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1. (WO2019005895) COMPLEXES DE CHITOSANE-MÉTAL FONGIQUES ET PROCÉDÉS POUR LES PRÉPARER ET LES UTILISER
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FUNGAL .CHITOSA - ETAL COMPLEXES AND

METHODS OF MAKING AND USES THEREOF

STATEMENT OF PRIORITY

This application claims the benefit, under 35 U.S.C, § 1 19 (c), of U.S. Provisional Application No, 62/527,440 filed on June 30, 2017, the entire contents of which is incorporated by reference herein.

FIELD OF THE INVENTION

The invention relates to chelated, fungal chitosan-metal compositions and methods for making the compositions as well as methods of using the same.

BACKGROUND OF THE INVENTIO

Wound healing is a complex bioiogical process that involves the restoration of the skin matrix due in response to disease or injury. This process can be complicated by

infection o f the wound/lesion site which can significantly inhibit healing time and also can affect the quality and strength of tissue that is regenerated. New methods and compositions are needed to improve wound healing and related physiological processes,

SUMMARY OF THE INVENTION

A first aspect of the invention provides a calionic solution comprising a chelated rungal chitosan-metal complex.

A second aspect of the invention provides a pharmaceutical composition comprising the cationic solution of the in venti on and a pharmaceutically acceptable carrier.

A third aspect of the invention provides a method of making a cationic fungal chitosan-metal chelate solution, comprising dispersing fungal chitosan in distilled water under agitation to produce a fungal chitosan solution; adding a citric acid solution to the fungal chitosan solution to produce a hydrolyzed fungal chitosan solution; and adding an aqueous sil ver-oxide solution to the hydrolyzed fungal chitosan solution, thereby producing the cationic fungal chitosan-metal chelate solution,

A fourth aspect of the invention provides a method of stimulating the production of dermal fibroblasts and suppressing the production of type I. and ill procollagen by

hypertropic scar fibroblasts (HSF) and keloids fibroblasts (KF) in a wound of a subject in need thereof, the method comprising; contacting the wound of the subject with a

therapeutic ally effective amount of a cationic solution or a pharmaceutical composition of the invention, thereby stimulating the production of dermal- fibroblasts and suppressing the production of type Ϊ and ΠΙ procollagen in the wound of the subject.

A fifth aspect of the. invention provides a method of reducing swelling in a subject in need thereof the method comprising: contacting the swelling in the subject with a

therapeutically effective amount of a cationic solution or a pharmaceutical composition of the invention, thereby reducing swelling in the subject,

A sixth aspect of the invention provides a method of enhancing healing of a wound in. a subject in need thereof, the method comprising: contacting a wound in the subject with a therapeutically efiective amount of a cationic solution or a pharmaceutical composition of the invention, thereby enhancing healing of the wound in the subject.

A seventh aspect of the invention provides method of keeping a wound substantially free of bacteria and/or fungi in a subject in need thereof or eliminating and/or reducing the presence bacteria or fungi in a wound of a subject in need thereof, the method comprising: contacting the wound of the subject with a therapeutically effective amount of a cationic solution or a pharmaceutical composition of the invention, thereby keeping the wound of the subject substanti lly free of bacteria and/or fungi or eliminating and/or reducing the presence bacteria or fungi in a wound of a subject,

A eighth aspect of the invention provides a method of increasing the rate of recovery of damaged neural tissue in a subj ect in need thereof, the method comprising: contacting the damaged neural tissue in the subj ect with a therapeutically effective amount of a -cationic solution or a pharmaceutical composition of the invention, thereby increasing the rate of recover of damaged neural tissue in a subject.

These and other aspects of the invention axe se forth in more detail in the description of the invention below.

DETAILED/DESCRIPTION

The present invention now will be described hereinafter with reference to the accompanying drawings and examples, in which embodiments of the invention are shown. This description Is not intended to be a detailed catalog of all the different ways in which the invention may be implemented, or all the features that may be added to the instant invention. For example, features illustrated with respect to one embodiment may be incorporated into other embodiments, and features illustrated with respect to a particular embodiment may be deleted from that embodiment. Thus, the invention contemplates that in some embodiments

of the invention, any feature or combination of features set forth herein can be excluded or omitted. In addition, numerous variations and additions to the various embodiments suggested herein will be apparent to those skilled in the art in light of the instant disclosure, which do not depart from the instant invention. Hence, the following descriptions are intended, to illustrate some particular embodiments of the invention, and not to exhaustively specify all permutations, combinations and variations thereof

Unless otherwise, defined, all technical and scientific terms used herein have the same meaning as commonl understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein, is for the purpose of describin particular embodiments only and is not intended to be limiting of the invention.

All publications, patent applications, patents and. other references cited herein are incorporated by reference in their entireties for the teachings relevant to the sentence and/or paragraph in which the reference is presented.

