Nitric oxide for use in the treatment of burns

Abstract

Disclosed herein is a method of treating a burn injury of a mammal requiring such treatment, comprising applying a topical composition comprising nitric oxide to the burn injury of the mammal.

Description

Attomey Docket No.: 274507 / 24-006WO / 573634Method of Treating Burns Using Nitric OxideBackground

[0001] Combined bum injuries heighten the need for point of injury wound healing agents that halt burn progression, promote wound healing, and fight infection. Such agents should also exhibit modes of action which stimulate the downstream stages of wound healing. The body’s natural molecule capable of meeting this high bar is nitric oxide (NO). NO is a powerful antimicrobial and wound healing agent that alters burn progression and advances the trajectory of a wound. Zhu H, Wei X, Bian K, Murad F. Effects of nitric oxide on skin bum wound healing. J Bum Care Res. 2008;29(5):804-14. doi: 10.1097 / BCR.0b013e3181848119. PubMed PMID: 18695618.

[0002] To this end, NO has three modes of action that address the complications of combined injuries. Firstly, NO is an endogenous cellular signaling molecule that governs the wound healing sequence from injury to inflammation resolution, cell proliferation, collagen deposition, and angiogenesis. Goedeke R. Decoding the benefits of nitric oxide for skin health. Biosphere Nutrition. Secondly, NO is a natural vasodilator that draws blood flow into otherwise ischemic environments. Poh WH, Rice SA. Recent Developments in Nitric Oxide Donors and Delivery for Antimicrobial and Anti-Biofilm Applications. Molecules. 2022;27(3). Epub 20220120. doi: 10.3390 / molecules27030674. PubMed PMID: 35163933; PMCID: PMC8839391. Thirdly, NO is a powerful broad-spectrum antiviral, antifungal, and antimicrobial agent with no known resistance that eradicates antibiotic resistant bacterial strains in both their planktonic and biofilm phenotypes. Schairer DO, Chouake JS, Nosanchuk JD, Friedman AJ. The potential of nitric oxide releasing therapies as antimicrobial agents. Virulence. 2012;3(3):271 -9. Epub 20120501. doi: 10.4161 / viru.20328. PubMed PMID: 22546899; PMCID: PMC3442839. Thus, NO therapies have multiple critical modes of action and stand apart from other wound healing agents.

[0003] However, despite the many benefits of nitric oxide for wound healing generally and burn injuries more particularly, there are currently no topical NO products in the mainstream wound care market. As a result, a need remains for a topical NO composition for the treatment of burn injuries.-1-61968708 1Attomey Docket No.: 274507 / 24-006WO / 573634Summary of the Invention

[0004] These and other needs are met by the present invention, which in one aspect is directed to a method of minimizing the severity of a bum injury of a mammal by halting the progression, comprising: applying a topical composition comprising nitric oxide to the bum injury of the mammal; wherein the amount of nitric oxide delivered from each application is from 0.001 pmol to 100 pmol over from 1 minute to 24 hour application time; and the topical composition is allowed to remain on the bum injury for from 30 seconds to 72 hours.

[0005] In another aspect, what is provided is a method of minimizing the progression of a bum injury infection of a mammal requiring such treatment, comprising: applying a topical composition comprising nitric oxide to the bum injury of the mammal; wherein the amount of nitric oxide delivered from each application is from 0.001 pmol to 100 pmol over a 1 to 10 minute application time; the topical composition is allowed to remain on the bum injury for from 30 seconds to 72 hours; the topical composition comprising nitric oxide is applied at least twice a week until the burn injury is healed; and wherein the infection is caused by bacteria, fungi, yeasts, or viruses, or combinations thereof.

[0006] In a further aspect, what is provided is a method of a eradicating a burn injury bioburden or biofilm of a mammal requiring such treatment, comprising: applying a topical composition comprising nitric oxide to the bum injury of the mammal; wherein the amount of nitric oxide delivered from each application is from 0.001 pmol to 100 pmol over a 1 to 10 minute application time; the topical composition comprising nitric oxide is allowed to remain on the bum injury for from 30 seconds to 72 hours; and the topical composition comprising nitric oxide is applied at least twice a week until the burn injury is healed.

[0007] In another aspect what is provided is a method of reducing burn injury inflammation and minimizing burn injury infection of a mammal requiring such treatment, comprising:-2-61968708 1Attomey Docket No.: 274507 / 24-006WO / 573634 applying a topical composition comprising nitric oxide to the bum injury of the mammal; wherein the amount of nitric oxide delivered from each application is from 0.001 pmol to 100 pmol over a 1 to 10 minute application time; the topical composition comprising nitric oxide is allowed to remain on the bum injury for from 30 seconds to 72 hours; and the topical composition comprising nitric oxide is applied at least twice a week until the burn injury is healed.Summary of the Figures

[0008] FIG. 1 provides a illustration of how the nitric oxide foam product allows for easy dermal absorption of nitric oxide.

[0009] FIG. 2 depicts total bacterial counts after treatment of a burn with NO Formula 1 and Formula 2 compared to vehicle.

[0010] FIG. 3 depicts wound epithelialization percent after treatment with NO Formula 1 and Formula 2 compared to vehicle.Detailed DescriptionDeflnitions

[0011] As used herein, “burn,” "bum wound" and "bum injury" can be used interchangeably.

[0012] As used herein, "bum wound progression" refers to factors that can induce a superficial burn wound to become a deep subcutaneous burn with severe consequences to bum victims, wherein a superficial partial thickness bum progresses in severity, to an deep partial-thickness burn to full thickness burns. As described by Salibian and coworkers, the concept of burn wound progression originated in the traditional description of burn injury zones in which three different zones of tissue damage of varying degrees can occur after a bum injury, including the central irreversibly damaged zone of coagulation, the surrounding damaged but threatened zone of stasis, and the outermost recoverable zone of hyperemia. The middle zone of stasis has been deemed a therapeutically critical section of bum surface area to address as it loses perfusion in its natural course, dies, and regresses into the inner zone of coagulation that cannot be salvaged. The issue of progression of injury in the zone of stasis is pivotal, since bum wound conversion often contributes to both greater bum surface area and burn depth. This larger, deeper wound has multiple local and systemic consequences that increase complications and morbidity. See-3-61968708 1Attomey Docket No.: 274507 / 24-006WO / 573634Salibian et.. al., Burns. 2016 Aug; 42(5): 1025-1035; published online 2016 Jan 17. doi: 10.1016 / j. burns.2015.11.007 (citing other references).

[0013] As used herein, "infection" refers to infections caused by bacteria, fungi, yeasts, viruses, as well as combinations thereof, and to morphological aspects of infection, for instance, as related to biofilms and bioburden.

[0014] The terms “first degree burn,” “second degree burn,” “third degree bum,” and “fourth degree burn.” Are the traditional classification of burn severity. This classification system has recently be replaced by a system reflecting the need for surgical intervention. Current designations of bum depth are superficial, superficial parti al -thickness, deep partial -thickness, and full-thickness. The term fourth degree is still used to describe the most severe burns, burns that extend beyond the skin into the subcutaneous soft tissue and can involve underlying vessels, nerves, muscle, bone, and joints. See https: / / www.uptodate.com / contents / assessment-and- classification-of-burn-injury / print (last visited August 18, 2025).Embodiments

[0015] As noted previously, in one aspect, what is provided is a method of minimizing the severity of a bum injury of a mammal by halting the progression, comprising: applying a topical composition comprising nitric oxide to the bum injury of the mammal; wherein the amount of nitric oxide delivered from each application is from 0.001 pmol to 100 pmol over from 1 minute to 24 hour application time; and the topical composition is allowed to remain on the burn injury for from 30 seconds to 72 hours.

[0016] In one embodiment of this aspect, the topical composition comprising nitric oxide is applied twice on the first day wherein the time between the first and second applications is 5 to 25 minutes.

[0017] In another embodiment, the topical composition comprising nitric oxide is applied at least every other day after the first day until the burn injury is healed.

[0018] In another embodiment, the topical composition comprising nitric oxide is applied at least twice a week after the first day until the burn injury is healed.

[0019] In a further embodiment, the burn is caused by extreme heat, extreme cold, high-voltage electricity, chemical exposure, electromagnetic energy, mechanical action, or radiation.-4-61968708 1Attomey Docket No.: 274507 / 24-006WO / 573634

[0020] In an additional embodiment, the topical composition comprising nitric oxide is applied every 12 hours; or every 24 hours; or 48 hours; or 72 hours; or once a week.

[0021] In an additional further embodiment, the amount of nitric oxide delivered from the topical composition to the bum injury over a 5 minute application time is 0.001 pmol to 0.01 pmol; 0.001 pmol to 0.1 pmol; 0.01 pmol to 10 pmol; 0.1 pmol to 1 pmol; 1 pmol to 10 pmol; or 10 pmol to 100 pmol.

[0022] In an additional further embodiment, the bum injury is infected by bacteria, fungi, yeasts, or viruses, or combinations thereof. In this and other embodiments, the infection is caused by gram positive bacteria or gram negative bacteria, wherein the gram positive bacteria or gram negative bacteria is selected form the group consisting of Staphylococcus epidermidis, Staphylococcus aureus, methicillin resistant Staphylococcus aureus, vancomycin intermediate Staphylococcus aureus, enterococcus, vancomycin resistant enterococcus, Pseudomonas aeruginosa, Acinetobacter, Escherichia coli, Klebsiella, Enterobacter, Serratia, Proteus, Bacteroides and Fusobacterium spp. Alternatively, in this and other embodiments, the infection is caused by fungi or yeast, wherein the fungi or yeast is Candida albicans, Aspergillus, or Fusarium.

