US20190307904A1 - Modified wound dressings - Google Patents

Modified wound dressings Download PDF

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US20190307904A1
US20190307904A1 US16/090,103 US201716090103A US2019307904A1 US 20190307904 A1 US20190307904 A1 US 20190307904A1 US 201716090103 A US201716090103 A US 201716090103A US 2019307904 A1 US2019307904 A1 US 2019307904A1
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wound
dressing material
mmp
polymer
canceled
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Lucy Ballamy
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Convatec Technologies Inc
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Convatec Technologies Inc
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Definitions

  • Embodiments described herein generally relate to wound healing, and in particular to compositions and methods for the detection and treatment of wounds.
  • Wound healing is a complex dynamic process that results in the restoration of anatomic continuity and function: an ideally healed wound is one that has returned to normal anatomic structure, function, and appearance.
  • Chronic wounds include, e.g., venous leg ulcers, diabetic foot ulcers and pressure sores (Krasner et al., Chronic Wound Care: A Clinical Source Book for Healthcare Professionals, HMP Communications, 2001).
  • Patients with chronic wounds require a great deal of care and the wound often leads to a reduction in quality of life; a chronic wound can become a problem that some patients must deal with throughout the rest of their life.
  • Patient co-morbidities can also have a significant effect on the wound healing process, limiting and even halting the process as well as being contributing factors to the loss of quality of life.
  • Factors which can lead to a wound being difficult to heal include pathophysiological issues, infection by microorganisms, presence of non-viable tissue, poor tissue perfusion, chronic inflammatory conditions and other underlying conditions such as diabetes (Bowler et al., Annals of Medicine 2002, 34, 419-427).
  • chronic wounds are colonized by bacterial flora and/or pathogens such as fungi and viruses, in which case they may become infected.
  • Infection of wounds by bacteria delays the healing process, since bacteria produce enzymes and toxins and also compete for nutrients and oxygen with macrophages and fibroblasts whose activities are essential for the healing of the wound. Infection is therefore a manifestation of a disturbed host/bacteria equilibrium in favor of the invading bacteria. This elicits a systemic septic response, and also inhibits the multiple processes involved in wound healing. The granulation phase of healing will only begin after the infection has subsided.
  • the inflammatory phase is particularly important to the wound healing process, wherein biochemical reactions at the wound situs facilitate healing but also cause tissue breakdown due to production of excess proteases.
  • proteases play an important role in breaking down dead tissue, in excess, they also have a detrimental effect on viable tissue, cause additional inflammation.
  • MMP matrix metalloproteases
  • elastase elastase
  • cathepsin G elastase
  • Elevated protease activity appears to be responsible for delaying wound repair and may be predictive of wound infection.
  • ECM extracellular matrix
  • MMP excess protease
  • Increased protease activity also leads to degradation of growth factors, thus inhibiting the healing process. Accordingly, infection and other problems are exacerbated in chronic wounds and the wound remains difficult to treat (Yager et al., Wound Repair Regen 1997, 5, 23-32; Widgerow et al., Wound Repair Regen 2011, 19, 287-291).
  • the technology disclosed herein provides for compositions and methods of detecting infected and/or chronic wounds.
  • the disclosed technology improves upon exiting assays by: increasing the sensitivity, precision and specificity of detection of infected wounds; providing for the ability of qualitative and quantitative measurements; and, increasing the speed of detection of infected wounds in situ and in real-time.
  • the assays and methods described herein are partly based on the use of specific reagents that detect biomarkers and/or probes which are present in infected or chronic wounds.
  • the detection process may involve use of reagents that are specific to the markers present in infected wounds but not non-infected or non-chronic wounds and the detection step may involve qualitative or quantitative measurements of the signal(s) that are generated when the probe is acted upon by the marker.
  • the probes preferably comprise modified enzyme substrates that are specific to the enzyme, which generate signals that may be optionally amplified. This greatly improves efficiency and specificity of detection.
  • a plurality of detection probes, each specific for one or more targets, e.g., enzymes that are specific to the wounds, may be employed.
  • the experimental results disclosed herein confirm that the novel probes and the assay techniques based thereon are capable of detecting and characterizing various types of wounds.
  • the reagents of the disclosed technology may be used together with therapeutic molecules such as antibiotics, antifungal agents, etc. to monitor and evaluate treatment and management of chronic wounds.
  • a wound-dressing material comprising a compound of Formula I comprising the structure M-L-R, wherein M is a gel-forming polymer; R is a reporter molecule; and L is a linker that is either absent or present, and L, when present, connects M and R.
  • a compound comprising the structure M-R, wherein M is a gel-forming polymer and R is a reporter molecule.
  • a compound comprising the structure M-R, wherein M is a gel-forming polymer and R is a reporter molecule and wherein M is covalently or non-covalently conjugated with R.
  • a wound-dressing material comprising a compound of Formula I comprising the structure M-L-R, wherein M is a gel-forming polymer; R is a reporter molecule; and L is a linker that is covalently or non-covalently conjugated, independently of each other, with M and R.
  • a wound-dressing material comprising a compound of Formula I comprising the structure M-L-R, wherein M is a gel-forming polymer; R is a reporter molecule; and L is a linker, wherein the reporter R comprises an enzyme substrate.
  • a wound-dressing material comprising a compound of Formula I comprising the structure M-L-R, wherein M is a gel-forming polymer; R is a reporter molecule; and L is a linker, wherein the reporter R comprises an enzyme substrate which is a sugar, a polysaccharide, a nucleic acid, an amide, a peptide, a protein, a lipid, or a derivative thereof or a combination thereof.
  • the substrate is a sugar, a polysaccharide, an amide, a peptide or a protein, or a derivative thereof.
  • the substrate is a peptide substrate (PEP) comprising an amino acid or a peptide comprising a plurality of amino acids.
  • a wound-dressing material comprising a compound of Formula I comprising the structure M-L-R, wherein M is a gel-forming polymer; R is a reporter molecule; and L is a linker that is either absent or present, and L, when present, connects M and R, wherein M is selected from cellulose, carboxymethylcellulose, pectin, alginate, chitosan, hyaluronic acid, polysaccharide, or gum-derived polymer, or a derivative thereof or any mixture or a combination thereof.
  • the polymer is carboxymethylcellulose (CMC) or a salt thereof.
  • a wound-dressing material comprising a compound of Formula I comprising the structure M-L-R, wherein M is a gel-forming polymer; R is a reporter molecule; and L is a linker that is either absent or present, and L, when present, connects M and R, wherein M comprises about 200 to about 4000 monomeric units. Particularly under this embodiment, M comprises about 500 to about 2000 monomeric units.
  • a wound-dressing material comprising a compound of Formula I comprising the structure M-L-R, wherein M is a gel-forming polymer; R is a reporter molecule; and L is a linker that is either absent or present, and L, when present, connects M and R, wherein L comprises a monomer or a neutral polymer which is an ethoxylated polyhydric alcohol, a polyvinyl pyrrolidone polymer, a polypropylene, a polyalkylene glycol, a polyamine or an ether, an amide, or an ester thereof.
  • L comprises 1-10 monomeric units of an ethoxylated polyhydric alcohol, a polyvinyl pyrrolidone polymer, a polypropylene, a polyalkylene glycol, a polyamine or an ether, an amide, or an ester thereof. More specifically under this embodiment, L comprises at least one polypropylene glycol subunit.
  • a wound-dressing material comprising a compound of Formula I comprising the structure M-L-R, wherein M is a gel-forming polymer; R is a reporter molecule; and L is a linker that is either absent or present, and L, when present, connects M and R, wherein R comprises a detectable label.