Unless the context indicates otherwise, it is specifically intended that the various features' of the invention described herein, can be used in any combination. Moreover, the presen invention also contemplates that in some embodiments of the invention, any .feature or combination of features set forth herein can be excluded or omitted. To illustrate, if the specification states that a composition comprises components A, B and C, it is specificall intended that any of A, B or C, or a combination thereof, ca be omitted and disclaimed singularly or in any combination.

As used in the description of the invention and the appended claims, the singular forms "a," "a " and "the" are intended to include. the plural forms as well, unless the context clearly indicates otherwise.

Also as used herein, "and/pf refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of

combinations when interpreted in the alternative ("or").

The term "about," as used herein when referring to a measurable value such as a amount or concentration and the like, is meant to encompass variations of ±· 10%, ± 5%, ± 1%, ± 0.5%, or even * 0.1% of the specified value as well as the specified value. For example, "about X" where X is the measurable value, is meant to include X as well as variations of ± 10%, ± 5%, ± 1%, ± 0.5%, or even ± 0.1% of X. A range provided herein for a.measureable value may include any other range and/or individual value therein.

As used herein, phrases such as "between X and Y" and "between about X and. Y" should be interpreted to include X and Y. As used herein, phrases such a "between about X

and Y " mean "between about X and about Y" and phrases such as "from about X to Y" mean "from about X to about Y."

The term "comprise,"' "comprises" and "comprising" as used herein, specify the presence of the stated features, integers, steps, operations, elements, and/or components, but do not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and/or groups thereof.

As used herein, the transitional phrase "consisting essentially of* means that the scope of a claim is to be interpreted to encompass the specified materials or steps recited in the claim and those that do not materially affect the basic and novel characteristic (s) of the claimed invention. Thus, the term "consisting essentially of when used in a claim of this invention is not intended to be interpreted to be equivalent to '-comprising."

As used herein, the terms "increase," "increasing,5' "increased," "enhance,"

"enhanced," "enhancing," and "enhancement" (and grammatical variations thereof) describe an elevation of at least about 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 110%, 120%, 130%, 140%, 150%, 200%, 250%, 300%, 350%, 400%, 500% or more as compared to a control (e.g., not treated with the chelated fungal chitosan-silver complex solution of the invention or a composition thereof).

As used herein, the terms "reduce," "reduced," "reducing," "reduction," "diminish," and "decrease" (and grammatical variations thereof), describe, for example, a decrease of at least about 5%, 10%, 15%, 20%, 25%, 35%, 50%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% as compared to a control. In particular embodiments, the reduction can result in no or essentially no- (i.e., an insignificant amount, e.g., less than about 10% or even 5%) detectable activity or amount.

The terms "contact" or "contacting" (or grammatical variations thereof) as used herein to refer to delivering to a surface, skin or fabric or delivering to or administering to a wound, a swelling, or damaged neural tissue a cationic solution of the invention comprising a chelated fungal chitosan-metal complex or a composition comprising the cationic solution of the invention using any method by which the solution or composition may be delivered to or placed in proximity to a surface, fabric, skin, a wound, a. swelling, or damaged neural tissue.

By the terms "treat," "treating," or "treatment of 5 (or grammatically equivalent terms) it is meant that the severity of the subject's condition is reduced or at least partially improved or ameliorated and/or that some alleviation, mitigation or decrease. in at least one clinical symptom is achieved and/or there is a delay in the progression of the condition.

As used herein, the terms "prevent," "prevents," or "prevention" and "inhibit," "inhibits," or "inhibition" (and grammatical equivalents thereof) are not meant to imply complete abolition of condition and encompasses any type of prophylactic treatment that reduces the incidence of the condition, delays the onset of the condition, and/or reduces the symptoms associated with the condition after onset.

An "effective ' "prophylactieal!y effective," or "therapeutically effective" amount as used herein is an amount that is sufficient to provide some improvement or benefit to the subject. Alternatively stated, an "effective," "prophylacticaily effective,'5 or "therapeutically effective" amount is an amount thai will provide some delay, alleviation, mitigation, or decrease in at least one clinical symptom in the subject Those skilled in the art will appreciate, that the effects need not be complete or curative, a long as some benefit is provided to the subject. An "effective amount" as used herein also refers to an amount that may be applied to a surface, skin, or textile to provide an antimicrobial, effect

A "subject" of the invention includes any animal that has or is suspected of having a wound, a swelling, and/or damaged neural tissue or that may have an infection or is suspected of having an infection. Such a subject is generally a mammalian subject (e.g., a laboratory animal such as a rat, mouse, guinea pig, rabbit, primate, etc), a farm or commercial animal (e.g., cattle, pig, horse, goat, donkey, sheep, etc.), or a domestic animal (e.g., cat, dog, ferret, gerbil, hamster etc.). In some embodiments, the subject may be a primate subject, a non-human primate -subject (e.g., a chimpanzee, baboon, monkey, gorilla, etc.) or a human. In some embodiments, a subject may be a reptile, an avian, an amphibian, or a fish. In some embodiments, an avian may be a chicken, a duck, a turkey, a goose, a quail, a pheasant, a parakeet, a parrot, a macaw, a cockatoo, or a canary.

