Method and compositions for treating burn wounds

Abstract

This disclosure provides the use of molecular complexes of polyammonium bisulfate alone and in combination with certain metals or metal salts to treat burn wounds.

Description

METHOD AND COMPOSITIONS FOR TREATING BURN WOUNDSCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] The present application claims the benefit of' and priority to U.S. Provisional Application No. 63 / 488,162, fried March 2, 2023; U.S. Provisional Application No. 63 / 544,137, filed October 13, 2023; and U.S. Provisional Application No. 63 / 544,140, filed October 13, 2023, each of which is hereby incorporated by reference in its entirety for ail purposes.FIELD OF THE INVENTION

[0002] The present disclosure relates to methods of treating a bum wound with, for example, topical application of a formulation comprising molecular clusters of poly ammonium bi sulfate (hereafter referred to as “PABS”), either on their own or in combination with a metal or salt thereof. Formulations comprising excipients and PABS or excipients and PABS in combination with selected metals or salt thereof are also provided and can be used to treat bum wounds, to enhance healing, and to prevent and / or treat infections in bum wounds.BACKGROUND OF THE INVENTION

[0003] The skin of humans and other mammals has multiple functions that range from protecting against pathogens to preventing excessive water loss to temperature regulation, among many others. Therefore, damage or other impairments to the skin and underlying tissues may have profound consequences to the overall health of a person or animal. Humans and animals are affected by a wide range of maladies and disorders of the skin and underlying tissues. Examples of such skin maladies and disorders vary from minor infections of one or more layers of the skin to trauma-induced wounds (e.g., cuts, abrasions, burns, puncture wounds, etc.).

[0004] Because wound healing is a highly complex process, there are multiple opportunities to impose external influences on selected aspects of this important process to increase the rate and quality of the healing process. However, severe burn wounds, e.g., 3rddegree burns, can be especially challenging to treat because, depending on the amount of skin surface area involved, infections are common and add complexity to the treatment approach.

[0005] Applying an antimicrobial agent to a bum wound can limit or decrease the number of potentially infectious cells, thereby lowering the probability of the wound becoming infected. Silver sulfadiazine (SSD) is the most common medicament used to prevent infections in bum wounds. SSD, however, may cause an allergic reaction in some bum wound patients and has been shown to be cytotoxic, depending on the amount applied. The biological activity of SSD is limited to antimicrobial effects. Some other metals have been reported to have antimicrobial activity and have other biological activities if used to treat burn wounds. As a non-limiting example, some metals such as copper and zinc have important roles in wound healing, especially as related to inhibition of infectious or potentially infectious microorganisms as well as being essential elements in some of the biochemical and immunological processes that are part of healing damaged skin and tissue.

[0006] The repair of wounds in mature skin and the underlying tissue is highly complex, involving numerous cell types and molecular processes. Wound repair processes often result in some scar formation, an outcome that reduces the functional capacity of the repaired skin and, in some cases, may limit joint motion as well as cause psychosocial harm, depending on the extent of scaring. Scar tissue is the result of activated fibroblasts producing excess levels of irregularly organized collagen. Scar tissue is weaker than normal skin, in terms of tensile strength, and is also more rigid versus normal skin. Scar formation has negative effects on the biomechanical properties of the skin.

[0007] There remains a need in the art for compositions and methods for treating bum wounds where the compositions and methods enhance healing quality and / or rate, reduce inflammation, and / or inhibit or prevent infection.BRIEF SUMMARY OF THE INVENTION

[0008] The present disclosure is directed to methods for treating bum wounds with molecular clusters of PABS. The compositions and methods of the present invention can be used to treat wounds of the skin and underlying tissues. As used herein, a PABS molecular cluster refers to an acidic molecular complex with a composition comprising about 1% to about 20% (w / w) polyammonium bisulfate, about 0% to about 30% (w / w) sulfuric acid (H2SO4), and about 50% to about 99% water (w / w). As will be appreciated by those of skilled in the art, the percentages of the PABS molecular clusters can vary over a wide range, e.g., from about 0.1% (weight basis) to about 40% (weight basis), depending on process andreaction conditions used when PABS is produced. A solution containing the PABS molecular complex may contain predominantly self-assembling molecular complexes that typically have average diameters of less than about 10 nm to more than about 2,000 nm, depending on the method of production, concentrations of reactants, and reaction conditions, as well as unassembled subunits. In one embodiment, the clusters preferably range in size, measured as average diameter, from about 10 nm to about 2,000 nm, such as from about 10 nm to about 1,000 nm, from about 50 nm to about 500 nm, or from about 100 nm to about 400 nm. Solutions of PABS clusters may also be comprised of self-assembled, three-dimensional clusters and unassembled subunits. In addition to using PABS solutions, water can be removed from the PABS solutions via evaporation, lyophilization, or other processes that produce a dry product. When dried, PABS is stable and can be rehydrated or used in a dry (powder) form. Thus, the PABS used in the compositions or formulations of the present invention can be in solution or dried and used in a solid or powdered form.

[0009] It has been unexpectedly discovered that PABS on their own or in combination with a metal or salt thereof is effective in treating burn wounds and improving healing. The compositions (used interchangeably herein with the terms “formulation” and “medicament”) described herein comprising PABS, pharmaceutically acceptable excipients and optionally metals or salts thereof can be applied to bum wounds to improve healing quality (e.g., lessen scar formation), to increase the rate of healing, and to inhibit or prevent infection. Importantly, it has been unexpectedly discovered that when a metal or salt thereof is included in the composition, the amount of the metal needed to treat a wound, when delivered in combination with PABS, is significantly lower than the amount of the same metal to achieve the same effect without PABS. For example, when silver is applied with PABS, the amount of silver required to achieve a desired effect is about one-tenth of the amount of silver required when SSD is applied. The ability to reduce the amount of metal provides advantages over commercially used products available at the time of the present invention.

[0010] The present disclosure also provides methods of applying molecular clusters of PABS and metals or salts thereof, such as salts of copper, selenium, and / or zinc, to a burn wound and the skin and tissue surrounding the wound. Such methods advantageously provide targeted delivery of important metals needed to treat wounds and surrounding tissues as a means of replenishing metals depleted during treatment recovery from burn wounds.

[0011] Disclosed herein are methods of treating an area of skin having a wound, the method comprising applying to the area of skin an effective amount of a composition comprising polyammonium bisulfate (“PABS”) and a pharmaceutically acceptable carrier or excipient to form a treated area of skin. In some examples, the wound is a bum wound. In some examples, applying an effective amount of the composition comprises topically applying a composition wherein the PABS is present in the composition in an amount from about 0.01 % to about 50 % by weight based on the weight of the composition. In some examples, the composition further comprises at least one metal or salt thereof. Optionally, the composition further comprises at least two metals or salts thereof. In some examples, the composition further comprises sulfuric acid in an amount from about 0 % to about 40 % by weight based on the weight of the composition. In examples where the composition includes at least one metal or salt thereof, the at least one metal or salt thereof optionally can include cobalt, iron, magnesium, manganese, molybdenum, bismuth, cerium, copper, zinc, silver, gold, gallium, or selenium. For example, the metal or salt thereof can include bismuth, cerium, copper, zinc, silver, gold, gallium, or selenium. In some examples, the at least one metal or salt thereof is present in the composition in an amount from about 1 x 10'7to about 5% by weight based on the weight of the composition. Optionally, the at least one metal or salt thereof is an antimicrobial metal. Optionally, the at least one metal or salt thereof is an essential metal. In some examples, the composition further comprises water in an amount from about 0.1 % to about 99 %. In some examples, the composition is a liquid, suspension, lotion, emulsion, powder, gel, ointment, cream, or paste.