[0023] In another embodiment, the topical composition is a gel or a foam.

[0024] In another embodiment, the topical composition is a gel.

[0025] In another embodiment, the topical composition is a foam.

[0026] In another embodiment, the topical dermatologic composition is defined as FormulationA and comprises: a first aqueous solution comprising:1-20 weight percent of at least one acid based on the total weight of the first solution;0.05 to 10.2 weight percent of a surfactant based on the total weight of the first solution; a second aqueous solution comprising:1-35 weight percent of a nitric oxide precursor based on the total weight of the second solution;0.05 to 10.2 weight percent of a surfactant based on the total weight of the second solution; wherein a resultant mixed medium containing nitric oxide is produced when the first and second solutions are mixed.

[0027] In one embodiment, the resultant mixed medium is a gel. In another embodiment, the-5-61968708 1Attomey Docket No.: 274507 / 24-006WO / 573634 resultant mixed medium is a foam.

[0028] In a further embodiment, the topical NO composition can be a composition of Formula A, which is Formulation B, an example of which is Advanox®. Advanox® is a composition of an acidified nitrite in a surfactant-containing aqueous foam formulation that is delivered in a two- part pump dispenser. See pmc.ncbi.nlm.nih.gov / articles / PMCl 1677572 / (last visited August 19, 2025).

[0029] Topical NO Foam is a foam based gaseous nitric oxide (NO) product where NO is delivered through a microbubble foam. See https: / / clinicaltrials.gov / study / NCT06020664. One pump each of Solution A (0.3g, containing Citric acid) and Solution B (0.3g, containing Sodium nitrite) will be dispensed, mixed, and applied immediately per square centimeter of wound length. Topical NO Foam is topically applied directly onto the wound bed and left on the wound bed for a 0.5 to 10 minute period. The composition of Topical NO Foam and its use is disclosed in, for example, US Pat. Nos. 10,052,348, 10,751,364, US 11,304,972, the entire contents of each of which is incorporated herein by reference.

[0030] In another embodiment, the composition can be a composition of Formula A, which is Formulation C, which is a topical liquid composition that contains an antiseptic such as but not limited to benzalkonium chloride, sodium nitrite, coco betaine, deionized water, and citric acid, an example of which is NoxyDerm®-benzalkonium liquid. Topical liquid compositions and their use are disclosed in, for example, USSN 18 / 667,467 (US2024 / 0382517), the entire contents of which is incorporated herein by reference. Also see dailymed. nlm.nih.gov / dailymed / druglnfo.cfm?setid=063ale99-dda6-9cb9-e063-6294a90aalb3 (last visited August 19, 2025).

[0031] In another aspect, what is provided is a method of minimizing the progression of a bum injury infection of a mammal requiring such treatment, comprising: applying a topical composition comprising nitric oxide to the bum injury of the mammal; wherein the amount of nitric oxide delivered from each application is from 0.001 pmol to 100 pmol over a 1 to 10 minute application time; the topical composition is allowed to remain on the bum injury for from 30 seconds to 72 hours; the topical composition comprising nitric oxide is applied at least twice a week until the burn injury is healed; and-6-61968708 1Attomey Docket No.: 274507 / 24-006WO / 573634 wherein the infection is caused by bacteria, fungi, yeasts, or viruses, or combinations thereof.

[0032] In one embodiment of this aspect, the infection is caused by gram positive bacteria or gram negative bacteria, wherein the gram positive bacteria or gram negative bacteria is selected form the group consisting of Staphylococcus epidermidis, Staphylococcus aureus, methicillin resistant Staphylococcus aureus, vancomycin intermediate Staphylococcus aureus, enterococcus, vancomycin resistant enterococcus, Pseudomonas aeruginosa, Acinetobacter, Escherichia coli, Klebsiella, Enterobacter, Serratia, Proteus, Bacteroides and Fusobacterium spp.

[0033] In another embodiment, the infection is caused by fungi or yeast, wherein the fungi or yeast is Candida albicans, Aspergillus, or Fusarium.

[0034] In a further embodiment, the infection relates to morphological aspects of infection, for instance, as related to wound biofdms and wound bioburdens. "Wound bioburden" refers to all of the dimensions of wound microbiology, including burn microbiology, believed to be important in the development of wound infection. These dimensions include microbial load, microbial diversity, and the presence of pathogenic organisms. Gardner, et. al. Biol Res Nurs. 2008 Jul; 10(1): 44-53; doi: 10.1177 / 1099800408319056. A "wound biofilm" refers to a microbial colony encased in a polysaccharide matrix which can become attached to a wound surface, including the surface of a bum wound. As noted by Percival and coworkers, because open wounds, including bum wounds, lack the protective covering of skin, they contain microorganisms from endogenous (the patient's own flora) or exogenous sources. Percival et. al. Adv Wound Care (New Rochelle). 2015 Jul 1; 4(7): 373-381; doi: 10.1089 / wound.2014.0557. In the early stages of the formation of a chronic wound, these microbes are generally held in check or destroyed by the host's immune system. However, if microbes attach to the wound surface and proliferate, a biofilm will begin to develop. When the biofilm is well established, it will exhibit resistance to destruction by the host immune system and antimicrobials. At this stage, the biofilm is considered mature and more difficult to eradicate. When this occurs, the wound is defined as being in a biofilm infected state.6 In this state, it is difficult to kill microorganisms and treatment will require specialized management practices. In these situations the risk of a wound not healing and becoming overtly clinically infected (i.e., showing signs of inflammation or purulence) is increased. Consequently, preventing a biofilm in the first place is fundamental for faster and more effective treatment of chronic wounds.-7-61968708 1Attomey Docket No.: 274507 / 24-006WO / 573634

[0035] In a further embodiment, the topical composition comprising nitric oxide is applied every 12 hours; or every 24 hours; or 48 hours; or 72 hours; or once a week.

[0036] In a further embodiment, the amount of nitric oxide delivered from the topical composition to the bum injury over a 5 minute application time is 0.001 pmol to 0.01 pmol; 0.001 pmol to 0.1 pmol; 0.01 pmol to 10 pmol; 0.1 pmol to 1 pmol; 1 pmol to 10 pmol; or 10 pmol to 100 qmol.

[0037] In another embodiment, the topical composition is a gel or a foam.

[0038] In another embodiment, the topical composition is a gel.

[0039] In another embodiment, the topical composition is a foam.

[0040] In a further embodiment, the topical composition can be Topical NO Foam (Advanox®). Topical NO Foam is a foam based gaseous nitric oxide (NO) product where NO is delivered through a microbubble foam. See https: / / clinicaltrials.gov / study / NCT06020664. One pump each of Solution A (0.3g, containing citric acid) and Solution B (0.3g, containing sodium nitrite) will be dispensed, mixed for five seconds and applied immediately per each square centimeter of wound area using any sterile applicator. Topical NO Foam is topically applied directly onto the wound bed and left on the wound bed for a 5-minute period. The composition of Topical NO Foam and its use is disclosed in, for example, US Pat. Nos. 10,052,348, 10,751,364, US 11,304,972, the entire contents of each of which is disclosed herein by reference.

[0041] In a further aspect, what is provided is a method of eradicating a bum injury bioburden or biofilm of a mammal requiring such treatment, comprising: applying a topical composition comprising nitric oxide to the bum injury of the mammal; wherein the amount of nitric oxide delivered from each application is from 0.001 pmol to 100 pmol over a 1 to 10 minute application time; the topical composition comprising nitric oxide is allowed to remain on the burn injury for from 30 seconds to 72 hours; the topical composition comprising nitric oxide is applied at least twice a week until the burn injury is healed.

[0042] In a further aspect, what is provided is a method of reducing bum injury inflammation and minimizing bum injury infection of a mammal requiring such treatment, comprising: applying a topical composition comprising nitric oxide to the bum injury of the mammal;-8-61968708 1Attomey Docket No.: 274507 / 24-006WO / 573634 wherein the amount of nitric oxide delivered from each application is from 0.001 pmol to 100 pmol over a 1 to 10 minute application time; the topical composition comprising nitric oxide is allowed to remain on the bum injury for from 30 seconds to 72 hours; the topical composition comprising nitric oxide is applied at least twice a week until the burn injury is healed.

[0043] The invention will now be illustrated by the following non-limiting examplesOther Aspects and Embodiments

[0044] In one aspect, what is provided is a method of treating a bum injury of a mammal comprising: applying a topical composition comprising nitric oxide to the burn injury of the mammal, and the topical composition is allowed to remain on the burn injury for a period of time.

[0045] In one embodiment of this aspect, the topical composition is applied for 10 seconds, 30 seconds, 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 10 minutes, 24 hours, 48 hours, 72 hours, or more than 72 hours.

[0046] In another embodiment the source for the NO in the topical composition is selected from the group consisting of a nitrite, a nitrate, NO gas, a diazeniumdiolate, a NO-impregnated polymer, nano-crystalline NO particles, NO-metal complexes, nitroprusside, nitrosamine, a nitrate ester, a nitrosothiols, L-arginine, or combinations thereof.