A "subject in need" of the methods .of the invention can be a subject known to have a wound, swelling, damaged neural tissue and/or a bacterial or fungal infection, suspected of having wound, swelling, damaged neural tissue and/or a bacterial or fungal infection, or having an increased risk of developing the same.

The present inventor has discovered that, the chitosan developed from fungal chi tin-has substantially different molecular characteristics from chitosan developed, from crustacean ehitki that results in the fungal chitosan having .an enhanced bioactivity. For example, the average particle size of the fungal chitosan is smaller enabling the fungal, chitosan to more-effectively chelate metal oxide ions. The fungal chitosan also may contain more receptor sites to which metal oxide ions can bind and binds the metal oxide more tightly.

Consequently, the compositions of the present invention comprising a fungal chitosan-metal

complex chelate as described herein can have enhanced bioactivity such as increased antimicrobial activity and increased wound healing activity.

Accordingly , a first aspect of the invention provides a cationic solution comprising a chelated fungal chitosan-metal complex. In some embodiments, the metal ma be a metal from silver oxide, copper oxide, gold oxide, or platinum oxide. In some embodiments, the metal may be silver from silver oxide.

As used herein, a "cationic solution" means a solution comprising a cationic fungal chitosan metal complex,

In some embodiments, fungal chitosan may he in a range of about 0.1% to about 30% by weight of the solution (e.g., about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.5, 2, 2.5, 3 , 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7,5, 8, 8,5, 9, 9.5, 10, 10.5, 1 1, 1 1.5, 12, 12.5, 13, 13.5, 14, 14.5, 15, 15.5, 16, 16.5, 17, 17.5, I S, 18,5, 19, 19.5, 20, 20.5, 21, 21.5, 22, 22,5, 23, 23.5, 24, 24.5, 25, 25.5, 26, 26.5, 27, 27.5, 28, 28.5, 29, 29.5, or 30% by weight of the solution or any range or value therein (e.g., about 0.1 % to 1 %, about 0J % to about 5%, about 0.1 % to about 10%, about 0.1 % to about 1 %, about 0.1 % to about 20%, about 0.1 % to about 25%, about 1 % to. about 5%, about 1% to abou 10%, about 1% to about 15%, about 1% to about 20%, about 1 % to about 25%, about 1% to about 30%, about 5% to about 10%, about 5% to about 15%, about 5% to about 25%, about 5% to about. 30%, about 10% to about 15%, about 10% to about. 20%, about 10% to about 25%, about 10% to about 30%, about 15% to about 20%, about 15% to about 25%, or about 1.5% to about 30% by weight of the solution and. the like, or any range or value therein)).

in some embodiments, a chelated fungal -chitosan-metal complex may comprise fimgal chitosan chelated with an amount of metal of a. metal oxide in range of about 1 ppm to about 10,000 ppm (e.g., about 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 65000, 7000, 7500, 8000, 8500, 9000, 9500, or 10,000 ppm metal, or any range or value therein (e.g., about 10 ppm to about 10,000 ppm, about 50 ppm to about 10.000 ppm, about 100 ppm to about 10,00.0 ppm, about 500 ppm to about 10,000 ppm, about 1000 ppm to about 10,000 ppm, about 1500 ppm to about 1 ,000 ppm, about 2000 ppm to about .10,000 ppm, about 2500 ppm to about 10,000 ppm, about 3000 ppm to about 10,000 ppm, about 3500 ppm to about 10,000 ppm, about 4000 ppm to about 10,000 ppm, about 4500 ppm to about 1.0,000 ppm, about 5000 ppm to about 10,000 ppm, about 5500 ppm to about 10,000 ppm, about 6000 ppm to about 10,000 ppm, about 6500 ppm to about 10,000 ppm, about 7000 ppm to about 1 ,000 ppm, about 7500 ppm to about 10,000 ppm, about

8000 ppm to about 10,000 ppm, about 8500 ppm to about 10,000 ppm, about 9000 ppm to about 10,000 ppm, about 9500 ppm to about 10,0(50 ppm, about 100 ppm to about 500 ppm, about 100 ρρκι to about 00 ppm, about 100 ppm to about 2000 ppm, about 500 ppm to about 1000 ppm, about 500 ppm to about 2000 ppm, about 500 ppm to about 3000 ppm, about 500 ppm to about 4000 ppm, about 1000 ppm to about 2000 ppm, about 1000 ppm to about 3000 ppm. about 1000 ppm to about 4000 ppm, about 1000 ppm to about 5000 ppm, about 1000 ppm to about 6000 ppm, about 1000 ppm to about 7000 ppm, or about 2000 ppm to about 4000 ppm, or the like, or any range or value therein)). In some embodiments, the metal of the metal oxide may be silver, copper, gold, or platinum. In some embodiments, the metal of the metal oxide is silver. Thus, in some embodiments, a chelated fungal chitosan-meta! complex may comprise fungal chitosan chelated, with about 1 ppm to about 10,000 ppm silver, copper, gold, or platinum as described above.