[0012] In some embodiments, a composition disclosed herein includes a metal or salt thereof and applying the composition increases an amount of the metal in a treated area of skin. For example, in some embodiments, the method comprises applying to a damaged area of skin an effective amount of a composition comprising PABS and at least one metal or salt thereof, wherein the damaged area of skin is depleted of the at least one metal, and wherein applying the composition increases the amount of the at least one metal in the area of skin to form a treated area of skin. In some examples, the damaged area of skin is depleted of the at least one metal due to initial wound trauma and / or subsequent wound treatment and / or healing. Optionally, the methods described herein are advantageous when the plasma concentration of copper, zinc, or selenium is lower than reference ranges for copper (about 12-20 pM), Zn (about 11-18 pM) or Se (about 0.75-1.80 pM). Optionally, the concentration of copper, zinc, and / or selenium in a formulation to be applied to a wound should be morethan the maximum of the reference ranges for copper (20 pM), Zn (18 pM) and / or Se (1.80 pM). In examples where the composition includes at least one metal or salt thereof, the metal optionally can be copper, selenium, or zinc, of from about 0.01 % to about 30 % by weight based on the weight of the composition. When the composition includes copper, the composition optionally comprises the copper in a concentration of from about 1 x 10'4gm / kg to about 1 gm / kg. When the composition includes zinc, the composition optionally comprises the zinc in a concentration of from about 1.5 x 10'4gm / kg to about 1 gm / kg. When the composition includes selenium, the composition optionally comprises the selenium in a concentration of from about 1.4 x 10'4to about 1 gm / kg. Optionally, the composition comprises at least two metals or salts thereof. In some examples, the composition is a liquid, suspension, lotion, emulsion, powder, gel, ointment, cream, or paste.

[0013] In some embodiments a method disclosed herein comprises applying to the wound an effective amount of a composition comprising PABS and cerium. In some examples, the wound comprises eschar, and applying the composition comprising PABS and cerium stabilizes the eschar. In some examples, the composition comprises the PABS in a concentration of from about 0.010 % to about 30 % by weight based on the weight of the composition. In some examples, the composition comprises the cerium in a concentration of from about 1 % to about 3 % by weight based on the weight of the composition. In some examples, the composition further comprises silver or bismuth. Optionally, the composition comprises the silver in a concentration of from about 0.1 % to about 2.5% by weight based on the weight of the composition. Optionally, the composition comprises the bismuth in a concentration of from about 0.01% to about 2.5% by weight based on the weight of the composition. In some examples, any composition described herein is a liquid, suspension, lotion, emulsion, powder, gel, ointment, cream, or paste.

[0014] Any composition described herein can be used in any method descried herein. In any example of a method disclosed herein, applying the composition comprises administering an effective amount of the composition to the wound and to skin and / or tissue surrounding the wound. Optionally, the effective amount comprises from about 0.1 mg to about 250 mg, or from about 50 mg to about 220 mg, or from about 100 mg to about 200 mg of the composition per cm2 of the surface area to which it is applied. Optionally, the step of applying the composition is repeated one or more times, based on medical necessity, for example until the wound has healed to a medically acceptable condition. Optionally, the step of applying the composition is repeated at one or more intervals from about one-hour to abouteight-hours, such as from about two-hours to about eight-hours. Optionally, the step of topically applying the composition is repeated between about three to about eight times a day for about one to about 28 days, preferably about 7 to about 21 days. In some examples, the method enhances healing quality and / or rate. In some examples, the method reduces inflammation. In some examples, the method reduces or prevents infection.

[0015] The disclosure further provides a topical medicament comprising a therapeutically effective amount of PABS in combination with at least one pharmaceutically acceptable excipient, filler, carrier or diluent. The topical medicament can be any composition disclosed herein for use in any method disclosed herein. In some examples, the medicament has a PABS content from about 0.01 % to about 30 % by weight based on the weight of the composition. In some examples, the medicament is a liquid, suspension, ointment, salve, lotion, paste, cream, gel, foam, emulsion, balm, or pomade. In some examples, the medicament further includes one or more metals or salts thereof. Optionally, the metal comprises cobalt, iron, magnesium, manganese, molybdenum, bismuth, cerium, copper, zinc, silver, gold, gallium, or selenium. Optionally, the metal comprises bismuth, cerium, copper, zinc, silver, gold, gallium, or selenium. Optionally, the pharmaceutically acceptable excipient, filler, carrier or diluent is water. In some examples, the medicament comprises from about 49 % to about 99 % water by weight based on the weight of the composition. In some examples, the medicament is an oil in water emulsion.BRIEF DESCRIPTION OF THE FIGURES

[0016] Figure 1 illustrates the bacterial population in the wounds increased in all treatments and the untreated control after a two-day (48 hours) incubation, but decreases by day 7 (168 hours), with the greatest improvement seen with PABS + Ag and PABS + Bi.

[0017] Figure 2 illustrates the changes in bacterial counts from Day 2 to Day 7.

[0018] Figure 3 illustrates the effect of DermaBase Cream amended with PABS versusDermaBase Cream and no treatment on wound healing.

[0019] Figure 4 provides an overview of the experimental design.

[0020] Figure 5 provides a summary of changes in wound size during the 45-day treatment trial. The insert illustrates the number of applications of the test formulations per day (gray = 2, no shading = 1 per day).

[0021] Figure 6 illustrates the grayscale images of 3rddegree burn wound in porcine skin treated with a topical cream containing a combination of PABS and copper.

[0022] Figure 7 illustrates the grayscale images of 3rd degree burn wound in porcine skin treated with a topical cream containing a combination of PABS and zinc.

[0023] Figure 8 illustrates the grayscale images of 3rd degree burn wound in porcine skin treated with a topical cream containing a combination of PABS, copper, and zinc.

[0024] Figure 9 illustrates the grayscale images of 3rd degree burn wound in porcine skin treated with the Vehicle Control.

[0025] Figure 10 illustrates grayscale images of the Untreated Control 3rd degree bum wounds in porcine skin.DETAILED DESCRIPTION OF THE INVENTIONA. Definitions

[0026] This specification includes references to “one embodiment” or “an embodiment.” The appearances of the phrases “in one embodiment” or “in an embodiment” do not necessarily refer to the same embodiment, although embodiments that include any combination of the features are generally contemplated, unless expressly disclaimed herein. Particular features, structures, or characteristics may be combined in any suitable manner consistent with this disclosure.

[0027] While the disclosure is susceptible to various modifications and alternative forms, specific embodiments thereof are shown by way of example in the drawings and will herein be described in detail. It should be understood, however, that the drawings and detailed description thereto are not intended to limit the disclosure to the particular form illustrated, but on the contrary, the intention is to cover all modifications, equivalents and alternatives falling within the spirit and scope of the present disclosure as defined by the appended claims. The headings used herein are for organizational purposes only and are not meant to be used to limit the scope of the description. As used throughout this application, the word “may” is used in a permissive sense (z.e., meaning having the potential to), rather than the mandatory sense (z.e., meaning must). Similarly, the words “include,” “including,” and “includes” mean including, but not limited to. Additionally, as used in this specification andthe appended claims, the singular forms “a”, “an”, and “the” include singular and plural referents unless the content clearly dictates otherwise.

[0028] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of ordinary skill in the art to which this invention pertains. Although methods and materials similar or equivalent to those described herein can be used in the practice of the present invention, suitable methods and materials are described below. All patents, applications, published applications and other publications referred to herein are incorporated by reference in their entirety. If a definition set forth in this section is contrary to or otherwise inconsistent with a definition set forth in the patents, applications, published applications and other publications that are herein incorporated by reference, the definition set forth in this section prevails over the definition that is incorporated herein by reference.

[0029] The terms “comprising,” “including,” “carrying,” “having,” “containing,” and “involving,” whether in the written description or the claims and the like, are open-ended terms, z.e., to mean “including but not limited to.” Thus, the use of such terms is meant to encompass the items listed thereafter, and equivalents thereof, as well as additional items. Only the transitional phrases “consisting of’ and “consisting essentially of,” are closed or semi -closed transitional phrases, respectively, with respect to the claims. Use of ordinal terms such as “first,” “second,” “third,” and the like in the claims to modify a claim element does not by itself connote any priority, precedence, or order of one claim element over another or the temporal order in which acts of a method are performed, but are used merely as labels to distinguish one claim element having a certain name from another element having a same name (but for use of the ordinal term) to distinguish the claim elements.

[0030] The term “molecular complex” refers to a heterogenous aggregate, cluster, or group of molecules and elements that a have a specific composition and arrangement.

[0031] “Pharmacologically-active compound”, as used herein, are those compounds, substances, or materials that are used to treat a disease and / or symptom or a disease, including pain. One skilled in the art will understand that pharmacologically-active compounds are used in different manners, depending on the disease or malady. For example, pharmacologically-active compounds can be ingested, injected, or applied to a surface of the body.

