RELATED APPLICATIONS

[0001] This PCT application claims the benefit of U.S. Provisional Patent Application

Serial No. 62/079,461, filed November 13, 2014. This application is incorporated herein by reference in its entirety for all purposes.

STATEMENT REGARDING FEDERALLY FUNDED RESEARCH

[0002] Embodiments disclosed herein have been supported in part by Defense Advanced

Research Projects Agency (DARPA), DARPA Grant No. HR 0011-11-1-0006. The government has certain rights to this invention.

FIELD

[0003] Embodiments of the present disclosure provide systems and devices for delivering gaseous Nitrous Oxide (gNO) under therapeutic parameters to reduce a targeted infection in a subject. Certain embodiments include devices and systems for delivering pressurized gNO to reduce bioburden and promote healing in the wounds of a subject having one or more health condition, including, but not limited to, skin and soft tissue infections (SSTIs) and/or osteomyelitis. Some embodiments disclosed herein relate to reducing pathogenic infections in soft tissue of a subject in order to promote wound healing of persistent or chronic wounds.

BACKGROUND

[0004] Nitric Oxide (NO) is a ubiquitous small molecule messenger involved in many pathological and physiological processes. NO plays critical roles in vascular and neuronal signal transduction pathways, smooth muscle contractility, bioenergetics, platelet adhesion and aggregation, immunity, and cell death regulation. NO deficiency is implicated in many pathophysiological processes such as hypertension, cardiovascular dysfunctions, neurodegeneration, arthritis, asthma and septic shock. NO is one of the few gaseous signaling molecules known and is additionally exceptional due to the fact that it is a radical gas. It is a known byproduct in almost all types of organisms, ranging from bacteria to plants, fungi, and animal cells. [0005] There are several mechanisms by which NO has been demonstrated to affect the biology of living cells. These include oxidation of iron-containing proteins such as ribonucleotide reductase and aconitase, activation of the soluble guanylate cyclase, ADP ribosylation of proteins, protein sulfhydryl group nitrosylation, and iron regulatory factor activation. NO has also been demonstrated to activate NF-κΒ in peripheral blood mononuclear cells, an important transcription factor in iNOS gene expression in response to inflammation. Additionally, during an immune response, NO is secreted as free radicals and is toxic to many bacteria and intracellular parasites; NO causes DNA damage and degradation of iron sulfur centers into iron ions and iron-nitrosyl compounds.

[0006] The administration of exogenous NO not only constitutes a powerful way to supplement NO when a subject cannot generate enough for normal biological functions, it also offers a means for accelerating wound healing and reducing bioburden in a subject. NO produced by both iNOS and eNOS plays many important roles in wound healing, from the inflammatory phase through to scar remodeling. In particular, NO exerts cytostatic, chemotactic, and vasodilatory effects during early wound repair, regulates proliferation and differentiation of several cell types, modulates collagen deposition and angiogenesis, and affects wound contraction. However, the timing, concentration, pressurization, and site of NO administration are all poorly understood critical factors affecting the ability of NO to facilitate wound repair.

SUMMARY

[0007] Embodiments of the present disclosure provide systems and devices for delivering gaseous Nitrous Oxide (gNO) under therapeutic parameters to reduce a targeted infection in a subject. Certain embodiments include devices and systems for delivering pressurized gNO to reduce bioburden and promote healing in the wounds of a subject having one or more health condition, including, but not limited to, skin and soft tissue infections (SSTIs) and/or osteomyelitis. Some embodiments disclosed herein relate to reducing pathogenic infections in soft tissue of a subject in order to promote wound healing of persistent or chronic wounds.

[0008] Embodiments of the present disclosure provide a gaseous nitric oxide (gNO) delivery device for delivering pressurized gNO to a subject. In some embodiments, the device includes a source of gNO functionally coupled to a subject interface unit, a gas flow regulator that measures flow rate of the gNO and a gas pressure regulator that measures pressure of the gNO as the gNO is delivered through the subject interface unit to the subject, wherein the gNO treats an infection in the subject.

[0009] The device according to paragraph [0008], wherein the pressure of the gNO delivered to the subject is from about 0.15 ATM to about 1.0 ATM.

[0010] The device according to either paragraph [0008] or [0009], wherein the gNO is delivered to the subject at a flow rate from about 0.1 liters/minute to about 1.0 liters/minute.

[0011] The device according to any of paragraphs [0008]-[0010], wherein the concentration of the gNO delivered to the subject is about 1.0%.

[0012] The device according to any of paragraphs [0008]-[0011], wherein the gNO is delivered to the subject continuously for about 30 minutes to about 120 minutes.

[0013] The device according to any of paragraphs [0008]-[0012], further includes one or more nitric oxide sensors.

[0014] The device according to any of paragraphs [0008]-[0013], further includes one or more oxygen sensors.

[0015] The device according to any of paragraphs [0008]-[0014], can further include a gas flushing mechanism to reduce the incidence of or prevent the subject from being exposed to gNO when the subject interface unit is removed.

[0016] The device according to any of paragraphs [0008]-[0015], wherein the subject interface unit can include a gas outlet to ensure continuous flow of the gNO and continuous exposure of gNO to the subject.

[0017] The device according to any of paragraphs [0008]-[0016], wherein the subject interface unit can include an attachment mechanism for maintaining a seal on the subject (or subject's appendage etc.) while the gNO gas is being delivered.

[0018] The device according to any of paragraphs [0008]-[0017], wherein treating the infection in the subject can include reducing bioburden in a wound located on the subject.

[0019] The device according to any of paragraphs [0008]-[0018], wherein treating the infection in the subject can include reducing one or more symptom(s) associated with the infection.

[0020] The device according to any of paragraphs [0008]-[0019], wherein treating the subject can include reducing the risk of developing an infection of one or more pathogenic organisms in the subject by pre-exposing them to gNO gas prior to onset of an infection at a wound site. In accordance with these embodiments, a subject can be treated with gNO gas upon presentation of a new wound.

[0021] The device according to any of paragraphs [0008]-[0020], wherein the infection includes an area of the subject's body infected by at least one pathogen selected from the group consisting of a bacterium, a virus, a fungus, a parasite, an arthropod, a protozoan, and an antibiotic resistant bacterium or a combination thereof.