  • the detectable label is selected from the group consisting of a luminescent molecule, a chemiluminescent molecule, a fluorochrome, a fluorescent quenching agent, a lipid, a colored molecule, a radioisotope, a scintillant, biotin, avidin, streptavidin, protein A, protein G, an antibody or a fragment thereof, a polyhistidine, Ni2+, a Flag tag, a myc tag, a heavy metal, and an enzyme.
  • a wound-dressing material comprising a compound of Formula I comprising the structure M-L-R, wherein M is a gel-forming polymer; R is a reporter molecule; and L is a linker that is either absent or present, and L, when present, connects M and R, wherein R comprises a fluorescent molecule selected from the group consisting of fluorescein, rhodamine, tetramethylrhodamine, R-phycoerythrin, Cy-3, Cy-5, Cy-7, Texas Red, Phar-Red, allophycocyanin (APC), fluorescein amine, eosin, dansyl, umbelliferone, 5-carboxyfluorescein (FAM), 2′7′-dimethoxy-4′5′-dichloro-6-carboxyfluorescein (JOE), 6 carboxyrhodamine (R6G), N,N,N′,N′-tetramethyl-6-carboxyrh
  • a wound-dressing material comprising a compound of Formula I comprising the structure M-L-R, wherein M is a gel-forming polymer; R is a reporter molecule; and L is a linker that is either absent or present, and L, when present, connects M and R, wherein R comprises a detectable label and a quencher molecule.
  • the reporter comprising the quencher molecule is activated by an enzyme or a product thereof
  • a wound-dressing material comprising a compound of Formula I comprising the structure M-L-R, wherein M is a gel-forming polymer; R is a reporter molecule; and L is a linker that is either absent or present, and L, when present, connects M and R, wherein the reporter molecule or a portion thereof is released upon interaction with an enzyme.
  • the reporter molecule comprises a detectable label which is released upon interaction with the enzyme.
  • a wound-dressing material comprising a compound of Formula I comprising the structure M-L-R, wherein M is a gel-forming polymer; R is a reporter molecule; and L is a linker that is either absent or present, and L, when present, connects M and R, wherein the reporter molecule comprises a substrate that is specific for a wound-specific enzyme, which forms a product when acted upon by the enzyme.
  • the wound-specific enzyme is a protease.
  • the reporter comprises a substrate that is specific for a wound-specific enzyme selected from the group consisting of MMP-1 (collagenase), MMP-2 (gelatinase A), MMP-3 (stomelysin 1), MMP-8 (neutrophil collagenase), MMP-9 (gelatinase B), human neutrophil elastase (HNE), cathepsin G, urokinase-type plasminogen activator (uPA), and lysozyme.
  • the reporter comprises a substrate that is specific for MMP-2 and MMP-9 or a combination thereof.
  • composition comprising a carrier and a wound-dressing material comprising a compound of Formula I comprising the structure M-L-R, wherein M is a gel-forming polymer; R is a reporter molecule; and L is a linker that is either absent or present, and L, when present, connects M and R.
  • the composition is a pharmaceutical composition. More particularly under this embodiment, the pharmaceutical composition comprises an antibiotic compound or a wound-healing peptide.
  • the antibiotic is selected from the group consisting of (3-lactams, fluoroquinolones, aminoglycosides, tetracyclines, glycylcyclines and polymyxins and/or the wound-healing peptide is fibroblast growth factor (FGF) or platelet derived growth factor (PDGF).
  • FGF fibroblast growth factor
  • PDGF platelet derived growth factor
  • composition comprising an article comprising a wound dressing material as hereinbefore described.
  • a method of diagnosing a status of a wound in a subject in need thereof comprising, contacting the wound with the wound dressing material as hereinbefore described to permit conversion of the reporter molecule into a detectable signal and detecting the signal.
  • the conversion of the reporter molecule into a detectable signal is carried out by a wound-specific protease, e.g., a wound specific protease selected from the group consisting of MMP-1 (collagenase), MMP-2 (gelatinase A), MMP-3 (stomelysin 1), MMP-8 (neutrophil collagenase), MMP-9 (gelatinase B), human neutrophil elastase (HNE), cathepsin G, urokinase-type plasminogen activator (uPA), and lysozyme.
  • the method comprises diagnosing a chronic wound or an infected wound.
  • a method of diagnosing a status of a wound in a subject in need thereof comprising, contacting the wound with the wound dressing material as hereinbefore described to permit conversion of the reporter molecule into a detectable signal and detecting the signal; assessing a parameter which is an activity or level of a wound-specific enzyme in the wound; comparing the parameter to a threshold level; and making a determination that the wound is chronic or infected if the level of the parameter in the wound is higher than the threshold level.
  • the parameter is an amount or activity of a wound-specific protease is selected from the group consisting of MMP-1 (collagenase), MMP-2 (gelatinase A), MMP-3 (stomelysin 1), MMP-8 (neutrophil collagenase), MMP-9 (gelatinase B), human neutrophil elastase (HNE), cathepsin G, urokinase-type plasminogen activator (uPA), and lysozyme.
  • MMP-1 collagenase
  • MMP-2 gelatinase A
  • MMP-3 stomelysin 1
  • MMP-8 neutral collagenase
  • MMP-9 gelatinase B
  • HNE human neutrophil elastase
  • cathepsin G cathepsin G
  • uPA urokinase-type plasminogen activator
  • lysozyme lysozyme.
  • the diagnostic method is performed in situ.
  • a method of treating a wound in a subject in need thereof comprising, contacting the wound with the wound dressing material as hereinbefore described.
  • the dressing material is topically or dermally applied at the site of the wound.
  • a method for making a compound of Formula I according to the foregoing, wherein L is absent comprising, conjugating the gel-forming polymer M with the reporter region R, wherein M and R are each, individually, as described previously.
  • the gel-forming polymer M is conjugated to the reporter region R via a covalent linkage selected from the group consisting of a peptide linkage, a glycosidic linkage, an ester linkage, an oxyester linkage, an amide linkage, an amido linkage, an oxyamido linkage, an ether linkage, a sulfonyl linkage, a sulfinyl linkage, a sulfonamide linkage, an alkoxy linkage, an alkylthio linkage, an alkylamino linkage, or a combination thereof.
  • the gel-forming polymer M is conjugated to the reporter region R via a glycosidic linkage or a peptide linkage.
  • a method for making a compound of Formula I according the foregoing, wherein L is present comprising, conjugating the gel-forming polymer M with the linker L to generate a precursor molecule M-L; conjugating the precursor molecule M-L to a reporter region R, wherein M, L and R are each, individually, as described previously.
  • the gel-forming polymer M is conjugated to linker L and/or the linker L is conjugated to the reporter region R via a covalent linkage selected from the group consisting of an ester linkage, an oxyester linkage, an amide linkage, an amido linkage, an oxyamido linkage, an ether linkage, a sulfonyl linkage, a sulfinyl linkage, a sulfonamide linkage, an alkoxy linkage, an alkylthio linkage, an alkylamino linkage, or a combination thereof.
  • a covalent linkage selected from the group consisting of an ester linkage, an oxyester linkage, an amide linkage, an amido linkage, an oxyamido linkage, an ether linkage, a sulfonyl linkage, a sulfinyl linkage, a sulfonamide linkage, an alkoxy linkage, an alkylthio
  • a method for making a compound of Formula I according to the foregoing, wherein L is present comprising, conjugating the linker L with the reporter R to generate a precursor molecule L-R; conjugating the precursor molecule L-R to a gel-forming polymer M, wherein M, L and R are each, individually, as described previously.
  • the gel-forming polymer M is conjugated to linker L and/or the linker L is conjugated to the reporter region R via a covalent linkage selected from the group consisting of an ester linkage, an oxyester linkage, an amide linkage, an amido linkage, an oxyamido linkage, an ether linkage, a sulfonyl linkage, a sulfinyl linkage, a sulfonamide linkage, an alkoxy linkage, an alkylthio linkage, an alkylamino linkage, or a combination thereof.
