WO2013096605A1 - Systems and methods for delivering an agent to a wound under negative pressure - Google Patents

Abstract

The invention provides biodegradable compositions and methods for using such biodegradable compositions for the local delivery of biologically active agents to an open fracture, complex wound or other site of infection. Advantageously, the degradation and drug elution profiles of the chitosan compositions can be tailored to the needs of particular patients at the point of care (e.g., in a surgical suite, clinic, physician's office, or other clinical setting).

Description

SYSTEMS AND METHODS FOR DELIVERING AN

AGENT TO A WOUND UNDER NEGATIVE PRESSURE

CROSS-REFERENCE TO RELATED APPLICATION This application claims the benefit of the following U.S. Provisional Application No.:

61/578,096, filed December 20, 2011, the entire contents of which are incorporated herein by reference.

STATEMENT OF RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED RESEARCH

This work was supported by the U.S. Government, U.S. Army Institute of Surgical Research. The Government may have certain rights in this invention.

BACKGROUND OF THE INVENTION

The skin serves as an important barrier to infection. Any trauma that breaks the skin creates an opportunity for pathogen entry and infection. To reduce the risk of infection, the current standard of care involves debridement, irrigation, and systemic antibiotic therapy. Even with aggressive therapies and systemic antibiotic treatment, infections remain a significant source of morbidity and mortality. Tissues compromised by trauma and infection often have reduced vascularization, which limits the delivery of circulating therapeutics.

Increased concentrations of systemic antibiotics are usually required to compensate for poor circulation in the damaged tissue. Negative pressure wound therapy provides certain advantages for the treatment of wounds, particularly for infected wounds and wounds prone to infection. The application of negative pressure to the wound provides for the removal of wound exudate and may increase circulation to the wound and surrounding tissue. Current methods for wound therapy rely primarily on dressings and films. Improved compositions and methods to prevent or treat an infection at a site of trauma are urgently required.

SUMMARY OF THE INVENTION

As described below, the present invention features a system for the treatment of wounds that involves the use of negative pressure together with sponges, pastes, hydrogels and other related polymeric compositions that provide for the delivery of biologically active agents for the treatment or prevention of a pathogen infection. In one aspect, the invention provides a method for treating a wound, the method involving implanting a biodegradable polymeric composition into the wound; sealably covering the wound; and applying negative pressure to the wound. In certain embodiments, the composition comprises an effective amount of at least one agent.

In another aspect, the invention provides a system for treating a wound, the system including a polymeric composition in the form of a biodegradable sponge, paste, or hydrogel; a sealable wound dressing comprising an opening for draining the wound; and a vacuum pump connected to the opening. In certain embodiments, the composition comprises an effective amount of at least one agent. In some embodiments, the system further includes a monitoring system for automatically adjusting the vaccum.

In a related aspect, the invention provides a kit for treating a wound, the kit containing a biodegradable chitosan composition; a wound dressing for use under negative pressure; and instructions for use in accordance with the methods of the invention. In certain embodiments, the composition comprises an effective amount of at least one agent.

In various embodiments of any of the aspects delineated herein, the wound is selected from the group consisting of an fracture, open fracture, complex wound, surgical site, closed wound, chronic wound, acute wound, diabetic ulcer, diabetic wound, and burns.

In various embodiments of any of the aspects delineated herein, the polymeric composition is in the form of a sponge, hydrogel, paste, dressing, plug, mesh, strip, suture, film or combinations thereof. In various embodiments of any of the aspects delineated herein, the sponge, paste, or hydrogel comprises polyvinyl alcohol - PVA, Poly(lactic-co- glycolic acid) - PLGA, Polyglycolic acid - PGA, Poly(lactic acid) - PLA, cellulose, chitosan, collagen, fibrin-based wound dressing (with polyethylene glycol), and gelatin.

In various embodiments of any of the aspects delineated herein,, the composition is a chitosan sponge, paste or hydrogel. In various embodiments of any of the aspects delineated herein, the chitosan sponge, paste, or hydrogel degrades within 1-35 days in vivo. In various embodiments of any of the aspects delineated herein, the chitosan has a uniform degree of deacetylation of at least about 51%, wherein the water content is about 0-90%. In various embodiments of any of the aspects delineated herein, the chitosan is treated with an acid selected from the group consisting of acetic, citric, oxalic, proprionic, ascorbic, hydrochloric, formic, salicylic, and lactic acids. In various embodiments of any of the aspects delineated herein, the chitosan degree of deacetylation is at least about 61, 71, or 80 percent.

In particular embodiments, the composition is 80% deacetylated chitosan treated with lactic or acetic acid. In specific embodiments, the chitosan composition is 61DDA or 71DDA chitosan that is neutralized with 0.175 or 0.5M NaOH. In various embodiments of any of the aspects delineated herein, the chitosan composition provides for the long term release of the agent.

In various embodiments of any of the aspects delineated herein, the composition comprises an effective amount of at least one agent. In various embodiments of any of the aspects delineated herein, the agent is one or more of an antimicrobial agent, angiogenic factor, growth factor, analgesic, anti-inflammatory, hemostatic agent, and anti-thrombotic. In particular embodiments, the antimicrobial agent is selected from the group consisting of antibacterial, anti-viral, and anti-fungal agents. In specific embodiments, the antimicrobial agent is an antibiotic selected from the group consisting of daptomycin, vancomycin, gentamicin, tobramycin, and amikacin. In various embodiments of any of the aspects delineated herein, the effective amount of the agent is sufficient to reduce the survival or proliferation of a bacterial cell (e.g., Pseudomonas aeruginosa or Staphylococcus aureus).

The invention provides compositions featuring chitosan and other polymers and methods of using such compositions in combination with negative wound therapy.

Compositions and articles defined by the invention were isolated or otherwise manufactured in connection with the examples provided below. Other features and advantages of the invention will be apparent from the detailed description, and from the claims. Definitions

By "chitosan" is meant a chitin-derived polymer that is at least 20% deacetylated. Preferably, chitosan is at least about 50% deacetylated. Chitin is a linear polysaccharide consisting of (l-4)-linked 2-acetamido-2-deoxy-b-D-glucopyranose. Chitosan is a linear polysaccharide consisting of (l-4)-linked 2-amino-2-deoxy-b-D-glucopyranose.

By "composite" is meant a mixture of materials. In one embodiment, a composite comprises sponge fragments dispersed within a hydrogel.

By "acid treated chitosan" is meant chitosan that is solubilized in an acidic solution.

By "degrades" is meant physically or chemically breaks down in whole or in part. Preferably, the degradation represents a physical reduction in the mass by at least about 10%, 25%, 50%, 75%, 80%, 85%, 90%, 95% or 100%.

By "film" is meant a thin layer of material.

By "long term release" is meant elution of an agent over the course of twenty-four - seventy two hours or longer. By "sponge" is meant a three-dimensional porous matrix. By "wound management device" or "wound healing device" is meant any material used to protect or promote healing at a site of trauma.

By "agent" is meant any small compound, antibody, nucleic acid molecule, or polypeptide, or fragments thereof.

By "ameliorate" is meant decrease, suppress, attenuate, diminish, arrest, or stabilize the development or progression of a disease.

By "alteration" is meant a change (increase or decrease) in the expression levels or activity of a gene or polypeptide as detected by standard art known methods such as those described herein. As used herein, an alteration includes a 10% change in expression levels, preferably a 25% change, more preferably a 40% change, and most preferably a 50% or greater change in expression levels.

By "analog" is meant a molecule that is not identical, but has analogous functional or structural features. For example, a chitosan analog retains the biological activity of a corresponding reference chitosan polymer (e.g., manufactured chitosan), while having certain biochemical modifications that enhance the analog's function relative to a reference chitosan polymer. Such biochemical modifications could increase the analog's ability to be degraded, to uptake or elute a therapeutic agent, or to increase or decrease mechanical strength.

By "antimicrobial" is meant an agent that inhibits or stabilizes the proliferation or survival of a microbe. In one embodiment, a bacteriostatic agent is an antimicrobial. In other embodiments, any agent that kills a microbe (e.g., bacterium, fungus, virus) is an

antimicrobial.

By "anti-inflammatory" is meant an agent that reduces the severity or symptoms of an inflammatory reaction in a tissue. An inflammatory reaction within tissue is generally characterized by leukocyte infiltration, edema, redness, pain, and/or neovascularization. Inflammation can also be measured by analyzing levels of cytokines or any other

inflammatory marker.

By "biodegradable" is meant susceptible to breakdown by biological activity. For example, biodegradable chitosan compositions are susceptible to breakdown by enzymes present in vivo (e.g., lysozyme, N-acetyl-o-glucosaminidase and lipases). Degradation of a chitosan composition of the invention need not be complete. A chitosan composition of the invention may be degraded, for example, by the cleavage of one or more chemical bonds (e.g., glycosidic bonds). By "clinician" is meant any healthcare provider. Exemplary clinicians include, but are not limited to, doctors, veterinarians, osteopaths, physician's assistants, emergency medical technicians, medics, nurse practitioners, and nurses.

In this disclosure, "comprises," "comprising," "containing" and "having" and the like can have the meaning ascribed to them in U.S. Patent law and can mean " includes,"

"including," and the like; "consisting essentially of" or "consists essentially" likewise has the meaning ascribed in U.S. Patent law and the term is open-ended, allowing for the presence of more than that which is recited so long as basic or novel characteristics of that which is recited is not changed by the presence of more than that which is recited, but excludes prior art embodiments.

"Detect" refers to identifying the presence, absence or amount of the analyte to be detected.

By "customize" is meant tailor to suit the needs of a particular subject.

By "degradation rate" is meant the time required to substantially degrade the composition. A composition is substantially degraded where at least about 75%, 85%, 90%, 95% or more has been degraded. Methods for measuring degradation of chitosan are known in the art and include measuring the amount of a sponge, film, composite or other

composition of the invention that remains following implantation in a subject or following in vitro exposure to an enzyme having chitosan-degrading activity.

By "disease" is meant any condition or disorder that damages or interferes with the normal function of a cell, tissue, or organ. In one example, a disease is a bacterial or other infection present in a wound site. In another embodiment, a disease is sepsis.

By "effective amount" is meant the amount of an agent required to ameliorate the symptoms of a disease relative to an untreated patient. The effective amount of active agent(s) used to practice the present invention for therapeutic treatment of a disease varies depending upon the manner of administration, the age, body weight, and general health of the subject. Ultimately, the attending physician or veterinarian will decide the appropriate amount and dosage regimen. Such amount is referred to as an "effective" amount.

By "elution rate" is meant the time required for an agent to be substantially released from a composition. Elution can be measured by determining how much of an agent remains within the composition or by measuring how much of an agent has been released into the composition's surroundings. Elution may be partial (10%, 25%, 50%, 75%, 80%, 85%, 90%, 95% or more) or complete. In one preferred embodiment, the agent continues to be released at an effective level for at least about 3, 4, 5, 6, 7, 8, 9, or 10 days. The invention provides a number of targets that are useful for the development of highly specific drugs to treat or a disorder characterized by the methods delineated herein. In addition, the methods of the invention provide a facile means to identify therapies that are safe for use in subjects. In addition, the methods of the invention provide a route for analyzing virtually any number of compounds for effects on wound healing or pathogen infection described herein with high-volume throughput, high sensitivity, and low

complexity.

By "fragment" is meant a portion of a polypeptide or nucleic acid molecule. This portion contains, preferably, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the entire length of the reference nucleic acid molecule or polypeptide. A fragment may contain 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 nucleotides or amino acids.

