plus SOC (25 subjects)o SOC (15 subjects)● All subjects who fail to meet eligibility criteria at T1 are considered screenfailures and are exited from the study without further evaluation. ●Subjects randomized to receive the foamed composition as disclosed herein willapply once daily applications of treatment for up to 12 weeks until epithelialization without drainage or dressing requirement of Target Ulcer, whichever comes first. Dressing changes will occur once daily, or more often if 30 59870042.1Attorney Docket No.: 274507 / NOXY24-001WO / 564918 drainage demands. For subjects requiring dressing changes more frequently that once daily, two SOC kits can be provided. Patient diaries will be used to record the dates and times of treatment applications and dressing changes (to confirm the dose application schedule). The study staff will review the diaries, interview the subject to obtain any missing information, and transfer complete compliance information to the appropriate eCRF at each study visit. In addition, sites can perform supervised video conferences to ensure proper treatment administration in between scheduled weekly clinic visits. ●Once Target Ulcer is 100% re-epithelialized without drainage or dressingrequirements, no more study treatment will be applied. All subjects, regardless of Ulcer Closure status, will enter the 4-week Follow-Up Phase. ●Subjects who achieved complete epithelialization and no drainage or dressingrequirement (clinical closure) of Target Ulcer at any time during the Treatment Phase prior to T13 / EOT will undergo the T13 / EOT assessments and then enter the Follow- Up Phase of the study. ●Subjects whose Target Ulcer has not closed by the end of the T13 / EOT visit willalso enter the 12-week Follow-Up Phase. ▪Follow-up Phase

[0144] All subjects who complete the Treatment Phase, either by duration or closure ofthe Target Ulcer, will complete the EOT assessments and enter the 4-week Follow-Up Phase. ●All subjects who enter the Follow-Up Phase are required to attend the FU1 (2-week) and FU2 (4- week), visits regardless of ulcer closure / re-open status. Note: Subjects who have failed to achieve complete closure of the Target Ulcer at the end of the 12-week Treatment Phase or have a re-opened Target Ulcer during the Follow-Up Phase will continue to receive standard of care treatment as deemed necessary by the PI during the Follow- Up Phase of the study. 1. STANDARD OF CARE

[0145] The goal of the SOC is to eliminate as many variables as possible, and to allowthe differences in healing to be attributed to the study drug. This will mean that the other wound treatments will need to be uniform across the various clinics and treating physicians. This study 31 59870042.1Attorney Docket No.: 274507 / NOXY24-001WO / 564918 is not comparing the best treatments provided by different providers. The study is to compare the study drug to internationally accepted norms for treatment of the DFU.

[0146] The SOC dressing is intended to promote a moist healing environment. However,the dressing needs to avoid being variable itself, be neutral and inert, and provide a moist environment but not insert itself into the healing mechanism.

[0147] As a result of these qualifiers, the dressing chosen was a calcium alginate. Allproviders need to use this dressing to obtain the data that would allow demonstration of the study drug effect. Based on clinical judgement, the alginate can be used dry for wounds with moderate to heavy exudate or moistened with Normal Saline for wounds with minimal to no exudate.

[0148] Debridement is a proven intervention for successful healing and wound closure.When required, sharp debridement using curette, scalpel, and tissue nipper should be used. The removal of biofilm, devitalized tissue, and necrotic tissue is an accepted norm in the community. It is also inert not adding any other chemical to the wound environment. For this reason, the protocol does not allow debridement other than sharp debridement.

[0149] Effective offloading is an essential component of diabetic foot ulcer healing. Toensure that subjects receive optimal and effective offloading, offloading will be used for all DFUs during the screening phase.

[0150] Study subjects must continuously wear their designated offloading device for theduration of their participation in the study. If subjects do not have an effective offloading device, the offloading device is excessively worn, or the subject cannot procure an effective device, the sponsor may dispense an appropriate offloading device to the subject.

[0151] During the course of the study, PI’s will need to study the offloading footwear toevaluate the wear to the device associated with its prescribed use. If the offloading footwear remains like new, the device is not being used. If the offloading footwear is excessively worn, the prescribed offloading is in question. Such noncompliance needs to be addressed and documented, and the study participant will be questioned and possibly removed from the study for non-compliance per study protocol. If the non-compliant behavior persists for several weeks, the patient should be removed from the study. New offloading footwear can be dispensed as needed for normal wear and tear. The standard of care will be used for all subjects during the screening, treatment, and follow-up phases. 32 59870042.1Attorney Docket No.: 274507 / NOXY24-001WO / 564918

[0152] The SOC for offloading will be a cast boot for DFUs. The SOC dressing in thisstudy is defined as a calcium alginate, a non-adherent foam dressing (depending on the amount of exudate), and an outer gauze wrap. For subjects, the SOC dressing change frequency will be at least once weekly or more frequently based on the condition of the wound. Subjects will be instructed on proper dressing care. Based on clinical judgement, the alginate can be used dry for wounds with moderate to heavy exudate or moistened with Normal Saline for wounds with minimal to no exudate.

[0153] During the Treatment phase, the following Treatment will be defined as “Standardof Care”: the study treatment, a secondary dressing, and an offloading device for wounds is required. The SOC secondary dressing in this study is defined as a calcium alginate, a non- adherent foam dressing (depending on the amount of exudate), and an outer gauze wrap. Subjects will be instructed on proper dressing care.