  • a covalent linkage selected from the group consisting of an ester linkage, an oxyester linkage, an amide linkage, an amido linkage, an oxyamido linkage, an ether linkage, a sulfonyl linkage, a sulfinyl linkage, a sulfonamide linkage, an alkoxy linkage, an alkylthio
  • wound dressing materials comprising a compound comprising the structure M-L-PEP (Formula II), wherein M is a gel-forming polymer; PEP is a peptide and at least one amino acid; and L is a linker that is either absent or present, and L, when present, connects M and PEP.
  • M is a gel-forming polymer
  • PEP is a peptide and at least one amino acid
  • L is a linker that is either absent or present, and L, when present, connects M and PEP.
  • wound dressing materials selected from the group consisting of:
  • FIG. 1 shows quantitation of enzyme efficacy of Polymer 12 using a fluorescence-based study.
  • FIG. 2 shows images of Polymer 12 samples used in the fluorescence-based study of FIG. 1 .
  • FIG. 2A shows samples as viewed under ambient light.
  • FIG. 2B shows samples as viewed under UV light.
  • FIG. 2C shows the labeling of individual samples.
  • FIG. 3 shows quantitation of enzyme efficacy of Polymer 17 (pretreated with PBS) using a fluorescence-based study: eluent after PBS-soaking period.
  • FIG. 4 shows quantitation of enzyme efficacy of Polymer 17 (pretreated with PBS) using a fluorescence-based study: solid re-suspended in PBS after initial PBS-soaking period.
  • FIG. 5 shows average time to closure during scratch model tests (Error bars show SD) for cell lines from Patients A, F and G.
  • the red bars highlight samples in fiber form; the blue bars represent samples in powder form.
  • FIG. 6A shows fibroblast proliferation as observed at 1 hour.
  • FIG. 6B shows fibroblast proliferation as observed at 30 hours.
  • FIG. 6C shows fibroblast proliferation as observed at 50 hours.
  • FIG. 6D shows fibroblast proliferation as observed at 68 hours.
  • FIG. 7A shows fibroblast proliferation as observed at 1 hour.
  • FIG. 7B shows fibroblast proliferation as observed at 30 hours.
  • FIG. 7C shows fibroblast proliferation as observed at 50 hours.
  • FIG. 7D shows fibroblast proliferation as observed at 68 hours.
  • FIG. 8 shows results of studies with collagen matrix model, plot showing the lattice diameter over 7 days—Patient A.
  • FIG. 9 shows results of studies with collagen matrix model, plot showing the lattice diameter over 7 days—Patient F.
  • FIG. 10 shows results of studies with collagen matrix model, plot showing the lattice diameter over 7 days—Patient G.
  • FIG. 11 shows results of studies with collagen matrix model, wherein photographs indicate the difference in lattice diameter at days 3 and 7—Patient A.
  • FIG. 12 shows results of studies with collagen matrix model, wherein photographs indicate the difference in lattice diameter at days 3 and 7—Patient F.
  • FIG. 13 shows results of studies with collagen matrix model, wherein photographs indicate the difference in lattice diameter at days 3 and 7—Patient G.
  • FIG. 14 shows a schematic diagram for a potential peptide modified CMC and LC system for detection of proteases, wherein (a) shows the initial system set up which would show homotropic LC alignment (dark if viewed under crossed polarized lenses), (b) shows the cleavage of the peptide releasing the lipid in progress, (c) shows the cleaved lipid in contact with LCs, which would initiate planar LC realignment (colored if viewed under crossed polarized lenses).
  • FIG. 15 shows micrographs showing liquid crystal 4′-n-pentyl-4-cyano-biphenyl (5CB) filled TEM (Transmission Electron Microscopy) grids upon application of CMC gel.
  • CB liquid crystal 4′-n-pentyl-4-cyano-biphenyl
  • FIG. 15A shows the 5CB filled TEM grids prior to application of CMC hydrogel-homeotropic LC alignment.
  • “Substantially” or “essentially” means nearly totally or completely, for instance, 80%-95% or greater of some given quantity, e.g., at least 85%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.9%, or more % by weight or volume or any other parameter being measured.
  • “Substantially free” means nearly totally or completely absent of some given quantity such as being present at a level of less than about 1% to about 20% of some given quantity, e.g., less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, less than 1%, less than 0.5%, less than 0.1%, or less % by weight or volume or any other parameter being measured.
  • “substantially free” means presence at a level of less than or equal to 1-5% by weight of the pharmaceutical composition.
  • wound dressing materials to be used in wound dressings for the therapy and diagnosis of wounds and wound management, wherein the wound dressing materials when in use indicate the presence of elevated enzyme levels in a wound in situ.
  • wound refers to physical disruption of the continuity or integrity of tissue structure.
  • Wild healing refers to the restoration of tissue integrity. It will be understood that this can refer to a partial or a full restoration of tissue integrity. Treatment of a wound thus refers to the promotion, improvement, progression, acceleration, or otherwise advancement of one or more stages or processes associated with the wound healing process.
  • the wound may be acute or chronic.
  • Chronic wounds including pressure sores, venous leg ulcers and diabetic foot ulcers, can simply be described as wounds that fail to heal. Whilst the exact molecular pathogenesis of chronic wounds is not fully understood, it is acknowledged to be multi-factorial. As the normal responses of resident and migratory cells during acute injury become impaired, these wounds are characterized by a prolonged inflammatory response, defective wound extracellular matrix (ECM) remodeling and a failure of re-epithelialization.
  • ECM extracellular matrix
  • the wound may be any internal wound, e.g., where the external structural integrity of the skin is maintained, such as in bruising or internal ulceration, or external wounds, particularly cutaneous wounds, and consequently the tissue may be any internal or external bodily tissue.
  • the tissue is skin (such as human skin), i.e. the wound is a cutaneous wound, such as a dermal or epidermal wound.
  • the human skin is composed of two distinct layers, the epidermis and the dermis, below which lies the subcutaneous tissue.
  • the primary functions of the skin are to provide protection to the internal organs and tissues from external trauma and pathogenic infection, sensation and thermoregulation.
  • the outermost layer of skin, the epidermis is approximately 0.04 mm thick, is avascular, is comprised of four cell types (keratinocytes, melanocytes, Langerhans cells, and Merkel cells), and is stratified into several epithelial cell layers.
  • the inner-most epithelial layer of the epidermis is the basement membrane, which is in direct contact with, and anchors the epidermis to, the dermis. All epithelial cell division occurring in skin takes place at the basement membrane. After cell division, the epithelial cells migrate towards the outer surface of the epidermis. During this migration, the cells undergo a process known as keratinization, whereby nuclei are lost and the cells are transformed into tough, flat, resistant non-living cells.
  • Migration is completed when the cells reach the outermost epidermal structure, the stratum corneum, a dry, waterproof squamous cell layer which helps to prevent desiccation of the underlying tissue.
  • This layer of dead epithelial cells is continuously being sloughed off and replaced by keratinized cells moving to the surface from the basement membrane. Because the epidermal epithelium is avascular, the basement membrane is dependent upon the dermis for its nutrient supply.
  • the dermis is a highly vascularized tissue layer supplying nutrients to the epidermis.
  • the dermis contains nerve endings, lymphatics, collagen protein, and connective tissue.
  • the dermis is approximately 0.5 mm thick and is composed predominantly of fibroblasts and macrophages. These cell types are largely responsible for the production and maintenance of collagen, the protein found in all animal connective tissue, including the skin. Collagen is primarily responsible for the skin's resilient, elastic nature.
  • the subcutaneous tissue, found beneath the collagen-rich dermis provides for skin mobility, insulation, calorie storage, and blood to the tissues above it.