By "inhibitory nucleic acid" is meant a double- stranded RNA, siRNA, shRNA, or antisense RNA, or a portion thereof, or a mimetic thereof, that when administered to a mammalian cell results in a decrease (e.g., by 10%, 25%, 50%, 75%, or even 90-100%) in the expression of a target gene. Chitosan compositions are useful for the delivery of

polynucleotides, such as inhibitory nucleic acid molecules, useful for the treatment or prevention of pathogen infection and related disease. Typically, a nucleic acid inhibitor comprises at least a portion of a target nucleic acid molecule, or an ortholog thereof, or comprises at least a portion of the complementary strand of a target nucleic acid molecule. For example, an inhibitory nucleic acid molecule comprises at least a portion of any or all of the nucleic acids delineated herein.

By "infection" is meant the presence of one or more pathogens in a tissue or organ of a host. An infection includes the proliferation of a microbe (e.g., bacteria, viruses, fungi) within a tissue of a subject at a site of trauma.

By "marker" is meant any protein or polynucleotide having an alteration in expression level or activity that is associated with a disease or disorder.

By "modulate" or "modulation" is meant any alteration (e.g., increase or decrease) in a biological function or activity. Such alterations are detected by standard art known methods such as those described herein.

As used herein, "negative pressure" is meant the difference between the pressure inside a wound dressing and atmospheric pressure. Measurement of negative pressure is indicated, for example, as a positive numerical value in mm Hg. In various embodiment, the negative pressure is at least about 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150 mm Hg up to about 200, 250, 300, 400, 500 mm Hg. In certain embodiments, the negative pressure is at least about 10 to about 150 mm Hg. In particular embodiments, the negative pressure is at least about 65 to about 135 mm Hg. In specific embodiments, the negative pressure is at least about 80 to about 120 mm Hg.

As used herein, "negative pressure wound therapy" or "NPWT" is a therapeutic technique using a negative pressure source or vacuum (e.g., at low pressure) to pull exudate out of the wound bed, introduce blood flow into the wound bed, remove infectious materials, and/or draw wound edges together, to treat acute and chronic wounds. Negative pressure may be applied continuously or intermittently, depending on the type of wound being treated and the clinical objectives. Negative pressure wound therapy promotes healing in wounds (e.g., acute and chronic), reduces edema, increases tissue granulation, and increases angiogenesis in the wound bed by introducing blood flow.

As used herein, "obtaining" as in "obtaining an agent" includes synthesizing, purchasing, or otherwise acquiring the agent.

By "physical interaction" is meant an association that does not require covalent bonding. In one embodiment, a physical interaction includes incorporation into a chitosan composition of the invention.

By "point of treatment" is meant the site where healthcare is delivered. A "point of treatment" includes, but is not limited to, a surgical suite, physician's office, clinic, or hospital.

By "polymer" is meant a natural or synthetic organic molecule formed by combining smaller molecules in a regular pattern. Exemplary polymers useful in the methods of the invention include, but are not limited to, polyvinyl alcohol - PVA, Poly(lactic-co-glycolic acid) - PLGA, Polyglycolic acid - PGA, Poly(lactic acid) - PLA, cellulose, chitosan, collagen, fibrin-based wound dressing (with polyethylene glycol), and gelatin. Such polymers may be used singly or in combination in the manufacture of sponges, hydrogels, and other compositions for the treatment of wounds.

By "profile" is meant a set of characteristics that define a composition or process. For example, a "biodegradation profile" refers to the biodegradation characteristics of a composition. In another example, an "elution profile" refers to elution characteristics of a composition.

By "prosthetic device" is meant an implanted medical device that substitutes for or supplements a missing or defective part of the body.

By "small molecule" is meant any chemical compound. By "trauma" is meant any injury that damages a tissue or organ of a subject. The injury need not be severe. Therefore, a trauma includes any injury that breaks the skin.

By "wound" is an injury to the outer body of a subject. A wound can result in the separation of the coherence of tissue of the outer body of the subject, including a loss of substance (e.g., blood).

By "subject" is meant a mammal, including, but not limited to, a human or non-human mammal, such as a bovine, equine, canine, ovine, or feline.

By "uniform degree of deacetylation" refers to a chitosan composition made from a single type of chitosan, (e.g., 61DDA, 71DDA, or 81DDA). In one embodiment, a chitosan composition having a uniform degree of deacetylation excludes chitosan compositions having a combination of types chitosans, where the chitosans have different degrees of deacetylation.

By "reference" is meant a standard or control condition.

By "siRNA" is meant a double stranded RNA. Optimally, an siRNA is 18, 19, 20, 21, 22, 23 or 24 nucleotides in length and has a 2 base overhang at its 3' end. These dsRNAs can be introduced to an individual cell or to a whole animal; for example, they may be introduced systemically via the bloodstream. Such siRNAs are used to downregulate mRNA levels or promoter activity.

As used herein, the terms "treat," treating," "treatment," and the like refer to reducing or ameliorating a disorder and/or symptoms associated therewith. It will be appreciated that, although not precluded, treating a disorder or condition does not require that the disorder, condition or symptoms associated therewith be completely eliminated.

As used herein, the terms "prevent," "preventing," "prevention," "prophylactic treatment" and the like refer to reducing the probability of developing a disorder or condition in a subject, who does not have, but is at risk of or susceptible to developing a disorder or condition.

BRIEF DESCRIPTION OF THE DRAWINGS

Figures 1A and IB are graphs depicting reduction in S. aureus in open fracture model when antibiotics were locally delivered via chitosan sponge and in combination with negative pressure wound therapy. After generation of an open fracture, goats were inoculated with luminescent S. aureus. At six hours after bacterial inoculation, the wounds were imaged to capture the quantitative and spatial distribution of the bacteria in the wound (6 hr pre;

baseline), and imaged after surgical debridement and irrigation (6 hr post). The wounds were dressed including local antibiotic delivery via polymethylmethacralate bone cement (PMMA) or chitosan sponge. Additionally, negative pressure wound therapy (NPWT) was used in combination with each type of antibody delivery. At 48 hours after bacterial inoculation, the wounds were imaged to capture the quantitative and spatial distribution of the bacteria in the wound (48 hr pre). Figure 1A depicts reductions in S. aureus in open fracture model at 48 hrs after bacterial inoculation when antibiotics were delivered via chitosan sponge, especially in combination with NPWT, compared to PMMA beads. Figure IB depicts reductions in S. aureus in open fracture model at 48 hrs after bacterial inoculation in PMMA, chitosan sponge, and chitosan sponge + NPWT groups.

Figures 2A-2C are representative images showing rebound bacterial growth up to the footprint of the PMMA beads in bead NPWT group. Figure 2A shows bead placement with superimposed bacterial photon emission. Figure 2B shows bacterial photon emission following bead removal. Figure 2C shows bacterial photon emission following bead removal without background photograph.

Figures 3A-3B are representative photographs showing post treatment location and intensity of bacteria in bead with NPWT (Figure 3 A) and sponge with NPWT (Figure 3B) groups.

Figure 4 is an image showing complex musculoskeletal wound immediately prior to debridement.

Figure 5 is an image showing representative wound bacterial measurement immediately prior to debridement.

DETAILED DESCRIPTION OF THE INVENTION

As described below, the present invention features a negative wound therapy system comprising polymeric compositions (e.g., solids, sponges, films, hydrogels, composites) that provide for the local delivery of biologically active agents in combination with devices apply negative pressure at a wound site. In addition, the invention provides methods of using such systems to treat or prevent an infection or otherwise promote wound healing.

The invention is based, at least in part, on the discovery that drug delivery via polymeric compositions (e.g., chitosan paste, chitosan sponge) in combination with negative pressure wound therapy (NPWT) is effective for wound treatment and/or wound

management. As reported in detail below, a combination of local antibiotic delivery via chitosan compositions (e.g., chitosan paste, chitosan sponge, chitosan "sponge-in-gel" composite) and negative pressure wound therapy (NPWT) was found to be surprisingly effective for enhancing wound healing and reducing bacterial growth in the wound bed. This is in contrast to skepticism from the medical community to the contrary that in an augmented NPWT technique the antibiotics eluted from the antibiotic sponge will not permeate throughout the wound, but rather will be removed from the wound via suction of the negative pressure device.

Negative Pressure Wound Therapy

Negative pressure wound therapy (NPWT; Wound Vac therapy; "reduced pressure therapy;" or "vacuum therapy") is a therapeutic technique using a vacuum dressing (e.g., polymeric sponge, polymeric hydrogel) to promote healing in acute or chronic wounds and enhance healing of first and second degree burns and other wounds. The therapy involves the controlled application of sub-atmospheric pressure to the local wound environment, e.g., using a sealed wound dressing connected to a vacuum pump. NPWT promotes wound healing by applying a vacuum through a special sealed dressing. The continued vacuum draws out fluid from the wound and increases blood flow to the area. The vacuum may be applied continuously or intermittently, depending on the type of wound being treated and the clinical objectives. Typically, the vacuum dressing is changed two to three times per week. The dressings used for the technique include polymeric sponges, hydrogels, pastes, and similar devices intended to contain the vacuum at the wound site.

Clinical studies and practice have shown that providing a reduced pressure in proximity to a tissue site augments and accelerates the growth of new tissue at the tissue site. The applications of this phenomenon are numerous, but application of reduced pressure has been particularly successful in treating wounds. Typically, reduced pressure is applied to tissue through a vacuum dressing (e.g., a polymeric sponge, polymeric hydrogel). The vacuum dressing can be incorporated into a wound dressing having other components that facilitate treatment, such as, for example, a drape (e.g., adhesive surgical drape). Instillation of fluids s (e.g., irrigation fluids and/or medicaments) may be used in conjunction with negative pressure wound therapy to promote healing and/or improve efficacy.

The general technique for NPWT is as follows: a vacuum dressing is fitted to the contours of a wound and sealed with a transparent film. In the methods of the invention, a biodegradable composition (e.g., a biodegradable sponge) is implanted into the wound.

Preferably, the wound is debrided and irrigated prior to applying the vacuum dressing. The biodegradable composition (e.g., a biodegradable sponge) may be cut to size for placement in the wound. The dressing includes a means for preventing the dressing from adhering to the biodegradable composition and/or tissue at the wound site. For example, the dressing may include a pad with non-adhesive gauze or non-adhesive gel that separates the dressing from the biodegradable composition and/or tissue at the wound site. The dressing sealably covers the wound and is adhered to the skin (e.g., using an adhesive transparent film). A drainage tube is connected to the dressing through an opening of the film. The drainage tube is also connected to a vacuum source, turning an open wound into a controlled, closed wound while removing excess fluid from the wound bed to enhance circulation and remove wound fluids. The wound may be sealed for up to 12 hrs, 1, 2, 3, 4, 5 days or more depending on the type of wound being treated and the clinical objectives. This creates a moist healing environment and reduces edema. The technique is usually used with chronic wounds or wounds that are expected to present difficulties while healing, such as those associated with diabetes or those otherwise prone to infection.

Conventional commercial products for the technique are generally divided into three categories based the type of dressing used over the wound surface: open-cell foam, gauze or honeycombed textiles with a dimpled wound contact surface. The present invention provides sponges and hydrogels comprising polymers of the invention (e.g., polyvinyl alcohol - PVA, Poly(lactic-co-glycolic acid) - PLGA, Polyglycolic acid - PGA, Poly(lactic acid) - PLA, cellulose, chitosan, collagen, fibrin-based wound dressing (with polyethylene glycol), and gelatin). Foam dressings are used to fill open cavity wounds and can be tailored in size to fit wounds. The foam dressing is applied, filling the wound and then a film drape is applied over the top to create a seal around the dressing. A vacuum tube is connected through an opening in the film drape to a canister on the side of a vacuum pump.