[0022] The device according to any of paragraphs [0008]-[0021], wherein the infection is a lesion including, but not limited to, a surgical wound, a trauma wound, a burn, an abscess, an actinic keratosis, a keloid, a scar, and skin cancer and a combination thereof.

[0023] Embodiments of the present disclosure can also include a gaseous nitric oxide

(gNO) delivery system for delivering pressurized gNO to a subject. In some embodiments, the system can include a source of gNO functionally coupled to a subject interface unit, a gas flow regulator that measures flow rate of the gNO and a gas pressure regulator that measures pressure of the gNO as the gNO is delivered through the subject interface unit to the subject, wherein the gNO treats, prevents the onset of, or reduces onset of, an infection in the subject.

[0024] The system according to paragraph [0023], wherein the pressure of gNO delivered to the subject is from about 0.15 ATM to about 1.0 ATM, wherein the flow rate of the gNO delivered to the subject is from about 0.1 liters/minute to about 1.0 liters/minute, and wherein the concentration of the gNO delivered to the subject is about 1.0%.

[0025] The system according to either paragraph [0023] or [0024], wherein the system can include one or more oxygen, nitric oxide, or nitric dioxide sensors.

[0026] The system according to any of paragraph [0023]-[0025], wherein treating an infection in the subject comprises reducing bioburden (e.g. pathogenic organisms) in a wound on the subject.

[0027] The system according to any of paragraph [0023]-[0026], further including a gas flushing mechanism to prevent the subject from being exposed to gNO when the subject interface unit is removed.

[0028] The system according to any of paragraph [0023]-[0027], wherein the subject interface unit can include a gas outlet to ensure continuous flow of the gNO and continuous exposure of gNO to the subject. [0029] Embodiments of the present disclosure can also include a method of treating a wound on a subject. In some embodiments, the method can include attaching a subject interface unit to a wound site on the subject, the subject interface unit functionally coupled to a source of gaseous nitric oxide (gNO), and delivering an effective amount of gNO to the wound site on the subject, wherein the gNO treats the wound site on the subject.

[0030] The method according to paragraph [0029], wherein the pressure of the gNO delivered to the wound site on the subject is from about 0.15 ATM to about 1.0 ATM, wherein the flow rate of the gNO delivered to the wound site on the subject is from about 0.1 liters/minute to about 1.0 liters/minute, and wherein the concentration of the gNO delivered to the wound site on the subject is about 1.0%.

[0031] The method according to either paragraph [0029] or [0030], wherein the gNO is delivered to the wound site on the subject continuously for about 30 minutes to about 120 minutes.

[0032] The method according to any of paragraphs [0029]-[0031], wherein treating the wound site can include reducing bioburden in an infection in the wound site.

[0033] The method according to any of paragraphs [0029]-[0032], wherein treating the wound site comprises reducing the risk of developing an infection in the wound site.

[0034] As used herein, the terms "subject," "user," and/or "patient" can include humans and other animals or mammals that are in need of treatment and capable of using or have assisted use of devices and systems as described herein. Additionally, the terms "subject," "user," and/or "patient" can include humans and other mammals treated in any type of environment such as a clinical setting, non-clinical setting, experimental setting, etc. In embodiments, a user may be incapable of effectively operating the various gNO delivery systems and may require the assistance of a third party. As such, functions performed by the "user" can include functions performed by a third-party provider, such as a healthcare provider and/or another authorized person associated with the user.

[0035] The terms "determine," "calculate," and "compute," and variations thereof, as used herein, are used interchangeably and include any type of methodology, process, mathematical operation or technique.

[0036] It is to be noted that the term "a" or "an" entity refers to one or more of that entity. As such, the terms "a" (or "an"), "one or more" and "at least one" can be used interchangeably herein. It is also to be noted that the terms "comprising," "including," and "having" can be used interchangeably.

[0037] As used herein, "at least one," "one or more," and "and/or" are open-ended expressions that are both conjunctive and disjunctive in operation. For example, each of the expressions "at least one of A, B and C," "at least one of A, B, or C," "one or more of A, B, and C," "one or more of A, B, or C," and "A, B, and/or C" means A alone, B alone, C alone, A and B together, A and C together, B and C together, or A, B and C together. When each one of A, B, and C in the above expressions refers to an element, such as X, Y, and Z, or class of elements, such as XI -Xn, Yl-Ym, and Zl-Zo, the phrase is intended to refer to a single element selected from X, Y, and Z, a combination of elements selected from the same class (e.g., XI and X2) as well as a combination of elements selected from two or more classes (e.g., Yl and Zo).

[0038] The term "means" as used herein shall be given its broadest possible interpretation in accordance with 35 U.S. C. § 112(f). Accordingly, a claim incorporating the term "means" shall cover all structures, materials, or acts set forth herein, and all of the equivalents thereof. Further, the structures, materials or acts and the equivalents thereof shall include all those described in the summary, brief description of the drawings, detailed description, abstract, and claims themselves.

[0039] It should be understood that every maximum numerical limitation given throughout this disclosure is deemed to include each and every lower numerical limitation as an alternative, as if such lower numerical limitations were expressly written herein. Every minimum numerical limitation given throughout this disclosure is deemed to include each and every higher numerical limitation as an alternative, as if such higher numerical limitations were expressly written herein. Every numerical range given throughout this disclosure is deemed to include each and every narrower numerical range that falls within such broader numerical range, as if such narrower numerical ranges were all expressly written herein.

[0040] The preceding is a simplified summary of the disclosure to provide an understanding of some aspects of the disclosure. This summary is neither an extensive nor exhaustive overview of the disclosure and its various aspects, embodiments, and configurations. It is intended neither to identify key or critical elements of the disclosure nor to delineate the scope of the disclosure but to present selected concepts of the disclosure in a simplified form as an introduction to the more detailed description presented below. As will be appreciated, other aspects, embodiments, and configurations of the disclosure are possible utilizing, alone or in combination, one or more of the features set forth above or described in detail below.