  • Wounds can be classified in one of two general categories, partial thickness wounds or full thickness wounds.
  • a partial thickness wound is limited to the epidermis and superficial dermis with no damage to the dermal blood vessels.
  • a full thickness wound involves disruption of the dermis and extends to deeper tissue layers, involving disruption of the dermal blood vessels.
  • the healing of the partial thickness wound occurs by simple regeneration of epithelial tissue. Wound healing in full thickness wounds is more complex. Cutaneous wounds contemplated herein may be either partial thickness or full thickness wounds.
  • Wounds contemplated herein include cuts and lacerations, surgical incisions or wounds, punctures, grazes, scratches, compression wounds, abrasions, friction wounds (e.g., nappy rash, friction blisters), decubitus ulcers (e.g., pressure or bed sores); thermal effect wounds (burns from cold and heat sources, either directly or through conduction, convection, or radiation, and electrical sources), chemical wounds (e.g.
  • pathogenic infections e.g., viral, bacterial or fungal
  • pathogenic infections including open or intact boils, skin eruptions, blemishes and acne, ulcers, chronic wounds, (including diabetic-associated wounds such as lower leg and foot ulcers, venous leg ulcers and pressure sores), skin graft/transplant donor and recipient sites, immune response conditions, e.g., psoriasis and eczema, stomach or intestinal ulcers, oral wounds, including a ulcers of the mouth, damaged cartilage or bone, amputation wounds and corneal lesions.
  • Embodiments described herein provide modified wound dressings, which may be used to diagnose and/or treat chronic wounds.
  • the dressings may comprise gel-forming polymers, non-gel-forming fibers, or a combination thereof.
  • the wound dressing materials described herein are used in methods to detect the level of one or more enzymes in a mammalian wound.
  • the wound dressing materials described herein are used in methods to diagnose a chronic wound in a mammal.
  • the wound dressing materials described herein are used in methods to diagnose an infected wound in a mammal.
  • the wound dressing materials described herein are used in methods to treat a wound in a mammal.
  • the wound dressing materials described herein are used in methods to treat a chronic wound in a mammal.
  • the wound dressing material has the structure of Formula I:
  • M is a gel-forming polymer
  • R is a region comprising a reporter molecule
  • L is a linker that connects M and R.
  • the linker (L) is present.
  • the linker (L) is absent, in which case, the wound dressing material comprises a compound of formula M-R, wherein M and R are each, individually, as described above.
  • the wound dressing material has the structure of Formula II:
  • M is a gel-forming polymer
  • PEP is a peptide region comprising a reporter molecule and at least one amino acid
  • L is a linker that connects M and PEP.
  • the linker (L) is present.
  • the linker (L) is absent, in which case, the wound dressing material comprises a compound of formula M-R, wherein M and R are each, individually, as described above.
  • the wound dressing material of Formula I or Formula II contains no linkers, wherein the reporter (R) or the peptide (PEP) is associated, either covalently or non-covalently, directly with the gel-forming polymer.
  • covalent bonds involve sharing of electrons between the bonded atoms.
  • non-covalent bonds may include, for example, ionic interactions, electrostatic interactions, hydrogen bonding interactions, physiochemical interactions, van der Waal forces, Lewis-acid/Lewis-base interactions, or combinations thereof.
  • the linker is absent, the peptide is attached or conjugated to the gel-forming polymer via covalent interaction.
  • peptide includes the peptide as well as pharmaceutically acceptable salts of the peptide.
  • a peptide comprises a plurality of amino acid residues, e.g., 2, 3, 4, 5, 6, 8, 10, or more amino acid residues which are bonded to each other via covalent bonds, e.g., a peptide bond.
  • Amino acid residue means the individual amino acid units incorporated into the peptides of the disclosure.
  • amino acid means a naturally occurring or synthetic amino acid, as well as amino acid analogs, stereoisomers, and amino acid mimetics that function similarly to the naturally occurring amino acids. Included by this definition are natural amino acids such as: 1. Histidine (His) 2.
  • Selenocysteine (Sec); Unnatural Amino Acids: Citrulline; Cystine; Gama-amino butyric acid (GABA); Ornithine; Theanine and Amino Acid Derivatives such as Betaine; Carnitine; Carnosine Creatine; Hydroxytryptophan; Hydroxyproline; N-acetyl cysteine; S-Adenosyl methionine (SAM-e); Taurine; Tyramine.
  • Amino acids containing reactive side chains e.g., cysteine, serine, threonine, lysine, arginine, aspartate/asparagine, glutamate/glutamine, glycine, alanine, etc. are particularly employed,
  • the peptide may be modified, e.g., via addition, deletion, substitution of one or more amino acids, via derivatization of one or more amino acids, or cyclization, etc.
  • the peptides are modified at the carboxy-terminal (C-terminus) or the amino-terminus (N-terminus) by adding, deleting or substituting one or more amino acids.
  • the peptides are modified at the C-terminus by adding at least one amino acid, especially, an amino acid containing reactive side chains, e.g., cysteine, serine, threonine, lysine, arginine, aspartate/asparagine, glutamate/glutamine, glycine, alanine, etc., wherein the reactive side chain may be employed in the conjugation with a label such as a dye.
  • the peptides are modified to contain additional cysteine or serine residues at the C-terminus, the sulfur group of cysteine or the hydroxyl group of serine being used to couple with fluorescent dyes.
  • the peptide containing an additional amino acid comprising a reactive side chain, e.g., SH group of cysteine may be coupled to a dye via click chemistry.
  • the reaction between a 1,2-aminothiol and a 2-cyanobenzothiazole (CBT) may be used to make luciferin, which is fluorescent.
  • the luciferin fluorescence can be then quantified by spectrometry following a wash, and used to determine the relative presence of the molecule bearing the 1,2-aminothiol.
  • the protein of interest can be cleaved to yield a fragment with a N′ Cys that is vulnerable to the 2-CBT. See Liang et al., J. Angew. Chem., Int. Ed., 48, 965, 2009.
  • the gel-forming polymer is a compound selected from cellulose, chemically modified cellulose, pectin, alginate, chitosan, modified chitosan, hyaluronic acid, polysaccharide, or gum-derived polymer, or a derivative thereof or any mixture or a combination thereof.
  • the gel-forming polymer is selected from cellulose, carboxymethylcellulose (CMC), oxidized cellulose (or a derivative thereof), cellulose ethyl sulfonate (CES), pectin, alginate, chitosan, modified chitosan, hyaluronic acid, polysaccharide, or gum-derived polymer, or any combination or mixture thereof.
  • the gel-forming polymer is cellulose or chemically modified cellulose, e.g., carboxymethylcellulose, an oxidized cellulose or a derivative thereof, cellulose ethyl sulfonate.
  • the gel forming polymer is a derivative of a polymeric compound, e.g., cellulose derivative.
  • derivative includes salts, amides, esters, enol ethers, enol esters, acetals, ketals, orthoesters, hemiacetals, hemiketals, acids, bases, solvates, hydrates or prodrugs of the gel forming polymer.
  • the polymer is cellulose
  • the hydroxyl groups (—OH) of cellulose can be partially or fully reacted with various reagents to form derivatives with useful properties, e.g., cellulose esters and cellulose ethers (—OR).
  • the derivative of cellulose is selected from carboxymethylcellulose, methylcellulose, ethylcellulose, methylethylcellulose, hydroxyethyl cellulose, hydroxypropylmethylcellulose and hydroxypropylcellulose.
  • Such derivatives may be readily prepared by those of skill in this art using known methods for such derivatization.
  • the derivatives may be administered to animals or humans without substantial toxic effects and either are pharmaceutically active or are prodrugs. Representative types of cellulosic derivatives are described in U.S. Pat. Nos. 7,544,640 and 9,561,188.