NPWT can be conducted using compositions of the invention alone or in combination with standard medical supplies such as open weave cotton gauze, transparent film, a flat drain and tubing that connects to a vacuum pump. The flat drain is sandwiched in gauze and this is then placed onto the wound. A film drape is used to cover the wound and create a complete seal, and then the drain is connected to the pump via the tubing. Another type of dressing involves layers of non- woven polyester, joined by a silicone elastomer, and has a nonadherent wound contact surface made up of numerous small semi-rigid dome structures. The dressing type used depends on the type of wound, clinical objectives and patient.

In accordance with the present invention, negative pressure wound therapy includes a means for the generating a negative pressure in the wound area. In a preferred embodiment, this is a means for the functional connection of the wound area with a negative pressure source outside of the cover material so that a negative pressure can be generated in the wound area and fluids can be sucked out of the wound area. The expression "negative pressure in the wound area" in the context of the invention describes an air pressure which is lower inside the wound dressing than the atmospheric pressure. "Within the wound dressing" refers to the cavity formed between the cover material and the wound.

Once the vacuum dressing is sealed the vacuum pump can be set to deliver continuous or intermittent pressures. The difference between the air pressure inside the wound dressing and the atmospheric pressure is stated in relation to the invention in mm Hg (millimeters of mercury), as this is the convention in the negative pressure wound therapy. 1 mm Hg corresponds to one torr or 133.322 Pa (Pascal). In relation to the invention, the negative pressure, i.e. the difference between the pressure inside the wound dressing and the atmospheric pressure, is stated as a positive numerical value in mm Hg. The levels of pressure vary between -125 and -75 mmHg depending on the material used and patient tolerance. Pressure can be applied constantly or intermittently, depending on the type of wound being treated and the clinical objectives. In one embodiment of the invention, the negative pressure is a maximum of 500 mm Hg, preferably a maximum of 250 mm Hg. This negative pressure range up to a maximum of 500 mm Hg, preferably a maximum of 250 mm Hg has proved suitable for wound healing. In a preferred embodiment of the invention, the negative pressure is at least 10 mm Hg up to a maximum of 150 mm Hg, more preferably of at least 65 and a maximum of 135 mm Hg. In one embodiment, the negative pressure is 80 mm Hg. In another embodiment, the negative pressure is 120 mm Hg. A monitoring system may be configured for automatically adjusting the vaccum.

Compositions of the invention are useful in methods and systems for negative pressure wound therapy, which are known in the art and described, for example, in US Patent Publication Nos: 20110275964, 20110172615, 20110213287, 20110087179, 2007167926, and 20110054810, each of which is incorporated herein by reference.

Wounds

The compositions and methods of the invention are useful for the treatment of wounds. In particular, the compositions and methods of the invention are effective in decreasing growth and survival of pathogens in a wound. Wounds include injury in which the dermis of the skin is torn, cut or punctured (an open wound), or where blunt force trauma causes a contusion (a closed wound). Open wounds may be classified according to the object that caused the wound. For example, types of open wounds include incisions or incised wounds, caused by a clean, sharp-edged object such as a knife, a razor or a glass splinter; lacerations, irregular tear-like wounds caused by some blunt trauma; abrasions (grazes), superficial wounds in which the topmost layer of the skin (the epidermis) is scraped off; puncture wounds, caused by an object puncturing the skin, such as a nail or needle;

penetration wounds, caused by an object such as a knife entering and coming out from the skin; or gunshot wounds, caused by a bullet or similar projectile driving into or through the body. For example, types of closed wounds include contusions, commonly known as bruises, caused by a blunt force trauma that damages tissue under the skin; hematomas or blood tumors, caused by damage to a blood vessel that causes blood to collect under the skin; or crush injury, caused by a great or extreme amount of force applied over a long period of time.

Wounds that heal in a predictable amount of time (e.g., within about two weeks in a healthy subject) are termed acute wounds. In contrast, a chronic wound is a wound that does not heal in an orderly set of stages and/or in a predictable amount of time (e.g., wounds that do not heal within three months). Chronic wounds may never heal or may take years to do so. Chronic wounds may be detained in one or more of the phases of wound healing. For example, chronic wounds often remain in the inflammatory stage for too long. In acute wounds, there is a precise balance between production and degradation of molecules such as collagen. In chronic wounds this balance is lost and degradation plays too large a role.

Acute wounds may fail to heal in a timely fashion and become a chronic wound. Chronic wound patients often report pain as dominant in their lives. Wound Healing Devices

The present invention provides wound healing devices that employ polymeric compositions, e.g., polyvinyl alcohol - PVA, Poly(lactic-co-glycolic acid) - PLGA,

Polyglycolic acid - PGA, Poly(lactic acid) - PLA, cellulose, chitosan, collagen, fibrin-based wound dressing (with polyethylene glycol), and gelatin compositions. A number of collagen dressings are commercially available (e.g., Puracol Plus, Collatamp, Innocoll). The wound healing devices may be configured by forming the polymeric composition into a shape and size sufficient to accommodate the wound being treated. If desired, the wound healing device comprises polymeric fibers. Wound healing devices are desirably produced in whatever shape and size is necessary to provide optimum treatment to the wound. These devices can be produced in forms that include, but are not limited to, plugs, meshes, strips, sutures, dressings, or any other form able to accommodate and assist in the repair of a wound. The damaged portions of the patient that may be treated with devices made of the polymeric composition of the present invention include, but are not limited to, bone, cartilage, skin, muscle and other tissues (nerve, brain, spinal cord, heart, lung). Other similar devices are administered to assist in the treatment repair and remodeling of a damaged tissue, bone, or cartilage. For some applications, it is desirable for the device to be incorporated into an existing tissue to facilitate wound repair. For other applications, it is desirable for the device to degrade over the course of days, weeks, or months. Such degradation may be

advantageously tailored to suit the needs of a particular subject using the methods described herein. The elution and/or degradation profile of a polymeric composition (e.g., polymeric sponge, polymeric hydrogel) can be altered as described herein.

Chitosan

As reported in detail below, chitosan compositions are particularly useful in combination with negative wound pressure therapy.

Chitosan is a naturally occurring linear polysaccharide composed of randomly distributed B-(l-4)-2-amino-2-D-glucosamine (deacetylated) and B-(l-4)-2-acetamido-2-D- glucoseamine (acetylated) units. Chitosan is derived from chitin, a naturally occurring polymer. Chitin is a white, hard, inelastic, nitrogenous polysaccharide isolated from fungi, mollusks, or from the exoskeletons of arthropods (e.g., crustaceans, insects). The major procedure for obtaining chitosan is the alkaline deacetylation of chitin with strong alkaline solution. Generally, the raw material is crushed, washed with water or detergent, and ground into small pieces. After grinding, the raw material is treated with alkali and acid to isolate the polymer from the raw crushed material. The polymer is then deacetylated by treatment with alkali. Chitin and chitosan differ in their degrees of deacetylation (DDA). Chitin has a degree of deacetylation of 0% while pure chitosan has a degree of deacetylation of 100%. Typically, when the degree of deacetylation is greater than about 50% the polymer is referred to as chitosan.

Chitosan is a cationic weak base that is substantially insoluble in water and organic solvents. Typically, chitosan is fairly soluble in dilute acid solutions, such as acetic, citric, oxalic, proprionic, ascorbic, hydrochloric, formic, and lactic acids, as well as other organic and inorganic acids. Chitosan' s charge gives it bioadhesive properties that allow it to bind to negatively charged surfaces, such as biological tissues present at a site of trauma or negatively charged implanted devices. Chitosan' s degree of deacetylation affects it resorption. Chitosan compositions having a 50% degree of deacetylation are highly degradable in vivo. As the degree of deacetylation increases, chitosan becomes increasingly resistant to degradation. Chitosan compositions having a degree of deacetylation that is higher than 95% degrade slowly over weeks or months. In the body chitosan is degraded by lysozyme, N-acetyl-o-glucosaminidase and lipases. Lysozyme degrades chitosan by cleaving the glycosidic bonds between the repeating chitosan units. The byproducts of chitosan degradation are saccharides and glucosamines that are gradually absorbed by the human body. Therefore, when chitosan is used for the local delivery of therapeutic or prophylactic agents, no secondary removal operation is required.

As reported herein, chitosan compositions (e.g., solids, sponges, pastes, films, hydrogels, composites) can be loaded with a biologically active agent at the site of care (e.g., in a surgical suite, clinic, or physician's office, trauma site, battlefield). This property allows the clinician to tailor the antibiotics or other agents used to load the chitosan wound management device to suit the needs of a particular patient. In one embodiment, the degree of deacetylation is adjusted to provide chitosan compositions that degrade in as little as about twenty-four, thirty-six, forty-eight, or seventy two hours or that are maintained for a longer period of time (e.g., 4, 5, 6, 7, 8, 9, 10 days). In other embodiments, chitosan compositions of the invention are maintained in the body for at least about two-six weeks or more (e.g., 2, 3, 4, 5, 6 weeks, two, three or four months). In still other embodiments, chitosan

compositions of the invention enhance blood clotting in a wound or other site of trauma (hemostasis). In other embodiments, the chitosan compositions are loaded with therapeutic or prophylactic agents that are clinician selected and that are delivered over at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days or for longer periods.

Chitosan Sponges, Pastes, and Hydrogels

Therefore, in certain embodiments, the invention provides a chitosan polymeric sponge or polymeric hydrogel comprising agents for delivery to a wound. If desired, the delivery profile of the chitosan is modulated by adjusting the following variables: degree of deacetylation, neutralization solution, solvent make-up, and chitosan weight %, and/or crystallinity.

Crystallinity indicates the degree of structural order in a compound. Polymers such as chitosan are either amorphous or semicrystalline. Chitosan's crystallinity, like other polymers, depends on its type, number, and regularity of polymer-chain, side group chemistry, the degree of matrix packing or density, and crosslinking. The crystallinity of chitosan or its products can be controlled or altered during manufacture through its molecular weight, degree of deacetylation, and crosslinking to affect thermal properties, such as melting point, and physical-mechanical properties, such as tensile strength, Young's modulus, swelling and degradation. Crosslinking is the process which links polymer chains together. In chitosan, crosslinking induces a three-dimensional matrix of interconnected, linear, polymeric chains. The degree or extent of crosslinking depends on the crosslinking agent. Exemplary crosslinking agents include sodium tripolyphosphate, ethylene glycol diglycidyl ether, ethylene oxide, glutaraldehyde, epichlorohydrin, diisocyanate, and genipin. Crosslinking can also be accomplished using microwave or ultraviolet exposure.

Chitosan' s properties can also be altered by modulating the degree of deacetylation. In one embodiment, the degree of deacetylation is adjusted between about 50-100%, wherein the bottom of the range is any integer between 50 and 99, and the top of the range is any integer between 51% and 100%. In particular embodiments, the degree of deacetylation is 51%, 55%, 60%, 61%, 65%, 70%, 71%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 85%, 90%, and 95%. In general, the higher the molecular weight, the slower the degradation of the chitosan composition.

If desired, chitosan is neutralized after acid treatment. Any base known in the art (e.g., NaOH, KOH, NH₄OH, Ca(OH)₂, Mg(OH)_2, or combinations thereof) may be used to neutralize an acid-treated chitosan composition. Preferably, a neutralization solution has a pH greater than 7.4 (e.g., 7.8, 8.0, 8.5, 9.0, 10, 11, and 12, 13, 14, 15, 16). The neutralization step is optional, and not strictly required. If desired, the chitosan is treated with water, PBS, or sterile saline following acid treatment. It may comprise 0.01-lO.OM of a base (e.g., 0.01, 0.025, 0.5, 0.75, 0.1, 0.25, 0.5, 0.75, 1.0, 1.5, 2.0, 2.5, 3, 4, 5, 6, 7, 8, 9, 10 M) (e.g., NaOH). Chitosan compositions neutralized in bases having lower molarity degrade more quickly. Chitosan compositions neutralized in bases of increased molarity degrade more slowly than those neutralized at lesser molarities. Thus, the degradation properties of chitosan can be modulated by altering the molarity of the neutralizing base.