[0154] The daily dressing during the treatment phase will consist of the following:2. Removal of the prior dressing3. Wash the wound with Normal Saline4. Pat dry5. Apply the Study Treatment (Investigative Drug). It is important to note the subjectneeds to keep the wound horizontal (that is, parallel to the ground and facing up). All enrolled subjects need to be able to keep the foam on the wound for 5 minutes, including those with plantar wounds. 6. Apply the calcium alginate dressing. Ensure that the alginate does not extendonto intact skin. It may be required to trim the calcium alginate dressing with a pair of sanitary scissors set aside for the purpose of administering SOC. Based on clinical judgement, the alginate can be used dry for wounds with moderate to heavy exudate or moistened with Normal Saline for wounds with minimal to no exudate . 7. Cover with the non-adherent foam dressing8. Apply outer gauze wrap9. Tape the outer gauze dressing10. Do not apply tape to skin33 59870042.1Attorney Docket No.: 274507 / NOXY24-001WO / 564918 11. Wear the offloading footgear as determined by the PI. Do not wear any other type ofshoe 12. Dressing changes will occur once daily, or more often if drainage demands.Standard of care also includes sharp debridement of the wound (as necessary) and infection management (systemic antibiotics). Topical antibiotics will not be used. STUDY TREATMENT

[0155] The topical, foam-based wound care product is a foam based gaseous nitric oxide(NO) product where NO is delivered through a microbubble foam. One pump each of Solution A containing an acid and a surfactant and Solution B containing a nitrite salt and a surfactant will be dispensed and mixed, then applied immediately to the wound and surrounding area using any sterile applicator. ADMINISTRATION

[0156] The topical, foam-based wound care product is topically applied directly onto thewound bed; and it is maintained on the wound over a treatment period. Subjects randomized to the foamed composition as disclosed herein treatment group will receive once a day application, for a total of 12 weeks. Drug administration should occur between 18-28 hours apart. For the first day of treatment, the study drug will be a double treatment, 10 minutes apart. Subjects / caregivers will be training on self- administration at the first treatment visit (T1).

[0157] On days where the subject is onsite for a clinic visit, the IP should beadministered on site. The administration should be done after debridement, photography, and measurement but before the ulcer dressings. a. CLINICAL LABORATORY ASSESSMENTS

[0158] All laboratory (See Lab Parameters Table following this paragraph)assessments will be performed at a central lab and reviewed by the Investigator. If clinically significant findings, as determined by the Investigator, are recorded for a particular symptom, sign or abnormal measurement, that measurement will be repeated at medically appropriate intervals until the value returns to an acceptable range, a specific diagnosis is established, or the condition is otherwise explained. Validated, quality-controlled laboratory data will be transferred to the main database for analyses. 34 59870042.1Attorney Docket No.: 274507 / NOXY24-001WO / 564918 Lab Parameters Hematology Parameters Biochemistry Urinalysis ParametersFINGERSTICK GLUCOSE

[0159] Subjects will measure glucose levels at each study visit.VASCULAR PERFUSION ASSESSMENT Adequate circulation to the affected foot as demonstrated by a dorsum transcutaneous 35 59870042.1Attorney Docket No.: 274507 / NOXY24-001WO / 564918 oxygen measurement (TCOM) or a skin perfusion pressure (SPP) measurement of ≥ 30 mmHg or an ABI between 0.7 and ≤ 1.3, or TBI of >0.6 within 3 months of the first Screening Visit. The assessment may also be performed between SV1 and SV2. IDENTIFICATION OF TARGET ULCER(S)

[0160] The Investigator will re-assess patient eligibility at T1, with emphasis onreduction of ulcer size since SV1. If a potential Target Ulcer has decreased > 25% between first Screening and T1 visits, then this potential Target Ulcer cannot be designated as Target Ulcer. If none of the potential Target Ulcer(s) meet eligibility criteria at T1, subject will be considered as screen failure and exit the study without further evaluation. If more than one potential Target Ulcer(s) meet eligibility criteria at T1, it is up to the clinical judgment of the Investigator to designate one of them as Target Ulcer. ASSESSMENT FOR OSTEOMYELITIS

[0161] The osteomyelitis should be ruled out by clinical examination (probing of thewound) or X- ray findings were found necessary by the Investigator based on availability of the investigation facility at the clinical sites. DEBRIDEMENT OF TARGET ULCER

[0162] Sharp debridement of target ulcer will be performed if required per theInvestigator’s judgment.

[0163] Subjects who have failed to achieve complete closure of the Target Ulcer or havea re-opened Target Ulcer during the Follow-Up Phase would require Target Ulcer cleaning and / or sharp debridement during the Follow-Up Phase. PHOTOGRAPH AND MEASUREMENT OF TARGET ULCER

[0164] All potential Target Ulcer(s) will be digitally photographed and measured usingthe Swift Imaging device and measured manually to include length, width, and depth, post- debridement if debridement is performed. At SV1 and T1, wound area reduction from the Swift imaging device will be used for target ulcer selection.

[0165] Photograph of the skin where the Target Ulcer was located, or of the Target Ulceritself (if the ulcer is not closed) is taken at all Follow-Up visits (post-debridement if performed). CLINICAL ASSESSMENT OF TARGET ULCER(S)

[0166] The following assessments of the Target Ulcer will be conducted as specified36 59870042.1Attorney Docket No.: 274507 / NOXY24-001WO / 564918 below. The assessments include the University of Texas Diabetic Wound Care Classification, Leg Edema Scale, Target Ulcer Margins Assessment (pre-debridement), Target Ulcer Appearance Assessment (pre-debridement), Target Ulcer Surround Skin Assessment, Target Ulcer Exudate Assessments University of Texas Diabetic Wound Classification Stage Grade0 I II IIILeg Edema ScaleTarget Ulcer Margins Assessment (pre-debridement) Target Ulcer Margins Assessment37 59870042.1Attorney Docket No.: 274507 / NOXY24-001WO / 564918 ●Sloping (Yes or No) ● Sinus (Yes or No)● Rolled (Yes or No) ● Inflamed (Yes or No)Target Ulcer Appearance Assessment (pre- debridement)arge cer urroun n ssessmen Target Ulcer Surrounding Skin AssessmentTarget Ulcer Exudate Assessments

[0167] The Investigator / blinded evaluator will determine the amount and type, if any, ofTarget Ulcer exudate. In determining the amount of Target Ulcer exudate, the Investigator / blinded evaluator must determine the amount of exudate absorbed into the Target Ulcer dressing. The following categories will be used to quantify the amount and describe the type of ulcer exudate: Target Ulcer Exudate Assessment59870042.1Attorney Docket No.: 274507 / NOXY24-001WO / 564918 ●Not applicable: no exudate present● Serous: clear or light-yellow watery plasma,TARG

[0168] Target Ulcer will be dressed in protocol-specified dressing. All other ulcers willbe treated as per Investigator discretion and per SOC.