  • the derivative is a salt of the polymeric compound, e.g., salts of Li + , Na + , K + , Rb + , Mg 2+ , Ca 2+ , Sr 2+ , or Ba 2+ , preferably Na + , K + , Mg 2+ , Ca 2+ .
  • Salts of cellulose, cellulose esters and cellulose ethers, such as sodium or calcium salts, are known in the art.
  • the gel-forming polymeric compound may contain a combination or mixture of one or more of the aforementioned compounds.
  • the term “combination” includes compounds containing more than one component, which may be conjugated or non-conjugated to one another.
  • the gel-forming polymeric compound comprises a combination of one or more of the aforementioned compounds which are conjugated to each other, e.g., via covalent or non-covalent interaction.
  • the gel-forming polymer may comprise a combination of pectin and carboxymethylcellulose. See, Ninan et al., Carbohydr Polym. 2013 Oct. 15; 98(1):877-85; PMID: 23987424.
  • the compounds include mixtures of the aforementioned polymeric compounds.
  • the term “mixture” refers to a mingling together of two or more substances without the occurrence of a reaction by which they would lose their individual properties.
  • a mixture of compound A and compound B may contain any weight ratio of compound A and compound B, such that the total weight of the mixture would amount to 100%, e.g., 99:1 weight ratio of compound A/compound B or 1:99 weight ratio of compound A/compound B.
  • a typical mixture may contain about 2, 3, 4, 5, or more of the aforementioned polymer compounds.
  • the gel-forming polymer is in the form of powder or fiber, or a combination thereof. In some embodiments of a wound dressing material of Formula I or Formula II, the gel-forming polymer is in the form of fiber.
  • Gel-forming fibers are hygroscopic fibers which upon the uptake of wound exudate become moist, slippery, or gelatinous and thus reduces the tendency for the surrounding fibers to adhere to the wound.
  • the gel-forming fibers can be of the type which retain their structural integrity on absorption of exudate or can be of the type which lose their fibrous form and become a structureless gel. Gel-forming fibers preferably have an absorbency of at least 2 grams 0.9% saline solution per gram of fiber (as measured by the free swell method).
  • the wound dressing material may comprise non-gel-forming fibers.
  • the non-gel-forming fibers are selected from cellulose fiber (e.g., cotton or lyocell/TENCEL), polyester, nylon, viscose, aramid, acrylic, elastane (LYCRA), polyolefin, polylactide, silk, and natural or synthetic wool.
  • the wound dressing material comprises gel-forming polymers and non-gel-forming fibers.
  • the gel-forming polymer is in the form of powder.
  • powder gel-forming polymer is preferred over fibrous gel-forming fiber because of the higher degree of substitution (DoS) of the powder gel-forming polymer.
  • fibrous gel-forming fiber is preferred over powder gel-forming polymer.
  • the gel-forming polymer has a DoS of at least 0.2, at least 0.3, at least 0.4, at least 0.5, at least 0.6, at least 0.7, at least 0.8, at least 0.9, at least 1.0, at least 1.1, at least 1.2, at least 1.3, at least 1.4, at least 1.5, at least 2.0, or more.
  • DoS is understood in the art. For instance, in the context of cellulose chemistry where each anhydroglucose ( ⁇ -glucopyranose) unit has three reactive (hydroxyl) groups; DoS may therefore range from zero (cellulose) to three (fully substituted cellulose).
  • the linker may be attached to the gel-forming polymer covalently or non-covalently.
  • covalent bonds involve sharing of electrons.
  • non-covalent bonds may include, for example, ionic interactions, electrostatic interactions, hydrogen bonding interactions, physiochemical interactions, van der Waal forces, Lewis-acid/Lewis-base interactions, or combinations thereof.
  • the linker is attached or conjugated to the gel-forming polymer via covalent interaction.
  • the chemical linker is a carboxylic acid having 2 to 10 carbon atoms, particularly 4 to 8 carbon atoms or especially about 4 to 6 carbon atoms.
  • the linker is a monomer or a neutral polymer selected from the group consisting of an ethoxylated polyhydric alcohol, a polyvinyl pyrrolidone polymer, a polypropylene, a polyalkylene glycol, a polyamine, including, ethers, amides, and esters thereof.
  • the neutral polymer is polypropylene, although a monomer thereof comprising propylene may also be used.
  • Polypropylene (PP) is one of the most important and widely used polyolefins as matrix material because of its low density, low production costs, design flexibility and recyclability. Because polyproylene is hydrophobic, it may be incompatible with polar surfaces, such as cellulose. This issue can be resolved by incorporating functionalized polypropylene, such as poly(propylene-graft-maleic anhydride) (PP-g-MA), into the composite, wherein the carboxylic anhydride groups can provide covalent bonding to the cellulose. See, Spoljaric et al., Composites: Part A 40, 791-799, 2009.
  • the neutral polymer linker is a polyalkylene glycol, although a monomer thereof comprising alkylene glycol may also be used.
  • polyalkylene glycol refers to straight or branched polyalkylene glycol polymers such as polyethylene glycol, polypropylene glycol, and polybutylene glycol.
  • a polyalkylene glycol subunit is a single polyalkylene glycol unit.
  • an example of a polyethylene glycol subunit would be an ethylene glycol, —O—CH2-CH2-O—, or propylene glycol, —O—CH2-CH2-CH2-O—, capped with a hydrogen at the chain termination point.
  • poly(alkylene glycol) examples include, but are not limited to, PEG, PEG derivatives such as methoxypoly(ethylene glycol) (mPEG), poly(ethylene oxide), PPG, poly(tetramethylene glycol), poly(ethylene oxide-co-propylene oxide), or copolymers and combinations thereof.
  • PEG PEG derivatives such as methoxypoly(ethylene glycol) (mPEG), poly(ethylene oxide), PPG, poly(tetramethylene glycol), poly(ethylene oxide-co-propylene oxide), or copolymers and combinations thereof.
  • the neutral polymer is a polyamine, although a monomer thereof comprising an amine may also be used.
  • polyamine refers to polymers having an amine functionality in the monomer unit, either incorporated into the backbone, as in polyalkyleneimines, or in a pendant group as in polyvinyl amines.
  • the linker is a PEG or a PEG derivative such as methoxypoly(ethylene glycol) (mPEG), poly(ethylene oxide), PPG, poly(tetramethylene glycol), poly(ethylene oxide-co-propylene oxide), or copolymers and combinations thereof.
  • linkers may also be used as linkers, as long as they are flexible, e.g., linkers that do not contain double bonds or cyclic structures or which contain only a few double bonds or cyclic structure.
  • linkers include, e.g., polyalkylene, polyhydroxyalkylene, polyalkylene succinate, polylactide, etc., with chain lengths from about 2 to about 20 chain atoms.
  • the chain length of the polyalkyleneglycols may vary from edgy 3 units (MW about 150 Da) up to e.g., about 100 (MW about 5000).
  • the relative amount of polyalkyleneglycol with respect to the polysaccharide may vary from about 1/200 to about 1/1, especially from about 1/50 to about 1/1.5, depending on the required thickness and the required flexibility of the product. See, U.S. Pat. No. 9,089,614 and US PGPUB No. 2005-0079155.
  • the linker L comprises 1 to about 20 monomeric units, e.g., of the natural polymer, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more monomer units.
  • the linker comprises from 1 to about 5 ethylene glycol units or a derivative thereof, from 2 to about 5 ethylene glycol units or a derivative thereof, from 2 to about 8 ethylene glycol units or a derivative thereof, from 2 to about 10 ethylene glycol units or a derivative thereof or from 5 to about 10 ethylene glycol units or a derivative thereof.