In other embodiments, the concentration of the acidic solvent used to dissolve the chitosan is adjusted or the time period used to dissolve the chitosan is altered. For example, a 0.1%, 0.5%, 1%, 2%, 3% or 5% acid solution is used. In particular embodiments, chitosan is dissolved in acetic, citric, oxalic, proprionic, ascorbic, hydrochloric, formic, salicylic and/or lactic acids, or a combination of those. In general, acidic solvents comprising increased levels of lactic acid form chitosan compositions that degrade more quickly and also have reduced strength and durability. In various embodiments, a combination of acetic and lactic acids are used. Acetic provides more strength and slower degradation. In contrast, lactic acid provides more flexibility. In one approach, the ratio of lactic to acetic acid is varied from 5:1, 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:4, to 1:5. In one embodiment, the blended acid solvent comprises 90%/10%, 80%/20% 75%/25%, 70%/30%, 60%/40%, 50%/50%. In still other embodiments, the chitosan weight % is altered from 0.25 - 10.0% (e.g., 0.1, 0.2, 0.25, 0.3, 0.4, 0.5, 1, 1.25, 1.5, 1.75, 2.0, 2.5, 3.0, 3.5, 4, 5, 6, 7, 8, 9, 10%). In one embodiment, a 1 wt% chitosan solution is preferred, where a 1 wt% chitosan solution contains 1 gram of chitosan per 100 ml solution. Typically, the higher the wt%, the slower the degradation.

If desired the chitosan composition is loaded with agents and the chitosan

composition is delivered to a wound to form a delivery system for the agent. Preferably, the chitosan composition contains an effective amount of a chemical or pharmaceutically active component. In one embodiment, the chitosan composition self-adheres to a site at which delivery is desired. In another embodiment, an adhesive or other adhering means may be applied to the outer edges of the chitosan composition to hold the composition in position during the delivery of the chemical or pharmaceutically active component. Such adherent means may be used alone or in combination with the self-adhering properties of chitosan. Chitosan compositions provide for the local administration of a desired amount of a therapeutic agent.

Other embodiments of the present invention include wound-healing devices configured and produced as biological fasteners, such as threads, sutures and woven sheets. Threads and sutures comprising various embodiments of the chitosan composition provide a biocompatible fastening and suturing function for temporarily treating and sealing an open wound. Additionally, the biological fasteners may include pharmacologically active agents that may assist in the healing and remodeling of the tissue within and around the wound. Advantageously, such fastening and suturing devices may be treated to degrade in vivo at a desired rate. In other embodiments, the chitosan composition is administered directly to an injured area. A chitosan composition of the invention is administered by sprinkling, packing, implanting, inserting or applying or by any other administration means to a site of trauma (e.g., open wound, open fracture, complex wound).

Delivery of Polymeric Compositions

Polymeric compositions (e.g., polymeric sponge, polymeric hydrogel) can be delivered by any method known to the skilled artisan. In one approach, a polymeric composition is locally delivered to a site of trauma in the form of a sponge or hydrogel. Such compositions are useful in combination with NPWT. The film, sponge, or other wound management device can be configured to fit a wound of virtually any size. In another approach, the composition is surgically implanted at a site where promotion of healing and/or treatment or prevention of infection is required. If desired, the polymeric composition is loaded with one or more antibiotics or other biologically active agents by a clinician within the surgical suite where treatment is to be provided. This advantageously allows the polymeric composition to be loaded with a specific agent or combination of agents tailored to the needs of a particular patient at the point at which care is to be provided.

Delivery of Agents via Polymeric Compositions

The invention provides a simple means for delivering biologically active agents (e.g., small compounds, nucleic acid molecules, polypeptides) using a polymeric composition. The polymeric composition is delivered to a subject and the biologically active agent is eluted from the composition in situ. In one embodiment, a chitosan composition is capable of delivering a therapeutic for the treatment of a disease or disorder that requires controlled and/or localized drug delivery over some period of time (e.g., 1, 3, 5, 7 days; 2, 3, 4 weeks; 1, 2, 3, 6, 12 months). Desirably, the chitosan composition comprises an effective amount of one or more antibiotics (e.g., amikacin, daptomycin, vancomycin), growth factors that promote wound healing, small molecules, hemostatic agents (e.g., thrombin and/or fibrinogen), anti-thrombotics (e.g., heparin), or cartilage or bone repair agents. The chitosan composition are administered in the form of solids, sponges, films, hydrogels, or composites (e.g., sponge fragments in a hydrogel matrix).

Preferably, the chitosan composition comprises at least about 1 μg, 25 μg, 50 μg, 100 μg, 250 μg, 500 μg, 750 μg, 1 mg, 5 mg, 10 mg, 25 mg, 50 mg, 75 mg, 100 mg, 200 mg, 250 mg, 300 mg, 400 mg, or 500 mg of an agent (e.g., an antibiotic). In another embodiment, the composition releases at least about 1 μg, 25 μg, 50 μg, 100 μg, 250 μg, 500 μg, 750 μg, 1 mg, 5 mg, 10 mg, 25 mg, 50 mg, 75 mg, 100 mg, 200 mg, 250 mg, 300 mg, 400 mg, or 500 mg of an agent (e.g., an antibiotic) over the course of at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 14, 21, 28, or 35 days. In still another embodiment, the composition comprises at least about 1 μg, 25 μg, 50 μg, 100 μg, 250 μg, 500 μg, 750 μg, 1 mg, 5 mg, 10 mg, 25 mg, 50 mg, 75 mg, 100 mg, 200 mg, 250 mg, 300 mg, 400 mg, or 500 mg of an agent (e.g., an antibiotic) per cm³.

A chitosan "sponge" is prepared using the lyophilized sponges (e.g., as described herein). The lyophilized sponges are neutralized by submerging in sodium hydroxide solution (various concentrations of NaOH may be used). Hydrated sponges are rinsed with water several times before re-freezing for at least 1 hour (-80°C). The frozen sponges are lyophilized again for 36-48 hours. The duration of lyophilization is dependent on lyophilizer and the size of sponge. The "double" lyophilized sponge samples are sterilized via gamma irradiation.

A composite containing chitosan sponge in chitosan gel (a paste or "sponge-in-gel" composite) can be made from a chitosan gel component and a chitosan sponge component. A gel "matrix" component is prepared by dissolving chitosan (e.g., as described herein), filtering particulate, and allowing the solution to de-gas overnight. Chitosan solution is transferred into a container and frozen for at least 1 hour (-80°C). The length of time the chitosan solution is frozen can be adapted to the size of the sponge (e.g., longer freezing time for larger sponges). After freezing, the frozen samples are lyophilized for -48 hours and sterilized via gamma irradiation. The lyophilized sponge is not neutralized and is used as the adhesive "gel" matrix.

A "sponge" component is prepared using the lyophilized sponges (e.g., as described herein). The lyophilized sponges are neutralized by submerging in sodium hydroxide solution (various concentrations of NaOH may be used). Hydrated sponges are rinsed with water several times before re-freezing for at least 1 hour (-80°C). The frozen sponges are lyophilized again for 36-48 hours. The duration of lyophilization is dependent on lyophilizer and the size of sponge. The "double" lyophilized sponge samples are sterilized via gamma irradiation.

To prepare the"sponge-in-gel" composite, a combination of "gel matrix" and

"sponge" components are coarsely ground (e.g., in a standard coffee grinder). The finer components are the single-lyophilized sponge pieces, and the larger components are the double lyophilized sponge pieces. In one embodiment, at least about 25%-95% of the composite is hydrogel component. In another embodiment, at least about 5%-75% of the composite is sponge. The composite is customized based on the adhesiveness required and/or the size of the wound. An increased amount of adhesiveness is desired if the wound is prone to drainage or has increased surface area. In another embodiment, an increased amount of sponge material is desired for a cavity wound. This blended mixture of single- and double- lyophilized chitosan sponge fragments is then hydrated with a solution (antibiotic, saline, antifungal, etc) to form a paste mixture. The resulting paste has a binding "gel" matrix (single-lyophilized sponge component) with larger, dispersed "sponge" fragments throughout the gel (double-lyophilized sponge component). The "sponge-in-gel" composite can be prepared in a short amount of time. In one embodiment, the paste is mixed and is delivered at the point-of-care. The agent is incorporated at the time the composite is hydrated. In one embodiment, the composite is delivered to a site of trauma via a sterile syringe. Advantageously, the composite provides for a complete void fill and prevents migration of the chitosan composition within the wound. This facilitates localized delivery of an agent to the site of trauma. The "gel matrix" typically has greater adherence properties than the sponge portion of the composite. Thus, the amount of "gel matrix" can be increased or decreased based on the needs of the patient. In one embodiment, an increased amount of gel matrix (e.g., greater than about 50%, 70%, 80%, 90%, 95%) is used to increase tissue adherence. In another embodiment, an increase amount of sponge fragments (e.g., greater than about 50%, 70%, 80%, 90%, 95%) to provide for sustained elution of an agent over time. Preferably, the composite provides for the bimodal delivery of an agent. In the first phase, an agent is quickly released from the gel matrix. This first phase of elution typically occurs over the course of hours (e.g., 1, 2, 3, 4, 5, 6 or 12 hours) or days (e.g., 1, 2, 3 days). The second phase of the biomodal elution involves the sustained release of an agent from the sponge portion of the composite. This phase typically occurs over the course of days, or weeks. Desirably, the composite provides for sustained elution of an agent during the course of the composite's degradation. In one embodiment, the composite comprises a non- neutralized gel portion. In another embodiment, the composite comprises a neutralized sponge portion.

Hemostatic Polymeric Compositions

The invention further provides polymeric compositions in the form of a hemostatic matrix (e.g., polymeric sponge, polymeric hydrogels). Such compositions are useful alone or may be used for the delivery of a therapeutic or prophylactic agent delineated herein. Such matrices generally comprise porous compositions formed from chitosan or other polymers (e.g., polyvinyl alcohol - PVA, Poly(lactic-co-glycolic acid) - PLGA, Polyglycolic acid - PGA, Poly(lactic acid) - PLA, cellulose, collagen, fibrin-based wound dressing (with polyethylene glycol), and gelatin). Such compositions are known in the art and are commercially available.

In one embodiment, chitosan sponges are formed by providing a liquid solution of chitosan capable of forming a porous three-dimensionally stable structure. In one

embodiment, a chitosan solution is prepared by dissolving deacetylated chitosan in an acidic solvent. A sponge is formed by casting the solution in a mold to achieve a desired shape. The chitosan solution is then frozen and lyophilized, thereby forming a chitosan sponge. Lyophilization is conducted to reduce the liquid (e.g. water) content of the matrix to less than about 5%, 10%, 20%, 25%, 30%, 40%, 50%, 75%, 80%, 90%, 95%, or 100% by weight. If desired, a second lyophilization step is carried out. This step is strictly optional. Following one or more lyophilizations, the chitosan composition may still include some amount of water. Typically, lypholization removes at least about 70%, 75%, 80%, 90%, 95, or 100% or the original water content of the chitosan composition. Chitosan compositions that retain some moisture may be packaged in sterile foil to maintain such moisture.

In one approach, the chitosan sponge is neutralized, for example, by treatment with a basic solution, re-lyophilized. The sponge matrix is stabilized structurally and remains in a highly dense and compacted state until contacted with a liquid susceptible to absorption by the matrix, for example, body fluids. For medical use, the compacted or compressed sponge is sterilized using any suitable means (e.g., radiation). The device is packaged in sterile packaging for medical use. Sponge elements or other devices of the invention may also contain one or more active therapeutic agents. For example, they include agents that promote clotting (e.g., thrombin and/or fibrinogen). Alternatively or in addition, sponge elements or other devices of the invention include antibiotics and/or growth factors that promote tissue growth and healing.