[0047] In another embodiment, the composition is a film, gel, cream, spray or foam.

[0048] In another embodiment, the bum is a first degree bum, second degree burn, third degree burn, or fourth degree burn. The traditional classification of burns as first, second, third, or fourth degree has been replaced by a system reflecting the need for surgical intervention. Current designations of bum depth are superficial, superficial partial -thickness, deep partial -thickness, and full-thickness. The term fourth degree is still used to describe the most severe burns, burns that extend beyond the skin into the subcutaneous soft tissue and can involve underlying vessels, nerves, muscle, bone, and joints. See https: / / www.uptodate.com / contents / assessment-and- classification-of-burn-injury / print (last visited August 18, 2025).

[0049] In another embodiment, the bum wound exhibits one or more of decreased edema, decreased eschar, decreased slough, decreased necrosis, stimulation of stem cell recruitment,-9-61968708 1Attomey Docket No.: 274507 / 24-006WO / 573634 keratinocyte differentiation, promote granulation, promote epithelialization, or increased perfusion. In another embodiment, the bum wound requires reduced hospitalization as compared to wounds or bums treated with the current standard of care.

[0050] In another aspect, what is provided is a method of minimizing the severity of a bum injury of a mammal, comprising: applying a topical composition comprising nitric oxide to the bum injury of the mammal; and the topical composition is allowed to remain on the bum injury for a period of time.

[0051] In one embodiment of this aspect, the topical composition is applied at the point of injury.

[0052] In another embodiment, the amount of nitric oxide delivered from each application is from 0.001 pmol to 100 pmol; and wherein the topical composition comprising nitric oxide is applied twice on the first treatment day wherein the time between the first and second applications is 5 to 25 minutes.

[0053] In another embodiment, the topical composition comprising nitric oxide is applied at least twice a week after the first day until the bum injury is healed.

[0054] In another embodiment, wherein the topical composition is applied after a delay of up to 72 hours.

[0055] In another embodiment, the bum is caused by extreme heat, extreme cold, high voltage electricity, chemical exposure, electromagnetic, mechanical action, or radiation.

[0056] In another embodiment, the topical composition comprising nitric oxide is applied every 12 hours; or every 24 hours; or 48 hours; or 72 hours; or once a week.

[0057] In another embodiment, the amount of nitric oxide delivered from the topical composition to the burn injury over five minutes, up to 24 hours, is 0.001 pmol to 0.01 pmol; 0.001 pmol to 0.1 pmol; 0.01 pmol to 10 pmol; 0.1 pmol to 1 pmol; 1 pmol to 10 pmol; or 10 pmol to 100 pmol.

[0058] In another embodiment, the bum injury is infected by bacteria, fungi, yeasts, or viruses, or combinations thereof.

[0059] In another embodiment, the method comprises applying a topical composition comprising nitric oxide to the bum injury of the mammal, and the topical composition is allowed to remain on the bum injury for 10 seconds, 30 seconds, 1 minute, 2 minutes, 3 minutes, 4 minutes, 5-10-61968708 1Attomey Docket No.: 274507 / 24-006WO / 573634 minutes, 10 minutes, 30 minutes, 1 hour, 2 hours, 3 hours, 6 hours, 8 hours, 12 hours, 24 hours, 48 hours, 72 hours, or more than 72 hours.

[0060] In another embodiment, the infection is caused by gram positive bacteria or gram negative bacteria.

[0061] In another embodiment, the gram positive bacteria or gram negative bacteria is selected from the group consisting of staph epidermidis, Staphylococcus aureus, methicillin resistant Staphylococcus aureus, vancomycin intermediate Staphylococcus aureus, enterococcus, vancomycin resistant enterococcus, Pseudomonas aeruginosa, Acinetobacter, Escherichia coli, Klebsiella, Enterobacter, Serratia, Proteus, Bacteroides and Fusobacterium spp.

[0062] In another embodiment, the infection is caused by fungi or yeast, wherein the fungi or yeast is Candida albicans, Aspergillus, or Fusarium.

[0063] In another embodiment, the topical composition is a foam.

[0064] In another embodiment, the topical composition is a gel.

[0065] In another aspect, what is provided is a method of managing or treating a burn infection of a mammal requiring such treatment, comprising applying a topical composition comprising nitric oxide to the injury of the mammal; the amount of nitric oxide delivered from each application is from 0.001 pmol to 100 pmol over a 1 to 10 minute application time; the topical composition is allowed to remain on the injury for from 30 seconds to 72 hours; wherein the topical composition comprising nitric oxide is applied at least twice a week until the injury is healed; and wherein the infection is caused by bacteria, fungi, yeasts, or viruses, or combinations thereof.

[0066] In one embodiment, the infection is caused by gram positive bacteria or gram negative bacteria, wherein the gram positive bacteria or gram negative bacteria is selected form the group consisting of staph epidermidis, Staphylococcus aureus, methicillin resistant Staphylococcus aureus, vancomycin intermediate Staphylococcus aureus, enterococcus, vancomycin resistant enterococcus, Pseudomonas aeruginosa, Acinetobacter, Escherichia coli, Klebsiella, Enterobacter, Serratia, Proteus, Bacteroides and Fusobacterium spp.

[0067] In another embodiment, the infection is caused by fungi or yeast, wherein the fungi or yeast is Candida albicans, Aspergillus, or Fusarium.

[0068] In another embodiment, the topical composition comprising nitric oxide is applied every 12 hours; or every 24 hours; or 48 hours; or 72 hours; or once a week.-11-61968708 1Attomey Docket No.: 274507 / 24-006WO / 573634

[0069] In another embodiment, the amount of nitric oxide delivered from the topical composition to the burn injury over 5 minutes is 0.001 pmol to 0.01 pmol; 0.001 pmol to 0.1 pmol; 0.01 pmol to 10 pmol; 0.1 pmol to 1 pmol; 1 pmol to 10 pmol; or 10 pmol to 100 pmol.

[0070] In another embodiment, the topical composition is a foam.

[0071] In another embodiment, the topical composition is a gel.

[0072] In a further aspect, what is provided is a method of eradicating a bum injury bioburden or biofdm of a mammal requiring such treatment, comprising applying a topical composition comprising nitric oxide to the burn injury of the mammal; wherein the amount of nitric oxide delivered from each application is from 0.001 pmol to 100 pmol over a 1 to 10 minute application time; wherein the topical composition comprising nitric oxide is allowed to remain on the bum injury for from 30 seconds to 72 hours; and wherein the topical composition comprising nitric oxide is applied at least twice a week until the burn injury is healed.

[0073] In a further aspect, what is provided is a method of modulating burn injury inflammation of a mammal requiring such treatment, comprising applying a topical composition comprising nitric oxide to the bum injury of the mammal; wherein the amount of nitric oxide delivered from each application is from 0.001 pmol to 100 pmol over a 1 to 10 minute application time; wherein the topical composition comprising nitric oxide is allowed to remain on the burn injury for from 30 seconds to 72 hours; and wherein the topical composition comprising nitric oxide is applied at least twice a week until the bum injury is healed.

[0074] In one embodiment of this aspect, the inflammation is inflammation dysregulation, proinflammation, anti-inflammation, or other unwanted inflammation.

[0075] In another embodiment, the NO promotes healing of the wound on a cellular level (phenotype) via one or more of immune cell infdtration, pro-inflammation, anti-inflammation, proliferation, or wound closure.

[0076] In another embodiment, the topical formulation mitigates bum sepsis

[0077] In another embodiment, the NO enhances immune modulation.

[0078] In another embodiment, the NO enhances tissue regeneration.

[0079] In another embodiment, the topical formulation limits necrosis.

[0080] In another embodiment, the topical formulation reduces necrosis.

[0081] In another embodiment, the delivered NO enhances cell proliferation, epithelialization, etc. for wound healing.-12-61968708 1Attomey Docket No.: 274507 / 24-006WO / 573634Examples

[0082] Example 1: Porcine Model Study to Determine the Optimal Nitric Oxide Route of Delivery and Dosing to a Burn InjuryResearch Plan:

[0083] Objective: This study is designed to determine the optimal nitric oxide (NO) route of delivery and dosing regimen. We will compare the efficacy of a foam-based vs. gel-based medium and daily vs. twice a week dosing for healing combined injury wounds involving full thickness bums, and for preventing infection of four anti -drug resistant strains. Given the complex biochemical pathway of which nitric oxide is a part, The Applicant hypothesizes that wound healing will not differ significantly in response to daily dosing of Topical NO compared to dosing twice a week.

[0084] Background: In modem wound care, wound healing agents that not only aid in wound healing or in halting bum progression, a type of trauma wound, but also in fighting infection are needed. The body’s natural molecule capable of meeting this high bar is nitric oxide (NO). NO is a powerful broad-spectrum antimicrobial agent with no known resistance, and is a primary cell signaling molecule in wound healing that alters bum progression and advances the trajectory of a wound. To this end, NO has three modes of action that address combat wounds at risk of infection: 1) NO is an endogenous cellular signaling molecule that governs the wound healing sequence from injury to inflammation resolution, cell proliferation, collagen deposition, and angiogenesis; 2) NO is a natural vasodilator that draws blood flow into otherwise ischemic environments; 3) NO is a powerful broad-spectrum antiviral, antifungal, and antimicrobial agent with no known resistance that eradicates antibiotic resistant bacterial and fungal strains. Thus, NO therapies are uniquely suited to treat combat wounds due to their multifaceted approach.