In some embodiments, the fungal chitosan may comprise an average particle size in a range of from about 10 pM to about 2000 μΜ (e.g., about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 1 10, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, or 2000 μΜ, or any range or value therein (e.g., about 10 μΜ to about 1500 μΜ, about 10 μΜ to about 1000 μΜ, about 10 μΜ to about 900 μΜ, about 10 μΜ to about 800 μΜ, about 10 μΜ to about 70 μΜ, about 10 μΜ to about 600 uM, about 10 μΜ ίο about 500 μΜ, about 10 μΜ ίο about 400 μΜ, about 10 uM to about 300 μ , about 10 μΜ to about 200 μΜ, about 10 μΜ to about 100 μΜ, about 10 uM to about 50 μΜ, about 10 uM to about 100 μΜ, about 10 μΜ to about 500 piVL about 10 μΜ to about 1000 μΜ, about 10 μΜ to about 1500 μΜ, about 50 μΜ to about 2000 μΜ, about 50 μΜ to about 1500 μΜ, about 50 μΜ to about 1000 μΜ, about 50 μΜ to about 900 μΜ, about 50 μΜ to about 800 μΜ, about 50 μΜ to about 700 μΜ, about 50 μΜ to about 500 μΜ, about 50 μΜ to about 400 μΜ, about 50 μΜ to about 300 μΜ, about 50 μΜ to about 200 μΜ, about 50 μΜ to about 00 μΜ, about 100 μΜ to about 500 μΜ, about 100 μΜ to about 1000 μΜ, about 100 μΜ to about 1500 μ , about 100 μΜ to about 2000 μΜ, about 500 μΜ to about 1 00 μΜ, about 500 μΜ to about 1500 μΜ, or 500 μΜ to about 2000 μ.Μ, or the like, or any range or value therein)).

The invention further provides a pharmaceutical composition comprising the catiome solution of the invention and a pharmaceutically acceptable carrier. "Pharmaceutically acceptable," as used herein, means a material that is not biologically or otherwise

undesirable, i.e., the material can be administered to an individual along with the compositions of this in vention, without causing; substantial deleterious biological effects or interacting in a deleterious manner with any of the other components of the composition in which it is contained. The material may be selected to minimize any degradation of the active ingredient and. to minimize any adverse side effects: in the subject, as would be well known to one of skill in the art (see, e.g. , Remington's Pharmaceutical' Science; 21st ed.

2005). Thus, a "pharmaceutically acceptable carrier' as used herein means any carrier, diluents or exeipi ent which is compatible with the other ingredients of the composition and not deleterious to the recipient.

in some embodiments, a composition of the invention may include a pharmaceutically

.acceptable carrier. Examples of carriers include, but are not limited to, petroleum jelly, polysaccharides, ianolines, polyethylene glycols, alcohols, transdermal enhancers, and combinations of two or more thereof.

Methods of treatment

Wound healing is a complex bio-process where damaged tissue is replaced by a healthy tissue. The four overlapping stages of wound healing include Hemostasia,

Inflammatory, Proliferation and Remodeling, In Hemostasis. the trauma of a wound causes the body to initiate blood vessel restriction and directs platelets to the site. Blood clotting forms a provisional temporary matrix at the wound site. Simultaneously, stimulation, migration and adhesion of fibroblasts, keritinoeytes and endothelial cells occur, The

Inflammatory phase occurs within 24 hours of the injury. Here, histamine is produced that is. accompanied by swelling and pain, A key type of cell involved in the inflammatory' phase is the macrophage. Macrophages are mature myeloid cells that derive from monocytes after infiltrating skin tissue. Macrophages increase the rate of dermal regeneration and skin repair at the site of .an injury, They are associated with cleansing the bacteria and removing debris remains and necrotic tissue fro the wound area as well as reducing inflammation. In part, the role of macrophages in wound healing may be related to their production of nitric oxide (NO), In the Proliferation phase, the rebuilding of damaged tissue occurs through the development of a temporary collagen matrix composed primaril of fibroblasts. This temporary matrix is known as granulation tissue. During the Remodeling phase, the granulation tissue matures to formed tissue matrix, which resembles the dermis. The tensile •strength of the tissue is increased with the formation of cross-linked fibers which compose the framework of the new dermal tissue.