A chitosan composition is incubated with a therapeutic agent such that the agent is incorporated into the chitosan. This incubation is typically carried out before or during a procedure to treat a subject using methods described herein. Sponge materials of the invention will advantageously be expandable when wetted. Preferably, the sponge has the capacity to expand at least about 10%-100% (10, 20, 30, 40, 50). In other embodiments, a sponge expands by about 200% by volume when wetted to saturation with deionized water, buffer, or an agent of the invention. Preferred sponge materials achieve rapid volume expansions (e.g., when immersed in aqueous solution). Hemostatic sponges are produced in any size required for application to a wound. In one embodiment, the expanded sponge exerts compression on surrounding tissues when implanted or delivers an active agent to the implantation site and surrounding tissue.

Antimicrobial Agents

Staphylococcus aureus, staphylococcus epidermidis, and Pseudomonas aeruginosa are pathogens that are commonly present at musculoskeletal wound sites. S aureus is one cause of osteomyelitis and nongonococcal bacterial arthritis, and is often associated with prosthetic joint infection. The invention provides chitosan compositions useful in treating or preventing infection in a wound, complex wound, open fraction, or other site of trauma. Any antimicrobial agent known in the art can be used in the chitosan compositions of the invention at concentrations generally used for such agents.

Antimicrobial agents useful in chitosan compositions of the invention include but are not limited to antibacterials, antifungals, and antivirals. An antimicrobial agent as used herein is an agent which reduces or stabilizes the survival, growth, or proliferation of a pathogen. Antimicrobial agents include but are not limited to Aztreonam; Chlorhexidine

Gluconate; Imidurea; Lycetamine; Nibroxane; Pirazmonam Sodium; Propionic Acid;

Pyrithione Sodium; Sanguinarium Chloride; Tigemonam Dicholine; Acedapsone;

Acetosulfone Sodium; Alamecin; Alexidine; Amdinocillin; Amdinocillin Pivoxil;

Amicycline; Amifloxacin; Amifloxacin Mesylate; Amikacin; Amikacin Sulfate;

Aminosalicylic acid; Aminosalicylate sodium; Amoxicillin; Amphomycin; Ampicillin;

Ampicillin Sodium; Apalcillin Sodium; Apramycin; Aspartocin; Astromicin Sulfate;

Avilamycin; Avoparcin; Azithromycin; Azlocillin; Azlocillin Sodium; Bacampicillin

Hydrochloride; Bacitracin; Bacitracin Methylene Disalicylate; Bacitracin Zinc;

Bambermycins; Benzoylpas Calcium; Berythromycin; Betamicin Sulfate; Biapenem;

Biniramycin; Biphenamine Hydrochloride; Bispyrithione Magsulfex; Butikacin; Butirosin

Sulfate; Capreomycin Sulfate; Carbadox; Carbenicillin Disodium; Carbenicillin Indanyl

Sodium; Carbenicillin Phenyl Sodium; Carbenicillin Potassium; Carumonam Sodium;

Cefaclor; Cefadroxil; Cefamandole; Cefamandole Nafate; Cefamandole Sodium; Cefaparole; Cefatrizine; Cefazaflur Sodium; Cefazolin; Cefazolin Sodium; Cefbuperazone; Cefdinir;

Cefepime; Cefepime Hydrochloride; Cefetecol; Cefixime; Cefinenoxime Hydrochloride;

Cefmetazole; Cefmetazole Sodium; Cefonicid Monosodium; Cefonicid Sodium;

Cefoperazone Sodium; Ceforanide; Cefotaxime Sodium; Cefotetan; Cefotetan Disodium;

Cefotiam Hydrochloride; Cefoxitin; Cefoxitin Sodium; Cefpimizole; Cefpimizole Sodium; Cefpiramide; Cefpiramide Sodium; Cefpirome Sulfate; Cefpodoxime Proxetil; Cefprozil;

Cefroxadine; Cefsulodin Sodium; Ceftazidime; Ceftibuten; Ceftizoxime Sodium; Ceftriaxone

Sodium; Cefuroxime; Cefuroxime Axetil; Cefuroxime Pivoxetil; Cefuroxime Sodium;

Cephacetrile Sodium; Cephalexin; Cephalexin Hydrochloride, Cephaloglycin; Cephaloridine;

Cephalothin Sodium; Cephapirin Sodium; Cephradine; Cetocycline Hydrochloride;

Cetophenicol; Chloramphenicol; Chloramphenicol Palmitate; Chloramphenicol Pantothenate

Complex; Chloramphenicol Sodium Succinate; Chlorhexidine Phosphanilate; Chloroxylenol;

Chlortetracycline Bisulfate; Chlortetracycline Hydrochloride; Cinoxacin; Ciprofloxacin;

Ciprofloxacin Hydrochloride; Cirolemycin; Clarithromycin; Clinafloxacin Hydrochloride;

Clindamycin; Clindamycin Hydrochloride; Clindamycin Palmitate Hydrochloride; Clindamycin Phosphate; Clofazimine; Cloxacillin Benzathine; Cloxacillin Sodium;

Cloxyquin; Colistimethate Sodium; Colistin Sulfate; Coumermycin; Coumermycin Sodium;

Cyclacillin; Cycloserine; Dalfopristin; Dapsone; Daptomycin; Demeclocycline;

Demeclocycline Hydrochloride; Demecycline; Denofungin; Diaveridine; Dicloxacillin;

Dicloxacillin Sodium; Dihydrostreptomycin Sulfate; Dipyrithione; Dirithromycin;

Doxycycline; Doxycycline Calcium; Doxycycline Fosfatex; Doxycycline Hyclate; Droxacin

Sodium; Enoxacin; Epicillin; Epitetracycline Hydrochloride; Erythromycin; Erythromycin

Acistrate; Erythromycin Estolate; Erythromycin Ethylsuccinate; Erythromycin Gluceptate;

Erythromycin Lactobionate; Erythromycin Propionate; Erythromycin Stearate; Ethambutol Hydrochloride; Ethionamide; Fleroxacin; Floxacillin; Fludalanine; Flumequine; Fosfomycin;

Fosfomycin Tromethamine; Fumoxicillin; Furazolium Chloride; Furazolium Tartrate;

Fusidate Sodium; Fusidic Acid; Gentamicin Sulfate; Gloximonam; Gramicidin; Haloprogin;

HetaciUin; HetaciUin Potassium; Hexedine; Ibafloxacin; Imipenem; Isoconazole; Isepamicin;

Isoniazid; Josamycin; Kanamycin Sulfate; Kitasamycin; Levofuraltadone; Levopropylcillin Potassium; Lexithromycin; Lincomycin; Lincomycin Hydrochloride; Lomefloxacin;

Lomefloxacin Hydrochloride; Lomefloxacin Mesylate; Loracarbef; Mafenide; Meclocycline;

Meclocycline Subsalicylate; Megalomicin Potassium Phosphate; Mequidox; Meropenem;

Methacycline; Methacycline Hydrochloride; Methenamine; Methenamine Hippurate;

Methenamine Mandelate; Methicillin Sodium; Metioprim; Metronidazole Hydrochloride; Metronidazole Phosphate; Mezlocillin; Mezlocillin Sodium; Minocycline; Minocycline

Hydrochloride; Mirincamycin lydrochloride; Monensin; Monensin Sodium; Nafcillin

Sodium; Nalidixate Sodium; Nalidixic Acid; Natamycin; Nebramycin; Neomycin Palmitate;

Neomycin Sulfate; Neomycin Undecylenate; Netilmicin Sulfate; Neutramycin; Nifuradene;

Nifuraldezone; Nifuratel; Nifuratrone; Nifurdazil; Nifurimide; Nifurpirinol; Nifurquinazol; Nifurthiazole; Nitrocycline; Nitrofurantoin; Nitromide; Norfloxacin; Novobiocin Sodium;

Ofloxacin; Ormetoprim; Oxacillin Sodium; Oximonam; Oximonam Sodium; Oxolinic Acid;

Oxytetracycline; Oxytetracycline Calcium; Oxytetracycline Hydrochloride; Paldimycin;

Parachlorophenol; Paulomycin; Pefloxacin; Pefloxacin Mesylate; Penamecillin; Penicillin G

Benzathine; Penicillin G Potassium; Penicillin G Procaine; Penicillin G Sodium; Penicillin V; Penicillin V Benzathine; Penicillin V Hydrabamine; Penicillin V Potassium; Pentizidone

Sodium; Phenyl Aminosalicylate; Piperacillin Sodium; Pirbenicillin Sodium; Piridicillin

Sodium; Pirlimycin Hydrochloride; Pivampicillin Hydrochloride; Pivampicillin Pamoate;

Pivampicillin Probenate; Polymyxin B Sulfate; Porfiromycin; Propikacin; Pyrazinamide;

Pyrithione Zinc; Quindecamine Acetate; Quinupristin; Racephenicol; Ramoplanin; Ranimycin; Relomycin; Repromicin; Rifabutin; Rifametane; Rifamexil; Rifamide; Rifampin;

Rifapentine; Rifaximin; Rolitetracycline; Rolitetracycline Nitrate; Rosaramicin; Rosaramicin

Butyrate; Rosaramicin Propionate; Rosaramicin Sodium Phosphate; Rosaramicin Stearate;

Rosoxacil; Roxarsone; Roxithromycin; Sancycline; Sanfetrinem Sodium; Sarmoxicillin; Sarpicillin; Scopafungin; Sisomicin; Sisomicin Sulfate; Sparfloxacin; Spectinomycin

Hydrochloride; Spiramycin; Stallimycin Hydrochloride; Steffimycin; Streptomycin Sulfate;

Streptonicozid; Sulfabenz: Sulfabenzamide; Sulfacetamide; Sulfacetamide Sodium;

Sulfacytine; Sulfadiazine; Sulfadiazine Sodium; Sulfadoxine; Sulfalene; Sulfamerazine;

Sulfameter; Sulfamethazine; Sulfamethizole; Sulfamethoxazole; Sulfamonomethoxine;

Sulfamoxole; Sulfanilate Zinc; Sulfanitran; Sulfasalazine; Sulfasomizole; Sulfathiazole;

Sulfazamet; Sulfisoxazole; Sulfisoxazole Acetyl; Sulfisoxazole Diolamine; Sulfomyxin;

Sulopenem; Sultamicillin; Suncillin Sodium; Talampicillin Hydrochloride; Teicoplanin;

Temafloxacin Hydrochloride; Temocillin; Tetracycline; Tetracycline Hydrochloride;

Tetracycline Phosphate Complex; Tetroxoprim; Thiamphenicol; Thiphencillin Potassium; Ticarcillin Cresyl Sodium: Ticarcillin Disodium; Ticarcillin Monosodium; Ticlatone;

Tiodonium Chloride; Tobramycin; Tobramycin Sulfate; Tosufloxacin; Trimethoprim;

Trimethoprim Sulfate; Trisulfapyrimidines; Troleandomycin; Trospectomycin Sulfate;

Tyrothricin; Vancomycin; Vancomycin Hydrochloride; Virginiamycin; Zorbamycin;

Difloxacin Hydrochloride; Lauryl Isoquinolinium Bromide; Moxalactam Disodium;

Ornidazole; Pentisomicin; and Sarafloxacin Hydrochloride. In particular embodiments, a chitosan composition comprises daptomycin.

In one preferred embodiment, a polymeric composition of the invention comprises an agent that treats a multidrug resistant bacteria. In one approach, linezolid may be used to treat multi-drug resistant Gram positive bacteria. Linezolid is commercially available under the trade name Zyvox (Pfizer).