  • the linker L comprises a chemical moiety that is a product of a nucleophilic reaction.
  • nucleophile is art-recognized to mean a chemical group having a reactive pair of electrons that reacts with a compound by displacing a leaving group (commonly another nucleophile), such as commonly occur in aliphatic chemistry as unimolecular (known as “SN1”) or bimolecular (“SN2”) reactions.
  • nucleophiles include uncharged compounds such as amines, mercaptans, and alcohols, and charged groups such as alkoxides, thiols, thiolates, carbanions, and a variety of organic and inorganic anions.
  • Illustrative anionic nucleophiles include, inter alia, simple anions such as azide, cyanide, thiocyanate, acetate, formate, or chloroformate, and bisulfite.
  • the linker L comprises a maleimide-thiol adduct.
  • Maleimides are particularly useful for conjugation to thiol-containing substances, e.g., thiol-containing amino acids such as cysteine.
  • a thiol group reacts with a maleimides by added across the double bond to form a thioether.
  • Maleimides are selective for the thiol of cysteine over methionine, histidine, or tyrosine. Reaction of maleimides with amines usually requires a higher pH than reaction of maleimides with thiols. Hydrolysis of maleimides competes significantly with thiol modification, particularly above pH 8. See, US PGPUB No. 2007-0087446.
  • L comprises a haloacetamide-thiol conjugation product.
  • Haloacetamides e.g., iodoacetamide or bromoacetamide, may also be used to bind covalently with the thiol group of amino acids, e.g., cysteine.
  • the linker L comprises a compound containing a thiol or a disulfide, which can be used analogously to maleimide. See, Zalipsky et al., Bioconjug. Chem. 6, 150-165, 1995; Greenwald et al. Crit. Rev. Ther. Drug Carrier Syst. 17, 101-161, 2000; and Herman et al., Macromol. Chem. Phys. 195, 203-209, 1994. See also, U.S. Pat. No. 7,432,330.
  • the wound dressing materials comprise a region comprising a reporter molecule.
  • the reporter is a substrate for one or more wound-specific markers, e.g., enzymes found in a wound environment.
  • a wound specific enzyme is an enzyme that is differentially expressed in a wound.
  • differential expression it is meant that the level or the activity of the enzyme is higher or lower in the wound microenvironment compared to other sites, e.g., normal tissue or surrounding tissue.
  • differential expression implies higher level of expression or activity of the enzyme in the wound microenvironment compared to normal or unwounded tissue. Differential expression of enzyme may be analyzed by routine means.
  • levels of enzyme in a sample may be analyzed by ELISA assays or other immunoassays. Activities of the enzyme may be analyzed by measuring rates of loss of a substrate and/or rates of formation of the product, e.g., using mass spectroscopy or HPLC. Such techniques are known in the art and are described in the Examples section.
  • the marker is an enzyme selected from the group consisting of hydrolases, proteases, esterases, and peroxidases.
  • the marker is a hydrolase.
  • a “hydrolase” or “hydrolytic enzyme” is an enzyme that catalyzes the hydrolysis of a chemical bond, e.g., esterases and nucleases (break ester bonds); glycolases (break glycosidic linkers); peptidases (break peptide bonds), etc.
  • the marker is a protease enzyme.
  • the protease may be a sequence-specific or a generic protease.
  • sequence-specific protease means a protease recognizing a specific sequence of a peptide for its digesting (for example, caspase), and is distinguished from a generic protease (for example, trypsin) that sequentially decomposes a peptide from one end thereof or digest a peptide in a sequence-nonspecific manner.
  • the amino acid sequence of the peptide substrate may comprise four or more amino acid (a.a.) residues.
  • the recognition site and the digestion site may be close to each other.
  • the term “substrate peptide for a protease” means a peptide comprising an amino acid sequence of a protein, which is recognized by the protease as a substrate for its protease activity, e.g., as a substrate that can be cleaved into one or more products.
  • the wound dressing materials comprise a peptide region comprising a peptide sequence comprising a plurality of amino acids.
  • plurality means two or more units, e.g., amino acids, although the individual units need not be structurally and/or functionally different.
  • the peptide comprises natural amino acids. In other embodiments, synthetic peptides containing one or more non-natural amino acids may also be used.
  • a plurality of substrates each of which is specific for a particular enzyme, may be used. In other embodiments, a plurality of substrates, each of which is specific for a plurality of enzymes, may also be used.
  • the protease enzyme is an exopeptidase or an endopeptidase.
  • Exopeptidases degrade the structure only near the ends of the peptide chain; endopeptidases are able to cleave internal bonds within the peptide.
  • These classes are also split into the subgroups: cysteine-protease, a serine-protease, a threonine-protease, aspartic-protease, a glutamic-protease, a metallo-protease etc. Each are able to digest specific protein linkages by hydrolysis of the peptide bond.
  • the protease is specific to a wound.
  • a “wound specific protease” is a protease that is differentially expressed in a wound.
  • differential expression it is meant that the level or the activity of the protease is higher or lower in the wound microenvironment compared to other sites, e.g., normal tissue or surrounding tissue. Particularly, differential expression implies higher level of expression or activity of the protease in the wound microenvironment compared to unwounded tissue.
  • Differential expression of proteases may be analyzed by routine means. For example, levels of proteases in a sample may be analyzed by ELISA assays or other immunoassays. Activities of the proteases may be analyzed by measuring rates of loss of a peptide substrate and/or rates of formation of the product, e.g., using mass spectroscopy or HPLC. Such techniques are known in the art and are described in the Examples section.
  • the wound specific protease is selected from the group consisting of MMP-1 (collagenase), MMP-2 (gelatinase A), MMP-3 (stomelysin 1), MMP-8 (neutrophil collagenase), MMP-9 (gelatinase B), human neutrophil elastase (HNE), cathepsin G, urokinase-type plasminogen activator (uPA), and lysozyme.
  • the substrate is a peptide sequence specific for collagenase. In some embodiments, the substrate is a peptide sequence specific for MMP-2. In some embodiments, the substrate is a peptide sequence specific for MMP-3. In some embodiments, the substrate is a peptide sequence specific for neutrophil collagenase. In some embodiments, the substrate is a peptide sequence specific for gelatinase. In some embodiments, the substrate is a peptide sequence specific for human neutrophil elastase. In some embodiments, the substrate is a peptide sequence specific for cathepsin G. In some embodiments, the substrate is a peptide sequence specific for urokinase-type plasminogen activator. In some embodiments, the substrate is a peptide sequence specific for lysozyme. In some embodiments, the substrate is a sugar that is cleavable by lysozyme.
  • the wound-specific protease is a matrix metalloproteinase (MMP) selected from the group consisting of MMP-1, MMP-2, MMP-8 and MMP-9 (collagenase), or a combination thereof.
  • MMP-1 (UNIPROT accession Nos. P03956 [human] and Q9EPL5 [mouse]) is also known as interstitial collagenase and fibroblast collagenase.
  • MMP-2 (UNIPROT accession Nos. P08253 [human] and P33434 [mouse]) is also known as gelatinase.
  • MMP-8 (UNIPROT accession Nos.
  • MMP-9 (UNIPROT accession Nos. P14780 [human] and P41245 [mouse]) is also known as gelatinase B (GELB).
  • the MMP is MMP-2 or MMP-9, or a combination thereof.
  • matrix metalloproteinase (MMP) activity levels of about 5 U/mL to about 30 U/mL, including all values in between, e.g., about 6 U/mL, about 7 U/mL, about 8 U/mL, about 9 U/mL, about 10 U/mL, about 11 U/mL, about 12 U/mL, about 13 U/mL, about 14 U/mL, about 15 U/mL, about 16 U/mL, about 17 U/mL, about 18 U/mL, about 19 U/mL, about 20 U/mL, about 21 U/mL, about 22 U/mL, about 23 U/mL, about 24 U/mL, about 25 U/mL, or more, indicate chronic wound infection.