[0169] Subjects who do not achieve complete closure during the Treatment Phase mustcontinue to use the dressing throughout the Follow-Up Phase. TARGET ULCER CLOSURE ASSESSMENT

[0170] Subjects who have failed to achieve complete closure of the Target Ulcer or havea re-opened Target Ulcer during the Follow-Up Phase would require clinical assessment of Target Ulcer to be performed by the Investigator / blinded evaluator during the Follow-Up Phase. SUBJECT DIARY

[0171] Patient diary will be used to record the dates and times of treatment applicationsand dressing changes (to confirm the dose application schedule). The study staff will review the diaries and complete dedicated worksheet to assess protocol compliance, interview the subject to obtain any missing information, and transfer complete compliance information to the appropriate eCRF at each study visit. WOUND-Q HEALTH-RELATED QUALITY OF LIFE (LIFE IMPACT, PSYCHOLOGICAL, SOCIAL)

[0172] Subject quality of life will be assessed using Wound-Q Health-Related Quality ofLife (Wound- Q) scales at T1, T5, and T13 / EOT. The Wound-Q measures the disease- specific, health-related quality of life of patients with chronic wounds. Additional details are available in the Wound- Q Manual of Instruction. METHEMOGLOBIN

[0173] Investigator to complete methemoglobin assessment via co-oximetry at SV1.Subjects should be excluded if metHb > 5% at SV1. Methemoglobin assessment via co-oximetry 39 59870042.1Attorney Docket No.: 274507 / NOXY24-001WO / 564918 within 10 minutes of first treatment and +5 minutes, +10 minutes (prior to second treatment), +30 minutes, and +60 minutes (after the first treatment) at T1. Methemoglobin assessment viaco- oximetry within 10 minutes prior to treatment and +5 minutes, +10 minutes, +30 minutes, and +60 minutes (after treatment) at T2, T5, T6. Methemoglobin screening via co-oximetry at T13 / EOT. Taking measurements at approximately the same time each day is recommended in case there is diurnal variability. Record assessment time. Participants should be evaluated by the study monitor if metHb is > 5% at T1, T2, T5, T6. STATISTICAL CONSIDERATIONS

[0174] This section presents general information about statistical considerations andconcepts such as randomization, covariates / stratification, statistical power, sample size, and a brief discussion on analysis methodology, as well as some data conventions. Detailed descriptions of the statistical analysis methods and data conventions will be included in a separate document; that is, the Statistical Analysis Plan (SAP). SAMPLE SIZE DIFFERENTIATION AND RATIONALE

[0175] The sample size of at least 40 subjects (25 in the treatment group and 15 in thecontrol group) will be used in this trial. This sample size is selected based on clinical judgment and not based on statistical power calculation; it is deemed adequate to provide clinically meaningful descriptive results consistent with study objectives. RANDOMIZATION

[0176] A total of 40 subjects will be randomized (25 in the treatment group and 15 in thecontrol). Post study analysis will be stratified by: ●Plantar vs. non-plantar● Baseline / T1 wound size: < 5 cm2 vs. ≥ 5 cm2

[0177] An individual, independent of the clinical trial team, will develop therandomization schedules. The actual randomization assignment will be made through a web- based system. Subjects who have provided written informed consent and have met all the inclusion criteria and none of the exclusion criteria will be randomized to one of the treatment groups. BLINDING AND PREVENTION OF BIAS

[0178] There will be a central blinded assessor provided by the sponsor to serve as the40 59870042.1Attorney Docket No.: 274507 / NOXY24-001WO / 564918 “blinded evaluator” to be responsible for assessing the efficacy endpoints such as wound measurements and complete wound closure. The blinded evaluator will not be involved in the clinical care of the subject. The Principal Investigator and the site team who are aware of treatment assignments will be considered “unblinded personnel”. The “unblinded personnel” will be responsible for clinical care including study treatment application, managing standard of care, and the like. To eliminate bias, the unblinded personnel should not be involved in any assessment of study endpoints. GENERAL STATISTICAL CONSIDERATIONS

[0179] All collected study data will be presented in subject data listings and / or will besummarized.

[0180] A Statistical Analysis Plan (SAP) will be developed and approved before thedatabase is locked. The SAP will present the detailed statistical methodology to be used in analyzing the efficacy and safety data from this trial. Analysis Populations ▪Intent to Treat (ITT) Populations

[0181] The Intent to Treat (ITT) population is defined as all randomized subjects. TheITT population will be the primary analysis population for the analysis of primary and secondary endpoints. ▪Per Protocol (PP) Populations

[0182] The Per Protocol (PP) population is defined as the set of subjects who meet theITT population requirements and were not associated with a major protocol violation. This population will be identified before the database lock. The PP population will be the secondary analysis population for the analysis of primary and secondary endpoints. ▪Safety Population

[0183] The Safety population is defined as all subjects receiving the treatment afterrandomization. This population will be used for the analysis of safety parameters. Covariates and Subgroups

[0184] For efficacy analyses, baseline values will be used as covariates in the analysismodels. Other covariates and subgroup analyses will be detailed in the SAP. ▪Missing Data41 59870042.1Attorney Docket No.: 274507 / NOXY24-001WO / 564918

[0185] All efforts will be made to minimize the amount of missing data for the study.The methods of handling missing data will be detailed in the SAP. a. STATISTICAL METHODS

[0186] All the efficacy analyses presented here will be conducted using ITT and PPpopulations. All safety analyses will be conducted using the Safety population.