[0085] In a healthy body, NO is produced in adequate supply; however, severe or multi-trauma wounds may suffer from localized ischemia and NO deficiency, and can become chronic or lifethreatening. Given its multiple modes of action, NO is an ideal candidate to address the needs of burn treatment in a single product. However, the difficulty in developing an effective topical NO product lies in the low molecular weight and highly reactive nature of NO. Due to its low density, NO is easily trapped in highly viscous mediums, posing a problem for typical cream embodiments of topical wound dressings. Additionally, NO reacts with oxygen in ambient air-13-61968708 1Attomey Docket No.: 274507 / 24-006WO / 573634 with a two-second half-life. This makes it difficult to trap or deliver NO by bandage, since bandages have no way of directing the therapeutic gas to the wound bed or preventing contact with air where NO’s therapeutic effect is neutralized.

[0086] The Applicant has developed a topical wound care product, as an innovative solution to these challenges of trans and delivering NO. The kit produces and delivers comparably high amounts of NO. Topical NO is applied by dispensing and adequately covering a wound and peri -wound. After just minutes, it can be wiped off. This ease of use makes it well suited for application at point-of-injury in emergency and field settings. Topical NO is disclosed in US 10,052,348, US 10,751,364, and US 11,304,972, the entire contents of which are disclosed herein by reference.

[0087] Topical NO can be used in conjunction with primary and secondary dressings, and it conforms to any wound size and shape. Owing to its three modes of action, Topical NO can operate as a three-in-one therapy to disrupt and even eradicate mature biofilms of drug-resistant strains, increase formation and deposition of fibroblasts and collagen, promote re- epithelialization, increase local blood flow, and reduce inflammation - all functions which are critical to early intervention, infection resolution, and wound healing.

[0088] To demonstrate the antimicrobial effects of Topical NO, an ex vivo porcine explant study was performed to test Topical NO in a prevention and eradication arm of 6 biofilms. For the prevention arm, the log reduction of colony forming units (CFU) relative to untreated controls ranged from 4 to 9 log CFU / explant for Topical NO, showing the ability to prevent biofilm proliferation. In the eradication arm, 72 hour matured biofilms were produced in the explants showed susceptibility to the Topical NO treatment. Results also indicated that two 5-minute treatments were more effective than a single treatment in all species tested.

[0089] Specific Aims:

[0090] Aim 1: Establish an optimal nitric oxide dose for preventing infection and healing full thickness burn wounds.

[0091] Aim 2: Identify the suitability and distinguish the foam form factor as the preferred delivery medium for treating infected combined injury wounds.

[0092] Rationale:

[0093] To achieve these aims, this study will analyze the rate of healing of infected full thickness burn wounds in a single porcine study. This study is designed to determine the following:-14-61968708 1Attomey Docket No.: 274507 / 24-006WO / 573634• The therapeutic effect of NO treatments to resolve drug resistant infection in full thickness burn wounds.• The effects of daily vs. twice a week treatment.• The effects of foam- vs. gel-based NO delivery vehicles on infection management and wound healing.• The amount of NO produced over 5 minutes (foam medium) or 24 hours (gel medium).

[0094] The ease of manufacture and applicability of its form factor make foam superior to gels.

[0095] Methods: A porcine model will be used for its similarity in skin healing to that of human physiology. NO-deficient animals, such as diabetic pigs, will be used to simulate NO-starved wounds. A split back design will be used, where each animal will be wounded with wounds, separated by the spine, with NO on one side and control (no treatment) on the other. Wounds will be treated with either foam or gel at 100% clinical dose 50% clinical dose, maximum dose of a 24 hr NO gel, a 1% dose of NO foam and its equivalent gel, and a 0% dose of NO (vehicle). Arms will be inoculated with four multi drug resistant bacterial strains most frequently encountered in burn wounds: Candida albicans, Acinetobacter baumannii, Staphylococcus aureus, and Pseudomonas aeruginosa5.

[0096] Wounds will be administered as follows: 1) animals will be anesthetized; 2) 10 full thickness wounds will be created on each side of the animal; 3) thermal burns will be administered by applying a heated rod to each wound site; 4) for infected arms, wounds will be inoculated by a cocktail of bioburden and incubated for 72 hours. Wounds will be randomized and treated with NO daily or twice a week with a double treatment 10 minutes apart on the first day (for foam) for 28 days or until full closure. During treatment, animals will be anesthetized. Wounds will be monitored daily for wound closure rate and percentage area reduction; clinically observed for eschar, slough, necrosis, and pain management; and imaged for oxygenation and perfusion.

[0097] Milestones:

[0098] Aim 1 : Establish an optimal nitric oxide dose for preventing infection and healing full thickness bum wounds. Aim 2: Identify the suitability and distinguish the foam form factor as the preferred delivery medium for treating infected combined injury wounds. Specifically, show that animals treated with higher concentrations of NO foam for 5 min treatments have a higher healing rate and higher oxygenation and perfusion than those treated with gel for 24 hr at any-15-61968708 1Attomey Docket No.: 274507 / 24-006WO / 573634 concentration or lower concentration NO foam.

[0099] Impact and Relevance: The proposed research will expand the scope of use for Topical NO, and build evidence to support NO supplementation for full thickness combined injury wounds, involving burns and infection.

[0100] One advantage of Topical NO is that it is easy to use. Topical NO comes in a self- contained kit that can be carried and deployed in any environment, simple treatment easily fits into field application and can be used in conjunction with standard dressings for prolonged care. It is believed that treatment with Topical NO will ultimately result in faster healing and fewer days hospitalized. Lastly, the multiple modes of action provided by NO may allow Topical NO to replace the multitude of products required to fulfill the basic standard care requirements of wound cleansing, debridement, and management of infection, which may also translate into cost savings associated with streamlined care.

[0101] Long term, this research will expand the indications of Topical NO to include acute, multi-trauma, and infected wounds. Furthermore, because of its many modes of action (antimicrobial, cell signaling, and vasodilating), Topical NO may serve as a treatment for additional wound etiologies including bum wounds of different sources and severity, puncture wounds, and wounds in the presence of contaminated, necrotic, and highly damaged tissue.References:1. Zhu H, Wei X, Bian K, Murad F. Effects of nitric oxide on skin bum wound healing. J Burn Care Res. 2008;29(5):804-14. doi: 10.1097 / BCR.0b013e3181848119. PubMed PMID: 18695618.2. Goedeke R. Decoding the benefits of nitric oxide for skin health. Biosphere Nutrition.3. Poh WH, Rice SA. Recent Developments in Nitric Oxide Donors and Delivery for Antimicrobial and Anti-Biofilm Applications. Molecules. 2022;27(3). Epub 20220120. doi: 10.3390 / molecules27030674. PubMed PMID: 35163933; PMCID: PMC8839391.4. Schairer DO, Chouake JS, Nosanchuk JD, Friedman AJ. The potential of nitric oxide releasing therapies as antimicrobial agents. Virulence. 2012;3(3):271-9. Epub 20120501. doi: 10.4161 / viru.20328. PubMed PMID: 22546899; PMCID: PMC3442839.

[0102] Example 2: Porcine Model Study to Determine the Optimal Nitric Oxide-16-61968708 1Attomey Docket No.: 274507 / 24-006WO / 573634Route of Delivery and Dosing to a Chemical Burn Injury

[0103] Background:

[0104] The Applicant has a topical, liquid foam-based wound care product (Topical NO) designed to restore the deficiency of nitric oxide (NO) experienced by traumatic wounds to promote faster healing using the body’s natural healing sequence. When in sufficient supply, NO is responsible for cell signaling and inflammation resolution, vasodilation, and infection management as a broad-spectrum antimicrobial agent. All of these modes of action are critical to the healing of bum wounds. When experiencing a full thickness burn, including atypical burns, the site of the bum is compromised by ischemia and necrotic tissue, which prevents sufficient NO from reaching the site. The Applicants proposes to measure the efficacy of its topical NO product, Topical NO, for the healing of infected full-thickness chemical burns using a porcine model and determine the underlying chemical or molecular mechanisms of action of Topical NO in this context. When combined with the results from other studies, Topical NO will be designated as a combination wound care product for healing of bum wounds as well as infection prevention and biofilm dismption. Topical NO is especially well suited for wound care as it is a one-product solution boasting multiple critical modes of action (cell signaling agent, vasodilatory agent, and antimicrobial agent) that contribute to its status as a combination wound product; and it can be easily deployed in environments without special equipment or training to treat burn wounds, including those resulting from chemical agents.

[0105] Scientific Rationale:

[0106] Challenges like combined burn injuries and limited air superiority heighten the need for wound healing agents that not only fight infection but also halt burn progression. Such an agent should also exhibit modes of action which stimulate the later stages of wound healing. The body’s natural molecule capable of meeting this high bar is nitric oxide (NO). NO is a powerful antimicrobial and wound healing agent that alters bum progression and advances the trajectory of a wound. To this end, NO has three modes of action that address the complications of combined injuries. 1) NO is an endogenous cellular signaling molecule that governs the wound healing sequence from injury to inflammation resolution, cell proliferation, collagen deposition, and angiogenesis. 2) NO is a natural vasodilator that draws blood flow into otherwise ischemic environments. 3) NO is a powerful broad-spectrum antiviral, antifungal, and antimicrobial agent with no known resistance that eradicates antibiotic resistant bacterial strains-17-61968708 1Attomey Docket No.: 274507 / 24-006WO / 573634 in both their planktonic and biofilm phenotypes. Thus, NO therapies have multiple critical modes of action and stand apart from other wound healing agents. Despite the many benefits of nitric oxide for wound healing, it is important to note that there are currently no topical NO products in the mainstream wound care market. Nevertheless, a NO-based treatment in wound care would be ground-breaking due to NO’s multiple modes of action that support and direct each phase of wound healing.