BRIEF DESCRIPTION OF THE DRAWINGS

[0041] The accompanying drawings are incorporated into and form a part of the specification to illustrate several examples of the present disclosure. These drawings, together with the description, explain the principles of the disclosure. The drawings simply illustrate preferred and alternative examples of how the disclosure can be made and used and are not to be construed as limiting the disclosure to only the illustrated and described examples. Further features and advantages will become apparent from the following, more detailed, description of the various aspects, embodiments, and configurations of the disclosure, as illustrated by the drawings referenced below.

[0042] FIG. 1 is a representation of a portable gaseous Nitric Oxide (gNO) delivery device, according to an embodiment of the present disclosure.

[0043] FIGS. 2A-2C are representations of three subject interface units that couple to the

NO delivery device, according to embodiments of the present disclosure.

[0044] FIG. 3 is a representative drawing of a portable gNO deliver device that does not require a manifold, according to an embodiment of the present disclosure.

[0045] FIG. 4 is a graph illustrating the effects of gNO treatment on bioburden after three hours of bacterial infection, according to an embodiment of the present disclosure.

[0046] FIGS. 5A-5C are representative histological sections stained to detect the presence of bacteria on treated and untreated tissue, according to embodiments of the present disclosure. (FIG. 5 A: wounded, uninfected tissue; FIG. 5B: tissue wounded and infected for 3 hours; and FIG. 5C: tissue wounded and infected for 24 hours)

[0047] FIGS. 6A-6F are representative histological sections stained to detect components of bacterial biofilm, according to an embodiment of the present disclosure. (FIG. 6A is stained with Hematoxylin and eosin; FIG. 6B is stained with Feulgen reaction; FIG. 6C is stained with Modified Congo Red; FIG. 6D is stained with Modified Congo Red with Carbol Fuchsin; FIG. 6E is stained with PAS; FIG. 6F is stained with Calcofluor)

[0048] While the disclosure is amenable to various modifications and alternative forms, specific embodiments have been illustrated by way of example in the drawings and are described in detail below. The intention, however, is not to limit the disclosure to the particular embodiments described. On the contrary, the disclosure is intended to cover all modifications, equivalents, and alternatives falling within the scope of the disclosure and/or the claims.

DETAILED DESCRIPTION

[0049] Embodiments of the present disclosure provide systems and devices for delivering gaseous Nitrous Oxide (gNO) under therapeutic parameters to reduce infection in a subject. Certain embodiments include devices and systems for delivering pressurized gNO to reduce bioburden and promote healing in the wounds of subjects having various disease conditions, including skin and soft tissue infections (SSTIs) and osteomyelitis.

[0050] FIG. 1 is a representation of a portable gNO delivery device, according to an embodiment of the present disclosure. The portable NO delivery device of the present disclosure can include a manifold 100 that includes a gas pressure regulator 105, a gas flow regulator 1 10, and a subject interface unit. The gNO delivery devices and systems allow for easy transport of the device to various settings where therapeutic intervention is not amenable to an in-patient hospital setting. As illustrated in FIG. 1 , gas manifolds 100 used with the gNO delivery devices and systems of the present disclosure can include various features to control the delivery of the gas to a subject (e.g., gas manifolds from ALICAT SCIENTIFIC). For example, gas manifolds used with the portable NO delivery devices and systems of the present disclosure can include a port for inserting a power source 1 15 and/or a port for inserting one or more rechargeable battery units (not illustrated), various digital readouts for gas concentration and pressure 120, various gas inlets 125 and outlets 130, and various gas concentration sensors 135. Gas manifolds used with the portable NO delivery devices and systems of the present disclosure can be functionally coupled with the subject interface unit to facilitate the administration of NO to a subject. Various type and models of gas manifolds can be used to deliver pressurized gNO to a subject, as would be recognized by one or ordinary skill in the art based on the present disclosure.

[0051 ] In some embodiments, the portable gNO delivery devices of the present disclosure include a source of gNO, such as a gNO storage container that houses the gNO prior to delivery to a subject. The gNO storage container can be any suitable tank or cylinder that contains medical grade compressed gNO for delivery to a subject. Suitable gNO storage cylinders can also be equipped with pressure gauges or regulators, flow gauges or regulators, adjustments knobs for adjusting both outlet pressure and flow rates, gas inlets/outlets, and the like. In some aspects, the gNO cylinder can include a specific amount of gNO (e.g., liters of gNO), such that the delivery of the gNO to a subject having a certain condition constitutes a single treatment. For example, a gNO storage cylinder can contain approximately 9.0 liters of gNO such that when it is delivered to a subject for 90 continuous minutes at a flow rate of 0.1 liters per minute (LPM), the gNO will be exhausted. In this manner of operation, the gNO storage cylinder can function as a safety feature to prevent gNO overexposure to the subject.

[0052] In some embodiments, the gNO delivered to a subject is part of a gas mixture that has a concentration of NO that ranges from about 1 ppm to about 1500 ppm, from about 1000 ppm to about 5000 ppm, from about 4000 ppm to about 10,000 ppm, from about 9,000 ppm to about 16,000 ppm, from about 15,000 ppm to about 22,000 ppm, from about 21,000 ppm to about 28,000 ppm, from about 27,000 ppm to about 34,000 ppm, and from about 33,000 ppm to about 40,000 ppm. In some aspects, the gNO delivered to the subject is 10,000 ppm, or about 1.0% of the gas mixture (1 ppm is about 0.0001%).