[0032] As used herein, “tissue” refers to the various groups of cells that make up organs and other body parts. It is well known and understood that the bodies of humans and many animals have bodies composed of well-organized tissues that are grouped according to cell type and function. These include muscle, epithelial, connective, and nervous tissue.

[0033] As used herein, “topical agent” refers to a pharmacologically-active compound, substance or material alone or in combination with one or more pharmacologically-inactive compound, substance, or material to treat a disease, wound or otherwise damaged area of skin and / or underlying tissue of a human or animal. “Pharmacologically-active” refers to a compound, substance, or material that has inherent properties that directly or indirectly affects the physiology, biology, or immunology of the body of a human or animal in a when used to treat a disease, infection, or other medical problem. As a non-limiting example, a pharmacologically-active compound, substance, or material may be inhibitory to an infectious microorganism. Other non-limiting examples include pain relievers, anticancer compounds, natural or synthetic hormones, and the like. In some cases, “dermatologically- acceptable” is used to convey that the components of a pharmacological composition are understood to be those that have proven to be safe for application to human and / or animal skin.

[0034] As used herein, “pharmacologically active substances” are often formulated with other ingredients that have no biological activity and such inactive ingredients as used herein, are referred to as “pharmaceutically acceptable excipient,” “dermatologically acceptable ingredient,” “biologically inert compounds” or “inactive ingredients.” Pharmaceutically acceptable excipients are added to formulations to impart desired characteristics on the final product. For example, topically applied pharmacological compositions may contain emulsifiers, penetration enhancers, absorption promoters, antioxidants, metal chelators, humectants, and fragrances, among others, to act as carriers of active substances, provide even spreading of a formulation, etc. In some embodiments, the pharmacological composition is applied as a coating on the surface of a wound. Such pharmacological compositions can be in the form of a solution, suspension, lotion, emulsion, powder, gel, ointment, cream, or paste. In preferred embodiments, the formulation is an emulsion in the form of a lotion or cream.

[0035] Pharmacological compositions are used to treat wounds and maladies of the skin and underlying tissue. As used herein, “biologically active” refers to a compound, substance,or material that has an effect or influence on the physiology or a biological function or process of a cell, tissue, or organism. Thus, a biologically active compound, substance, or material may have a beneficial or detrimental effect on a cell, tissue, or organism, depending on the desired outcome.

[0036] As used herein, “inflammatory process” refers to the body’s natural response to trauma, injury or other physical damage that cause cellular damage and / or introduction of materials or substances foreign to the body. As used herein, foreign materials and substances refer to any substance, material, chemical substance not produced by the body as well as any virus, bacteria, fungus, or other type of microorganism or macroorganism. “Topically applied” or “topical application”, as used herein, refers to the placement of a substance, material, or formulation to the outermost layer of the skin of a human or animal. Topical application is a common procedure for use of pharmacologically active substances to treat a wide range of skin diseases and maladies. One skilled in the art will recognize that other methods of application are possible, for example, injection under the skin.

[0037] The term “biofilm disruptor” is used herein as means to convey the effect of a chemical, compound, or substance on an existing biofilm. It is well known that a biofilm is a three-dimensional microhabitat that some microorganisms produce as a means of enhancing their survival. For example, in a wound, a biofilm provides protection from the host’s immune system as well as antimicrobial agents used to treat a wound that includes a biofilm. One aspect of biofilms is that most are composed of an extracellular matrix with a composition that is species specific and requires maintenance and repair. A biofilm disruptor is a substance, compound, or material that either causes loss of structural integrity of the extracellular matrix, prevents maintenance and repair of said matrix, or prevent production and / or assembly of new matrix components. The result of the activity of a biofilm disruptor is eventual loss of the extracellular matrix so that the biofilm is eliminated from a wound.

[0038] The term “about,” as used herein when referring to a measurable value such as an amount or concentration and the like, is meant to encompass variations of 20%, 10%, 5%, 1%, 0.5%, or even 0.1% of the specified amount.

[0039] As used herein the term “bum wound” is defined as skin and tissue injury caused by any source of heat, cold, electromagnetic energy, chemical substances, and radiation. The four types of burn wounds are designated as first-degree, second-degree, third-degree, and fourth-degree, depending on the depth of the damage into the skin and underlying tissues.

[0040] As used herein, “skin” refers to the thin layer of tissue forming the natural outer covering of the body of a person or animal. It is generally accepted that skin refers to the two cellular layers known as the epidermis and the dermis. The epidermis and dermis can be subclassified by cell type in each sublayer. As used herein, “skin” is used to include all cell types and sublayers of the epidermis and dermis. “Tissue” is used herein as a generic term that includes cells and natural structures such as blood vessels, fat, muscle, ligaments, and the like.

[0041] As used herein, “wound” refers to an injury to the skin and underlying tissue caused by a cut, abrasion, impact, or contact with an object or material. The term “wound” also includes damage to the skin and underlying tissue by contact with an object or material at a high temperature so that thermal damage to cells and tissue occurs. In the context of this disclosure, sustained contact with an object or material includes that experienced by a body confined to a bed, wheelchair, or other situation in which prolonged contact results in injuries to the skin and underlying tissues because of prolonged pressure on the skin. Such wounds are referred to as pressure ulcers or bedsores.

[0042] As used herein “bum” or “burns” refer to any detectable injury to skin caused by energy applied to the skin. The terms further refer to any burning, or charring of the skin, including thermal burns caused by contact with flames, hot liquids, hot surfaces, and other sources of high heat as well as chemical burns and electrical burns. “Bum” or “bums” includes first degree burns which may cause skin manifestations such as reddening, pain, and / or mild swelling. One non-limiting example of first-degree burn is a sunburn. Burn or burns further refers to second-degree burns involving the first two layers of skin. Signs of second degree burning include, among other things, deep reddening of the skin, blisters, pain, glossy appearance from leaking fluid, and possible skin loss. Burn or burns further refers to third-degree burns which penetrate the entire thickness of the skin and may destroy tissue. Signs of third degree burning include, among other things, loss of skin, dry skin, leathery skin, charred skin having a mottled appearance, and combinations thereof. In some case, skin with a third-degree burn may be painless.B. Overview of the Invention and Embodiments

[0043] While certain aspects of the present disclosure are shown and described herein, it is understood that such aspects are merely exemplary. The present disclosure is not intended to be limited to these specific embodiments and may encompass other aspects or embodiments.Therefore, specific compositional, process, and method details disclosed herein are not to be interpreted or inferred as limiting, but merely as a basis for the amended claims and as a representative basis for teaching one skilled in the art how to make and use the disclosed subject matter.

[0044] The present invention provides methods and pharmacological compositions comprising PABS and a pharmacologically acceptable excipient(s). In some embodiments, the present invention provides a composition / formulation comprising PABS and water or another pharmaceutically acceptable excipient, such as a biologically inert liquid, that can be topically applied by a suitable means as necessary to an area of the skin comprising a bum wound to treat the bum wound. It has been discovered that topical application of this formulation advantageously decreases the amount of time needed for burn wounds to heal and, in addition, it improves the overall quality of the tissue and skin after the wound has healed. Another embodiment of the invention is a formulation comprising PABS and one or more excipients in a composition that can be applied topically to an area of the skin, wherein the formulation is a dosage form including, but not limited to, a suspension, lotion, emulsion, powder, gel, ointment, cream, or paste to treat a burn wound to enhance healing. In one embodiment, the compositions disclosed herein are formulated as an emulsion, such as an oil- in-water emulsion or a water-in-oil emulsion, and the compositions can be in the form of a lotion or a cream.

[0045] PABS, which is described in U.S. Patents Nos. 9,938,171, 10,544,055, and 10,807,889, the teachings of each of which are incorporated by reference, is a molecular complex of ammonium sulfate and ammonium bisulfate. PABS is formed by mixing anhydrous liquid ammonia and sulfuric acid with water flowing through a process line to form a mixed fluid, cooling the mixed fluid by, for example, flowing the mixed fluid through a heat exchanger, and combining the cooled mixed fluid with a second portion of sulfuric acid. The product is an aqueous solution including sulfuric acid and stable, three-dimensional molecular structures composed of ammonium sulfate, ammonium bisulfate, sulfuric acid, and water. PABS can be represented by Formula I as follows:((NH4)2SO4)a(H2SO4)b(H2O)c(NH4HSO4)x. (I) wherein a is at least 1, b is at least 1, c is at least 1, and x is at least 1. Alternatively, in some examples, a is from 1 to 5 or from 1 to 3; b is from 1 to 5 or from 1 to 3; c is from 0 to 5, from 1 to 5, or from 1 to 3; and x is from 1 to 20, from 1 to 10, or from 1 to 6. Molecularclusters comprised of molecules of Formula I are referred to herein as polyammonium bi sulfates or “PABS.”