[0107] The Applicant has developed a form factor which can be easily deployed by non- medically trained personnel, and which offers advantages over other NO concepts to date, as described below. Therefore, the Applicant's product, Topical NO holds promise as an early treatment to meet wound management requirements. This proposal seeks to expound on the preclinical support of Topical NO for acute burns and infected wounds by studying the effectiveness of Topical NO on healing full thickness chemical burn wounds with and without infection.

[0108] In a healthy body, NO is produced in adequate supply; however, severe wounds such as bums which may suffer from localized ischemia and NO deficiency can become chronic or life-threatening. Given its multipronged modes of action, NO is an ideal candidate to address the needs of burn treatment. Recent attempts to deliver NO in gels and creams, whether in gaseous form or as one of its innovative precursors, have failed because NO remains trapped in the high viscosity of these media, inhibiting delivery to the wound and requiring prohibitively long treatment times. NO-generating bandages have been considered; however, they have no way of directing the NO down into the wound, and instead rely on serendipitous absorption, resulting in lower therapeutic effect.

[0109] Although the benefits of nitric oxide have been well-known for decades, there are several limitations that have prevented the development and application of NO-based treatments for dermal administration. First, due to the molecule’s reactive nature, NO has a short half-life of approximately two seconds. This makes NO difficult to capture, transport, and apply in an effective and economic manner. Early approaches attempting to use compressed gas proved to be economically unfeasible, cumbersome to store and transport, particularly for emergency scenarios, and carried unnecessary safety risks. Attempts to capture NO in gels and creams have failed because their high viscosity traps the generated NO, inhibiting delivery to the wound. Several groups have created bandages which generate NO. While these are innovative-18-61968708 1Attomey Docket No.: 274507 / 24-006WO / 573634 approaches, they still cannot force or direct the NO to diffuse into the wound before it reacts and dissipates to ambient air. Furthermore, delivering NO via bandage may not meet the various geometries, depths, and sizes of wounds, nor can it be used with other primary dressings.

[0110] To overcome these challenges, the Applicant has developed Topical NO as a topical, wound care product designed to restore the deficiency of NO experienced by traumatic wounds to promote faster healing using the body’s natural healing sequence. Topical NO is applied by dispensing a sufficient amount to adequately cover the wound and peri-wound. Topical NO is similar in administration to other topical wound treatments. Compared to creams or gels that must stay on the skin for up to 24 hours for a single application, Topical NO only needs a matter of minutes for application. It is then wiped off or patted dry, which allows easy workflow in field settings.

[0111] Compared to cream and gel mediums which trap the NO in the medium and have poor absorption into the wound, the foam-based Topical NO creates an air-tight dome trapping the produced NO against the wound tissue for ease of absorption (FIG. 1). prevents NO gas from escaping to the atmosphere or oxidizing before it diffuses into the tissue.

[0112] In addition, Topical NO is clinically flexible, making it compatible with other therapies and secondary dressings. Topical NO conforms to any wound size and shape, increases formation and deposition of fibroblasts and collagen, promotes re-epithelialization, increases local blood flow, reduces inflammation, and prevents or clears biofilm infections - all functions which are critical to early intervention and wound healing. Preliminary Data. The Applicant performed several pre-clinical studies and obtained promising results that demonstrated the robust safety profile of Topical NO. These include a toxicity study and pharmacokinetic (PK) test, an in vitro phototoxicity study, an in vitro Ames genotoxicity study and micronucleus test, and an in vitro sensitization test. All studies reported the high safety profile of the treatment.

[0113] Objective:

[0114] Topical NO is an efficacious product for fast, easy, and efficient delivery of NO to the wound bed to speed healing of atypical full thickness burn wounds.

[0115] Specific Aims:

[0116] Specific Aim #1: Measure the efficacy of Topical NO for the healing of infected chemical burns using a porcine model.

[0117] Healing effects will be measured, including the time to closure of 2 cm diameter-19-61968708 1Attomey Docket No.: 274507 / 24-006WO / 573634 atypical chemical burn wounds with infection comparing healing of uninfected vs. infected bums in a porcine model over a 28-day treatment period. Secondly, the time to closure of infected chemical burn wounds treated daily vs. Monday / Wednesday / Friday (M / W / F) vs. Monday / Friday (M / F) in the same porcine model will be measured.

[0118] Specific Aim #2: Determine the cellular and / or molecular mechanisms of action of Topical NO in the context of chemical burn wounds in biopsies taken over 9 days to validate primary modes of action of Topical NO in burn wounds.

[0119] The effect of daily, M / W / F, or M / F Topical NO on wound cellular and / or molecular activity in a porcine model will be evaluated by assessing markers of hemostasis, inflammation, and proliferation, and by using imaging devices to capture oxygenation and perfusion data. In addition, the effect of immediate vs. delayed treatment of Topical NO on bum progression will be determined, as well as the effect of Topical NO for treating uninfected vs. infected full thickness chemical burn wounds on the cellular and / or molecular level.

[0120] Research Strategy and Feasibility:

[0121] Research Design:

[0122] The proposed study aims to determine the effectiveness of Topical NO in the treatment and healing of complex chemical burn wounds. To do so, a porcine model will be used due to its similarity in skin healing to that of human skin, and each animal will be wounded with full thickness chemical bums in a split back design where each animal will serve as its own control.

[0123] Burn wounds, often characterized by localized ischemia and necrotic tissue, are particularly suitable for treatment by topical NO, which, in addition to wound healing properties, has vasodilatory and debridement capabilities

[0124] The study is designed to determine the following:• The effect of Topical NO compared to control on wound healing;• The effect of Topical NO on preventing and eradicating infection of multidrug resistant strains of bacteria;• The effect of daily, Monday / Wednesday / Friday (M / W / F), and Monday / Friday (2x / week) treatment of Topical NO; and• The effect of immediate treatment, simulating point-of-injury treatment, compared to treatment after a two-day delay-20-61968708 1Attomey Docket No.: 274507 / 24-006WO / 573634

[0125] The study will be split into two groups: a 28-day group aimed to study the effects of Topical NO to full wound closure of infected and non-infected full thickness chemical bums; and an 8-day group aimed to study the cellular and / or molecular effects of Topical NO on the various arms by taking frequent biopsies, as described below. Aims 1.2-1.3 and Aim 2 will be conducted in parallel. Data from the 28-day group and 8-day group of animals will be used to answer the research questions of Aims 1.2-1.3 and Aim 2, respectively. Biopsied wounds will only be monitored for the first 8-days in order to provide microbial enumeration (if applicable) and measurements of wound healing cytokines and infection response responsive to NO.

[0126] Wound measurements and clinical assessments will be performed according to the same schedule as treatment for the study duration. At the time of termination, select wounds will be excised for histological evaluation.

[0127] Specific Aim #1:

[0128] Measure the efficacy of Topical NO for the healing of infected chemical burns using a porcine model.

[0129] Rationale:

[0130] Specific Aim #1 proposes to demonstrate the effect of nitric oxide in the context of complete wound healing of full thickness chemical bum wounds, as well as in infection management on the same wound type. Data generated will support its use as a point-of-injury care product. To investigate Topical NO’s efficacy in wound healing, animals (20 wounds each) will be studied over 28 days in order to allow for complete wound closure and observation.

[0131] Treatment arms will be used to compare the effect of Topical NO treatment on non-infected vs. infected full thickness chemical bum wounds, and the effect of daily vs. M / W / F vs. 2x week Topical NO treatment on infected full thickness chemical burn wounds. Thus, this aim will: determine whether the rate of healing for treated animals after 28 days is equal to or greater than that of control wounds for both non-infected and infected full thickness chemical burn wounds; and determine an optimized dosing regimen between daily, M / W / F, or 2x / week treatments.

[0132] 1.1 Measure time to closure of 2 cm diameter chemical burn wounds comparing healing of uninfected vs. infected burns in a porcine model over a 28-day-21-61968708 1Attomey Docket No.: 274507 / 24-006WO / 573634 treatment period.

[0133] This sub-aim is designed to demonstrate the healing rate of chemical bum wounds with and without drug resistant biofilms treated with Topical NO compared to control. To measure the time to closure of full thickness infected vs. non-infected chemical bum wounds using Topical NO, a porcine animal model has been selected due to its proximity to human skin in terms of wound healing. Each animal will follow a split back design, and which reduces the need for more animals. In this design, twenty wounds will be created per animal (10 per side). Half of the animal’s back will be treated with Topical NO and half will be reserved as control. In this way, each animal will serve as its own control to compare healing rates and time to healing without being confounded by anatomical anomalies.