[0053] In some embodiments, the NO delivery device is equipped to deliver gNO under various parameters, including delivering gNO at various pressures. In some aspects, gNO can be delivered to a subject at pressures anywhere between about 0 atmospheres (ATM) to about 1 ATM (i.e., the pressure within the subject interface unit). The delivery of gNO to a subject in this range is independent of, and in addition to, the pressure of the external environment (e.g., barometric pressure). As would be recognized by one of ordinary skill in the art based on the present disclosure, units of pressure can be expressed using various metrics, including ATMs, pounds-force per square inch (e.g., lbf/in ² or psi), bar (e.g., Mbar, kilobar, millibar, etc.), pascal (e.g., Pa, kPa, MPa, etc.) and/or torr (e.g., Torr, mTorr, etc.). For example, 1 ATM can be expressed as 14.695 psi. In some aspects of the present disclosure, pressure can be measured and expressed in increments that are tenths, hundredths and/or thousandths of these various metrics. In some aspects, the gNO is delivered at various ranges. For example, the gNO gas can be delivered at pressures from about 0 ATM to about 1.0 ATM, from about 0 ATM to about 0.9 ATM, from about 0 ATM to about 0.8 ATM, from about 0 ATM to about 0.7 ATM, from about 0 ATM to about 0.6 ATM, from about 0 ATM to about 0.5 ATM, from about 0 ATM to about 0.4 ATM, from about 0 ATM to about 0.3 ATM, from about 0 ATM to about 0.2 ATM, and from about 0 ATM to about 0.1 ATM. In some aspects, the gNO can be delivered at pressures from about 0.1 ATM to about 0.5 ATM, from about 0.15 ATM to about 1.0 ATM, from about 0.15 ATM to about 0.5 ATM, from about 0.15 ATM to about 0.25 ATM, and from about 0.25 ATM to about 0.5 ATM. In some aspects, the gNO can be delivered at pressures of about 0.1 ATM, about 0.15 ATM, about 0.2 ATM, about 0.25 ATM, about 0.3 ATM, about 0.35 ATM, about 0.4 ATM, about 0.45 ATM, about 0.5 ATM, about 0.55 ATM, about 0.6 ATM, about 0.65 ATM, about 0.7 ATM, about 0.75 ATM, about 0.8 ATM, about 0.85 ATM, about 0.9 ATM, and about 0.95 ATM.

[0054] FIGS. 2A-2B are representations of two subject interface units that couple to the

NO delivery device, according to an embodiment of the present disclosure. The subject interface units are the portions of the NO delivery system that are placed directly on the subject, for example, around a wound site, such that NO is delivered to the wound site. The subject interface unit generally includes an attachment mechanism for securing the subject interface unit onto the subject and/or maintaining or creating a seal on the subject. In some aspects, as illustrated in FIG. 2A, the subject interface unit 205 is cup-shaped and includes mounting straps 210 that are light, easy to manipulate, and are made from materials suitable for attachment to a subject. In some aspects, as illustrated in FIG. 2B, the subject interface unit 215 is more rigid, and includes a peripheral vacuum unit (not illustrated) that aids in the establishment and maintenance of a seal around the NO delivery port.

[0055] In other aspects, as illustrated in FIG. 2C, the subject interface unit 220 is configured to fit around a larger area of a subject's appendage. The subject interface unit 220 can be a flexible transparent enclosure that covers, for example, the entire foot or hand of a subject. The subject interface unit 220 can also be a rigid box-like or cylindrical structure that encloses most of the subject's leg or arm, for example, and is configured to form a seal around the more proximal region of the leg or arm. Other configurations of the subject interface unit and the attachment mechanisms can be employed in order to secure the subject interface unit on the subject and to ensure the effective deliver of NO the subject. As would be recognized by one of ordinary skill in the art based on the present disclosure, such configurations depend on factors such as, but not limited to, the size of the treatment site, its location on the subject, the type of infection, and the like.

[0056] In some embodiments, the gNO delivery devices and systems of the present disclosure can be configured such that the gas manifold is not required, as illustrated in FIG. 3. For example, the gNO delivery device 300 can include a source for the gNO 305 (e.g., cylinder or tank) that is directly and functionally coupled to mechanisms for regulating gNO pressure and flow to the subject interface unit, such as a gas pressure regulator 310 and/or a gas flow regulator 315. The gNO delivery device 300 can also include digital and/or analog readouts or displays for visualizing the pressure and flow of the gNO being delivered to the subject interface unit (e.g., pressure gauge, flow gauge). The gNO delivery device 300 can also include fine and/or coarse adjustment knobs for adjusting the pressure and flow of the gNO being delivered. The gNO delivery device 300 can also include gas inlets 320 and gas outlets 325 coupled to the pressure and flow regulators, as well as gas inlets 330 and gas outlets 335 coupled to the subject interface unit 340.

[0057] Various embodiments of the gNO delivery device 300 can provide the added benefits of not requiring a manifold or any electronic components; therefore, they can be more easily deployed in emergency medical situations. In some aspects, the gNO cylinder used with these gNO devices can include a specific amount of gNO (e.g., liters of gNO), such that the delivery of the gNO to a subject constitutes a single treatment. For example, a gNO storage cylinder can contain approximately 9.0 liters of gNO such that when it is delivered to a subject for 90 continuous minutes at a flow rate of 0.1 liters per minute (LPM), the gNO will be exhausted. In this manner of operation, the gNO storage cylinder can function as a safety feature to prevent gNO overexposure to the subject.

[0058] The subject interface units of the present disclosure can be coated with substances that help to prevent or reduce contamination from microorganisms, bacteria, fungi, viruses, and the like. The coatings can be active pharmaceutical agents that reduce the growth and/or survival of these harmful microorganisms (e.g., anti-bacterial substances), and/or the coatings can function passively to prevent or reduce contamination, for example, by preventing adherence of these microorganism to the various surfaces of the subject interface units (e.g., wetting agents).

[0059] Suitable materials that can be used to construct the subject interface units of the present disclosure include, but are not limited to, various plastics and polymers materials, such as polystyrene (PS), polycarbonate (PC), acrylonitrile-butadiene-styrene (ABS), polybutylene terephthalate (PBTP), styrene acrylonitrile (SAN), polyamide (PA), polyoxymethylene (POM), polyphenylene oxide (PPO), PE, PP, PTFE and homopolymers and copolymers of these plastics and similar materials known in the art and based on the present disclosure. The plastics may also be used in a filled or fiber-reinforced form, and/or coupled to portions of metals or metal alloys, such as aluminum, titanium, steel, and combinations thereof. The materials used to construct the subject interface units can be surface-coated, for example with paints, varnishes or lacquers. The use of color plastics, for example colored with pigments, is also possible.

[0060] Various other subject interface units can be constructed, depending on the characteristics of the wound site, the location of the wound site, the condition of the subject, the environment in which the subject is to be treated, and the like. In some aspects, the various subject interface units can be adapted to model use on a subject, but in an in vitro setting, for experimental purposes (see, e.g., FIG. 2C). For example, a prosthetic leg or arm can be placed in a sealed, cylindrically-shaped subject interface unit configured to receive cultured cells and/or tissues {e.g., full-thickness skin tissue). Such configurations can be used to conduct experiments prior to use on an actual living subject.