[0046] In some embodiments, PABS -containing compositions can be prepared comprising water in an amount ranging from about 49% to about 99% by weight of the total composition and an aqueous solution comprising PABS and sulfuric acid, wherein the concentration of the PABS ranges from about 0.001% to about 50% on a weight basis and wherein the concentration of the sulfuric acid ranges from about 0.5% to about 40% on a weight basis.

[0047] Essential metals are helpful in the wound healing process. Metals considered essential for normal biological functioning include, but are not limited to, manganese, magnesium, iron, cobalt, copper, zinc, and molybdenum. For example, copper plays a role in many physiological processes in humans and other animals, including wound healing. Likewise, zinc is another key metal that is essential in many metabolic and immune system- related processes. Zinc has been shown to be essential to all phases of wound healing. Copper and zinc are essential elements and are required by the body to produce collagen, elastin, tropoelastin and / or elastic fibers in the dermis, one of the layers of skin. The present invention provides formulations comprising PABS and optionally a biologically active essential metal, such as biologically active copper and / or zinc, and methods of using these formulations to stimulate faster healing and to improve the quality (e.g., less scar formation) of the healed wounds.

[0048] In some embodiments, a formulation comprising PABS, one or more metals or salts thereof, and a pharmaceutically acceptable excipient(s) can be applied to burned skin to inhibit infectious microorganisms in the affected area. Optionally the metal or salt thereof includes cobalt, iron, magnesium, manganese, molybdenum, copper (Cu), zinc (Zn), silver (Ag), bismuth (Bi), gold (Au), cerium (Ce), selenium (Se), and gallium (Ga). Optionally the metal or salt thereof includes copper (Cu), zinc (Zn), silver (Ag), bismuth (Bi), gold (Au), cerium (Ce), selenium (Se), and gallium (Ga). An advantage is a safe and effective delivery of biologically available metal ions to a bum wound to inhibit infectious microorganisms, including those in biofilms.

[0049] In some embodiments, a formulation comprising PABS, an aqueous solution of bismuth or a bismuth salt, and a pharmaceutically acceptable excipient is provided. Bismuth is known to be antibacterial in relatively high concentrations (e.g., about 12.5 mg / L to about 50 mg / L as Bi+3) and an effective biofilm disruptor in low concentrations (e.g., about 1 mg / Las Bi+3), depending on the bacterial species used in laboratory studies. In one embodiment, a wound in which a biofilm has formed can be treated with a composition comprising PABS and bismuth (or a bismuth salt), wherein infectious cells are inhibited by a high concentration of Bi ions or, if a low concentration is used, the Bi ions are not biocidal per se, but do prevent production and assembly of the biofilm matrix. A key aspect of biofilms in wounds is that they require maintenance by the resident microorganisms. Therefore, low concentrations of Bi will gradually allow the biofilm matrix to not be maintained so that it becomes structurally unstable and new biofilm is not produced. This, therefore, allows biofilm-associated bacterial cells to be unprotected by the biofilm matrix and more susceptible to inhibitory agents.

[0050] In some embodiments, a formulation comprising PABS, an aqueous solution of a low concentration of bismuth or a bismuth salt, and a pharmaceutically acceptable excipient is provided. As explained above, the present invention provides methods for preventing biofilm formation by treating a wound in need thereof with PABS and a low concentration of Bi. It has unexpectedly been discovered that an aqueous solution of PABS can be used to prepare stable solutions of Bi and Ag that can be used in preparing pharmacological compositions, such as emulsions, for treating wounds and skin maladies as well as preventing infections of fresh wounds.

[0051] In some embodiments, a formulation comprising PABS, a metal or salt thereof, where the metal optionally is Zn, Cu, or Zn and Cu, and a pharmaceutically acceptable excipient(s) can be used to treat first-degree, second-degree, and third-degree bum wounds. Optionally, the formulation is topically applied at about 4-hour intervals to the damaged skin and onto the surrounding undamaged skin. In some embodiments, the formulation is topically applied at an interval including, but not limited to, about a 1-hour interval, a 2-hour interval, a 3-hour interval, a 4-hour interval, a 5-hour interval, or a 6-hour interval. In some embodiments, the formulation is topically applied at about 4-hour intervals. In some embodiments, the formulation is topically applied once daily, twice daily, or thrice daily until the wound has healed. Typically, the application method involves coating the burn wound and at least a one-to-two-inch perimeter of healthy skin surrounding the burn wound. The formulation provides the skin and underlying tissue with a non-toxic amount of Zn, Cu, or Zn and Cu ions as a means of reducing inflammation and increasing the rate of healing as well as the quality of the skin once healed.

[0052] Some embodiments of the present invention provide a formulation comprising PABS, Bi, a second antimicrobial metal or metal salt, and a pharmaceutically acceptable ingredient to treat a burn wound covered partially or completely with a biofilm. An infected wound can be treated with the formulation so that the infectious microorganisms outside the biofilm or near the surface of the biofilm will be inhibited by the antimicrobial metal or metal salt and Bi, preventing the formation and maintenance of the extracellular biofilm matrix so that, as the biofilm loses structural integrity, the infectious cells will be without the protection provided by an intact biofilm matrix and, as a result, will be inhibited by the second metal or metal salt. An added benefit of this embodiments is that the PABS will permeate into the underlying tissue and act as a stimulant to the immune response and subsequent wound healing process.

[0053] An advantage of the compositions and methods disclosed herein is that, in addition to the wound healing processes being enhanced by the metals, the wound heals faster and with less scar formation.

[0054] Some embodiments are methods for treating a bum wound by topically applying to the wound and related areas a PABS -containing formulation that includes cerium, silver, or another metal. The burn wound can be treated with this composition to stabilize the eschar, to improve healing, and to prevent infections.

[0055] The disclosure provides pharmaceutical compositions comprising PABS on its own or PABS in combination with a metal or salt thereof, and a pharmaceutically acceptable excipient.

[0056] The pharmaceutical compositions can be prepared by any of the methods well known in the art of pharmacy and drug delivery. In general, methods of preparing the compositions include the step of bringing the active ingredient, such as PABS or PABS in combination with a metal or salt thereof, into association with a carrier containing one or more accessory ingredients. The pharmaceutical compositions are typically prepared by uniformly and intimately bringing the active ingredient into association with a liquid carrier or an emulsion base or both, and then, if necessary, shaping the product into the desired formulation (e.g., cream, lotion, or gel) having the desired viscosity. The compositions can be conveniently prepared and / or packaged in unit dosage form.

[0057] Optionally, a topical pharmaceutical composition comprising PABS on its own or in combination with a metal or salt thereof is in the dosage form of a gel, an emulsion(including lotion and cream), a foam, a suspension, a liquid, a spray, a paste, or an ointment. Optionally, a topical pharmaceutical composition of the present invention is an oil-in-water emulsion, in which an internal oil phase is dispersed in a continuous aqueous phase. Water- in-oil-in-water, water-in-oil and oil-in-water-in-oil emulsions are also contemplated. The emulsion may be a macroemulsion, a microemulsion, or a nanoemulsion.

[0058] In addition to the active ingredients as disclosed herein, the compositions of the present invention optionally includes one or more pharmaceutically acceptable excipients, especially one or more dermatologically acceptable excipients, such as liquid oils, waxes, viscosity-modifying agents, thickening agents, gelling agents, alcohols, surfactants, chelating agents, buffers, preservatives, humectants, emollients, stabilizers, diluents, dispersing agents, emulsifiers, wetting agents, stabilizers, pH adjusters, solvents or cosolvents.