[0134] Methods. On day 0, pigs will be anesthetized. A split back paradigm will be used in which a single animal will serve as its own control. Twenty 2.0 cm diameter full thickness circular wounds (10 wounds on each side of the spine) will be created in a split back design. Following wound creation, chemical bums will be administered to each wound site. To test the broad-spectrum antimicrobial effects of Topical NO, a randomized subset of the testing arms will be inoculated with four multidrug resistant bacterial strains, Acinetobacter baumannii, Staphylococcus aureus, Candida albicans, and Pseudomonas aeruginosa, which are most frequently encountered in burn wounds. For the infected arms, inoculation will occur following the chemical bum administration.

[0135] Next, split backs will be randomized, treated, and studied over 28 days in order to allow for complete wound closure. Animals will be treated according to the following treatment arms: daily administration of treatment (Topical NO) on full thickness chemical bum wounds, M / W7F administration on full thickness chemical burn wounds, M / W / F administration on infected full thickness chemical burn wounds, and M / F treatment on full thickness chemical burn wounds. Animals will receive a double treatment 10 minutes apart on the first day for 28 days or until full closure. A subset of animals with inoculated wounds will be observed for 48 hours to allow bioburden to incubate and form biofilms prior to first treatment.

[0136] During treatment, animals will be anesthetized, and treatments will be administered by laying each animal on its side, applying the test article, allowing it to sit on the wound, then wiping away the residual foam. Following wounding and each treatment application, wounds will be dressed with a saline-moistened non-adherent dressing. Pigs will be-22-61968708 1Attomey Docket No.: 274507 / 24-006WO / 573634 wrapped with a protective pad to secure the dressings. Pain and irritation will be monitored, and animals will receive oral pain medication for at least the first seven days of healing. All animals will be euthanized on day 28, and wounds will be collected en bloc (as one slab) per side (10 wounds each) for histology.

[0137] Wounds will be photographed and monitored Mondays, Wednesdays, and Fridays or Mondays and Fridays for wound closure rate and percentage area reduction (measured by caliper); assessed clinically (4 point scale) for erythema, edema, and granulation; observed for eschar, slough, necrosis, and pain management; imaged for bioburden; and for oxygenation and perfusion. Animal weights will be collected on days 0, 7, 14, 21, and 28.

[0138] 1.2 Measure time to closure of 2 cm diameter infected chemical burn wounds treated at the different dosing frequencies of daily vs. Monday / Wednesday / Friday vs. Monday / Friday.

[0139] This sub-aim is designed to determine the optimum dosing frequency of Topical NO to heal full thickness chemical burn wounds. For this sub-aim, the same 28-day chemical burn wound porcine model described above will be used due to its ability to mimic the healing of human skin,

[0140] Methods:

[0141] To determine the effectiveness of daily, M / W / F, or 2x / week treatments of Topical NO on bum wounds, the same porcine model as described in sub-aim 1.2 will be used. Animals (20 wounds each) will be studied over 28 days in order to allow for complete wound closure with the following treatment arms: daily administration of treatment (Topical NO) on full thickness chemical burn wounds, M / W / F administration on full thickness chemical bum wounds, and 2x / week treatment on full thickness chemical bum wounds. Animals will be randomized to each arm, and split backs will be used to serve as control arms. The ‘daily’ treatment arm will receive

[0142] Topical NO every day for 28 days, the M / W / F arm will receive treatments on Mondays, Wednesdays, and Fridays, and the 2x / week arm will receive treatment on Mondays and Fridays until the end of the study or until full closure. During treatment, animals will be anesthetized, and treatments will be administered by laying each animal on its side, applying the test article, allowing it to sit on the wound then wiping away the residual. Following wounding and treatments, wounds will be dressed with a saline-moistened non-adherent dressing. Pigs will be wrapped with a protective pad to secure the dressings. Wounds will be photographed and-23-61968708 1Attomey Docket No.: 274507 / 24-006WO / 573634 monitored Mondays, Wednesdays, and Fridays or Mondays and Fridays for wound closure rate and percentage area reduction (measured by caliper); assessed clinically (4 point scale) for erythema, edema, and granulation; observed for eschar, slough, necrosis, and pain management; imaged for bioburden; and for oxygenation and perfusion. Animal weights will be collected on days 0, 7, 14, 21, and 28.

[0143] Specific Aim #2: Determine the chemical or molecular mechanisms of action of Topical NO in the context of chemical burn wounds over 9 days.

[0144] Rationale:

[0145] Though the mechanisms of action of NO regarding wound healing have been documented, the specific molecular and / or cellular processes that it governs during bum wound and combined wound healing have not been identified. The goal of Specific Aim #2 is to elucidate the cellular and / or molecular mechanisms of action of nitric oxide, the active ingredient in Topical NO, in the context of full thickness chemical bum wound healing and burn progression, as well as in infection management. For this purpose, punch biopsies will be taken daily to measure 19 cytokines of inflammation, infection resolution, and wound healing. These biomarkers will be used to evaluate the wound’s progress as it advances through the wound healing sequence. To study the bum progression, biopsies will also be taken of the peri-wounds.

[0146] Animals will be studied over 8 days. Though full wound closure is not expected for this study, 8 days is sufficient time for a wound to progress through the inflammatory stage, which is the stage at which NO has its highest therapeutic effect due to its role in inflammation regulation, and into the proliferation stage. Treatment arms will be completed simultaneously and used to compare: 1) treatment of non-infected vs. infected full thickness chemical burn wounds to understand the impact of NO in infection management of such combined wounds; 2) immediate treatment (representing point-of-injury application) with delayed treatment (representing delay in evacuation to a medical facility) to demonstrate the impact of point-of- injury treatment on burn wound progression; and 3) daily treatment with M / WZF and 2x / week treatment to show the cellular effects of wound healing with varying frequencies of topical NO supplementation.

[0147] 2.1 Assess the effect of daily, M / WZF, and 2x / week Topical NO on wound cellular and / or molecular activity.-24-61968708 1Attomey Docket No.: 274507 / 24-006WO / 573634

[0148] An 8-day study using the full thickness chemical burn wound porcine model will be used to demonstrate the cellular and / or molecular effects of daily vs. M / W / F vs. 2x / week treatment of Topical NO compared to control. The effects will be measured through the taking of frequent punch biopsies of the wound wherein cytokines indicative of wound healing will be measured and correlated to the wound healing sequence starting immediately before and after the burn injury. This will provide a key primary representation of the tissue specific bum-injury and wound healing process while assessing the molecular impact of Topical NO treatment. Biopsies will be taken of both the wound and peri-wound to determine bum progression. The study consists of five arms, and the data collected will also be used for both sub-aims 2.2 and 2.3 for ethical and economic considerations.

[0149] Methods:

[0150] To elucidate the cellular and / or molecular effects of NO on wound healing, the wounded animal model described in 1.2 will be used. Briefly, on day 0, animals will be anesthetized; 20 2.0 cm diameter full thickness circular wounds will be created on each side of the animal in a split back design (10 wounds on each side of the spine). Following wound creation, chemical burns will be administered to each wound site. Animals will then receive treatment, and be monitored for 8 days. During treatment, animals will be anesthetized, and treatments will be administered by laying each animal on its side, applying the test article, allowing it to sit on the wound, then wiping away the residual foam. Following wounding and treatments, wounds will be dressed after treatment with a saline-moistened non-adherent dressing. Pigs will be wrapped with a protective pad to secure the dressings. This study will involve five arms in the same split back paradigm described in Aim 1 (n=5 animals): daily administration of treatment on full thickness chemical bum wounds starting on day 2; daily administration on infected full thickness chemical burn wounds; M / W / F administration of treatment on full thickness chemical bum wounds; M / W / F administration of treatment on infected full thickness chemical burn wounds treated starting on day 2 in order to simulate delayed treatment under evacuation scenarios; and 2x / week treatment on full thickness chemical burn wounds starting on day 2. Imaging devices will be used M / W / F or M / F to capture oxygenation and perfusion data, indicative of revascularization.Biopsies of the wounds and peri-wounds will be taken every two days over 8 days to test for 19 cytokines and anolytes.-25-61968708 1Attomey Docket No.: 274507 / 24-006WO / 573634

[0151] At each timepoints, two (2) wounds will be sampled from each animal: one from the appropriate Topical NO-treated group and one from the corresponding control group. From each wound, four (4) biopsies will be collected using a 4 mm biopsy punch:One biopsy will be placed into a pre-weighed vessel containing an appropriate neutralizer and processed for microbial enumeration on site.Three biopsies will be shipped for histological analysis.

[0152] After biopsy, each biopsied wound will be removed from further treatment or analysis as not to confound the wound healing data.

[0153] The cytokines and anolytes were selected to demonstrate increased wound healing. The following process will be involved:

[0154] Stage 1 : Establish and validate the assays. In this stage, pre and post wound porcine biopsy tissues at 40 mg to 80 mg will be used to test three different tissue lysis buffers to find one with the best signal to noise ratio. The reproducibility and sensitivity of the assay will be validated.

[0155] Stage 2: Test data collection. Process and test the tissue biopsy samples, each in duplicate. The quantity of cytokines under different conditions will be compared.

[0156] 2.2 Determine the effect of immediate vs. delayed Topical NO on burn progression.