The anti-infective and anti-inilaramatory properties of a eationic solution comprising chelated fungal chitosan- metal complex of invention, and compositions thereof, may assist in wound healing by providing protection of the wound site. The chelated fungal chitosan-metal complex of the invention may inhibit the production of histamine and therefore, reduce swelling during the inflammatory phase, which in turn results i a decrease in pain. In addition, a chelated fungal chitosan-metal complex of invention may stimulate NO

production by macrophages via theN-acctyiglucosamine unit of the fungal chitosan-metal oxide complex,, thereby improving healing. Application of the chelated fungal chitosan-metal complex, of the invention may also encourage the production of normal dermal fibroblasts (NDF) (as opposed to hypertropic scar fibroblasts (HSF) and keloid fibroblasts (KF) that promote development of sear tissue), and may suppress type I and ΠΙ procollagen production during the proliferation phase. The. chelated fungal chitosan-metal complex of invention may therefore play a role in encouraging the proliferation of epithelial cells that are involved in restoring the integrity of the epithelial barrier rather than weaker extracellular matrix scar tissue.

Wound healing and regeneration of tissue may also be impacted by a naturally occurring electrical current in the body thai moves ions toward the site of a wound. The flow of the electrical current may be enhanced by contacting the tissue with a chelated fungal chitosan-metal complex of the invention, thereby assisting in the healing process (e.g., increasing the rate of healing and increasing, the quality of the regenerated tissue produced in. the healing process).

As described above, the present invention enhances the body's natural ability to heal itself, i some embodiments, the present invention utilizes citric acid to create the chelated fungal chitosan-metal complex of the invention, thro ugh the chemical hydrolysis of the constituents, thereby producing a biodegradable, non-toxic, natural polymeric-metal molecule tha can promote wound healing, lacks irritant or allergic effects, and Is compatible with both healthy as well as infected and/or injured skin. In some aspects, the eationic solutions comprising chelated fungal chitosan-metal complex of invention, and compositions thereof, may be used to promote healing of soft tissue or ulcers, encourage the migration of epithelial and fibroblasts to wound sites, accelerate the recovery of damaged neural tissue, reduce swelling, and inhibit the growth of pathogens.

Accordingly, in some embodiments, the presen invention provides a. method of stimulating the production of dermal fibroblasts and suppressing the production of type ! and ΙΪΙ procollagen by hypertropic scar fibroblasts (HSF) and keloid fibroblasts (KF) in a wound of a subject in need thereof, the method comprising; contacting the wound of the subject with a therapeutically effective amount of a cationic solution or a pharmaceutical composition of the invention, thereby stimulating the production of dermal, fibroblasts and suppressing the production of type Ϊ and ΙΠ procollagen in the wound of the subject.

In some embodiments, a method of reducing swelling in a subject in need thereof is provided, the method comprising: contacting the swelling in the subject with a therapeutically effective amount of a cationic solution or a pharmaceutical composition of the invention, thereby reducing swelling in the subject. In some aspects, swelling may be the result of inflammation due to an injury. In some embodiments, swelling ma be reduced by 5%, 10%, 15%. 20%, 25%, 30%. 35%, 40%, 45%, 50%, 55%, 60%, 65%. 70%, 75%, 80%, 85%, 90%, 95%, 100% as compared to a swelling not contacted with the cationic solution or a pharmaceutical composition of the invention.

In some embodiments, a method of enhancing ion channel electrical current in a wound of a subject in need thereof is provided, the method comprising; contacting the wound/lesion of the subj ect with a therapeutically effective amount of a cationic solution or a pharmaceutical composition of the invention, thereby enhancing ion channel electrical current in the wound of the subject. In some embodiments, the solutions and compositions of the invention may enhance or increase the ion channel electrical current (flow of electrons) at a wound site by 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100% as compared to a wound site not contacted with the cationic solution or a pharmaceutical .composition of the invention,

in some embodiments, a method of enhancing healing of a wound in a. subject in need thereof is provided, the method composing: contacting a wound in the subject with a therapeutically effective amount of a cationic solution or a pharmaceutical composition of the invention, thereby enhancing healing of the wound in the subject. As used herein, "enhanced or enhancing healing of a wound" comprises elimination and prevention of bacterial and fungal infection and increasing the rate of tissue regeneration. In some embodiments, the solution and compositions of the invention may enhance healing of a wound lesion by 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%,.80%, 85%, 90%, 95%, 1.00% as compared to a wound/lesion not contacted with the cationic solution or a pharmaceutical composition of the invention.

In some embodiments, a method of keeping a wound substantially free of bacteria and/or fungi in a subject in need thereof, or eliminating and/or reducing the presence bacteria and/or fungi in a wound of a subject in need thereof is pro vided, the method comprising:

contacting the wound of the subject with a therapeutically effective amount of a cationic solution or a pharmaceutical composition of the invention, thereby keeping the wound of the subject substantially free of bacteria and or fungi in a subject and/or eliminating and/or reducing the presence bacteria or fungi in the wound of the subject as compared to. a wound that is not contacted with the therapeutically effective amount of a cationic solution or a pharmaceutical composition of the invention. In some embodiments, the bacteria and/or fungi will be reduced by about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 9.0% or more as compared to the amount of bacteria or fungi in a wound of a subject thai is not contacted with, the therapeutically effective amount of a cationic solution or a

pharmaceutical composition of the invention.