[0061] Whatever the configuration of the subject interface unit, it is beneficial to establish and maintain a seal on the subject so that NO can be administered at sufficiently elevated pressures to provide therapeutic benefits to the subject {e.g., reducing bioburden, reduce infection, accelerate wound healing and the like). As used herein, bioburden generally refers to the number of bacteria or other pathogens present on a surface, for example, the surface of a tissue or wound {e.g., skin and/or bone). Reducing bioburden generally correlates with reducing or minimizing an infection, as well as the various symptoms that accompany an infection {e.g., pain, swelling, redness, foul odor, blood or pus being released, etc.). Reducing bioburden and reducing infection also tend to correlate with accelerated wound healing, tissue repair, and the growth of healthy tissue. The application of pressurized NO for a given amount of time at a given flow rate can reduce bioburden in the wound of a subject, which in turn promotes healing.

[0062] The NO delivery systems of the present disclosure can include subject interface units having the ability to maintain a seal on a subject when administering NO between about 0.1 ATM (1.47 psi) to about 1.0 ATM (14.695 psi). In some aspects, administering NO at a pressure ranging from about 0.1 ATM (1.47 psi) to about 0.25 ATM (3.674 psi) is sufficient to establish and maintain a seal on the wound of a subject and reduce bioburden in the wound, thereby accelerating healing. In other aspects, administering NO at a pressure ranging from about 0.1 ATM (1.47 psi) to about 0.15 ATM (2.20 psi) is sufficient to establish and maintain a seal on the wound of a subject and to reduce bioburden in the wound, thereby accelerating healing. Administering NO in these pressure ranges is sufficient to reduce bioburden without significantly compromising the viability of the cells and tissues of the subject.

[0063] In some embodiments, the NO delivery systems of the present disclosure can be used to administer NO to the site of a subject's wound at a certain flow rate. As would be recognized by one of ordinary skill in the art based on the present disclosure, units of flow rate can be expressed using various metrics, including liters/minute (LPM) and/or cubic centimeters per minute (cm ³ /min or cc/min). For example, NO can be delivered to a subject at a flow rate ranging from about 0.1 liters/minute to about 2.0 liters/minute, from about 0.1 liters/minute to about 1.9 liters/minute, from about 0.1 liters/minute to about 1.8 liters/minute, from about 0.1 liters/minute to about 1.7 liters/minute, from about 0.1 liters/minute to about 1.6 liters/minute, from about 0.1 liters/minute to about 1.5 liters/minute, from about 0.1 liters/minute to about 1.4 liters/minute, from about 0.1 liters/minute to about 1.3 liters/minute, from about 0.1 liters/minute to about 1.2 liters/minute, from about 0.1 to about 1.1 liters/minute, from about 0.1 liters/minute to about 1.0 liters/minute, from about 0.1 liters/minute to about 0.9 liters/minute, from about 0.1 liters/minute to about 0.8 liters/minute, from about 0.1 liters/minute to about 0.7 liters/minute, from about 0.1 liters/minute to about 0.6 liters/minute, from about 0.1 liters/minute to about 0.5 liters/minute, from about 0.1 liters/minute to about 0.4 liters/minute, from about 0.1 liters/minute to about 0.3 liters/minute, and from about 0.1 liters/minute to about 0.2 liters/minute. In some aspects, the NO can be delivered to a subject at a flow rate of about 0.1 liters/minute, about 0.2 liters/minute, about 0.3 liters/minute, about 0.4 liters/minute, about 0.5 liters/minute, about 0.6 liters/minute, about 0.7 liters/minute, about 0.8 liters/minute, and about 0.9 liters/minute, about 1.0 liters/minute, about 1.2 liters/minute, about 1.3 liters/minute, about 1.4 liters/minute, about 1.5 liters/minute, about 1.6 liters/minute, about 1.7 liters/minute, about 1.8 liters/minute, about 1.9 liters/minute, and about 2.0 liters/minute or equivalent.

[0064] In some embodiments, the NO delivery systems of the present disclosure can be used to administer NO to the site of a subject's wound for a certain period of time. For example, NO can be delivered to a subject for a period of time ranging from about 30 minutes to about 180 minutes, from about 30 minutes to about 170 minutes, from about 30 minutes to about 160 minutes, from about 30 minutes to about 150 minutes, from about 30 minutes to about 140 minutes, from about 30 minutes to about 130 minutes, from about 30 minutes to about 120 minutes, from about 30 minutes to about 110 minutes, from about 30 minutes to about 90 minutes, from about 30 minutes to about 80 minutes, from about 30 minutes to about 70 minutes, from about 30 minutes to about 60 minutes, from about 30 minutes to about 50 minutes, and from about 30 minutes to about 40 minutes. In some aspects, NO can be delivered to a subject for a period of time of about 110 minutes, about 105 minutes, about 100 minutes, about 95 minutes, about 90 minutes, about 85 minutes, about 80 minutes, about 75 minutes, about 70 minutes, about 65 minutes, about 60 minutes, about 55 minutes, about 50 minutes, about 45 minutes, about 40 minutes, about 35 minutes, about 30 minutes, about 25 minutes, about 20 minutes, about 15 minutes, about 10 minutes, and about 5 minutes or as determined to be appropriate for the subject and wound under consideration.

[0065] In some embodiments, NO delivery devices and systems of the present disclosure can include one or more gas sensors (e.g., electrochemical sensors) for measuring the concentration of one or more gases being delivered to the subject (FIG. 1, 135). For example, the delivery systems of the present disclosure can include nitric oxide sensors, nitric dioxide sensors, and/or oxygen sensors. These sensors can be functionally coupled to the source of the gas (e.g., NO tank or cylinder) and/or they can be coupled to the subject interface unit to measure gas concentrations at the site of the wound or infection. In some aspects, gas sensors can help to maintain a constant flow rate and concentration of NO over a given treatment period. In some aspects, the sensors can indicate to a user (e.g., health care provider) that the concentration of the gas is above or below a prescribed threshold for a given treatment protocol for a subject, in which case the user can adjust one or more parameters, such as the gas pressure regulator or the gas flow regulator, or the user can stop the treatment and replace the source of the gas with one having the appropriate concentrations of gases. In other aspects, the gas sensors can be used as part of a protocol to purge the system, including the subject interface unit, of oxygen, such that gNO can be delivered to a subject in a manner that is substantially free of oxygen. For example, a continuous stream of gNO and/or a bolus of gNO can be delivered to a subject through the subject interface unit while an oxygen sensor measures the decreasing concentration of oxygen in the subject interface unit. Once the sensor indicates that little to no oxygen is present in the system, the gNO treatment protocol can commence.