In other embodiments, a polymeric composition comprises one or more of the following: Benzalkonium Chloride, Cetylpyridinium Chloride, and Chlorhexidine

Digluconate. In still other embodiments, a chitosan composition comprises one or more of antimicrobials: Polyhexamethylene Biguanide, Octenidine Dihydrochloride, Mild Silver Protein, Povidone Iodine (solution or ointment), Silver Nitrate, Silver Sulfadiazine, Triclosan,

Cetalkonium Chloride, Myristalkonium Chloride, Tigecycline, Lactoferrin,

Quinupristin/dalfopristin, Linezolid, Dalbavancin, Doripenem, Imipenem, Meropenem, and

Iclaprim. In still other embodiments, the polymeric composition comprises an essential oil having antimicrobial properties. Exemplary essential oils include Oregano oil, tea tree oil, mint oil, sandalwood oil, clove oil, nigella sativa oil, onion oil, leleshwa oil, lavender oil, lemon oil, lemon myrtle oil, neem oil, garlic, eucalyptus oil, peppermint oil, cinnamon oil, and thyme oil.

In still other embodiments, the antimicrobial is a fatty acid (e.g., Cis-2-Decenoic

Acid).

Antivirals are agents capable of inhibiting the replication of viruses. Examples of anti-viral agents include but are not limited to l,-D-ribofuranosyl-l,2,4-triazole-3

carboxamide, 9-2-hydroxy-ethoxy methylguanine, adamantanamine, 5-iodo-2'-deoxyuridine, trifluorothymidine, interferon, adenine arabinoside, protease inhibitors, thymidine kinase inhibitors, sugar or glycoprotein synthesis inhibitors, structural protein synthesis inhibitors, attachment and adsorption inhibitors, and nucleoside analogues such as acyclovir, penciclovir, valacyclovir, and ganciclovir.

Antifungal agents useful in chitosan compositions of the invention include fungicidal and fungistatic agents such as, for example, benzoic acid, undecylenic alkanolamide, ciclopirox olamine, polyenes, imidazoles, allylamine, thicarbamates, amphotericin B, butylparaben, clindamycin, econaxole, fluconazole, flucytosine, griseofulvin, nystatin, and ketoconazole. In one preferred embodiment, the antifungal is amphotericin.

In one embodiment, the invention provides polymeric compositions comprising a combination of one or more antimicrobials and antivirals or antifungals.

Growth factors

Growth factors are typically polypeptides or fragments thereof that support the survival, growth, or differentiation of a cell. Such agents may be used to promote wound healing. A chitosan composition described herein can be used to deliver virtually any growth factor known in the art. Such growth factors include but are not limited to angiopoietin, acidic fibroblast growth factors (aFGF) (GenBank Accession No. NP_149127) and basic FGF (GenBank Accession No. AAA52448), bone morphogenic protein (BMP)(GenBank Accession No. BAD92827), vascular endothelial growth factor (VEGF) (GenBank Accession No. AAA35789 or NP_001020539), epidermal growth factor (EGF)(GenBank Accession No. NP_001954), transforming growth factor a (TGF-a) (GenBank Accession No. NP_003227) and transforming growth factor β (TFG-β) (GenBank Accession No. 1109243 A), platelet- derived endothelial cell growth factor (PD-ECGF)(GenBank Accession No. NP_001944), platelet-derived growth factor (PDGF)( GenBank Accession No. 1109245 A), tumor necrosis factor a (TNF- a) (GenBank Accession No. CAA26669), hepatocyte growth factor

(HGF)(GenBank Accession No. BAA14348), insulin like growth factor (IGF)(GenBank Accession No. P08833), erythropoietin (GenBank Accession No. P01588), colony stimulating factor (CSF), macrophage-CSF (M-CSF)(GenBank Accession No. AAB59527), granulocyte/macrophage CSF (GM-CSF) (GenBank Accession No. NP_000749) and nitric oxide synthase (NOS)(GenBank Accession No. AAA36365). In one preferred embodiment, the growth factor is BMP.

Angiogenic Factors

Angiogenesis is a complex multistep process that involves interactions between endothelial cells (EC), pericytes, vascular smooth muscle cells, and stromal cells (e.g., stem cells and parenchymal cells). These interactions occur through secreted factors, such as vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF), basic fibroblast growth factor (bFGF or FGF-2) and angiopoietins, as well as through cell-cell and cell-extracellular matrix (ECM) interactions. Endothelial cell-ECM interactions regulate numerous processes that are critical for angiogenesis, including endothelial cell migration, proliferation, differentiation and apoptosis. Angiogenic processes include network stabilization and remodeling that may involve the recruitment of stromal cells, as well as the pruning of some vessels. In many cases, angiogenesis occurs as a response to hypoxia. A transcription factor called hypoxia-inducible factor, HIFl , has been demonstrated to act as an oxygen sensor whose activity leads to upregulation of VEGF in parenchymal and stromal cells (Semenza, Physiology (Bethesda), 19:176-82, 2004). VEGF is secreted as a homodimer in the form of several heparin-binding and non-heparin-binding splice- variant isoforms; it diffuses through the interstitial space and can bind to the endothelial cell receptors VEGFR1 and VEGFR2, as well as co-receptors such as Neuropilin-1, thus initiating a signal transduction cascade that leads to endothelial cell proliferation and migration. The production of endothelial cell matrix metalloproteinases, MMPs, increases as a result of endothelial cell activation; MMPs are necessary for selectively clipping the capillary basement membrane and the ECM, which constitute physical barriers to endothelial cell migration and capillary sprouting. MMPs and their associated molecules also play a crucial role in uncovering cryptic sites of the ECM proteins, a number of which have been identified as anti- angiogenic (Davis et al., Anat Rec, 268:252-75, 2002; Folkman, Annu Rev Med, 57:1- 18, 2006; Rundhaug, J Cell Mol Med, 9:267-85, 2005; Schenk and Quaranta, Trends Cell Biol, 13:366-75, 2003), and in processing cell-surface receptors (Mott and Werb, Curr Opin Cell Biol, 16:558-64, 2004). Vasculogenesis is the development of new blood vessels originating from stem cells, angioblasts, or other precursor cells, such as endothelial progenitor cells (EPCs). These stem cells can be recruited from bone marrow endogenously or implanted therapeutically.

Analgesics

Chitosan compositions of the invention can be used for the delivery of one or more agents that ameliorate pain, such agents include but are not limited to opioid analgesics (e.g. morphine, hydromorphone, oxymorphone, levorphanol, levallorphan, methadone,

meperidine, fentanyl, codeine, dihydrocodeine, oxycodone, hydrocodone, propoxyphene, nalmefene, nalorphine, naloxone, naltrexone, buprenorphine, butorphanol, nalbuphine or pentazocine; a nonsteroidal antiinflammatory drug (NSAID) (e.g., aspirin, diclofenac, diflusinal, etodolac, fenbufen, fenoprofen, flufenisal, flurbiprofen, ibuprofen, indomethacin, ketoprofen, ketorolac, meclofenamic acid, mefenamic acid, nabumetone, naproxen, oxaprozin, phenylbutazone, piroxicam, sulindac, tolmetin or zomepirac, or a

pharmaceutically acceptable salt thereof; a barbiturate sedative, e.g. amobarbital,

aprobarbital, butabarbital, butabital, mephobarbital, metharbital, methohexital, pentobarbital, phenobartital, secobarbital, talbutal, theamylal or thiopental or a pharmaceutically acceptable salt thereof; a COX-2 inhibitor (e.g. celecoxib, rofecoxib or valdecoxib).

Anti-thrombotic

Chitosan compositions of the invention are also useful for inhibiting, reducing or ameliorating clot formation. In one embodiment, a chitosan composition contains one or more anti-thrombotids (e.g., thrombin, fibrinogen, cumidin, heparin).

Anti-inflammatories

In other embodiments, a chitosan composition is used to deliver an anti-inflammatory agent. Such anti-inflammatory agents include, but are not limited to, Alclofenac;

Alclometasone Dipropionate; Algestone Acetonide; Alpha Amylase; Amcinafal; Amcinafide; Amfenac Sodium; Amiprilose Hydrochloride; Anakinra; Anirolac; Anitrazafen; Apazone; Balsalazide Disodium; Bendazac; Benoxaprofen; Benzydamine Hydrochloride; Bromelains; Broperamole; Budesonide; Carprofen; Cicloprofen; Cintazone; Cliprofen; Clobetasol

Propionate; Clobetasone Butyrate; Clopirac; Cloticasone Propionate; Cormethasone Acetate;

Cortodoxone; Deflazacort; Desonide; Desoximetasone; Dexamethasone Dipropionate;

Diclofenac Potassium; Diclofenac Sodium; Diflorasone Diacetate; Diflumidone Sodium; Diflunisal; Difluprednate; Diftalone; Dimethyl Sulfoxide; Drocinonide; Endrysone;

Enlimomab; Enolicam Sodium; Epirizole; Etodolac; Etofenamate; Felbinac; Fenamole;

Fenbufen; Fenclofenac; Fenclorac; Fendosal; Fenpipalone; Fentiazac; Flazalone; Fluazacort;

Flufenamic Acid; Flumizole; Flunisolide Acetate; Flunixin; Flunixin Meglumine; Fluocortin

Butyl; Fluorometholone Acetate; Fluquazone; Flurbiprofen; Fluretofen; Fluticasone

Propionate; Furaprofen; Furobufen; Halcinonide; Halobetasol Propionate; Halopredone

Acetate; Ibufenac; Ibuprofen; Ibuprofen Aluminum; Ibuprofen Piconol; Ilonidap;

Indomethacin; Indomethacin Sodium; Indoprofen; Indoxole; Intrazole; Isoflupredone

Acetate; Isoxepac; Isoxicam; Ketoprofen; Ketorolac; Lofemizole Hydrochloride;

Lornoxicam; Loteprednol Etabonate; Meclofenamate Sodium; Meclofenamic Acid;

Meclorisone Dibutyrate; Mefenamic Acid; Mesalamine; Meseclazone; Methylprednisolone

Suleptanate; Morniflumate; Nabumetone; Naproxen; Naproxen Sodium; Naproxol;

Nimazone; Olsalazine Sodium; Orgotein; Orpanoxin; Oxaprozin; Oxyphenbutazone;

Paranyline Hydrochloride; Pentosan Polysulfate Sodium; Phenbutazone Sodium Glycerate;

Pirfenidone; Piroxicam; Piroxicam Cinnamate; Piroxicam Olamine; Pirprofen; Prednazate; Prifelone; Prodolic Acid; Proquazone; Proxazole; Proxazole Citrate; Rimexolone; Romazarit;

Salcolex; Salnacedin; Salsalate; Sanguinarium Chloride; Seclazone; Sermetacin; Sudoxicam;

Sulindac; Suprofen; Talmetacin; Talniflumate; Talosalate; Tebufelone; Tenidap; Tenidap

Sodium; Tenoxicam; Tesicam; Tesimide; Tetrydamine; Tiopinac; Tixocortol Pivalate;

Tolmetin; Tolmetin Sodium; Triclonide; Triflumidate; Zidometacin; and Zomepirac Sodium.

Kits

The invention provides kits that include polymeric compositions for use in NPWT. In one embodiment, the kit includes a polymeric composition containing a therapeutic or prophylactic agent that that prevents or treats infection (e.g., an antimicrobial agent) or that promotes healing (e.g., growth factor, anti-inflammatory, clot promoting agent, antithrombotic). In other embodiments, the kit contains a therapeutic polymeric device, such as a chitosan sponge or hydrogel. If desired, the aforementioned chitosan compositions further comprise an agent described herein. In some embodiments, the kit comprises a sterile container which contains a polymeric composition; such containers can be boxes, ampoules, bottles, vials, tubes, bags, pouches, blister-packs, or other suitable container forms known in the art. Such containers can be made of plastic, glass, laminated paper, metal foil, or other materials suitable for holding medicaments.