[0059] In some embodiments, the compositions of the present invention are an oil-in-water emulsion comprising an oil phase and an aqueous phase, an emulsifier, and one or more of the pharmaceutically or dermatologically acceptable excipients as described above. The oily phase can be a vegetable oil, for example olive oil or arachis oil, or a mineral oil, for example liquid paraffin or mixtures of these. Suitable emulsifying agents can be naturally occurring gums, such as gum acacia or gum tragacanth; naturally occurring phospholipids, such as soy lecithin; esters or partial esters derived from fatty acids and hexitol anhydrides, such as sorbitan monooleate; and condensation products of said partial esters with ethylene oxide, such as polyoxyethylene sorbitan monooleate. Other oils and emulsifying agents suitable for use in the compositions or formulations of the present invention will be known to and used by those of skill in the art.

[0060] In some embodiments, the emulsion for the PABS or PABS in combination with a metal or salt thereof is a commercially available emulsion, wherein the PABS or PABS in combination with the metal or salt thereof is added to or mixed with the commercially available emulsion. Suitable commercially available emulsions for use in the compositions disclosed herein include, DermaBase Cream (NDC 0574-0071-16), which is an oil-in-water emulsion base that is commercially available from Padlock Laboratories, and from Macelle. Any suitable emulsion base, which is often referred to as a “DermaBase” by those in the art, can be used in combination with PABS or PABS in combination with a metal or salt thereof to form the compositions or formulations of the present invention.

[0061] Pharmaceutical compositions according to the invention can also include one or more additional active agents useful in the treatment of bum wounds. Suitable additional active agents include, for example, anti-bacterial or anti-microbial agents.

[0062] Having now described some illustrative embodiments of the invention, it should be apparent to those skilled in the art that the foregoing is merely illustrative and not limiting, having been presented by way of example only. Numerous modifications and other embodiments are within the scope of one of ordinary skill in the art and are contemplated as falling within the scope of the invention. Although many of the examples presented herein involve specific combinations of method acts or system elements, it should be understood that those acts and those elements may be combined in other ways to accomplish the same objectives.EXAMPLES1. Example 1

[0063] Example 1 illustrates the process for preparing formulations according to the present invention, some of which are used in the other examples, such as Example 2 and Example 3, infra. It will be appreciated by those of ordinary skill in the art that other formulations in accordance with the teachings of the present invention can be made using similar methods.

[0064] The Bismuth formulation and the Silver Formulation used in Example 2 were prepared as follows:Bismuth Formulation:

[0065] Bismuth nitrate pentahydrate (0.47 g) was added to a solution of PABS (24.03 g) and distilled water (56.01 g). This yielded a solution containing 0.25 Wt. % bismuth and 29.8 Wt. % PABS. A portion of the resulting 0.25 Wt. % Bismuth solution (4.12 g) was added to a Derma Base (50 g) and thoroughly mixed to provide a uniform dispersion of the bismuth nitrate / PABS in the Derma Base. The bismuth nitrate / PABS Derma Base formulation contains 0.019 Wt. % bismuth and 2.27 Wt. % PABS.Silver Formulation:

[0066] Silver Nitrate (0.273 g) was added to a solution of PABS (8.13 g) and distilled water (59.75 g). This yielded a solution containing 0.254 Wt. % silver and 11.9 Wt. % PABS.A portion of the resulting 0.254 Wt. % silver solution (5.37 g) was added to a Derma Base (50 g) and thoroughly mixed to provide a uniform dispersion of the silver nitrate / PABS in the Derma Base. The silver / DermaBase formulation contains 0.025 Wt. % silver and 1.15 Wt. % PABS.

[0067] The Copper Formulation, the Zinc Formulation, and the Copper + Zinc Formulation used in Example 3 were prepared as follows:Copper Formulation:

[0068] To prepare a 100 g sample of a copper-containing formulation, 228.99 uL of EarthTec, a 5% wt. % commercially available product, was added to 100 gm of the Vehicle formulation and thoroughly mixed. This yielded a formulation containing 134 mg Cu / kg product.Zinc Formulation:

[0069] Zinc sulfate monohydrate (24.8 g) was added to a solution of PABS (10.5 g) and distilled water (64.4) gm of water. This yielded a solution of 9.4% Wt. % Zn. A portion of the 9.4 Wt. % zinc solution (110.88 uL) was added to the Vehicle formulation and thoroughly mixed. This yielded a formulation containing 134 mg Zn / kg product.Copper + Zinc Formulation:

[0070] Aliquots of the copper and zinc formulations were added to the Vehicle formulation and thoroughly mixed. This yielded a formulation containing 134 mg Cu / kg and 134 mg Zn / kg product.Vehicle Control

[0071] It is noted that the Vehicle Control is a Perrigo DermaBase Cream (NDC 0574- 0071-16), a proprietary emulsion, amended with amended with 2.5% (by weight) PABS, a solution comprised of water (50% by weight), sulfuric acid (30% by weight), and PABS (20% by weight).2. Example 2

[0072] Example 2 illustrates the ability of exemplary formulations of the present invention comprising PABS and selected metals to reduce numbers of Pseudomonas aeruginosa ATCC27312 (hereafter referred to as PA 27312) cells and their healing effects in non-infected wounds using a deep partial thickness porcine wound model. This pilot study was carried out in a laboratory on the campus of a major medical school of a university in the Southeastern region of the United States.

[0073] The protocol used for this study was approved by the University’s Animal Care and Use Committee and all the procedures followed the federal guidelines for the care and use of laboratory animals (U.S. Department of Health and Human Services, U.S. Department of Agriculture). The study was conducted in compliance with the University’s School of Medicine Standard Operating Procedure (SOPs). The animal was monitored daily for any observable signs of pain or discomfort. To help minimize possible discomfort, analgesics were used during the entire experiment.

[0074] Experimental Animals. Swine were used for the experimental research animal since their skin is morphologically and biochemically similar to human skin. One animal was used for this study. The young female specific pathogen free pig (B. G. Looper Farm 4673 Petra Mill Road Granite Falls, NC) 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 the animal facilities (American Association for Accreditation of Laboratory Animal Care [AAALAC] accredited) with controlled temperature (19-21°C) and lighting (12h / 12h LD).

[0075] Animal Preparation, Wounding and Treatment. The animal was anesthetized with Telazol HC1 (1.4 mg / kg), Xylazine (2 mg / kg), Atropine (0.05 mg / kg) I.M. and inhalation of an isoflurane and oxygen combination. The hair on the back of the pig was clipped with standard animal clippers and the skin was prepared by washing with a non-antibiotic soap (Neutrogena®) and sterile water. The area was blotted dry with sterile gauze.

[0076] Wounding. Fifty-seven (57) deep partial thickness wounds measuring (12 mm x 12 mm x 1mm deep) were made in the paravertebral and thoracic area with a specialized electrokeratome. The wounds were separated from one another by 5-7cm of unwounded skin, thirty (30) wounds were inoculated, and the remaining wounds were kept uninoculated as described below.

[0077] Wound Inoculation. A fresh culture of PA 27312 was used for this study. The challenge inoculum suspension was prepared by scraping the overnight growth from a culture plate into 5 ml of Tryptic Soy Broth (TSB). This resulted in a suspension concentration of approximately 1010colony forming units / ml (CFU / ml) for the bacteria. Serial dilutions weremade until a concentration of 106CFU / mL (critically colonized level of bacteria associated in wound infection) was achieved. The inoculum was vortexed and 25 pL of the suspension was inoculated into thirty (30) wounds. In addition, serial dilutions of the suspension were plated onto selective media (see below) and plates were incubated aerobically overnight (16-24 hours) at 37°C, in order to quantify the exact concentration of viable organisms used for this experiment.

[0078] Treatment Regimen. Immediately after wounding and inoculation the thirty (30) wounds were randomly divided into five groups of six wounds each. The remaining twentyseven (27) non-infected wounds were divided into 3 treatments groups of 9 wounds each. All wounds were treated with 200 pL by applying the formulations with a sterile syringe.

[0079] After treatment application, all wounds were then covered with Tegaderm dressings. Afterwards, all sites were secured with surgical tape then loosely wrapped with Coban self-adhesive elastic wrap (3M, St. Paul MN). All wounds were treated once daily.

[0080] The Untreated Control wounds were only covered with polyurethane film dressing (Tegaderm; 3M, St. Paul, MN).