In some embodiments, a method of increasing the rate of recovery of damaged neural tissue in a subject in need thereof is provided, the method comprising: contacting the damaged, neural tissue in the subject with a therapeutically effective amount of a cationic solution or a pharmaceutical composition of the invention, thereby increasing the rate. of recovery of damaged neural tissue in a subject. In some embodiments, the damaged neural tissue is in a wound in the subject.

Examples of wounds that may be treated using the methods: and compositions of the invention .include, but are not limited to, a cut, a scrape, an abrasion, a laceration, a lesion, a rash, a scald, a burn, an ulcer, a surgical incision, a graft and/or a skin flap, or any

combination thereof. Examples of ul cers treatable using the methods and compositions of the invention include, but are not limited to, a pressure ulcer, a venous leg nicer, an arterial ulcer, and/or a diabetic foot ulcer, or any combination thereof, in some embodiments, a wound may be in, for example, a soft, tissue.

In some embodiments, a cationic solution or a pharmaceutical composition of the invention may be administered locally to, for example, skin or tissue usin a topical route, formulated as, for example, applicator sticks, solutions, suspensions, emulsions, gels, oils, lotions, creams, ointments, pastes, jellies, paints, powders, sprays and aerosols, or any combinatio thereof.

In some embodiments, an effective amount of the composition of the invention -may be in a range from about 0.001 % to -about 1% metal oxide (i.e., about 10 ppm to 10,000 ppm) (e.g., about 0,001 , 0,002, 0.003, 0.004, 0.005, 0.006, 0.007, 0.008, 0.009, 0.01 , 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0,3, 0,4, 0.5, 0.6, 0,7, 0,8, 0.9, or 1%, or the like or any value or range therein). Thus, in some embodiments, an effective amount of a composition of the inventio may be in a range from about 0.001% to about 0.01%, about

0.001% to about 0.1%, about 0.00.5% (50 ppm) to about 02% (about 200 ppm )5 about.0.01% to about 1%, or about 0.1% to about 1%, or the like or any range or value therein.

In some embodiments, a cationie solution comprising a chelated fungal ehitosan-metal may be used as a disinfectant for a surface, skin or a fabric. In some embodiments, a cationie solution for use as a disinfectant for a surface, skin or a fabric may be provided as a composition in the form of. for example, a liquid, a lotion, a gel, a spray, a foam, and/or an ointment. In some embodiments., example liquids include but are not limited to a detergent, a shampoo,, a conditioner, a hand sanitizer, a body wash, a deodorant, and/or a carpet shampoo. In some embodiments, the composition may be impregnated in, for example, a bandage, a dryer sheet, and/or a wipe. In some embodiments, a cationie solution of the invention may be provided as a spray, a paint, or a coating that may be applied to an surface. In some embodiments, a cationie solution of the invention may be adsorbed onto fine dry particles (powder) or may be incorporated into oils. In some embodiments, a cationie solution of the invention may be applied to a continuous cellular fabric (such as human or animal tissue used for skin graft). When used for such purposes, a cationie solution, or composition: of the invention may be used at a concentration of about 5ppm to about 500 ppm (about 0.0005% to about 0.05%),

Methods of making

Chitosan is a deaeetylaied homopol.ym.er of acetyl-glucosamine. Long chain molecules of chitosan are associated with proteins by covaleni bonds, which forms a complex structural network. The present inventor has surprisingly discovered that fungal chitosan differs from chitosan. isolated from crustaceans by, for example, its molecular weight, degree of acetylation and distribution of charged groups. Moreover, chitosan from fungal mycelia has lower levels of inorganic materials compared to that obtained from crustacean shells and no demlnerallzation treatment is required during processing for fungal, chitosan, These differences and more play a role in the functional properties of the fungal chitosan, which result in an enhanced bioaciivlty over crustacean chitosan.

For example, in crustaceans chitosan comprises calcium carbonate deposits, which contribute to the strength of the animal ' s shell. In contrast fungal chitosan does not. contain calcium carbonate but. instead is associated with glucan molecules in the form of microfibrils that, are embedded in an amorphous matrix. This association between glucan molecules and fungal chitosan occurs through covaleni and hydrogen bonds, and in combination with the lack of calcium carbonate, can result In a greater proliferation of positively charged receptor sites. Having greater number of positively charged receptor sites provides, for example, a

chitosan that may be a more complete chelating agent for the metal oxide ions as compared to crustacean derived chitosan.