[0187] For continuous variables, descriptive statistics (n, mean, standard deviation,median, minimum and maximum) will be presented by treatment group. For categorical variables both frequencies and percentages will be presented by treatment group. Subject Disposition

[0188] The disposition of all subjects who signed an ICF will be provided. The numbersof subjects screened, randomized, received treatment, completed, and discontinued during the study, as well as the reasons for screen failures and discontinuation will be summarized. Disposition and reason for study discontinuation will also be provided as a by-subject listing. Demographics and Baseline Characteristics Analysis

[0189] Demographics and baseline characteristics will be summarized using appropriatedescriptive statistics. Concomitant Medications / Therapies

[0190] All prior and concomitant medications recorded in the case report form will becoded to all matching Anatomic Therapeutic Classification codes using the most recent version of the WHO Drug Dictionary. Descriptive summaries will be prepared using the coded term. All concomitant medications recorded in the case report form will be listed. Efficacy Analysis

[0191] The primary analysis of primary and secondary endpoint will be conducted on theITT population. PP population will be used for supportive analysis. The primary outcome variable for the study is the proportion of subjects with complete wound closure during the 12 weeks of the Treatment Phase. Logit model will be used for the primary endpoint and all other secondary efficacy endpoints that are categorical in nature. The Repeated Measures Analysis of Covariance (Mixed model) will be used for analysis of percent change in surface area. Safety Analysis 42 59870042.1Attorney Docket No.: 274507 / NOXY24-001WO / 564918 ▪Adverse Events

[0192] Adverse events will be classified by system organ class (SOC) and preferred term(PT) according to the most recent version of MedDRA dictionary.

[0193] TEAE are defined as adverse events with onset date on or after the firstrandomized treatment. TEAEs will be summarized by treatment group, System Organ Class, and preferred term. The following TEAE summaries will be provided: ●Overall (that is, regardless of severity or relationship to treatment)● Adverse events by severity● Related adverse events by severity● Adverse events leading to treatment discontinuation by severity● Adverse events leading to death by severityClinical Laboratory Data

[0194] Tabulations of raw data and change from baseline values will be presented bytime point for each lab parameter. Tabulations will include the number of observations, mean, standard deviation, median, and minimum and maximum values. Abnormal laboratory results will be listed and summarized. Vital Signs

[0195] Tabulations of raw data and change from baseline values will be presented bytime point for each vital sign parameter. Tabulations will include the number of observations, mean, standard deviation, median, and minimum and maximum values. Physical Examination

[0196] Physical Examination findings will be presented as by-subject listing.Methemoglobin

[0197] Methemoglobin raw data will be presented for SV1, T1, T2, T5, T6, andT13 / EOT. Participants should be evaluated by the study monitor if Methemoglobin is > 5% at SV1 and evaluated by the study monitor from treatment if Methemoglobin is > 5% at T1, T2, T5, T6. Other Safety Data

[0198] All data from all other safety assessment will be presented as a by-subject listing43 59870042.1Attorney Docket No.: 274507 / NOXY24-001WO / 564918 and / or summarized. a. ADVERSE EVENT (AE) DEFINITION

[0199] An adverse event (AE) is defined as any unfavorable or unintended sign,symptom, or disease that occurs or is reported by the patient to have occurred, or a worsening of a pre-existing condition. An adverse event may or may not be related to the study treatment.

[0200] AEs will be elicited through direct questioning and subject reports. Anyabnormality in physical examination findings or laboratory results that the investigator believes is clinically significant (CS) to the research subject and that occurred after initiation of the first study treatment will be reported as AEs. Abnormal findings that are NOT clinically significant should not be recorded as an AE. ▪CTCAE Grade (Intensity) Assessment

[0201] The guidelines outlined in CTCAE v5.0 will be used for assessing the intensity ofthe event. The general guidelines for assessing the AE grade appear below. Grade Description )*. ; ga. SERIOUS ADVERSE EVENT (SAE) DEFINITION

[0202] A SAE is defined as any AE that:● Results in death44 59870042.1Attorney Docket No.: 274507 / NOXY24-001WO / 564918 ●Is life threatening (the subject is at immediate risk of dying from the adverse experience)● Requires subject hospitalization or prolongs existing hospitalization● Results in persistent or significant disability / incapacity● Is a congenital anomaly / birth defect● Important medical events that may not result in death, be life-threatening, or requirehospitalization may be considered a serious adverse effect when, based upon appropriate medical judgment, they may jeopardize the subject or subject and may require medical or surgical intervention to prevent one of the outcomes listed in this definition. Example 2 In Vitro Testing of the Protease Modulating Activity of Nitric Oxide Generating Foam

[0203] The purpose of this study was to test a two-component foaming liquid's effects ona variety of proteases.

[0204] One of the molecular hypotheses for wound chronicity is that there is a disruptionof healing due to the activities of proteases. The protein destroying activities hypothetically leadto a lack of pro-healing growth factors. growth factor receptors being stripped from the surface of cells, and extracellular matrix destruction. Host-derived elastases have been found to be associated with growth factor destruction and receptor striping, whereas host-derived matrix metalloproteases have been associated with matrix destruction. Bacterial proteases have a mixture of activities, though P aeruginosa's acid protease is sufficient to generate a corneal ulcer (FIG.2).

[0205] A novel 2-part nitric oxide generating foam is in pre-clinical testing to determinethe effects on known barriers to healing. Canonically, nitric oxide is a known vasodilator, and the increased blood flow is anticipated to improve healing. A non-exhaustive list of activities of nitric oxide include regulation of apoptosis(1,2,7), neuroprotection(3), antibacterial(4), and antiviral(5). These activities are commonly considered to be "double edged" in that they seem to affect both the pro- and con- side of biological activities. The same can be said in the case of proteases: where there is evidence of activation(6) and of inhibition(7).