[0081] Microbiology Recovery Methods. Three wounds from each group were cultured on Days 2 and 7 after inoculation and treatment. A sterile cylinder was placed over the wounds and 1 mL of Scrub Solution was pipetted into it. The site was then scrubbed with a sterile Teflon spatula for 30 seconds. Serial dilutions were made from all culture samples and the extent of microbiological contamination was assessed using the Spiral Plater System (Spiral Biotech, Norwood, MA). This system deposits a 50 pL aliquot of the scrub suspension over the surface of a rotating agar plate. Pseudomonas Agar-base with CN supplement was used to isolate and enumerate cells of PA 27312. All plates were incubated aerobically for 24 hours at 37°C, after which the number of viable colonies were counted.

[0082] Histological Assessment. Three biopsies were taken from each treatment group (uninoculated wounds) on Days 2, 4 and 6 after wounding and treatment. Incisional biopsies were obtained through the center of the wound including normal adjacent skin on both sides. These specimens were placed in formalin then stained with hematoxylin and eosin (H&E). One section per block was analyzed. The specimens were evaluated blinded via light microscopy and examined for the following selected histological parameters as described in Table 1, infra.

[0083] Treatment compositions. Commercially available Silver Sulfadiazine was used as the benchmark for comparing treatment efficacies. The Ag concentration was calculated to be 0.00302 mg / mg product formulation. The Ag-containing formulation was prepared with DermaBase Cream, PABS, and a solution of silver nitrate. The final concentration of Ag in the experimental formulation was 0.00025 mg Ag / mg product formulation. The Bi-containing formulation was prepared with bismuth nitrate and had a Bi content of 0.00019 mg Bi / mg product formulation.

[0084] Results - Efficacy against P. aeruginosa ATCC27312, As illustrated in Figure 1, the size of the bacterial population in the wounds increased in all treatments and the untreated control after a two-day incubation. The smallest increase in numbers of viable bacteria occurred in wounds treated with the DermaBase Cream formulation amended with PABS and Bi. Numbers of viable bacteria decreased in all wounds from Day 2 until Day 7. As illustrated in Figure 2, a comparison of percent changes in bacterial counts from Day 2 to Day 7 can be made by subtracting the log-transformed counts and calculating the percent reduction in counts. The untreated control had an increase in numbers of PA27312 approximately the same as that in the DermaBase Cream carrier, e.g., about 8 to about 10% reductions.Numbers of viable PA27312 decreased in wounds treated with formulations comprising Ag or Bi as the active ingredient. The percent logic reductions in numbers of PA27312 approximately 29% for the Ag treatment and approximately 33% for the Bi treatment. As is well known in the field of microbiology, the percent decreased are within an expected error range, these values are not significantly different and are similar to the SSD treatment counts. However, if the concentrations of the metals are compared on a mg metal per mg formulation, DermaBase Cream amended with PABS and Ag contained about 1 / 12ththe amount of Ag in silver sulfadiazine. Likewise, a comparison of the concentration of Bi in DermaBase Cream amended with PABS and Bi with the concentration of Ag in the silver sulfadiazine formulation demonstrates Bi was about 1 / 16ththe concentration of silver on a mg metal to mg formulation basis. These comparisons demonstrate how much more effective the PABS-containing formulation were versus the widely used silver sulfadiazine product.

[0085] As is well known, the dermal reconstitution begins in about 3 to 4 days of injury with the hallmark of granulation tissue formation, which include new blood vessel formation (angiogenesis), and the accumulation of fibroblasts and collagen extracellular matrices. The granulation tissue formation measures the percent of wound bed filled with newly formed granulation tissue. On Day 4, Wound Healing Agent - 2021-BC1 had the highest granulationtissue score compared to Vehicle Control - DermaBase Cream. At Day 6 the Wound Healing Agent - 2021-BC1 scored 5 while the other 2 groups had scores of 4.5

[0086] Figure 3 illustrates changes in skin wounds treated with DermaBase Cream amended with PABS versus DermaBase Cream and no treatment. By Day 6, there was an obvious difference in the healing process in the wound treated with DermaBase Cream. Key observations noted in the final project report were: “Wounds treated with DermaBase Cream amended with PABS appeared to re-epithelialize faster with more granulation tissue and angiogenesis than those untreated or treated with DermaBase Cream.”3. Example 3

[0087] Example 3 illustrates the ability of 3 PABS + metal formulations of the present invention, PABS+Ag, PABS+Bi, and PABS+Ag and Bi, to enhance the healing and scar using a porcine 3rddegree wound model.MATERIALS AND METHODSExperimental Animals

[0088] A porcine model was used for our experimental research due to the morphological similarities between swine skin and human skin see, e.g., Sullivan TP, Eaglstein WH, Davis SC, and Mertz PM). The pig as a model for human wound healing. Wound Repair and Regeneration 9, 2, 2001, 66-76, the teachings of which are incorporated herein by reference). Two (2) female specific pathogen -free pigs (B. G. Looper Farm 4673 Petra Mill Road Granite Falls, NC 28630) weighing 40-45 kg were kept in house for at least 5 days prior to initiating the experiment to allow the animals to acclimatize. The animals were fed a basal diet ad libitum and were housed individually in our animal facilities (meeting American Association for Accreditation of Laboratory Animal Care [AAALAC] accredited) with controlled temperature (19-21°C) and lighting (12h / 12h LD).Wounding Technique

[0089] Thirty (30) third degree burn wounds were made on the paravertebral and thoracic area. Bums were created by using a branding iron (L & H Manufacturing Company Mandan, North Dakota 58554) with a heat controller that was set to 300° C. The iron was held at a vertical position on the skin for 15 seconds, with pressure supplied by gravity, to make a bum wound of 27 mm diameter and with a depth of approximately 3 mm (to subcutaneous tissue). The wounds were separated from one another by 5 - 7 cm of unwounded skin. The wounds were randomly assigned to five treatment groups with 6 wounds per treatment as seen in Figure 4. One animal had three wounds from each treatment group assessed on Days 7 & 14 and the other animal was evaluated on Days 21 and 45,Treatment Regimen

[0090] Immediately after wounding, all wounds except for Untreated Control wounds were treated with 1000 pL of their assigned test article to cover the wounded area and surrounding unwounded skin. After treatment application, all treatment groups including Untreated Control wounds were then covered with polyurethane film dressing (Tegaderm; 3M, St. Paul, MN)

[0091] Test articles, which were prepared as described in Example 1, were as follows:• 134 mg Cu / kg formulation;• 134 mg Zn / kg formulation;• 134 mg Cu / kg + 134 mg Zn / kg formulation; and• Vehicle Control = Perrigo DermaBase Cream (NDC 0574-0071-16) - Composition is proprietary — amended with 2.5% (by weight) PABS, a solution comprised of water (50% by weight), sulfuric acid (30% by weight), and PABS (20% by weight).

[0092] All wounds were treated twice daily from Day 0 to 4 and on Day 7 and 8 (weekdays). On Days 5, 6 and 9 - 13 wounds were treated only once daily. Starting on Day 9 when all wounds were just treated once, instead of being covered with Tegaderm, all wounds were covered with non-adherent gauze. All dressings were secured in place with tape and covered with Coban wrap (3M, St. Paul MN).Observations and Analysis

[0093] Wounds from each group were photographed and the area of wound was traced to measure wound contraction. The wound circumferences were traced by digital imaging with ImageJ, an open-source image processing program designed for scientific multidimensional images developed at the National Institutes of Health and the Laboratory for Optical and Computational Instrumentation, and compared to Day 0 to determine the degree of wound contraction.