[0157] The cellular and / or molecular effect of immediate vs. delayed administration of Topical NO on the burn progression of full thickness chemical bum wounds will be demonstrated using a porcine model as described above. To accomplish this, treatment arms will be divided into groups that receive treatment (Topical NO) immediately after wounding (representing point-of-injury application) compared to those that receive treatment with a 2-day delay (representing treatment after delayed evacuation). Among the cytokines tested, burn progression can be positively correlated with certain levels of cytokines The dysregulation of hemostasis factors. Indication of burn progression can be assessed by comparison of those cytokine levels between the wound and peri-wound. Data collected in sub-aim 2.1 will be used in conjunction with this study to reduce the wounding of additional animals. Data collected from this sub-aim will support the value of administering Topical NO immediately at the point-of- injury, as well as improve the understanding of the impact of treatment after 48hr delay when point-of-injury treatment is not feasible.-26-61968708 1Attomey Docket No.: 274507 / 24-006WO / 573634

[0158] Methods:

[0159] The data generated from the wounded animal model described in 2.1 will be used with a focus on data from three of the treatment arms: daily administration of treatment on full thickness chemical burn wounds starting on day 2; M / W / F administration of treatment on infected full thickness chemical burn wounds treated starting on day 2; and 2x / week treatment on full thickness chemical bum wounds starting on day 2. Biopsies of the wounds and peri-wounds will be taken using a 4 mm biopsy punch and wound healing assays will be performed to test for 19 wound healing cytokines to determine the wounds’ progress through hemostasis, inflammation, and proliferation phases. Wounds and peri-wounds will be sampled at the timepoints specified in 2.1, collecting four biopsies each for the same testing as described in 2.1. Imaging devices will be used Mondays, Wednesdays, and Fridays or Mondays and Fridays to capture oxygenation and perfusion data, indicative of revascularization. Biopsies and wound healing assays will be taken on wounds and peri-wounds to understand bum progression of the comparator groups.

[0160] 2.3 Determine the effect of Topical NO on treating uninfected vs. infected full thickness chemical burn wounds on a cellular and / or molecular level.

[0161] The cellular and / or molecular effects of Topical NO will be compared to control in treating uninfected vs. infected full thickness chemical bums. These findings will support the preliminary data showing the effect of Topical NO in disrupting and eradicating bioburden, even of drug-resistant strains, and will contribute to the support of Topical NO for multi-trauma wounds (full thickness, chemical bums, infection). The porcine skin healing model is again used in this sub-aim for its proximity to human skin healing.

[0162] Methods:

[0163] The wounded animal model described in Aim 2.1 will be used including the infection model described in Aim 1.2. Briefly, on day 0, animals will be anesthetized; 10 2.0 cm diameter full thickness circular wounds will be created on each side of the animal, and chemical burns will be administered. The five treatment arms as described in 2.1 will be used. For the subset of infected arms, following the chemical burn administration, wounds will be inoculated by a solution of A. baumannii, S. aureus, C. albicans, and P. aeruginosa bacteria then wiped dry thoroughly. Biopsies of the wounds and peri -wounds will be taken, and wound healing assays performed to test for 19 wound healing cytokines to determine the wounds’ progress through-27-61968708 1Attomey Docket No.: 274507 / 24-006WO / 573634 hemostasis, inflammation, and proliferation phases. Wounds will be sampled at the timepoints as defined in 2.1. At each of the timepoints, two (2) wounds will be sampled from each animal - one from the appropriate Topical NO-treated group and one from the corresponding control group - with four biopsies from each. Imaging devices will be used Mondays, Wednesdays, and Fridays or Mondays and Fridays to capture the degree of bioburden, as well as oxygenation and perfusion data, indicative of revascularization.

[0164] The biopsies and wound healing assays as described in 2.1 will be taken on wounds and peri-wounds to understand the effects of Topical NO on the molecular and / or cellular healing of bum wounds in the presence and absence of infection.References1. Zhu H, Wei X, Bian K, Murad F. Effects of nitric oxide on skin bum wound healing. J Burn Care Res. 2008;29(5):804-14. doi: 10.1097 / BCR.0b013e3181848119. PubMed PMID: 18695618.2. Goedeke R. Decoding the benefits of nitric oxide for skin health. Biosphere Nutrition.3. Poh WH, Rice SA. Recent Developments in Nitric Oxide Donors and Delivery for Antimicrobial and Anti-Biofilm Applications. Molecules. 2022;27(3). Epub 20220120. doi: 10.3390 / molecules27030674. PubMed PMID: 35163933; PMCID: PMC8839391.4. Schairer DO, Chouake JS, Nosanchuk JD, Friedman AJ. The potential of nitric oxide releasing therapies as antimicrobial agents. Virulence. 2012;3(3):271-9. Epub 20120501. doi: 10.4161 / viru.20328. PubMed PMID: 22546899; PMCID: PMC3442839.5. Miller CM, Lantz E, Strickland A, Bell DA, Schultz G. Acidified Nitrite Foam Antimicrobial Action in an Ex-vivo Porcine Dermal Model. Journal of Wound Managem ent2022.6. Ja G-E, Vb A-A, Eh O-V, Garcia-Manzano R, Barker Antonio A, Aron J, Garcia- Espinoza J. Bums: Definition, Classification, Pathophysiology and Initial Approach.International Journal of General Medicine. 2020; Volume 5:2327-5146. doi: 10.4172 / 2327- 5146.1000298.

[0165] Example 3. Efficacy Evaluation of NO formulations in the treatment of third-degree burn wounds in a Porcine Model

[0166] A study evaluated the efficacy of two topical NO formulations in the treatment of third-degree burn wounds. Wounds were assessed on days 2 and 9 post-injury; two wounds were-28-61968708 1Attomey Docket No.: 274507 / 24-006WO / 573634 assessed for total bacterial counts and histological analysis in each assessment time. The untreated control showed the highest bacterial counts on both assessment days (7.21 and 6.62 Log CFU / g, respectively). Wounds treated with Nitric Oxide Foam had the lowest bacterial counts on day 9, 4.62 and 4.96 Log CFU / g, respectively. These values represent a more than 99.0% and 97.8% bacterial reduction for NODF formulations, respectively. Pilot histology demonstrated that treatment with NODF yielded thicker granulation tissue, accelerated re-epithelialization, and a reduction in wound area by Day 9 versus untreated controls. No erythema was noted with any of the treatments.

[0167] A porcine model was used for experimental research due to the morphological similarities between swine skin and human skin. One female animal specific pathogen free pig weighing 40-45 kg was kept in house for at least 5 days prior to initiating the experiment to allow the animal to acclimatize. The animal was fed a basal diet ad libitum and housed individually in animal facilities (meeting American Association for Accreditation of Laboratory Animal Care [AAALAC] accredited) with controlled temperature and lighting.

[0168] Twenty-eight (28) third degree bum wounds were made on the paravertebral and thoracic area. Bums were created by using a branding iron with a heat controller that was set to 300o C. The iron was held at a vertical position on the skin for 15 seconds, with pressure supplied by gravity, to make a burn wound of 27mm diameter and with a depth of approximately 3mm (to subcutaneous tissue). The wounds were separated from one another by 5-7 cm of unwounded skin and treatments randomly assigned.

[0169] Thirty minutes after a burn was administered, NO Formula 1, NO Formula 2, and vehicle (water containing coco betaine) treatments were applied. NO Formula 1 and NO Formula 2 are compositions of Formula A. Prior to application, each bottle was pumped three times for priming. Each wound was treated then with enough test material (approximately 1.5-2.0mL) to completely cover the wound and surrounding area, with the exception of Untreated Control wounds.

[0170] All wounds, including Untreated Control, were then covered with a polyurethane fdm dressing. Dressings were secured with surgical tape and wrapped with Coban elastic wrap. Wounds were subsequently treated daily until harvesting at days 2 and 9. Two (2) wounds were recovered from each treatment group, by harvesting an incisional biopsy using a sterile scalpel through the center of the wound, making sure to include normal adjacent skin on both sides for-29-61968708 1Attomey Docket No.: 274507 / 24-006WO / 573634 histological assessment.

[0171] The depth of injury was assessed two days post burn induction by measuring the amount of thermal damage (identified regions of damaged collagen and elastic fibers, cellular necrosis and tissues at risk for ischemia / reperfusion injury) from five equidistant points in the biopsy and averaged and reported as relative size (in micrometers) for tissue necrosis and burn progression.

[0172] The wounds harvested on day 9 post bum induction were evaluated for the following parameters to determine whether the proposed treatments affect wound healing responses, 4:• Percentage of wound re-epithelialization (%) - Measurement of the length of the wound surface that has been covered with epithelium.• Epithelial thickness (pm) - The epithelial thickness may vary from area to area within the biopsy. The thickness of the epithelium was measured on five equidistant points biopsy and reported as an average value (in pm).• White cell infiltrate - Measured by the presence and amount of dermal leukocytic infiltrates. Mean Score: 1 = absent, 2 = mild, 3 = moderate, 4 = marked, 5 = exuberant.• Granulation Tissue Formation - The approximate amount of new granulation tissue formed in the dermis: 0 = 0, 0.5 = 1-10%, 1 = 11-30%, 2 = 31-50%, 3 = 51-70%, 4 = 71- 90%, 5= 91-100%. On days 2 and 9 after treatment, wounds were also biopsied for microbiological assessment.Results

[0173] NO Formula 1 and NO Formula 2 were tested. NO Formula 1 and NO Formula 2 are compositions of Formula A.