If desired a composition of the invention is provided together with instructions for using it in a prophylactic or therapeutic method described herein. The instructions will generally include information about the use of the composition for the treatment of a trauma, infection or related disease in a subject in need thereof. The instructions may be printed directly on the container (when present), or as a label applied to the container, or as a separate sheet, pamphlet, card, or folder supplied in or with the container.

The practice of the present invention employs, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology, which are well within the purview of the skilled artisan. Such techniques are explained fully in the literature, such as,

"Molecular Cloning: A Laboratory Manual", second edition (Sambrook, 1989);

"Oligonucleotide Synthesis" (Gait, 1984); "Animal Cell Culture" (Freshney, 1987);

"Methods in Enzymology" "Handbook of Experimental Immunology" (Weir, 1996); "Gene Transfer Vectors for Mammalian Cells" (Miller and Calos, 1987); "Current Protocols in Molecular Biology" (Ausubel, 1987); "PCR: The Polymerase Chain Reaction", (Mullis,

1994); "Current Protocols in Immunology" (Coligan, 1991). These techniques are applicable to the production of the polynucleotides and polypeptides of the invention, and, as such, may be considered in making and practicing the invention. Particularly useful techniques for particular embodiments will be discussed in the sections that follow.

The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the assay, screening, and therapeutic methods of the invention, and are not intended to limit the scope of what the inventors regard as their invention. EXAMPLES

Example 1: Antibiotic delivery by chitosan sponge inhibited bacterial growth and promoted wound healing.

Antibiotic-loaded chitosan sponges were evaluated in a model of negative pressure wound therapy. Surprisingly and unexpectedly, antibiotic-loaded chitosan sponges were effective in decreasing bacteria levels either with or without negative pressure wound therapy, despite skepticism from the medical community to the contrary about such outcomes. This is in contrast to the prevailing opinion of the medical community that with an augmented NPWT technique the antibiotics eluted from the antibiotic sponge will not permeate throughout the wound, but rather will be removed from the wound via suction of the negative pressure device.

In the study, 4 test groups (n=8) were tested under the following conditions:

antibiotic-loaded PMMA beads without vac, antibiotic-loaded PMMA beads with vac, antibiotic-loaded chitosan sponge without vac, antibiotic-loaded chitosan sponge with vac. Chitosan sponges and chitosan paste were loaded with vancomycin (250 mg in 50 mL sterile saline solution) immediately prior to implantation into the wound. The sponges were 4"x6" in size and the paste was equal in mass to the amount of chitosan in a single sponge. Wounds were closed for 42 hrs after the initial debridement and irrigation. The chitosan sponges (not paste) were removed and frozen for further evaluation of degradation and drug release kinetics.

To measure bacterial growth, a bacterial luminescent image was superimposed on the a gray-scale image of the site. This allowed for both the location and intensity, in terms of photon number, of the bacteria to be quantified within the wound. These results are provided at Table 1. The baseline photon count before the debridement and irrigation were similar between the two groups (ABP = 6.89 x 105 + 1.17 x 10 4, Augmented NPWT = 4.99 x 105 + 7.93 x 104). Debridment and irrigation reduced the bacteria levels in the wounds of the ABP and augmented NPWT groups a similar amount (29 + 2% and 30 + 4% of baseline values, respectively) (p = 0.92). The antibiotic bead pouch resulted in much less bacteria within the wounds than the augmented NPWT, 11 + 2% versus 67 + 11%, respectively, (p=0.01) (Figure 1). Table 1

Percent of Baseline Absolute Value

Beads Beads + NPV Chitosan Spoi:Chitosan Spoi Beads Beads + NPV Chitosan Spoi Chitosan Spo

6hr pre 100.0% 100.0%; i oo.o%! 100.0% 3.99E+05 4.50E+05; 5.26E+05 4.50E+05 i AVG 6hr post 32.1 % 30.3%! 26.2%! 29.1 % 1 .26E+05 1.23E+05! 1.41 E+05 1 .33E+05

48hr pre 15.2% 56.7%! 6.4%; 2.4% 6.40E+04 2.24E+05; 4.15E+04 1 .16E+04

6hr pre 0.0% 0.0%; 0.0%^; 0.0% 2.62E+04 5.67E+04; 3.41 E+04 2.11 E+04

; SEM 6hr post 2.0% 4.9%; 3.2%^; 4.4% 8.21 E+03 1.44E+04; 2.30E+04 2.25E+04

48 hr pre 6.6% 14.6%; 4.0%; 1.1% 3.19E+04 4.37E+04; 2.92E+04 6.03E+03

Unadjusted significance testing showing significance of results of Table 1 by inter group comparison is provided at Table 2

Table 2

Thus, vancomycin chitosan sponge either with or without NPWT treatment was statistically significantly better than vancomycin impregnated PMMA beads when used either with or without NPWT treatment.

Qualitative analysis of the spatial distribution of the wounds corroborates these findings. There was much more bacteria within the wounds of the beads with NPWT group (see, e.g., Figures 2A-2C), and the bacteria were concentrated away from wound bead contact points. There was much less bacteria within the wounds of the Sponge with NPWT group (see, e.g., Figure 3A-3B) and the bacteria were generally found in very small clusters throughout the wound.

Further, the systemic levels of antibiotic appear to remain safe. For example, the serum concentration of vancomycin stayed well under the toxic level for humans (20 μg/ml in humans). As shown in Table 3, the sponge group had the highest levels, which is

consistent with the observation that the chitosan sponge releases more of the antibiotic than PMMA beads. TABLE 3. Vancomycin serum concentration ^g/ml)

Sponge with

Bead Pouch Beads with NPWT Sponge Pouch NPWT

6 hours 0.07 + 0.02 0.14 + 0.05 0.43 + 0.12 0.18 + 0.05

24hours 0.07 ± 0.03 0.04 ± 0.02 0.28 ± 0.08 0.38 ± 0.20

The vancomycin concentration recovered from the NPWT canisters was also higher for the sponge group, as shown in Table 4.

TABLE 4. Vancomycin exudate concentration recovered from canister ^g/ml)

Beads with NPWT Sponge with NPWT 6 hours 27 + 3 978 + 155

24 hours 31 + 2 186 + 49

42 hours 27 + 3 97 + 22

Thus, the chitosan system is superior in terms of its ability to release more drug (e.g., an antibiotic) to the patient.

Example 2: Chitosan paste compositions comprising antibiotics inhibited bacterial growth and promoted wound healing.

Antibiotic-loaded chitosan paste and antibiotic-loaded chitosan sponges were evaluated in a model of negative pressure wound therapy. Surprisingly and unexpectedly, antibiotic-loaded chitosan paste and antibiotic-loaded chitosan sponges were effective in decreasing bacteria levels either with or without negative pressure wound therapy, despite skepticism from the medical community to the contrary about such outcomes. This is in contrast to the prevailing opinion of the medical community that with an augmented NPWT technique the antibiotics eluted from the antibiotic sponge will not permeate throughout the wound, but rather will be removed from the wound via suction of the negative pressure device.

To measure bacterial growth, a bacterial luminescent image was superimposed on the a gray-scale image of the site. This allowed for both the location and intensity, in terms of photon number, of the bacteria to be quantified within the wound. These results are provided at Table 5. In this experiment, the animal treated with beads and NPWT died before conclusion of the experiment. However, similarly infected animals in groups treated with chitosan paste and NPWT, chitosan sponge and NPWT, and chitosan sponge alone showed significant reductions in bacterial levels 48 hrs after inoculation (15%, 7%, and 3%, respectively). Thus, vancomycin-loaded chitosan paste and sponge with or without NPWT treatment significantly reduced bacteria levels in this wound model.

Table 5

Goat Date Image Treatment Total Pet

06.20.1 1 Background — 8.03E+02

720 06.20.1 1 6hr pre Beads+NPWT 6.32E+05 100%

06.20.1 1 6hr post Beads+NPWT 3.25E+05 51 %

.... 06.21 .1 1 Background — dead n/a

17 48hr pre Beads+NPWT in cage n/a

06.21 .1 1 Background 7.29E+02

17 06.21 .1 1 6hr pre Chitosan Paste+NPWT 5.77E+05 100%

17 06.21 .1 1 6hr post Chitosan Paste+NPWT 5.01 E+04 9%

.... 06.23.1 1 Background 1 .00E+03

17 06.23.1 1 48hr pre Chitosan Paste+NPWT 8.49E+04 15%

06.21 .1 1 Background 7.74E+02

1 181 9 06.21 .1 1 6hr pre Chitosan Sponge+NPWT 4.83E+05 100%

1 181 9 06.21 .1 1 6hr post Chitosan Sponge+NPWT 4.14E+04 9%

06.23.1 1 Background 9.55E+02

1 1819 06.23.1 1 48hr pre Chitosan Sponge+NPWT 3.24E+04 7%

06.21 .1 1 Background 7.45E+02

18 06.21 .1 1 6hr pre Chitosan Sponge 4.84E+05 100%

18 06.21 .1 1 6hr post Chitosan Sponge 1 .61 E+05 33%

06.23.1 1 Background 9.88E+02

18 06.23.1 1 48hr pre Chitosan Sponge 1 .45E+04 3%

The results described herein were obtained using the following methods and materials.

Wound Creation

All animals were fasted prior to surgical procedures. After adequate anesthesia utilizing both general anesthetic and epidural injection, a complex, contaminated

musculoskeletal wound was created on the hindlimb of 32 castrated, adult male Boer goats (Capra hircus). As previously described, a 35 cm trapezoidal portion of skin and fascia covering the anterior tibia was removed. After the anterior tibia and musculature was exposed, a portion of the periosteum was removed, leaving behind a 6-mm strip laterally. A 10-mm cortical defect was created in the metaphyseal region of the proximal tibia using a core reamer. Approximately 13 g of muscle was then removed from the tibialis anterior with bovie electrocautery to maintain hemostasis and a freeze injury was performed to a portion of the remaining muscle by applying a 1 cm x 4 cm metal bar, cooled in liquid nitrogen, for two iterations of 30 seconds. Finally, a thermal injury was performed to all exposed muscle, fascia and periosteum with bovie electrocautery; thus rendering a complex musculoskeletal wound (see, e.g., Figure 4).

This technique resulted in a reproducible complex musculoskeletal wound intended to mimic an open fracture without the need for skeletal stabilization. Following creation of the wound, the wound was contaminated with 1 mL of > 10 cfu/mL Staphylococcusaureus (Xenogen 29; Caliper Life Science, Hopkinton, MA), which was spread evenly over the wound surface. These bacteria are genetically engineered to emit photons, allowing for quantification with a photon-counting camera system.

After surgery, the goats were recovered in their pens and allowed activity ad libitum for 6 hours. The goats were re-anesthetized and placed supine on an operating table in a custom, light-free imaging chamber. As described previously, a photon counting camera (Charge Couple Device (CCD) Imaging System Model C2400, Hamamatsu Photonics, Inc, Hamamatsu-City, Japan) was used to capture the quantitative and spatial distribution of the bacteria within the wound (see, e.g., Figure 5). After collection of baseline luminescent data, standard debridement and irrigation was performed with 9 L of normal saline using gravity flow low-pressure irrigation. The imaging sequence was then repeated to obtain post debridement and irrigation data.