[0206] Here, the impact on in vitro protease activity using wound-relevant proteases isassessed for the two individual parts and the 1:1 combined final mixture. 45 59870042.1Attorney Docket No.: 274507 / NOXY24-001WO / 564918 Materials & Methods

[0207] A mix-and-read fluorescent substrate-based assay was used to measure theprotease activity of matrix metalloprotease (MMP) -1, -2, -8, -9, human neutrophil elastase (hNE), and a type-I collagenase mixture from Clostridium histolyticum. The assay was run in a 96-well plate format using a phosphate buffered saline buffer augmented with 1mM CaCI2 and 5pµM ZnCI2 + 0.05% polysorbate 20. The enzymes were tested at levels associated with extremely high proteolytic activity in human chronic wounds (5-10 µg / ml). A standard curve was generated for each enzyme to ensure a proportional response. The test product comes as a two-part unmixed pair of solutions (Part A and Part B); when applied the two solutions as foams are mixed 1:1 prior to application to the wound and periphery. Three test groups were utilized consisting of Part A, Part B, and a 1:1 mixture of Part A:B. Dilutions of each agent with diH2O resulting in 100%, 50%.25%. and 12.5% active agent were tested. The reaction was monitored for fluorescence generation with 485nm excitation and 528nm emission, 1 reading per minute for 10 minutes. The relative fluorescence unit (RFU) per minute slope was calculated for eachsample while the reaction was still in the linear phase. The residual activity was reported as theRFU / min observed divided by the RFU / min of the non-treated control. Results

[0208] As depicted in FIGS. 3 and 4, An effect was found among the control, Part A,Part B, and 1:1 mixture (multiple p-values, all p ≤ 1.69 x10-8). All components and the final mixture reduced or eliminated detectable protease activity. Dilution had no apparent effect on activity reduction for Part A, and there was a dose-dependent loss of inhibition with Part B. The dilution of the 1:1 mixture had a slight reduction of residual protease activity; with the most pronounced effect being with the C. histolyticum collagenase (0.7% residual activity in the 100%, down to 0.1% in the 12.5% diluted sample). Conclusions

[0209] These initial results indicate definite broad-spectrum anti-protease activity whentested in vitro. The anti-protease activity appears to be stable up to at least a 12.5% dilution. Additional testing in wound fluids or wound fluid surrogates is necessary to determine if the effects will be diminished in the presence of additional biomolecules. 46 59870042.1Attorney Docket No.: 274507 / NOXY24-001WO / 564918 Example 3 Nitric Oxide Delivering Foam Outperforms Other Commercially Available Actives for Wound Healing

[0210] Nitric oxide (NO) plays a critical role in the regulation of various wound healingprocesses, including perfusion, inflammatory response, cell proliferation, collagen formation, antimicrobial action, angiogenesis, wound contraction, and epithelialization.1-3For patients with chronic or critical illness, NO bioavailability is reduced, resulting in delayed wound healing.1,2Despite a large body of evidence supporting the benefits of supplemental NO. there were no available NO delivering products available for wound treatment prior to introduction of the study product.

[0211] The aim of this study was to compare wound healing rates among patients treatedwith a topical nitric oxide-delivering foam (NODF) alone, and those treated with the NODF plus commercially available actives (NODF+A) Nitric Oxide Delivering Foam ^FDA-registered OTC product^ Liquid foam^ Benzalkonium Chloride (an antiseptic)^ Formulated to deliver Nitric Oxide^ 5-minute topical applicationMethods

[0212] A research protocol, data collection form, and informed consent were developed.A data registry was designed to automate the data collection form. Product and data registry training was provided to facility staff by facility leadership. A topical nitric oxide delivering foam was implemented in 21 skilled nursing facilities across California. Clinical case patient selection was guided by physician judgement and data were entered by facility staff. Cases were categorized and data analyzed based on the use of the NODF alone, compared to the NODF used in conjunction with another wound product with active properties. Results and Discussion

[0213] Active wound therapies are commonly deployed for the treatment of chronic orcomplex wounds and are intended to activate wound healing. While commercially available actives may perform their single function consistently, NO delivers its vasodilatory, 47 59870042.1Attorney Docket No.: 274507 / NOXY24-001WO / 564918 antimicrobial, and cell signaling properties to heal more wounds faster, as suggested by the results of the study as depicted in FIG.5. The data demonstrates that the topical NODE catalyzed wound healing is better than the NODFA in terms of percentage of wounds healed. This suggests that the use of often costly actives may offer no additional wound healing benefits compared to using NODF alone, even among the most severe wounds. Treatment Cost

[0214] Based on common use of the NODF (three pump / three times per week), theaverage cost of NODE for complete healing of severe wounds was $45, compared the cost of other actives such as NPWT ($212 per dressing), or collagenase ($309.15 per 30g tube). Example 4 A Registry-based Study Assessing the Efficacy of a Nitric Oxide Delivering Foam for Wound Healing

[0215] Nitric oxide is a highly diffusible, endogenous vasodilator, antimicrobial, and cellsignaling molecule. Nitric oxide also modulates hemostasis, Inflammation, immune response, debridement. matrix metalloprotease activity, perfusion, angiogenesis, collagen synthesis, granulation formulation, wound contraction, epithelialization, and reduced scarring1-3; all processes necessary for effective wound healing.

[0216] In the healthy adult, nitric oxide is produced in sufficient amounts for effectivewound healing. However, persons with advanced age, critical illness, malnutrition, or chronic illnesses such as cardiovascular disease, diabetes, and obesity may be deficient in nitric oxide, leading to delayed wound healing.1

[0001] The aim of the study was to assess the effectiveness of a first-of-its-kind topical nitricoxide-delivering foam for wound healing. The nitric oxide delivering foam is depicted in FIG. 6.

[0217] Research protocol, data collection form, and informed consent documents weredeveloped. A data registry was designed to automate the data collection form. Product and data registry training were provided to facility staff by facility leadership. A topical nitric oxide delivering foam was implemented in 21 skilled nursing facilities across California. Patient selection for product use was guided by physician judgement. Clinical cases were submitted to 48 59870042.1Attorney Docket No.: 274507 / NOXY24-001WO / 564918 the registry by facility staff following the subjects last visit, due to complete closure, discharge, or treatment with the NO delivering foam stopped for any reason. Data collection included healthcare provider demographics, patient demographics, wound and treatment history, and wound assessment and treatment details. No protected health information was collected. Data were analyzed in aggregate form. Results and Discussion

[0218] The results are summarized in FIGS. 7 and 8. The NO delivering foam costapproximately $5.00 per treatment. Based on MWF application for 2 weeks, the most common dose and duration across the registered wound cases; the total cost to heal even severe wounds was $30.00 but could be as little as $10.00.