[0066] Additionally, the NO delivery devices and systems of the present disclosure can include a flushing mechanism. In some aspects, the flushing mechanism can be used to flush out the NO used during treatment and restore oxygen levels to normal so that the subject is not exposed to elevated levels of NO after the subject interface unit is removed. The flushing mechanism can be coupled to the source of the NO gas, such that a user can replace the source of the gNO used for treatment with a source of gas having a lower concentration of NO and a higher concentration of oxygen (e.g., cylinder having compressed ambient air or pure oxygen). In some aspects, the user can then activate the flushing mechanism to displace the previously administered gNO out of the subject interface unit. The flushing mechanism can be performed according to a prescribed protocol, for example, the flushing gas can be administered over a certain duration of time while the subject interface unit is still attached to the subject. In some aspects, the flushing mechanism can include the injection of a bolus of air or purified oxygen into the delivery device soon after the gNO treatment has ended. In this manner of operation, there is no significant need for a flushing protocol, as the bolus of air will in injected and travel through the delivery device rapidly. Other flushing protocols can be used according to accepted medical standards and practices, as would be recognized by one of ordinary skill in the art based on the present disclosure.

[0067] Embodiments of the NO delivery systems of the present disclosure can be used to alleviate disease symptoms or health conditions and improve therapeutic outcomes in a subject regarding wounds. For example, the NO delivery systems of the present disclosure can be used to treat subjects having health conditions, for example, diabetes mellitus, as these subjects are known to be at risk for developing acute as well as chronic dermal ulcers (e.g., foot ulcers), in the presence of established long-term complications of the health condition or disease. Infections due to diabetic ulcers can range in severity from superficial paronychia to deep infections involving bone. It is contemplated that any and all of these types of ulcers can be treated using systems and methods disclosed herein. In certain embodiments, types of infection can include, but are not limited to, cellulitis, myositis, abscesses, necrotizing fasciitis, septic arthritis, tendinitis, and osteomyelitis.

[0068] Additionally, embodiments of the NO delivery systems of the present disclosure can be used to treat skin and soft tissue infections (SSTIs). SSTIs are common, and complicated SSTIs (cSSTIs) can be the more extreme end of this indication. SSTIs can encompass a range of clinical presentations, including but not limited to, deep-seated infection, which typically requires surgical intervention, the presence of systemic signs of sepsis, the presence of complicating co-morbidities, accompanying neutropenia, accompanying ischemia, tissue necrosis, burns and bites. Staphylococcus aureus is the most common cause of SSTI; however, its epidemiology {e.g. , causative strains) and antibiotic susceptibility are not currently able to be accurately predicted. It is contemplated that systems and methods disclosed herein can be used to reduce the risk of SSTIs as well as treat them.

[0069] Various embodiments of the NO delivery systems disclosed in the present application can provide the means to minimize infection by reducing bioburden using pressurized gNO delivered to a wound site. Unlike antibiotic-related treatments against pathogenic organisms such as bacteria, bacteria are incapable of developing resistance to gNO. Therefore, NO delivery systems of the present disclosure represent a universally effective means to treat and/or prevent infections by reducing bioburden in a wound and accelerate healing. Embodiments of the present application regarding NO delivery systems can be used to reduce infections already present in a subject, and/or used prophylactically to prevent the development of pathogenic infection, for example, by being applied to the site of a surgical wound or incision. Additionally, gNO delivered using the systems and devices of the present disclosure can be used to treat infection in a wide range of wounds and disease indications in a subject, including, but not limited to, sites of pathogen infections such as bacterial infections, fungal infections, viral infections, protozoan infections, burns, wounds, wrinkles, lesions, and the like. Lesions can include, but are not limited to, a surgical wound, a trauma wound, a burn, an abscess, an actinic keratosis, a keloid, a scar, and skin cancer and a combination thereof.

[0070] Additionally, embodiments of the present disclosure regarding NO delivery systems include uses in combination with other therapies to alleviate disease symptoms and improve therapeutic outcomes in a subject in need of such a treatment. For example, in the case of osteomyelitis, NO administration according to embodiments of the present disclosure can be combined with other known treatment methods, including but not limited to, antibiotic administration, hyperbaric oxygen therapy (HBO), maggot debridement therapy, and granulocyte colony-stimulating factor administration. In some aspects, combinatorial therapy can have synergistic effects and can preclude the need for more serious surgical intervention, such as amputation.

EXAMPLES

[0071] Examples of the present disclosure are included to demonstrate certain embodiments presented herein. It should be appreciated by those of skill in the art that the techniques disclosed in the examples that follow represent techniques discovered to function well in the practices disclosed herein. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the certain embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope herein.

Reduction in bioburden

[0072] FIG. 4 is a graph illustrating the effects of NO treatment on bioburden using an infection assay, after three hours of infection, according to an embodiment of the present disclosure. Skin samples infected for 3 hours with Staphylococcus, MRSA, Acinetobacter or Pseudomonas and subsequently exposed to 1% gNO at 1 ATM (14.695 psi) for 90 minutes at a flow rate of 0.1 liters/minute. Staphylococcus aureus, subspecies aureus Rosenbach (ATCC# 12600); Staphylococcus aureus, subspecies aureus Rosenbach (ATCC# 33591), methicillin- resistant strain of Staphylococcus aureus, phage type 92; Acinetobacter baumannii (ATCC# BAA-747); and Pseudomonas aeruginosa (ATTC# BAA-47). Control = infected, untreated tissue. Three sets of triplicates were performed per experiment.