  • MMP matrix metalloproteinase
  • Units of activity (U) are typically used to describe enzyme catalytic activity, where a unit (U) refers to the amount of enzyme that catalyzes the conversion of 1 micromole ( ⁇ mole) of substrate per minute.
  • 1 enzyme unit (U) 1 ⁇ mol/min, where ⁇ mol refers to the amount of substrate converted.
  • the MMP is MMP-2 or MMP-9, wherein MMP-2 and MMP-9 activity levels of at least 10.5 U/mL indicate chronic wound infection.
  • Any peptide cleavable by MMP may be used in accordance with the embodiment described herein. For instance, See Table 1 of U.S. Pat. No. 7,148,194, herein incorporated by reference for this subject matter.
  • Table 1 shows the various substrates and their specificity to different isoforms of human MMPs. The data are presented in Table 3 of Nagase et al. (“Substrate specificity of MMPs,” in Matrix Metalloproteinase Inhibitors in Cancer Therapy , Clendeninn & Appelt Eds., Springer Science Media New York, 2001), which is incorporated by reference herein.
  • the wound-specific protease of the invention is human neutrophil elastase (HNE) (UNIPROT accession Nos. P08246 [human] and Q3UP87 [mouse]) is a serine proteinase in the same family as chymotrypsin and has broad substrate specificity. Secreted by neutrophils and macrophages during inflammation, it destroys bacteria and host tissue.
  • the substrate for detecting HNE has a core sequence Alanine-Alanine-Proline-Valine (AAPV).
  • the substrate for HNE is Ala-Pro-Glu-Glu-Ile/Met-Arg-Arg-Gln (APEEI/MRRQ) (Kasperkiewicz et al., PNAS USA, 111(7): 2518-2523, 2014; Korkmaz et al., Methods Mol Biol., 844:125-138, 2012).
  • human neutrophil elastase activity levels of at least 9.6 indicate chronic wound infection.
  • human neutrophil elastase activity levels of at least 22.9 U/mL indicate chronic wound infection.
  • the MMP and the HNE subgroups have different mechanisms when interacting with the proteins in a wound and therefore as one would expect, each has a different method of inhibition of wound healing.
  • the wound-specific enzyme is lysozyme.
  • Lysozyme (UNIPROT accession Nos. P61626 [human] and P08905 [mouse]) is a glycoside hydrolase and its main function is to destroy the cell walls of bacteria. It hydrolyses the (1 ⁇ 4)- ⁇ -linkages between N-acetylmuramic acid and N-acetyl-D-glucosamine residues in peptidoglycan and also between N-acetyl-D glucosamine residues in chitodextrin.
  • the natural substrate for lysozyme is the peptidoglycan layer of bacterial cell walls.
  • low molecular mass substrates including murein degradation products as well as synthetic compounds have been used for various photometric, isotopic, and immunological lysozyme assays. Holtje et al., EXS, 75:105-10, 1996.
  • the following low molecular mass lysozyme substrates are available from Sigma Aldrich, Saint Louis, Mo.: 4-Methylumbelliferyl ⁇ -D-N,N′,N′′-triacetyl-chitotrioside (Sigma Catalog Number M5639) and 4-Nitrophenyl ⁇ -D-N,N′,N′′-triacetyl-chitotrioside (Sigma Catalog Number N8638).
  • lysozyme activity levels of about 1000 U/mL to about 10000 U/mL including all values in between, e.g., about 1100 U/mL, about 1200 U/mL, about 1300 U/mL, about 1400 U/mL, about 1500 U/mL, about 1600 U/mL, about 1700 U/mL, about 1800 U/mL, about 1900 U/mL, about 2000 U/mL, about 2100 U/mL, about 2200 U/mL, about 2300 U/mL, about 2400 U/mL, about 2500 U/mL, about 2600 U/mL, about 2700 U/mL, about 2800 U/mL, about 2900 U/mL, about 3000 U/mL, about 3250 U/mL, about 3500 U/mL, about 3750 U/mL, about 4000 U/mL, about 4250 U/mL, about 4500 U/mL, about 4750 U/mL, about 5000
  • the wound-specific enzyme is peroxidase, more specifically, a myeloperoxidase (MPO).
  • MPO UNIPROT accession Nos. P05164 [human] and P11247 [mouse]
  • MPO myeloperoxidase
  • H 2 O 2 hydrogen peroxide
  • a halide most commonly chloride
  • MPO can be detected using tetramethylbenzidine or 4-Benzoylamino-2,5-dimethoxyaniline. See, Andrews et al., Anal Biochem, 127(2):346-50, 1982; Klebanoff et al., J. Leukocyte Biol., 77, 598-625, 2005.
  • the wound-specific enzyme is cathepsin G (UNIPROT accession Nos. P08311 [human] and P28293 [mouse]), which is one of the three serine proteases of the chymotrypsin family that are stored in the azurophil granules.
  • Cathepsin G-specific substrates have the sequence Ala-Ala-Pro-Phe or Ala-Ala-Pro-Met (Sigma Aldrich Catalog Nos. S7388 and M7771).
  • cathepsin G activity levels of about 10 U/mL to about 100 U/mL including all values in between, e.g., about 15 U/mL, about 20 U/mL, about 25 U/mL, about 30 U/mL, about 35 U/mL, about 40 U/mL, about 45 U/mL, about 50 U/mL, about 55 U/mL, about 60 U/mL, about 65 U/mL, about 70 U/mL, about 75 U/mL, about 80 U/mL, about 85 U/mL, about 90 U/mL, about 95 U/mL, about 100 U/mL, about 110 U/mL, about 120 U/mL, or more, indicate chronic wound infection.
  • cathepsin G activity levels of at least 50 U/mL, at least 40 U/mL, at least 30 U/mL, at least 20 U/mL, at least 15 U/mL or at least 10 U/mL indicates chronic wound infection.
  • the wound-specific enzyme is urokinase-type plasminogen activator (UNIPROT accession Nos. P00749 [human] and P06869 [mouse]), which is a serine protease involved in degradation of the extracellular matrix and possibly tumor cell migration and proliferation.
  • a substrate specific for urokinase has a basic motif Arg-Val or Lys-Val. See, Rijken et al., Biochem Biophys Res Commun., 174(2):432-8, 1991.
  • the one or more enzymes are esterases.
  • An esterase is a hydrolase that splits esters into an acid and an alcohol in a chemical reaction with water.
  • the substrate for esterase is fluorescein diacetate-5-maleimide.
  • compositions comprise substrates that are capable of detecting a plurality of enzymes, e.g., at least 2, at least 3, at least 4, or more of the aforementioned enzymes.
  • Such compositions may include, for example, a plurality of substrates conjugated to the same gel polymer or different gel polymers.
  • the substrates are labeled.
  • label refers to any substance attached to an epitope binding agent, or other substrate material, in which the substance is detectable by a detection method.
  • suitable labels include luminescent molecules, chemiluminescent molecules, fluorochromes, fluorescent quenching agents, colored molecules, radioisotopes, scintillants, biotin, avidin, streptavidin, protein A, protein G, antibodies or fragments thereof, polyhistidine, Ni2+, Flag tags, myc tags, heavy metals, and enzymes (including alkaline phosphatase, peroxidase, and luciferase). Such methods are well-known in the art.
  • the substrates are labeled with label which is a detectable label.
  • a detectable label is a moiety, the presence of which can be ascertained directly or indirectly.
  • detection of the label involves the creation of a detectable signal such as for example an emission of energy.