In addition, the interaction between the positively charged fungal chitosan molecules and a negatively charged microbial surface may be enhanced as compared to chitosan from crustaceans, Such an interaction may result in better disruption of microbial cell

walls/membranes and killing of microbes. Further, the greater percentage of cationic sites observed in metal oxide chelated with fungal chitosan may provide in a stronger electrostatic attraction io the negative charges on the surface of cells and/or tiss ues, in. addition, the chelated fungal chitosan metal oxide complex of the invention shows improved mechanical , water absorption and lysozyme degradation properties as compared to crustacean chitosan chelated with metal oxide.

Additionally, fungal derived chitosan has a lower molecular weight and a lower polydispersity (particle size) as compared to chitosan derived from crustaceans. The smaller particle size means that there is a greater surface area available for chelation with metal oxide and a more complete chelation, sn addition to a greater affinity (higher positive charge) fo adhesion to negatively charged tissue when compared to crustacean derived chitosan.

Further, the ability of fungal chitosan to absorb more water than crustacean chitosan allows it to hydrate more effectively, which in turn increases the surface area of the chitosan. This increases cell migration and fibroblastic interactions, which leads to enhanced tissue regeneration (increased rate of healing, increased quality (e.g., strength) of regenerated tissue.

Accordingly, in some embodiments, a method of making a cationic chelated fungal chitosan-metal solution is provided, comprising dispersing fungal chitosan in distilled water under agitation to produce a fungal chitosan solution; adding a citric acid solution to the fungal chitosan solution to produce a hydroiyzed fungal chitosan solution; and adding an aqueous metal oxide solution to the hydroiyzed fungal chitosan solution, thereby producing the cationic chelated fungal chitosan-metal solution.

In some embodiments, a citric acid solution may be prepared at an elevated temperature of about 90 - 212 Fahrenheit (32 to 100 Celsius).

In some embodiments, a citric acid solution may be a saturated citric acid solution having a citric acid concentration of about 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%,

43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51 %, 52%, .53%, 54%, 55%, .56%, 57%, 58%, 59%, or 60% by weight of the solution or the like or any range or value therein. Thus in some embodiments;, the citric acid solution may have a citric acid concentration of about 35% to about 45%, about 35% to about 50%, about 40% to about 45%, about 40 to about 50%,

about 4.0% to about 55%, about 40% to about 60%, about 45% to about 50%, about 45% to about 55%, about 45% to about 60%, about 50% to about 55%. or about 50% to about 60% by weight of the solution or the like, or any value or range therein. In some embodiments, the concentration of citric acid in the citric acid solution is about 50% by weight of the solution.

in some embodiments, a fungal chitosan solution may comprise fungal chitosan at a concentration in a range of about 0.1 % to about 30% by weight of the solution (e.g., about 0.1 , 0.2, 0.3, 0.4, 0.5, 0.6, .7, 0.8, 0.9, 1 , 1.5, 2, 2.5, 3, 3,5, 4, 4.5, 5, 5.5, 6, .6.5, 7, .7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 1 1.5, 12, 12.5, 13, 13.5, 14, 14.5, 1.5, 15.5, 16, 16.5, 17, .17.5, 18, 18.5, 19, 19.5, 20, 20,5, 21, 21.5, 22, 22,5, 23, 23.5, 24, 24,5, 25, 25.5, 26, 26.5, 27, 27.5, 28, 28.5, 29, 29.5, or 30% by weight of the solution or any range or value therein). Thus in some embodiments, the fungal, chitosan solution may comprise chitosan at a concentration in a range of about 0.1% to about 1%, about 0.1% to about 5%. about 0.1% to about 10%, about 0.1% to about 15%, about 0.1% to about 20%, about 0.1% to about 25%, about 1% to about 5%, about 1 % to about 1 %, about 1 % to about 15%, about 1 % to about 20%, about 1 % to about 25%, about 5% to about 1 %, about 5% to about 15%, about 5% to about 25%, about 5% to about 30%, about 10% to about 15%, about 10% to about 20%, about 10 to about 25%. about 1 % to about 30%, about 15% to about 20%, about 15% to about 25%, or about 15% to about 30% by weight of the solution, or the like, or any range or value therein.

In some embodiments, fungal chitosan may have an average particle size in a range from about 10 μΜ to about 200 μΜ (e.g., about 10, 1 1, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 1,00, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 575, 600, 625, 650, 675, 700, 7.25, 750, 775, 800, 825, 850, 875, 900, 925, 950, 975, 1000, 1100, 1200, 1300, 1.400, 1500, 1 00, 1700, 1800, 1900, or 2000 μΜ or the like, or any range or value therein). Thus in some embodiments, the fungal chitosan may have an average particle size in a . range from about 1.0 μΜ to about 50 μΜ, about 10 μΜ to about 100 μΜ, about 10 μΜ to about 150 μΜ, about 1.0 μΜ to about 200 μΜ, about 10 uM to about 250 μΜ, about 10 μΜ to about 300 μΜ, about 1.0 μΜ to about 350 μΜ, about 10 μΜ ΐο about 500 μίνΐ, about 10 μΜ ίο about 550 uM, about 10 μΜ to about 600 μΜ, about 10 μΜ to about 650 μΜ, about 10 μΜ to about 700 μΜ, about 10 μΜ to about. 800 μΜ, about 10 μΜ to about 850 μ , about 1 μ to about 900 uM, about 10 μΜ to about 1000 μΜ, about 100 μΜ to about 300 μ , about 100 μΜ to about 400 μΜ, about 100 u to about 500 μΜ, about 100 μΜ to about 600 μΜ, about 100 μΜ to about 700 μΜ, about: 100 Μ to about 800 μΜ, about 100 μΜ to about 900