[0094] As illustrated in Figures 5-10, some key findings from the experiment are reported here. Treated and untreated wounds increased in size during Days 0 and 1. After Day 1, the treated wounds had almost no increase in wound size, with the Vehicle Control wounds having a greater increase in size than those wounds treated with the Vehicle Control + Cuand / or Zn. The wounds were treated only once during Days 5 and 6. The wounds sizes started to increase after 2 days of treatment once per day; it appears resuming the twice per day treatments slowed the increases in wounds sizes. Subsequent once a day treatment showed increased healing (as indicated by smaller wound sizes) in the treated wounds on days 10-13. Overall, the Vehicle + Cu formulation had the fastest healing rate, and the wounds were -50% smaller verses other treatments by Day 30. Eventually, all wounds were in the same size range by Day 45, but the wounds treated with the Vehicle + Cu formulation had the smallest wound area.4. Example 4

[0095] Example 4 illustrates the benefits of the PABS formulations of the present invention as a topical wound therapeutic. The use of PABS clusters has advantages for prolonged field care applications due to their stability at a wide range of temperatures, small volumes of use, and effectiveness against common burn wound pathogens found in the field. These characteristics are ideal for austere environments that military personnel face, and further support an expeditious mission ready response when needed. In this example, PABS, individually or in combination with metals (AgNCE, Ag2SO4, and Ce(NO3)3), were characterized for their ability to influence both prokaryotic (bacteria) and eukaryotic (yeast and mammalian skin cells) cells. It was hypothesized that PABS complexed with antimicrobial metals may be an effective broad spectrum antimicrobial agent while concurrently not causing cytotoxicity to mammalian host cells.MethodsAgar Well Diffusion Assay

[0096] The agar well diffusion assay utilized isolates of Klebsiella pneumoniae (CDC 0347), Pseudomonas aeruginosa (ATCC 10145), Staphylococcus aureus (ATCC 4330), Candida auris (CDC 0381), and Aspergillus fumigatus, comprising 3 of the 6 ESKAPE pathogens. For each of these species, a 10 mL starter culture was created and shaken overnight for 18-24 hours at 37° C with species-specific broth. The mixture was diluted with 150 mL of fresh broth the next morning and was shaken for another 5-6 hours depending on turbidity. The bacteria were plated on species-specific agar. A 10 mm biopsy punch was used to create three wells in the agar. Treatments included a positive (Povidone iodine) and negative (diELO) control, PABS alone, AgNCL-loaded PABS, Ag2SO4-loaded PABS, andCe(NO3)3-loaded PABS. A volume of 100 pL of each treatment was added to the wells.Plates were incubated for 18-24 hours. Digital images were taken of the plates and uploaded to ImageJ Software. For each well, the zone of inhibition (ZOI) was measured, and the radius was calculated. Data were analyzed using a two-way ANOVA with p<0.05 considered significant using GraphPad Prism software.Mammalian Cell Culture and Treatment

[0097] Normal human epidermal keratinocytes (NHEK) and normal human dermal fibroblasts (NHDF) were cultured and passaged. Cells were seeded in 96-well flat clear bottom black polystyrene tissue culture-treated microplates at an average cell density of 2.5 xlO4cells / well or 8.0 x 103cells / well, respectively. NHEK and NHDF were allowed to adhere, rinsed, then treated for 1 h with media containing diluted test articles in equivalent volumes of a distilled water (“dH2O”) vehicle control. Povidone Iodine was used as a positive control.Cell Viability Assay

[0098] NHEK and NHDF viability was measured using the MTT [3-(4,5-dimethylthiazol- 2-yl)-2,5-diphenyltetrazolium bromide] assay. Following treatment with test articles, cells were incubated at 37° C for 3 h in 50 pL MTT reagent + 50 pL serum-free media. The reagent solution was then aspirated and replaced with 150 pL MTT solvent and shaken on an orbital shaker, protected from light, at room temperature for 15 minutes. The absorbance of each well was read at 595 nm in a plate reader.ResultsAgar Well Diffusion Assay

[0099] As produced for this study, an aqueous solution of PABS clusters has an apparent pH of -0.55. The low pH of the aqueous solution of PABS clusters was inhibitory to the test organisms. To measure any inhibition of the test organisms by PABS alone or PABS with a metal, dilutions of the PABS solution were made and tested. This allowed measuring efficacy of test solutions of PABS alone and PABS + metals; inhibitory effects beyond that caused by acidic pHs is referred to as the “true effect.” After the pH effect was eliminated by dilution, a dilution range was empirically determined that is termed below as the “true effect”. This was the dilution that resulted in a difference between the PABS alone and PABS + metalformulation. Dilutions of 1 :2, 1 :5, 1 : 10, 1 :25, 1 :50, and 1 : 100 of each test article were performed for each species to understand the trend of pH effect and true effect.

[0100] For K. pneumoniae, PABS AgNCh formulation had significantly larger zones of inhibition compared to PABS Ag2SO4 formulation and PABS Ce(NO3)3 formulation and PABS alone (p=0.0318, pO.OOOl, pO.OOOl).

[0101] P. aeruginosa showed PABS AgNCh formulation had significantly larger zones of inhibition compared to PABS Ag2SO4 formulation, Ce(NO3)3-loaded PABS, and PABS alone (p=0.0015, pO.OOOl, and pO.OOOl).

[0102] For S. aureus, PABS AgNCh formulation and PABS Ag2SO4 formulation ZOIs were significantly larger than PABS alone (pO.OOOl, pO.OOOl and pO.OOOl, pO.OOOl).

[0103] For A. fumigatus, there was not a significant difference between PABS, and metal loaded PABS, nor did any metal with PABS out-perform PABS alone.

[0104] C. auris show two effective dilution ranges. At 1 :2 dilution, PABS AgNCh formulation produced significantly larger zones of inhibition compared to PABS Ag2SO4 formulation (pO.OOOl) and PABS Ce(NO3)3 formulation (pO.OOOl). PABS Ag2SO4 formulation and PABS Ce(NO3)3 formulation did not produce significantly different ZOIs from the PABS alone. At the 1 :5 dilution, AgNOs (p<0.0001) and PABS Ag2SO4 formulation (pO.OOOl) created significantly greater ZOIs than PABS alone. Ce(NO3)3-loaded PABS did not create a ZOI significantly greater than PABS alone.Cell Viability Assay

[0105] NHEK and NHDF treated with AgNOs-loaded PABS and Ag2SO4-loaded PABS were nonviable at a 1 : 100 dilution (pO.OOOl, pO.OOOl and pO.OOOl, pO.OOOl) whereas NHEK and NHDF treated with PABS and Ce(NO3)3 + PABS had similar viability to untreated cells and cells treated with a dH2O vehicle control at the same dilution (pO.9832, pO.8155 and pO.9444, pO.1865). NHEK treated with AgNCE-loaded PABS and Ag2SO4- loaded PABS were less viable than cells treated with a povidone iodine positive control at 1 : 100 and 1 :500 dilutions. The inherent acidity of PABS produced a pH effect in NHEK and NHDF similar to that seen in the agar well diffusion assay at dilutions of 1 : 10 and 1 :25 for NHEK and 1 : 10, 1 :25 and 1 :50 for NHDF treated with PABS and Ce(NO3)3-loaded PABS. Following these dilutions, NHEK and NHDF had similar viability to untreated cells and cells treated with a dH2O vehicle control, indicating a loss of the pH effect.Conclusions

[0106] Ag2SO4-loaded PABS and AgNCE-loaded PABS were significantly more effective than Ce(NO3)3-loaded PABS at preventing the growth of bacteria and yeast including K. pneumoniae, P. aeruginosa, S. aureus, and C. auris.

[0107] Ce(NO3)3-loaded PABS proved to be minimally cytotoxic compared to its silver- loaded PABS counterparts. These findings demonstrate that compositions of the present invention can be used to prevent pathogen proliferation while remaining non-cytotoxic to dermal and epidermal skin cells. Ongoing studies continue to investigate bacterial growth inhibition and mammalian cell cytotoxicity with clinically equivalent concentrations of cerium in Ce(NO3)3-loaded PABS as it will be useful to understand the similarities and differences of Cerium Sulfadiazine and Silver Sulfadiazine as compared to Ce(NO3)3-loaded PABS and silver-loaded PABS, respectfully. These findings will further highlight the effectiveness of metal loaded PABS versus commercially available topicals for wound healing.

[0108] Metal-loaded PABS and PABS alone are highly effective antimicrobial agent useful for fighting the ESKAPE pathogens commonly faced in combat-related burn wounds or other surgical infections. By utilizing this treatment in the field, a patient could be evacuated already having received infection prevention, which is beneficial not only to the patient’s health, but could ultimately lead to fewer days unfit for duty.5. Example 5

[0109] As described herein, a pharmacologically active composition comprising PABS, Cu, Zn, and inert ingredients can be prepared for topical application to treat bum wounds. In one embodiment, the concentrations of Cu and / or Zn in the formulation is preferably in the range of 0.0001 to about 0.5% on a weight basis for each metal. Advantageously, in this embodiment, in addition to the wound healing processes being enhanced by the metals, PABS permeating into the wound and underlying tissues will result in the formation of ammonium sulfate which has an analgesic effect, so the amount of pain experienced by a burn victim is decreased. Furthermore, as described herein, this embodiment can be used to treat bum wounds so that the wound heals faster and with less scar formation.