[0073] As illustrated in FIG. 4, treatment that included 1% gNO at 1 ATM (14.695 psi) for 90 minutes significantly reduced bioburden in each of the four types of bacteria tested. S. aureus was reduced 5 -fold, and the other bacteria strains were nearly completely eradicated. These results demonstrate the efficacy of NO administration for reducing infection by reducing bioburden in the wound of a subject. Additionally, because these experiments were performed using an acute 3 hour infection model, these data also indicate that NO treatment according to the above therapeutic parameters can be used to prevent infections as well as to treat infections.

Reduction in bioburden at pressures below 1.0 ATM

[0074] Experiments were also conducted to determine the infection-reducing capability of gNO administration at various pressures below 1.0 ATM (14.695 psi) (independent of and in addition to the pressure applied by the external environment) using both a Franz cell tissue culture device (i.e., 3-Ring) and a portable NO deliver device (i.e., Leg Device). The results of one set of experiments are illustrated below in Table 1. NO was administered at a 1% concentration after 24 hours of infection with S. aureus. Flow Rates (cm ³ /min or cc) varied from about 100 to about 1500, Purge Flow (liters/min or LPM) varied from about 0.1 to about 1.5, and NO Exposure Time varied between about 45 minutes to about 105 minutes among the different treatment groups and controls. Colony counts (log CFU) were performed at various dilutions, which are represented in the last eight columns labeled 0, lxl 0 ^"1 through lxl 0 ^"7 ("0" indicates no dilution). The results in Table 1 demonstrate that bioburden was significantly reduced (i.e., total kill) after NO administration at pressures as low as about 0.15 ATM (2.2 psi) for exposure times of about 105 minutes.

[0075] Additionally, MTT assays were performed to assess cell viability after NO administration. MTT assays are colorimetric-based assays used to assess cell viability as a function of color variation. As illustrated below in Table 2, cell viability after the administration of pressurized NO at about 0.15 ATM (2.2 psi) for about 105 minutes varied from about 40%- 50%. Therefore, the administration of NO at pressures less than 1.0 ATM (14.695 psi) is effective for reducing bioburden and does not significantly compromise cell viability.

[0076] The results in Tables 2 and 3 indicate that NO can be delivered at pressures below

1.0 ATM (14.695 psi) (independent of and in addition to the pressure applied by the external environment) and effectively reduce infection by reducing bioburden without significantly compromising the health of the subject's cells. The use of pressures below 1.0 ATM (14.695 psi) have the additional benefit of requiring less external pressure on the subject interface unit to maintain a seal, thus making the seal easier to establish and maintain, as well as providing a greater degree of comfortable for the subject. Striking the balance between delivering NO at a high enough pressure to reduce bioburden and yet still maintain an effective seal on the subject is one important contribution of the present disclosure.

Table 1 : The effect of pressurized NO administration on reducing bioburden

Purge Pressure Exposure

Franz cell Flow Flow rate Delivered Time

Trial Sample Version (LPM) (cc) (ATM) (min) 0 1x10-1 1x10-2 1x10-3 1x10-4 1x10-5 1x10-6 1x10-7