[0174] Data for Formulas 1 and 2 were collected 0.5 hrs after burning, and 24 hrs after burning (NO Formulation l_0.5, NO Formulation 2_0.5, NO Formulation 1_24 or NO Formulation 2_24). After counting the colonies, the data was tabulated, the Log of colony forming units / ml (Log CFU / g) determined, and the average calculated for each time and treatment. Due to the limited number of samples, statistical analysis was not performed.

[0175] On day 2, untreated wounds showed the highest microbial load (7.21 Log CFU / g). Early treatment (within 30 minutes) with a topical nitric oxide composition as disclosed herein; for example, NO Formulation 1 0.5 or NO Formulation 2 0.5 reduced bacterial counts to 5.53-30-61968708 1Attomey Docket No.: 274507 / 24-006WO / 573634 and 5.35 Log CFU / g, respectively, representing 97.94% and 98.63% reductions compared to untreated controls. These early applications also achieved greater than 32.4% greater reduction than the same treatments applied 24 hours post-injury. Treatments at 24 hours (NO Formula 1_24 and NO Formula 2_24) still achieved 96.95% and 97.60% reductions versus untreated controls. Against vehicle controls, NO Formula 1 0.5 and NO Formula 2_0.5 reduced counts by 0.61 and 0.79 Log CFU / g, while NO Formula 1_24 and NO Formula 2_24 reduced counts by 0.78 and 0.88 Log CFU / g. These corresponded to higher relative percentage reductions for the 24-hour treatments compared to the vehicle groups than for the 30-minute treatments.

[0176] On day 9, untreated wounds again had the highest bacterial load (6.62 Log CFU / g), while NO Formula l_0.5 achieved the lowest in the study (4.62 Log CFU / g), representing a 54.5% reduction compared to NO Formula 2_0.5. Early Formula 1 treatment reduced counts by 2.0, 1.5, and 1.35 Log CFU / g versus untreated control, Vehicle_24, and Vehicle_0.5, corresponding to 99.0%, 96.87%, and 95.48% reductions, respectively. NO Formula 2_0.5 and NO Formula 2_24 showed similar counts (4.96 and 5.08 Log CFU / g), with 97.8% and 97.1% reductions versus untreated controls. All treatments showed lower bacterial counts on day 9 compared to Day 2, with NO Formula 1 applied early showing the greatest decrease (0.91 Log CFU / g; 87.79% reduction), followed by NO Formula 2_24 (69.52% reduction).

[0177] FIG. 2 further illustrates total bacterial counts after treatment of a bum with NO Formula 1 and No Formula 2 compared to vehicle.

[0178] The percent of re-epithelialization represents the percent of the wound area covered by newly formed epidermis with one or more layers of keratinocytes, which is a good index for the speed of keratinocyte migration and the first step of the re-epithelialization. To assess re-epithelialization, day 9 wounds were quantified from each treatment. NO Formula 1 and NO Formula 2 samples treated for 24 hours exhibited demonstrable increases in wound closure in comparison to both untreated and vehicle (24 hour) controls.

[0179] FIG. 3 further illustrates wound epithelialization percent after treatment with NO Formula 1 and Formula 2 compared to vehicle.

[0180] The foregoing disclosure has been described in some detail by way of illustration and example, for purposes of clarity and understanding. The invention has been described with-31-61968708 1Attomey Docket No.: 274507 / 24-006WO / 573634 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 understood that the above description is intended to be illustrative and not restrictive. The scope of the invention should, therefore, be determined not with reference to the 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.-32-61968708 1

Claims

Attomey Docket No.: 274507 / 24-006WO / 573634Claims1. A method of minimizing the severity of a bum injury of a mammal, comprising: applying a topical composition comprising nitric oxide to the bum injury of the mammal; and the topical composition is allowed to remain on the bum injury for at least 30 seconds.

2. The method of claim 1, wherein the topical composition comprising nitric oxide is applied over from 1 minute to 24 hours and wherein the amount of nitric oxide delivered from each application is from 0.001 pmol to 1000 pmol per gram.

3. The method of claim 1, wherein the topical composition comprising nitric oxide is applied twice on the first treatment day wherein the time between the first and second applications is 5 to 25 minutes.

4. The method of claim 1, wherein the topical composition comprising nitric oxide is applied at least twice a week after the first day and until the burn injury is healed.

5. The method of claim 1, wherein the topical composition is applied after a delay of up to 72 hours, wherein delay is measured from the time the burn occurred.

6. The method of claim 1, wherein the topical composition is allowed to remain on the bum injury for 10 seconds, 30 seconds, 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 10 minutes, 30 minutes, 1 hour, 2 hours, 3 hours, 6 hours, 8 hours, 12 hours, 24 hours, 48 hours, 72 hours, or more than 72 hours.

7. The method of claim 1, wherein the bum is caused by extreme heat, extreme cold, high- voltage electricity, chemical exposure, electromagnetic, mechanical action, or radiation.

8. The method of claim 1, wherein the topical composition comprising nitric oxide is applied every 12 hours; or every 24 hours; or 48 hours; or 72 hours; or once a week.-33-61968708 1Attomey Docket No.: 274507 / 24-006WO / 5736349. The method of claim 1, wherein the amount of nitric oxide delivered per gram from the topical composition to the bum injury is 0.001 pmol to 0.01 pmol; 0.001 pmol to 0.1 pmol; 0.01 pmol to 10 pmol; 0.1 pmol to 1 pmol; 1 pmol to 10 pmol; or 10 pmol to 100 pmol, 100-500, 500-1000.

10. The method of claim 1, wherein the burn injury is infected by bacteria, fungi, yeasts, or viruses, or combinations thereof.

11. The method of claim 1, wherein the infection is caused by gram positive bacteria or gram negative bacteria.

12. The method of claim 11, wherein the gram positive bacteria or gram negative bacteria is selected form the group consisting of Staphylococcus aureus, methicillin resistant Staphylococcus aureus, vancomycin intermediate Staphylococcus aureus, enterococcus, vancomycin resistant enterococcus, Pseudomonas aeruginosa, Acinetobacter, Escherichia coli, Klebsiella, Enterobacter, Serratia, Proteus, Bacteroides and Fusobacterium spp.

13. The method of claim 8, wherein the infection is caused by fungi or yeast, wherein the fungi or yeast is Candida albicans, Aspergillus, or Fusarium.

14. The method of claim 1, wherein the topical composition is a foam.

15. The method of claim 1, wherein the topical composition is a gel.

16. A method of managing or treating a burn infection of a mammal requiring such treatment, comprising: applying a topical composition comprising nitric oxide to the bum injury of the mammal; wherein the amount of nitric oxide delivered from each application is from 0.001 pmol to 100 pmol per gram; the topical composition is allowed to remain on the bum injury for a period of time;-34-61968708 1Attomey Docket No.: 274507 / 24-006WO / 573634 the topical composition comprising nitric oxide is applied at least twice a week until the burn injury is healed; and wherein the infection is caused by bacteria, fungi, yeasts, or viruses, or combinations thereof.

17. The method of claim 16, wherein the nitric oxide is delivered over a 1 to 10 minute application time.

18. The method of claim 16, wherein the infection is caused by gram positive bacteria or gram negative bacteria, wherein the gram positive bacteria or gram negative bacteria is selected form the group consisting of Staphylococcus aureus, methicillin resistant Staphylococcus aureus, vancomycin intermediate Staphylococcus aureus, enterococcus, vancomycin resistant enterococcus, Pseudomonas aeruginosa, Acinetobacter, Escherichia coli, Klebsiella, Enterobacter, Serratia, Proteus, Bacteroides and Fusobacterium spp.

19. The method of 16, wherein the infection is caused by fungi or yeast, wherein the fungi or yeast is Candida albicans, Aspergillus, or Fusarium.

20. The method of claim 16, wherein the topical composition comprising nitric oxide is applied every 12 hours; or every 24 hours; or 48 hours; or 72 hours; or once a week.

21. The method of claim 16, wherein the amount of nitric oxide delivered from the topical composition to the bum injury over a 5 minute application time is 0.001 pmol to 0.01 pmol;0.001 pmol to 0.1 pmol; 0.01 pmol to 10 pmol; 0.1 pmol to 1 pmol; 1 pmol to 10 pmol; or 10 pmol to 100 pmol.

22. The method of claim 16, wherein the topical composition is a foam.

23. The method of claim 16, wherein the topical composition is a gel.-35-61968708 1Attomey Docket No.: 274507 / 24-006WO / 57363424. A method of eradicating bioburden or biofilm from a bum of a mammal requiring such treatment, comprising: applying a topical composition comprising nitric oxide to the bum injury of the mammal; wherein the amount of nitric oxide delivered from each application is from 0.001 pmol to 100 pmol over a 1 to 10 minute application time; the topical composition comprising nitric oxide is allowed to remain on the bum injury for from 30 seconds to 72 hours; the topical composition comprising nitric oxide is applied at least twice a week until the burn injury is healed.

25. A method of modulating burn injury inflammation of a mammal requiring such treatment, comprising: applying a topical composition comprising nitric oxide to the bum injury of the mammal; wherein the amount of nitric oxide delivered from each application is from 0.001 pmol to 100 pmol per gram; the topical composition comprising nitric oxide is allowed to remain on the burn injury for from at least 30 seconds; the topical composition comprising nitric oxide is reapplied until the bum injury is healed.-36-61968708 1

Images