[0219] Data collection is ongoing, but this data demonstrates that the topical nitric oxide-delivering foam is a cost-effective option for timely wound healing across various wound etiologies and severities of injury. Example 5 High Risk Healing: Use of a Nitric Oxide Delivering Foam for Wound Healing Among Patients with Obesity and Diabetes

[0220] The prevalence of obesity has more than doubled in the last two decades and type2 diabetes is the most devastating complication of obesity. Decreased nitric oxide bioavailability is a feature of both obesity and diabetes. A deficiency in nitric oxide (NO) increases the risk of wound development, can slow the healing process, increases recovery duration, and heightens the risk of infections and complications associated with wounds.

[0221] The aim of this study is to access the effectiveness of a first-of-its-kind topicalnitric oxide-delivering foam (NODF) for wound healing. Materials and Methods

[0222] The nitric oxide delivering foam is described in Biological Example 3. A researchprotocol, data collection form, and informed consent were developed. A data registry was designed to automate the data collection form. Product and data registry training was provided to facility staff by facility leadership. A topical nitric oxide delivering foam was implemented in 21 skilled nursing facilities across California. Clinical case patient selection was guided by physician judgement and data were entered by facility staff. Cases were categorized and data 49 59870042.1Attorney Docket No.: 274507 / NOXY24-001WO / 564918 analyzed based on the presence of diabetes mellitus (DM) and / or obesity (0) versus no diabetes mellitus or obesity.

[0223] Results and Discussion

[0224] The results are summarized in FIG. 9. Wounds occurring among patients withDM and / or obesity traditionally heal slower, are more severe or complex. and present increased risks of complications, including infection and amputation. Conversely, the outcomes of this study confirm that NO supplementation can catalyze superior wound healing rates among patients with DM and / or obesity, compared to those without DM or obesity. The caveat is that patients with DM and / or obesity may require more NO supplementation to heal.

[0225] Due to the cost-effectiveness of the NODF, the average treatment cost was $27for patients without DM or obesity, compared to $42 for those with DM and / or obesity. However, both costs are significantly lower than published treatment costs for similar wounds.4

[0226] This study demonstrates that the NODF can optimize rapid healing, which is ahallmark of effective treatment, even among traditionally hard to heal patients. Embodiments

[0227] The following listing of exemplary embodiments supports and is supported by thedisclosure provided herein.

[0228] Embodiment 1. A method of treating a wound in a human or animal patientin need thereof, wherein the method comprised applying a foamed composition comprising nitric oxide to the wound.

[0229] Embodiment 2. The method of claim 1 wherein the wound is selected fromthe group consisting of diabetic foot ulcers (DFUs), veinous leg ulcers (VLUs), pressure injuries, burns, traumatic wounds, deep tissue injuries (DTIs), abrasions, lacerations, skin tears, bruises, and arterial ulcers.

[0230] Embodiment 3. The method of embodiments 1 to 2 wherein the foamedcomposition comprising nitric oxide is administered at least once a day, twice a day, three times a day, four times a day, 6 times a day, 12 times a day, 24 times a day, every other day, twice a week, once a week, for at least one week and up to 12 months.

[0231] Embodiment 4. The method of any of embodiments 1 to 3, wherein thefoamed composition is applied to the wound each day for from 1 day to 12 weeks, 1 week to 12 50 59870042.1Attorney Docket No.: 274507 / NOXY24-001WO / 564918 weeks, 2 to 12 weeks, or from 2 to 10 weeks, or from 2 to 8 weeks, or from 2 to 6 weeks, or from 2 to 4 weeks, or from 4 to 12 weeks, or from 4 to 10 weeks, or from 4 to 8 weeks, or from 4 to 6 weeks, or from 6 to 12 weeks, or from 6 to 10 weeks, or from 6 to 8 weeks, or from 8 to 12 weeks or from 10 to 12 weeks.

[0232] Embodiment 5. The method of any of embodiments 1 to 4, wherein thefoamed composition comprises: a first solution comprising water, at least one nitrite reactant, and a cationic surfactant; a second solution comprising water, at least one acidic reactant, and a cationic surfactant; and a collapsible foam which is the foamed composition, obtained by mixing the first solution and the second solution.

[0233] Embodiment 6. The method of any of embodiments 1 to 5, wherein thefoamed composition comprises: a first solution comprising water, at least one nitrite reactant, and a cationic surfactant; a second solution comprising water, at least one acidic reactant, and a cationic surfactant; a first foam obtained by aerating the first solution; a second foam obtained by aerating the second solution; and a collapsible foam which is the foamed composition, obtained by mixing the first foam and the second foam.

[0234] Embodiment 7. The method of either of embodiments 5 or 6, wherein thenitrite reactant is sodium nitrite, the acidic reactant is citric acid, and the surfactant is coco betaine.

[0235] Embodiment 8. The method of any of embodiments 5 to 7 wherein theeffective amount of the foamed composition per cm2comprises 0.1 to 0.4 g of an acidic solution comprising a cationic surfactant and 0.1 to 0.4 g of a sodium nitrite solution comprising a cationic surfactant.

[0236] Embodiment 9. The method of any one of embodiments 1 to 8, wherein thefoamed composition is applied to the wound for from one second to up to 24 hours.

[0237] Embodiment 10. The method of any of embodiments 1 to 9, wherein thefoamed composition overlays the wound to be treated without completely collapsing, from more 51 59870042.1Attorney Docket No.: 274507 / NOXY24-001WO / 564918 than 1 minute to up to an hour.

[0238] Embodiment 11. The method of any one of the foregoing embodimentswherein the wound to be treated is reduced in area and / or volume by from 50% to 100%, compared to before treatment, after 4 weeks of treatment, or after 5 weeks, or after 6 weeks, or after 7 weeks, or after 8 weeks, or after 9 weeks, or from 10 weeks, or after 11 weeks, or after 12 weeks.