Control TNTC TNTC TNTC TNTC TNTC >250 47 3

Leg l A .eg Devi 1.5 LPM 1500 1 ATM 90m in 0 0 0 0 0 0 0 0

Leg l B .eg Devia 1.5 LPM 1500 1 ATM 90m in 0 0 0 0 0 0 0 0

Leg l C .eg Devici 1.5 LPM 1500 1 ATM 90m in 0 0 0 0 0 0 0 0

0.5 ATM A 3-Ring 0.1 100 0.5 ATM 90m in 0 0 0 0 0 0 0 0

0.5 ATM B 3-Ring 0.1 100 0.5 ATM 90m in 16 2 0 0 0 0 0 0

0.5 ATM C 3-Ring 0.1 100 0.5 ATM 90m in 0 0 0 0 0 0 0 0

Control TNTC TNTC TNTC TNTC TNTC >250 46 3

Leg 2 A .eg Devia 1.5 LPM 100 0.5 ATM 90m in 0 0 0 0 0 0 0 0

Leg 2 B .eg Devia 1.5 LPM 100 0.5 ATM 90m in 0 0 0 0 0 0 0 0

Leg 2 C .eg Devici 1.5 LPM 100 0.5 ATM 90m in 0 0 0 0 0 0 0 0

Control TNTC TNTC TNTC TNTC TNTC >250 56 5

Calf A .eg Devici 0.1 100 0.5 ATM 90m in 0 0 0 0 0 0 0 0

Heel B .eg Devici 0.1 100 0.5 ATM 90m in 0 0 0 0 0 0 0 0

Toe C .eg Devici 0.1 100 0.5 ATM 90m in 0 0 0 0 0 0 0 0

0.25 ATM A 3-Ring 0.1 100 0.25 ATM 90m in 0 0 0 0 0 0 0 0

0.25 ATM B 3-Ring 0.1 100 0.25 ATM 90m in 0 0 0 0 0 0 0 0

0.25 ATM C 3-Ring 0.1 100 0.25 ATM 90m in 0 0 0 0 5 0 0 0

Control TNTC TNTC TNTC TNTC TNTC 180 16 0

0.5 ATM A .eg Devici 1.0 LPM 500 0.5 ATM 45m in 0 0 0 0 0 0 0 0

0.5 ATM B .eg Devici 1.0 LPM 500 0.5 ATM 45m in 0 0 0 0 0 0 0 0

0.5 ATM C .eg Devici 1.0 LPM 500 0.5 ATM 45m in 0 0 0 0 0 0 0 0

Control TNTC TNTC TNTC TNTC TNTC 67 4 0

1 Pressure A .eg Devici 0.1 100 0.5 ATM 45m in TNTC TNTC TNTC TNTC 28

1 Pressure B .eg Devici 0.1 100 0.5 ATM 45m in TNTC TNTC TNTC TNTC 14

1 Pressure C .eg Devici 0.1 100 0.5 ATM 45m in TNTC TNTC TNTC TNTC 40

Control TNTC TNTC TNTC TNTC TNTC TNTC 59 6

0.25 ATM A 3-Ring 0.1 100 0.25 ATM 90m in 0 0 0 0 0 0 0 0

0.25 ATM B 3-Ring 0.1 100 0.25 ATM 90m in 0 0 0 0 0 0 0 0

0.25 ATM C 3-Ring 0.1 100 0.25 ATM 90m in 0 0 0 0 0 0 0 0

0.25 ATM A 3-Ring 0.1 100 0.25 ATM 90m in TNTC TNTC TNTC TNTC >150

0.25 ATM B 3-Ring 0.1 100 0.25 ATM 90m in TNTC TNTC TNTC TNTC 66

0.25 ATM C 3-Ring 0.1 100 0.25 ATM 90m in TNTC TNTC TNTC TNTC 59

Control TNTC TNTC TNTC TNTC TNTC TNTC 54 11

9 attempt A 3-Ring 0.1 100 0.25 ATM 90m in TNTC TNTC TNTC TNTC 97

9 attempt B 3-Ring 0.1 100 0.25 ATM 90m in TNTC TNTC TNTC 38 0

9 attempt C 3-Ring 0.1 100 0.25 ATM 90m in TNTC TNTC TNTC TNTC 188

Leg purge A .eg Devici 0.5 LPM 100 0.25 ATM 90m in 0 0 0 0 0

Leg purge B .eg Devici 0.5 LPM 100 0.25 ATM 90m in 0 0 0 0 0

Leg purge C .eg Devici 0.5 LPM 100 0.25 ATM 90m in 0 0 0 0 0

Control TNTC TNTC TNTC TNTC TNTC TNTC 142 14

0.15ATM A 3-Ring 0.1 100 0.15 ATM 105m in TNTC TNTC 181* 20* 1 0 0 0

0.15ATM B 3-Ring 0.1 100 0.15 ATM 105m in 0 0 0 0 0 0 0 0

0.15ATM C 3-Ring 0.1 100 0.15 ATM 105m in 0 0 0 0 0 0 0 0

Control TNTC TNTC TNTC TNTC TNTC TNTC 124 14

0.15 ATM A 3-Ring 0.1 100 0.15 ATM 105m in 0 0 0 0 0 0 0 0

0.15 ATM B 3-Ring 0.1 100 0.15 ATM 105m in TNTC TNTC 24 3 0 0 0 0

0.15 ATM C 3-Ring 0.1 100 0.15 ATM 105m in 5* 0 0 0 0 0 0 0

Table 2: The effect of pressurized NO administration on cell viability Detection of S. aureus in skin infections

[0077] FIGS. 5A-5C are representative histological sections stained to detect the presence of the bacteria S. aureus on treated and untreated skin sample tissue, according to an embodiment of the present disclosure. FIG. 5A was taken from tissue that was wounded but left uninfected. FIG. 5B was taken from tissue that was wounded and infected for 3 hours with S. aureus. FIG. 5C was taken from tissue that was wounded and infected for 24 hours with S. aureus. The representative histological sections were stained with Giesma, which identifies S. aureus bacteria present in the skin sample tissue. S. aureus bacteria could be seen on the on the surface of the wound after 3 hour of infection, but by 24 hours of infection, the bacteria had infiltrated further into the tissue.

Detection biofilm production by of S. aureus in skin infections

[0078] Biofilms can play a role in histopathology and are complex structures consisting of bacterial cells embedded in an extracellular matrix that contains polysaccharides, proteins and DNA. The biofilm matrix can limit the effectiveness of topical antibiotic treatment in infected wounds and can impede wound healing and immune responses. The various components of biofilm and associated extracellular matrix can be visualized using histological techniques, and can provide a basis for assessing the efficacy of NO administration for reducing bioburden for bacteria that form biofilms.

[0079] FIGS. 6A-6F are representative histological sections stained to detect components of bacterial biofilm, according to an embodiment of the present disclosure. The skin samples used in FIGS. 6A-6F were infected for 24 hours with S. aureus bacteria, then fixed in formaline, embedded, and sliced at 5-6 μιη increments. FIG. 6A was taken from tissue stained with Hematoxylin and eosin. FIG. 6B was taken from tissue stained with Feulgen reaction. FIG. 6C was taken from tissue stained with Modified Congo Red. FIG. 6D was taken from tissue stained with Modified Congo Red with Carbol Fuchsin. FIG. 6E was taken from tissue stained with PAS. FIG. 6F is stained with Calcofluor. Using these histological techniques, skin tissue sample can be taken before and after treatment with pressurized NO to determine reductions in bioburden for bacteria that form biofilm. [0080] The present disclosure, in various aspects, embodiments, and configurations, includes components, methods, processes, systems and/or apparatus substantially as depicted and described herein, including various aspects, embodiments, configurations, sub combinations, and subsets thereof. Those of skill in the art will understand how to make and use the various aspects, aspects, embodiments, and configurations, after understanding the present disclosure. The present disclosure, in various aspects, embodiments, and configurations, includes providing devices and processes in the absence of items not depicted and/or described herein or in various aspects, embodiments, and configurations hereof, including in the absence of such items as may have been used in previous devices or processes, e.g., for improving performance, achieving ease and\or reducing cost of implementation.

[0081] The foregoing discussion of the disclosure has been presented for purposes of illustration and description. The foregoing is not intended to limit the disclosure to the form or forms disclosed herein. In the foregoing Detailed Description for example, various features of the disclosure are grouped together in one or more, aspects, embodiments, and configurations for the purpose of streamlining the disclosure. The features of the aspects, embodiments, and configurations of the disclosure may be combined in alternate aspects, embodiments, and configurations other than those discussed above. This method of disclosure is not to be interpreted as reflecting an intention that the claimed disclosure requires more features than are expressly recited in each claim. Rather, as the following claims reflect, inventive aspects lie in less than all features of a single foregoing disclosed aspects, embodiments, and configurations. Thus, the following claims are hereby incorporated into this Detailed Description, with each claim standing on its own as a separate preferred embodiment of the disclosure.

[0082] Moreover, though the description of the disclosure has included description of one or more aspects, embodiments, or configurations and certain variations and modifications, other variations, combinations, and modifications are within the scope of the disclosure, e.g., as may be within the skill and knowledge of those in the art, after understanding the present disclosure. It is intended to obtain rights which include alternative aspects, embodiments, and configurations to the extent permitted, including alternate, interchangeable and/or equivalent structures, functions, ranges or steps to those claimed, whether or not such alternate, interchangeable and/or equivalent structures, functions, ranges or steps are disclosed herein, and without intending to publicly dedicate any patentable subject matter.