μΜ, about 100 μΜ to about 1000 μΜ, about 100 μΜ to about 1500 μΜ, about 100 μΜ to about 2000 μΜ, about 200 μ.Μ to about 500 μΜ, about 200 μΜ to about 7500 μΜ, about 200 μΜ to about 1000 μΜ, about 200 μΜ to about 1500 μΜ,. about 200 μΜ to about 2000 μΜ, abou 500 μΜ to about 1000 μΜ, about 500 μΜ to about 1500 μΜ, about 500 μΜ to about 2000 μ , about 750 μΜ to about 1000 μΜ, about 750 μΜ ΐο about 1500 μΜ, or about 750 μΜ to about 2000 μΜ, or the like* or any range or value therein, in some embodiments, fungal chitosan may have an average particle size from about 100 μΜ to about 1000 μΜ. In some embodiments, fungal chitosan may have an average particle size from about 200. μΜ to about 750 μΜ,

In some embodiments, an aqueous metal oxide solution may comprise metal oxide in water. In some embodiments, an aqueous metal oxide (e.g., silver oxide, copper oxide, gold oxide, or platinum oxide) solution may comprise a concentration of metal, oxide in a range from .about 0.000.1 % to about 1 % by weight (i.e., 1 ppm-10,000 ppm) (e.g., about 0.001 , 0.002, 0.003, 0.004, 0.005, 0.006, 0.007, 0.008, 0.009,. 0.01 , 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1 , 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1%, or the like or any value or range therein). Thus, in some embodiments, the aqueous metal oxide solution may comprise a. concentration of metal oxide in a range from about. 0,001% to about 0.01%, about 0.001% to about 0, 1%, about 0.01 to about 1%, or about 0.1% to about 1%, or the like, by weight or any range or value therein.

In some embodiments, an aqueous metal oxide solution may be prepared by agitating metal oxide in water. As is understood, agitation can. be a combination of rotation speed and time. Thus, in some embodiments, agitation may comprise a mild stirring to high shear (e.g., about 10 rpm to about 4000 rpm for about 1 min to about 15 min; e.g., about 10, I I , 12, 13, 14, 15, 16, 17, IS, 19, 20, 21 , 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, SO, 8.5, 90, 95, .100, 125, 1 50, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 575, 600, 625, 650, 675, 700, 725, 750, 775, 800, 825, 850, 875, 900, 925, 950, 975, 1000., 1 100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3 100, 3200, 3300, 3400, 3500, 3600, 3700, 3800, 3900, or 4000 rpm, or the like, or any range or value therein, for about 1, 2, 3, 4, 5, 6, 7, 8,. 9, 10, 1 1 , 12, 13, 14, or 15 min, or the like, or any range or value therein). In some embodiments, agitation may comprise about 1000 rpm to about 4000 rpm for about 1 min to about 3 min.

The invention will now be described with reference to the following examples, it should be. appreciated that these examples are not intended to limit the scope of the claims to the invention, but are rather intended to be exemplary of certain embodiments. Any variations

In the exemplified nietiiods that occur to the skilled artisan are intended to fall within the scope of the invention.

EXAMPLE

A fungal chiiosan solution (e.g., a 2% fungal chiiosan solution) is prepared at ambient temperature by dispersing the fungal chiiosan In distilled water under agitation. A solution of citric acid is made at elevated temperature: and introduced into the fungal chitosan/water dispersion, thereby hydrolyzing the chiiosan. A slurry of silver oxide and distilled water is prepared using high, speed agitation fo at least two minutes. The silver oxide/distilled water slurry is introduced into the hydrolyzed fungal ehitosan solution.

Utilizing citric acid in water causes the release of citrate ions that upon contact with Ag20 in water (Ag (+) H2Q + H20 ~ AgOH + H30 + Ag ( ) causes a reduction and complexing of the Ag20 to [Ag3(C6H50?) n+l]3n-. The. citric acid in water acts as a lixiviant (natural leaching solution) to the Ag20, tints enabling the fungal chitosan-citxic acid water solution to more effectively chelate the silver ions.

The foregoing is illustrative of the present invention, and is not to be construed as. limiting thereof. The in vention, is defined by the follo wing claims, with equivalents of the claims to be included therein.