[0239] Embodiment 12. The method of any one of the foregoing embodimentswherein the volume or the area, or both the volume and the area, of the wound decreases over time in a period from less than a week to 4 months.

[0240] Embodiment 13. The method of embodiment 12, wherein the volume or thearea, or both the volume and the area, of the wound is measured according to the University of Texas Staging System for Diabetic Foot Ulcers, the Wagner–Meggitt classification system, the Saint Elian Wound Score System (SEWSS), the SINBAD Classification System and Score, and / or the Society for Vascular Surgery Lower Extremity Threatened Limb Classification System (WIfI).

[0241] Embodiment 14. The method of any one of the foregoing embodimentswherein the area and / or the volume of the wound to be treated decreases to complete wound closure, wherein complete wound closure is defined as substantially complete re-epithelialization without drainage or dressing requirements confirmed at two observations two weeks apart.

[0242] Embodiment 15. The method of embodiment 14, wherein complete re-epithelialization means from 70% to 100%, or from 80% to 100%, or from 90% to 100%, or from 70% to 90%, or from 80% to 90%, or from 70% to 80%, or 100% re-epithelialization.

[0243] Embodiment 16. The method of any one of the foregoing embodimentswherein the patient’s level of methemoglobin does not change by more than +-1% of the patient’s pre-treatment level of methemoglobin during the treatment.

[0244] Embodiment 17. The method of any one of the foregoing embodimentswherein the wound is also treated by the standard of care, wherein the standard of care comprises at least one and optionally more than one of a therapy selected from one or more of moisture control, debridement, dressing changes, wound off-loading, revascularization, infection control, glycemic control, and multidisciplinary care. 52 59870042.1Attorney Docket No.: 274507 / NOXY24-001WO / 564918

[0245] Embodiment 18. The method of any one of the foregoing embodimentswherein a concentration of ambient nitric oxide gas produced from the foamed composition within 1 foot of the wound is at or below 25 ppm over an eight-hour period.

[0246] Embodiment 19. The method of embodiment 18 wherein the concentrationof ambient nitric oxide gas within 1 foot of the wound is at or below 20 ppm, 15 ppm, 10 ppm, or 5 ppm over an eight-hour period.

[0247] Embodiment 20. The method of any of the foregoing embodiments whereinthe foamed composition reduces pathogen concentration at the wound to be treated.

[0248] Embodiment 21. The method of any of the foregoing embodiments whereinthe foamed composition reduces the need for debridement of the wound.

[0249] Embodiment 22. The method of any of the foregoing embodiments, whereinthe treatment is safe and tolerable to the subject. REFERENCES Adler, B. L., & Friedman, A. J. (2015). Nitric oxide therapy for dermatologic disease. Future Science OA, 1(1). Armstrong, D. G., Boulton, A. J., & Bus, S. A. (2017). Diabetic foot ulcers and their recurrence. New England Journal of Medicine, 376(24), 2367-2375. Armstrong, D. G., Swerdlow, M. A., Armstrong, A. A., Conte, M. S., Padula, W. V., & Bus, S. A. (2020). Five-year mortality and direct costs of care for people with diabetic foot complications are comparable to cancer. Journal of foot and ankle research, 13(1), 1-4. Armstrong D. G., Basic principles of wound healing, In: UpToDate, Post TW (Ed), UpToDate, Waltham, MA. (Accessed on March 30, 2022.) Barraud, N., J Kelso, M., A Rice, S., & Kjelleberg, S. (2015). Nitric oxide: a key mediator of biofilm dispersal with applications in infectious diseases. Current pharmaceutical design, 21(1), 31-42. Castilla, D. M., Liu, Z. J., & Velazquez, O. C. (2012). Oxygen: implications for wound healing. Advances in wound care, 1(6), 225-230. Frank, S., Kämpfer, H., Wetzler, C., & Pfeilschifter, J. (2002). Nitric oxide drives skin repair: novel functions of an established mediator. Kidney international, 61(3), 882-888. 53 59870042.1Attorney Docket No.: 274507 / NOXY24-001WO / 564918 Gregg, E. W., Sorlie, P., Paulose-Ram, R., Gu, Q., Eberhardt, M. S., Wolz, M., ... & Geiss, L. (2004). Prevalence of lower-extremity disease in the US adult population ≥ 40 years of age with and without diabetes: 1999–2000 national health and nutrition examination survey. Diabetes care, 27(7), 1591-1597. Guzik, T., Korbut, R., & Adamek-Guzik, T. (2003). Nitric oxide and superoxide in inflammation. J physiol pharmacol, 54(4), 469-487. Krzyszczyk, P., Schloss, R., Palmer, A., & Berthiaume, F. (2018). The role of macrophages in acute and chronic wound healing and interventions to promote pro-wound healing phenotypes. Frontiers in physiology, 9, 419. Malone‐Povolny, M. J., Maloney, S. E., & Schoenfisch, M. H. (2019). Nitric oxide therapy for diabetic wound healing. Advanced healthcare materials, 8(12), 1801210. Murray, P. J., & Wynn, T. A. (2011). Protective and pathogenic functions of macrophage subsets. Nature reviews immunology, 11(11), 723-737. Ramsey, S. D., Newton, K., Blough, D., Mcculloch, D. K., Sandhu, N., Reiber, G. E., & Wagner, E. H. (1999). Incidence, outcomes, and cost of foot ulcers in patients with diabetes. Diabetes care, 22(3), 382-387. Schairer, D., Chouake, J., Nosanchuk, J., Friedman, A., (2012). The potential of nitric oxide releasing therapies as antimicrobial agents. Virulence 3:3, 271-279; May / June 2012; Landes Bioscience. Miller CM et al. Acidified nitrite foam anti-microbial action in an ex vivo porcine dermal model. Journal of Wound Management 2023;24(3):81-85. DOI https: / / doi.org / 10.35279 / jowm2024.25.01.02 Other Embodiments

[0250] The foregoing disclosure has been described in some detail by way of illustrationand example, for purposes of clarity and understanding. The invention has been described with reference to various specific and preferred embodiments and techniques. However, it should be understood that many variations and modifications can be made while remaining within the spirit and scope of the invention. It will be obvious to one of skill in the art that changes and modifications can be practiced within the scope of the appended claims. Therefore, it is to be 54 59870042.1Attorney Docket No.: 274507 / NOXY24-001WO / 564918 understood that the above description is intended to be illustrative and not restrictive.