[0110] Having now described some illustrative embodiments of the invention, it should be apparent to those skilled in the art that the foregoing is merely illustrative and not limiting,having been presented by way of example only. Numerous modifications and other embodiments are within the scope of one of ordinary skill in the art and are contemplated as falling within the scope of the invention. Although many of the examples presented herein involve specific combinations of method acts or system elements, it should be understood that those acts and those elements may be combined in other ways to accomplish the same objectives.[OHl] The scope of the present disclosure includes any feature or combination of features disclosed herein (either explicitly or implicitly), or any generalization thereof, whether or not it mitigates any or all the problems addressed herein. Accordingly, new claims may be formulated during prosecution of this application (or an application claiming priority thereto) to any such combination of features. With reference to the appended claims, features from dependent claims may be combined with those of the independent claims and features from respective independent claims may be combined in any appropriate manner and not merely in the specific combinations enumerated in the appended claims.

[0112] Those skilled in the art should appreciate that the parameters and configurations described herein are exemplary embodiments of the invention and that modifications may be made without departing from the spirit and scope of the present disclosure. Those skilled in the art should recognize that actual parameters and treatment programs will depend on the specific application in which the systems and techniques of the invention are used. Those skilled in the art should also recognize or be able to ascertain, using no more than routine experimentation, equivalents to the specific embodiments of the invention. Therefore, it is to be understood that the embodiments described herein are presented by way of example only and that, within the scope of the appended claims and equivalents thereto; the invention may be practiced otherwise than as specifically described.

[0113] Moreover, it should also be appreciated that the invention is directed to each feature, system, subsystem, or technique described herein and any combination of two or more features, systems, subsystems, or techniques described herein and any combination of two or more features, systems, subsystems, and / or methods, if such features, systems, subsystems, and techniques are not mutually inconsistent, is considered to be within the scope of the invention as embodied in the claims. Further, acts, elements, and features discussed only in connection with one embodiment are not intended to be excluded from a similar role in other embodiments.

[0114] The above disclosure is intended to be illustrative and not exhaustive. This description will suggest many variations and alternatives to one of ordinary skill in this art. All these alternatives and variations are intended to be included within the scope of the claims where the term “comprising” means “including, but not limited to”. Those familiar with the art may recognize other equivalents to the specific embodiments described herein which equivalents are also intended to be encompassed by the claims.

[0115] All patents, publications, and abstracts cited above are incorporated herein by reference in their entireties. Various embodiments of the invention have been described in fulfillment of the various objectives of the invention. It should be recognized that these embodiments are merely illustrative of the principles of the present invention. Numerous modifications and adaptions thereof will be readily apparent to those skilled in the art without departing from the spirit and scope of the present invention as defined in the following claims.

Claims

WHAT IS CLAIMED IS1. A method of treating an area of skin having a wound, the method comprising applying to the area of skin an effective amount of a composition comprising polyammonium bisulfate (“PABS”) and a pharmaceutically acceptable carrier or excipient to form a treated area of skin.

2. The method of claim 1, wherein the wound is a bum wound.

3. The method of claim 1, wherein the composition further comprises at least one metal or salt thereof.

4. The method of claim 3, wherein the metal or salt thereof comprises bismuth, cerium, cobalt, copper, iron, magnesium, manganese, molybdenum, zinc, silver, gold, gallium, or selenium.

5. The method of claim 3, wherein the metal or salt thereof comprises bismuth, cerium, copper, zinc, silver, gold, gallium, or selenium.

6. The method of claim 3, wherein the metal or salt thereof is present in the composition in an amount from about 1 x 10'7to about 5 % by weight based on the weight of the composition.

7. The method of claim 3, wherein the composition comprises the metal or salt thereof, and wherein applying the composition increases the amount of the metal in the treated area of skin.

8. The method of claim 7, wherein a. the metal comprises copper and the composition comprises the copper in a concentration of from about 1 x 10'4gm / kg to about 1 gm / kg, b. the metal comprises zinc and the composition comprises the zinc in a concentration of from about 1.5 x 10'4gm / kg to about 1 gm / kg, or c. the metal comprises selenium and the composition comprises the selenium in a concentration of about 1.4 x 10'4to about 1 gm / kg.

9. The method of claim 7, wherein a. the metal comprises copper and applying the composition increases a plasma concentration of copper from less than 12 pM to greater than 20 pM, b. the metal comprises zinc and applying the composition increases a plasma concentration of zinc from less than 11 pM to greater than 18 pM, or c. the metal comprises selenium and applying the composition increases a plasma concentration of selenium from less than 0.75 pM to greater than 1.8 pM.

10. The method of claim 1, wherein the composition comprises cerium in a concentration of from about 1 % to about 3 % by weight based on the weight of the composition.

11. The method of claim 10, wherein the composition further comprises silver in a concentration of from about 0.1 % to about 2.5% by weight based on the weight of the composition or bismuth in a concentration of from about 0.01% to about 2.5% by weight based on the weight of the composition.

12. The method of claim 10, wherein the burn wound comprises eschar.

13. The method of any preceding claim, wherein the composition comprises the PABS in a concentration of from about 0.010 % to about 50 % by weight based on the weight of the composition.

14. The method of any preceding claim, wherein the composition is a liquid, suspension, lotion, emulsion, powder, gel, ointment, cream, or paste.

15. The method of any preceding claim, wherein applying the composition comprises topically applying the composition to the area of skin and to skin and / or tissue surrounding the area of skin.

16. The method of any preceding claim, wherein the effective amount comprises from about 0.1 mg to about 250 mg, or from about 50 mg to about 220 mg, or from about 100 mg to about 200 mg of the composition per cm2of the surface area to which it is applied.

17. The method of any preceding claim, wherein the step of applying the composition is repeated one or more times.

18. The method of any preceding claim, wherein the treating comprises enhances healing quality or rate, reducing inflammation, or reducing or preventing infection.

19. A topical medicament comprising a therapeutically effective amount of PABS in combination with at least one pharmaceutically acceptable excipient, filler, carrier or diluent, wherein the medicament has a PABS content from about 0.01 % to about 30 % by weight based on the weight of the composition.

20. The medicament of claim 19, further comprising at least one metal or salt thereof.

21. The medicament of claim 20, wherein the metal or salt thereof comprises bismuth, cerium, cobalt, copper, iron, magnesium, manganese, molybdenum, zinc, silver, gold, gallium, or selenium.

22. The medicament of claim 20, wherein the metal or salt thereof comprises bismuth, cerium, copper, zinc, silver, gold, gallium, or selenium.

23. The medicament of claim 20, wherein the metal or salt thereof is present in the composition in an amount from about 1 x 10'7to about 5 % by weight based on the weight of the composition.

24. The medicament of claim 20, wherein a. the metal or salt thereof comprises copper and the composition comprises the copper in a concentration of from about 1 x 10'4gm / kg to about 1 gm / kg, b. the metal or salt thereof comprises zinc and the composition comprises the zinc in a concentration of from about 1.5 x 10'4gm / kg to about 1 gm / kg, or c. the metal or salt thereof comprises selenium and the composition comprises the selenium in a concentration of about 1.4 x 10'4to about 1 gm / kg.

25. The medicament of claim 20, wherein the metal or salt thereof comprises cerium in a concentration of from about 1 % to about 3 % by weight based on the weight of the composition.

26. The medicament of claim 25, wherein the metal or salt thereof comprises silver in a concentration of from about 0.1 % to about 2.5% by weight based on the weight ofthe composition, or wherein the metal or salt thereof comprises bismuth in a concentration of from about 0.01% to about 2.5% by weight based on the weight of the composition.

27. The medicament of claim 19, wherein the medicament is a liquid, suspension, ointment, salve, lotion, paste, cream, gel, foam, emulsion, balm, or pomade.

28. The medicament of claim 19, wherein the medicament is an oil in water emulsion or a water in oil emulsion.

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