For animals receiving treatment with beads, two strands of eight antibiotic- impregnated beads (approximately 258 mg of vancomycin) were placed within the wound bed. For animals receiving treatment with sponges or paste, chitosan sponges and chitosan paste were loaded with vancomycin (250 mg in 50 mL sterile saline solution) immediately prior to implantation into the wound. The sponges were 4"x6" in size and the paste was equal in mass to the amount of chitosan in a single sponge. The wounds in the groups without NPWT were sealed with a semipermeable membrane; the augmented NPWT groups received a standard NPWT dressing. The goats were returned to their cages and were allowed water, food, and activity ad libitum. The augmented NPWT group had the device suspended 5 feet above the floor to prevent the animals from tampering with it. The animals were euthanized at 48 hours post injury, and the bacteria within the wound were quantified. Sponge Preparation

Chitosan sponges were prepared as follows. In one approach, chitosan sponges were prepared by dissolving 4.5 grams (g) of chitosan into 295.5 milliliters (ml) of 1% (v/v) acidic solvent (lactic and/or acetic acids). The chitosan was 71% deacetylated (DDA) acquired from Primex (Siglufjordur, Iceland). In another approach, a chitosan solution was prepared by dissolving 5.0 grams (g) of chitosan into 500 milliliters (ml) of 1% (v/v) acidic solvent. The chitosan used was 61 and 71% deacetylated (DDA) from Primex (Iceland). 25 ml of aqueous chitosan was cast into aluminum dishes and frozen for one hour at -80 C. Samples were lyophilized for 48 hours. Sponges were neutralized in sodium hydroxide and washed in distilled water until pH-neutral. Samples were re-frozen and re-lyophilized before sterilization. The sponges were sterilized using low-dose gamma irradiation (25-32 kGy).

In a preferred approach adopted for use in the goat wound treatment model, 2.5 grams of chitosan was dissolved in 247.5 ml of 1 (v/v)% blended acid solvent. The mixture contained 75%/25% lactic to acetic acid. This mixture was stirred for 4-6 hours at maximum allowable speed on a stir plate. The chitosan solution was filtered to remove undissolved chitosan. 25 ml of chitosan solution was pipetted into small aluminum weigh dishes (6 mm diameter). Each dish was frozen at -80 C freezer for 1 hour. The frozen samples were then placed into a freeze-dryer and lyophilize for 48 hours. Sponge samples were neutralized in 0.175 M NaOH- solution for -30-40 seconds. The sponges were rinsed repeatedly in containers filled with distilled water and pH changes were monitored until the rinsing water was neutral in pH. The re-hydrated sponges were then frozen in a -80 C freezer for 1 hour and re-lyophilized for 24-48 hours. The sponges were then sterilized using low-dose gamma irradiation (25-32 kGy).

A composite containing chitosan sponge in chitosan gel (a "sponge-in-gel" composite) can be made from a chitosan gel component and a chitosan sponge component. A gel "matrix" component is prepared by dissolving chitosan (e.g., as described herein), filtering particulate, and allowing the solution to de-gas overnight. Chitosan solution is transferred into a container and frozen for at least 1 hour (-80°C). The length of time the chitosan solution is frozen can be adapted to the size of the sponge (e.g., longer freezing time for larger sponges). After freezing, the frozen samples are lyophilized for -48 hours and sterilized via gamma irradiation. The lyophilized sponge is not neutralized and is used as the adhesive "gel" matrix.

A "sponge" component is prepared using the lyophilized sponges (e.g., as described herein). The lyophilized sponges are neutralized by submerging in sodium hydroxide solution (various concentrations of NaOH may be used). Hydrated sponges are rinsed with water several times before re-freezing for at least 1 hour (-80°C). The frozen sponges are lyophilized again for 36-48 hours. The duration of lyophilization is dependent on lyophilizer and the size of sponge. The "double" lyophilized sponge samples are sterilized via gamma irradiation.

To prepare the"sponge-in-gel" composite, a combination of "gel matrix" and

"sponge" components are coarsely ground (e.g., in a standard coffee grinder). The finer components are the single-lyophilized sponge pieces, and the larger components are the double lyophilized sponge pieces. In one embodiment, at least about 25%-95% of the composite is hydrogel component. In another embodiment, at least about 5%-75% of the composite is sponge. The composite is customized based on the adhesiveness required and/or the size of the wound. An increased amount of adhesiveness is desired if the wound is prone to drainage or has increased surface area. In another embodiment, an increased amount of sponge material is desired for a cavity wound. This blended mixture of single- and double- lyophilized chitosan sponge fragments is then hydrated with a solution (antibiotic, saline, antifungal, etc) to form a paste mixture. The resulting paste has a binding "gel" matrix (single-lyophilized sponge component) with larger, dispersed "sponge" fragments throughout the gel (double-lyophilized sponge component). The "sponge-in-gel" composite can be prepared in a short amount of time. In one embodiment, the paste is mixed and is delivered at the point-of-care. The agent is incorporated at the time the composite is hydrated. In one embodiment, the composite is delivered to a site of trauma via a sterile syringe.

Advantageously, the composite provides for a complete void fill and prevents migration of the chitosan composition within the wound. This facilitates localized delivery of an agent to the site of trauma. The "gel matrix" typically has greater adherence properties than the sponge portion of the composite. Thus, the amount of "gel matrix" can be increased or decreased based on the needs of the patient. In one embodiment, an increased amount of gel matrix (e.g., greater than about 50%, 70%, 80%, 90%, 95%) is used to increase tissue adherence. In another embodiment, an increase amount of sponge fragments (e.g., greater than about 50%, 70%, 80%, 90%, 95%) to provide for sustained elution of an agent over time. Preferably, the composite provides for the bimodal delivery of an agent. In the first phase, an agent is quickly released from the gel matrix. This first phase of elution typically occurs over the course of hours (e.g., 1, 2, 3, 4, 5, 6 or 12 hours) or days (e.g., 1, 2, 3 days). The second phase of the biomodal elution involves the sustained release of an agent from the sponge portion of the composite. This phase typically occurs over the course of days, or weeks. Desirably, the composite provides for sustained elution of an agent during the course of the composite's degradation. In one embodiment, the composite comprises a non- neutralized gel portion. In another embodiment, the composite comprises a neutralized sponge portion.

Data Analysis

Gray-scale images were first obtained. The bacterial luminescent picture was then superimposed on the gray-scale image. This allowed for both the location and intensity, in terms of photon number, of the bacteria to be quantified within the wound. Photon counts at each time point were compared to the baseline photon counts (six hour pre-debridement and irrigation).

Data is reported as the mean + standard deviation. All data were analyzed using oneway analysis of variance with repeated measures using SAS statistical software (SAS Institute, Cary, NC) with significance set at p < 0.05.

Other Embodiments

From the foregoing description, it will be apparent that variations and modifications may be made to the invention described herein to adopt it to various usages and conditions. Such embodiments are also within the scope of the following claims.

The recitation of a listing of elements in any definition of a variable herein includes definitions of that variable as any single element or combination (or subcombination) of listed elements. The recitation of an embodiment herein includes that embodiment as any single embodiment or in combination with any other embodiments or portions thereof.

This application may be related to U.S. Patent Application Ser. No. 13/256,585, filed September 14, 2011, which is a U.S. national phase application, pursuant to 35 U.S.C. §371, of PCT/US2010/027481, filed on March, 16, 2010, which claims the benefit of the following U.S. Provisional Application Ser. Nos.: 61/160,539, filed March 16, 2009, 61/171,805, filed April 22, 2009, and 61/227,606, filed July 22, 2009; the entire contents of each of which are incorporated herein by this reference.

All patents and publications mentioned in this specification are herein incorporated by reference to the same extent as if each independent patent and publication was specifically and individually indicated to be incorporated by reference.

Claims

What is claimed is:

1. A method for treating a wound, the method comprising:

(a) implanting a biodegradable polymeric composition into the wound;

(b) sealably covering the wound; and

(c) applying negative pressure to the wound.

2. The method of claim 1, wherein the composition comprises an effective amount of at least one agent.

3. The method of claim 1, wherein the polymeric composition is in the form of a sponge, hydrogel, paste, dressing, plug, mesh, strip, suture, film or combinations thereof.

4. The method of claim 1, wherein the sponge, paste, or hydrogel comprises polyvinyl alcohol - PVA, Poly(lactic-co-glycolic acid) - PLGA, Polyglycolic acid - PGA, Poly(lactic acid) - PLA, cellulose, chitosan, collagen, fibrin-based wound dressing (with polyethylene glycol), and gelatin.

5. The method of claim 1, wherein the wound is selected from the group consisting of an fracture, open fracture, complex wound, surgical site, closed wound, chronic wound, acute wound, diabetic ulcer, diabetic wound, and burns.

6. The method of any one of claims 1-5, wherein the composition is a chitosan sponge, paste, or hydrogel.

7. The method of any one of claims 1-6, wherein the chitosan sponge, paste, or hydrogel degrades within 1-35 days in vivo.

8. The method of any one of claims 1-6, wherein the chitosan has a uniform degree of deacetylation of at least about 51%, wherein the water content is about 0-90%,

9. The method of any one of claims 1-7, wherein the chitosan is treated with an acid selected from the group consisting of acetic, citric, oxalic, proprionic, ascorbic, hydrochloric, formic, salicylic, and lactic acids.

10. The method of any one of claims 1-9, wherein the chitosan degree of deacetylation is at least about 61, 71, or 80 percent.

11. The method of any one of claims 1-10, wherein the composition is 80% deacetylated chitosan treated with lactic or acetic acid.

12. The method of any one of claims 1-11, wherein the chitosan composition is 61DDA or 71DDA chitosan that is neutralized with 0.175 or 0.5M NaOH.

13. The method of any one of claims 1-12, wherein the chitosan composition provides for the long term release of the agent.

14. The method of any one of claims 1-13, wherein the agent is one or more of an antimicrobial agent, angiogenic factor, growth factor, analgesic, anti-inflammatory, hemostatic agent, and anti-thrombotic.

15. The method of claim 14, wherein the antimicrobial agent is selected from the group consisting of anti-bacterial, anti-viral, and anti-fungal agents.

16. The method of claim 14, wherein the antimicrobial agent is an antibiotic selected from the group consisting of daptomycin, vancomycin, gentamicin, tobramycin, and amikacin.

17. The method of claim 14, wherein the effective amount of the agent is sufficient to reduce the survival or proliferation of a bacterial cell.

18. The method of claim 17, wherein the bacterial cell is Pseudomonas aeruginosa or Staphylococcus aureus.

19. A system for treating a wound, the system comprising:

(a) a polymeric composition in the form of a biodegradable sponge, paste or hydrogel;

(b) a sealable wound dressing comprising an opening for draining the wound; and

(c) a vacuum pump connected to the opening.

20. The system of claim 19, wherein the composition comprises an effective amount of at least one agent.

21. The system for claim 19, further comprising a monitoring system for automatically adjusting the vaccum.

22. The system of claim 19, wherein the sponge, paste, or hydrogel comprises a polymer selected from the group consisting of polyvinyl alcohol - PVA, Poly(lactic-co-glycolic acid) - PLGA, Polyglycolic acid - PGA, Poly(lactic acid) - PLA, cellulose, chitosan, collagen, fibrin-based wound dressing (with polyethylene glycol), and gelatin.

23. The system of claim 19, wherein the agent is selected from the group consisting of antimicrobial agent, angiogenic factor, growth factor, analgesic, anti-inflammatory, hemostatic agent, and anti-thrombotic.

24. A kit for treating a wound, the kit comprising a polymer composition; a wound dressing for use under negative pressure; and instructions for use in the method of claim 1.

25. The kit of claim 24, wherein the sponge, paste, or hydrogel comprises polyvinyl alcohol - PVA, Poly(lactic-co-glycolic acid) - PLGA, Polyglycolic acid - PGA, Poly(lactic acid) - PLA, cellulose, chitosan, collagen, fibrin-based wound dressing (with polyethylene glycol), and gelatin.