[0251] The scope of the invention should, therefore, be determined not with reference tothe above description, but should instead be determined with reference to the following appended claims, along with the full scope of equivalents to which such claims are entitled. 55 59870042.1

Claims

Attorney Docket No.: 274507 / NOXY24-001WO / 564918 Claims 1. A method of treating a wound in a human or animal patient in need thereof, wherein the method comprises applying a foamed composition comprising nitric oxide to the wound.

2. The method of claim 1 wherein the wound is selected from the group consisting of diabetic foot ulcers (DFUs), veinous leg ulcers (VLUs), pressure injuries, burns, traumatic wounds, deep tissue injuries (DTIs), abrasions, lacerations, skin tears, bruises, and arterial ulcers.

3. The method of any of claim 1 wherein the foamed composition comprising nitric oxide is administered at least once a day, twice a day, three times a day, four times a day, 6 times a day, 12 times a day, 24 times a day, every other day, twice a week, once a week, for at least one week and up to 12 months.

4. The method of any of claims 1 to 3, wherein the foamed composition is applied to the wound each day for from 1 day to 12 weeks, 1 week to 12 weeks, 2 to 12 weeks, or from 2 to 10 weeks, or from 2 to 8 weeks, or from 2 to 6 weeks, or from 2 to 4 weeks, or from 4 to 12 weeks, or from 4 to 10 weeks, or from 4 to 8 weeks, or from 4 to 6 weeks, or from 6 to 12 weeks, or from 6 to 10 weeks, or from 6 to 8 weeks, or from 8 to 12 weeks or from 10 to 12 weeks.

5. The method of any of claims 1 to 4, wherein the foamed composition comprises: a first solution comprising water, at least one nitrite reactant, and a cationic surfactant; a second solution comprising water, at least one acidic reactant, and a cationic surfactant; and a foam which is the foamed composition, obtained by mixing the first solution and the second solution.

6. The method of any of claims 1 to 5, wherein the foamed composition comprises: a first solution comprising water, at least one nitrite reactant, and a cationic surfactant; a second solution comprising water, at least one acidic reactant, and a cationic surfactant; a first foam obtained by aerating the first solution; a second foam obtained by aerating the second solution; and 56 59870042.1Attorney Docket No.: 274507 / NOXY24-001WO / 564918 a foam which is the foamed composition, obtained by mixing the first foam and the second foam.

7. The method of either of claims 5 or 6, wherein the nitrite reactant is sodium nitrite, the acidic reactant is citric acid, and the surfactant is coco betaine.

8. The method of any of claims 5 to 7 wherein the effective amount of the foamed composition comprises 0.1 to 0.4 g of an acidic solution comprising a cationic surfactant and 0.1 to 0.4 g of a sodium nitrite solution comprising a cationic surfactant.

9. The method of any one of claims 1 to 8, wherein the foamed composition is applied to the wound for from one second to up to 24 hours.

10. The method of any of claims 1 to 9, wherein the foamed composition overlays the wound to be treated without completely collapsing, from more than 1 minute to up to an hour.

11. The method of any one of the foregoing claims wherein the wound to be treated is reduced in area and / or volume by from 50% to 100%, compared to before treatment, after 4 weeks of treatment, or after 5 weeks, or after 6 weeks, or after 7 weeks, or after 8 weeks, or after 9 weeks, or from 10 weeks, or after 11 weeks, or after 12 weeks.

12. The method of any one of the foregoing claims wherein the volume or the area, or both the volume and the area, of the wound decreases over time in a period from less than a week to 4 months.

13. The method of claim 12, wherein the volume or the area, or both the volume and the area, of the wound is measured according to the University of Texas Staging System for Diabetic Foot Ulcers, the Wagner–Meggitt classification system, the Saint Elian Wound Score System (SEWSS), the SINBAD Classification System and Score, and / or the Society for Vascular Surgery Lower Extremity Threatened Limb Classification System (WIfI). 57 59870042.1Attorney Docket No.: 274507 / NOXY24-001WO / 564918 14. The method of any one of the foregoing claims wherein the area and / or the volume of the wound to be treated decreases to complete wound closure, wherein complete wound closure is defined as substantially complete re-epithelialization without drainage or dressing requirements confirmed at two observations two weeks apart.

15. The method of claim 14, wherein complete re-epithelialization means from 70% to 100%, or from 80% to 100%, or from 90% to 100%, or from 70% to 90%, or from 80% to 90%, or from 70% to 80%, or 100% re-epithelialization.

16. The method of any one of the foregoing claims wherein the patient’s average level ofmethemoglobin does not change by more than +-1% of the patient’s pre-treatment level of methemoglobin during the treatment.

17. The method of any one of the foregoing claims wherein the wound is also treated by the standard of care, wherein the standard of care comprises at least one and optionally more than one of a therapy selected from one or more of moisture control, debridement, dressing changes, wound off-loading, revascularization, infection control, glycemic control, and multidisciplinary care.

18. The method of any one of the foregoing claims wherein a concentration of ambient nitricoxide gas produced from the foamed composition within 1 foot of the wound is at or below 25 ppm over an eight-hour period.

19. The method of claim 18 wherein the concentration of ambient nitric oxide gas within 1foot of the wound is at or below 20 ppm, 15 ppm, 10 ppm, or 5 ppm over an eight-hour period.

20. The method of any of the foregoing claims wherein the foamed composition reduces pathogen concentration at the wound to be treated.

21. The method of any of the foregoing claims wherein the foamed composition reduces the need for debridement of the wound. 58 59870042.1Attorney Docket No.: 274507 / NOXY24-001WO / 564918 22. The method of any of the foregoing claims, wherein the treatment is safe and tolerable to the subject. 59 59870042.1

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