  • the label may be of a chemical, peptide or nucleic acid nature although it is not so limited. The nature of label used will depend on a variety of factors, including the nature of the analysis being conducted, the type of the energy source and detector used and the type of polymer, analyte, probe and primary and secondary analyte-specific binding partners.
  • the label should be sterically and chemically compatible with the constituents to which it is bound.
  • the label can be detected directly for example by its ability to emit and/or absorb electromagnetic radiation of a particular wavelength.
  • a label can be detected indirectly for example by its ability to bind, recruit and, in some cases, cleave another moiety which itself may emit or absorb light of a particular wavelength (e.g., an epitope tag such as the FLAG epitope, an enzyme tag such as horseradish peroxidase, etc.).
  • the detectable label can be selected from the group consisting of directly detectable labels such as a fluorescent molecule (e.g., fluorescein, rhodamine, tetramethylrhodamine, R-phycoerythrin, Cy-3, Cy-5, Cy-7, Texas Red, Phar-Red, allophycocyanin (APC), fluorescein amine, eosin, dansyl, umbelliferone, 5-carboxyfluorescein (FAM), 2′7′-dimethoxy-4′5′-dichloro-6-carboxyfluorescein (JOE), 6 carboxyrhodamine (R6G), N,N,N′,N′-tetramethyl-6-carboxyrhodamine (TAMRA), 6-carboxy-X-rhodamine (ROX), 4-(4′-dimethylaminophenylazo) benzoic acid (DABCYL), 5-(2′-aminoethyl)
  • the detectable label can also be selected from the group consisting of indirectly detectable labels such as an enzyme (e.g., alkaline phosphatase, horseradish peroxidase, p-galactosidase, glucoamylase, lysozyme, luciferases such as firefly luciferase and bacterial luciferase (U.S. Pat. No.
  • an enzyme e.g., alkaline phosphatase, horseradish peroxidase, p-galactosidase, glucoamylase, lysozyme
  • luciferases such as firefly luciferase and bacterial luciferase
  • saccharide oxidases such as glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase
  • heterocyclic oxidases such as uricase and xanthine oxidase coupled to an enzyme that uses hydrogen peroxide to oxidize a dye precursor such as HRP, lactoperoxidase, or microperoxidase
  • an enzyme substrate an affinity molecule, a ligand, a receptor, a biotin molecule, an avidin molecule, a streptavidin molecule, an antigen (e.g., epitope tags such as the FLAG or HA epitope), a hapten (e.g., biotin, pyridoxal, digoxigenin fluorescein and dinitrophenol), an antibody, an antibody fragment, a microbead, etc.
  • Antibody fragments include Fab, F(ab)2, Fd and antibody fragments which include a CDR3 region.
  • the substrates are conjugated with donor and acceptor fluorophores, respectively, that form a FRET pair.
  • FRET can be used, for example, in an array format in order to determine if a particular secondary antibody is bound regardless of the identity of the analyte to which it binds.
  • the secondary binding partner may be labeled detectably without labeling of the primary binding partner. Labeling of the secondary binding partner is also useful for establishing the orientation of the nucleic acid attached thereto.
  • FRET alone generally requires only one excitation source (and thus wavelength) and usually only one detector. The detector may be set to either the emission spectrum of the donor or acceptor fluorophore.
  • FRET emission spectrum if FRET is detected by quenching of donor fluorescence.
  • acceptor fluorophore emission spectrum if FRET is detected by acceptor fluorophore emission.
  • FRET emissions of both donor and acceptor fluorophores can be detected.
  • the donor is excited with polarized light and polarization of both emission spectra is detected.
  • the detectable label is compatible with FRET-based assays.
  • FRET requires the use of a FRET fluorophore pair.
  • FRET fluorophore pairs are two fluorophores that are capable of undergoing FRET to produce or eliminate a detectable signal when positioned in proximity to one another. Examples of donors include Alexa 488, Alexa 546, BODIPY 493, Oyster 556, Fluor (FAM), Cy3 and TMR (Tamra). Examples of acceptors include CyS, Alexa 594, Alexa 647 and Oyster 656. Cy5 can work as a donor with Cy3, TMR or Alexa 546, as an example. FRET should be possible with any fluorophore pair having fluorescence maxima spaced at 50-100 nm from each other.
  • the substrate may be labeled in a sequence non-specific manner in addition to the barcode labeling discussed herein.
  • the polymer is a nucleic acid such as DNA
  • its backbone may be stained with a backbone label.
  • backbone stains that label nucleic acids in a sequence non-specific manner include intercalating dyes such as phenanthridines and acridines (e.g., ethidium bromide, propidium iodide, hexidium iodide, dihydroethidium, ethidium homodimer-1 and -2, ethidium monoazide, and ACMA); minor grove binders such as indoles and imidazoles (e.g., Hoechst 33258, Hoechst 33342, Hoechst 34580 and DAPI); and miscellaneous nucleic acid stains such as acridine orange (also capable of intercalating), 7-AAD, actinomycin D
  • nucleic acid stains include the following dyes from Molecular Probes: cyanine dyes such as SYTOX BLUE, SYTOX GREEN, SYTOX ORANGE, POPO-1, POPO-3, YOYO-1, YOYO-3, TOTO-1, TOTO-3, JOJO-1, LOLO-1, BOBO-1, BOBO-3, PO-PRO-1, PO-PRO-3, BO-PRO-1, BO-PRO-3, TO-PRO-1, TO-PRO-3, TO-PRO-5, JO-PRO-1, LO-PRO-1, YO-PRO-1, YO-PRO-3, PICOGREEN, OLIGREEN, RIBOGREEN, SYBR GOLD, SYBR GREEN I, SYBR GREEN II, SYBR DX, SYTO-40, -41, -42, -43, -44, -45 (BLUE), SYTO-13, -16, -24, -21, -23, -12, -11
  • the reporter molecule comprises a chromophore or a fluorophore.
  • the chromophore is an azo moiety, a nitro moiety, a triarylmethane moiety, a methine, anthraquinone, a polyene moiety, or phthalocyanine.
  • the reporter molecule is a dye.
  • the dye can be, but is not limited to, rhodamine, coumarin, cyanine, xanthene, polymethine, pyrene, dipyrromethene borondifluoride, naphthalimide, a phycobiliprotein, peridinium chlorophyll proteins, conjugates thereof, and combinations thereof.
  • Non-limiting examples of dyes include, fluorescein, 6-FAM, rhodamine, Texas Red, California Red, iFluor594, tetramethylrhodamine, a carboxyrhodamine, carboxyrhodamine 6F, carboxyrhodol, carboxyrhodamine 110, Cascade Blue, Cascade Yellow, coumarin, Cy2®, Cy3®, Cy3.5®, Cy5®, Cy5.5®, Cy7®, Cy-Chrome, DyLight 350, DyLight 405, DyLight 488, DyLight 549, DyLight 594, DyLight 633, DyLight 649, DyLight 680, DyLight 750, DyLight 800, phycoerythrin, PerCP (peridinin chlorophyll-a Protein), PerCP-Cy5.5, JOE (6-carboxy-4′,5′-dichloro-2′,7′-dimethoxyfluorescein), NED, ROX
  • the reporter molecule is dimethylaminoazobenzenesulfonic acid (dabsyl) or a dabsyl derivative. In some embodiments, the reporter molecule is fluorescein, a fluorescein derivative, or a fluorescein-containing compound.
  • the reporter molecule is a lipid.
  • the lipid is a synthetic phospholipid derivative.
  • the synthetic phospholipid derivative is DDPC, DLPC, DMPC, DPPC, DSPC, DOPC, POPC, or DEPC.
  • the synthetic phospholipid derivative is DLPC, DMPC, or DPPC.
  • the synthetic phospholipid derivative is DLPC.
  • the synthetic phospholipid derivative is DMPC.
  • the synthetic phospholipid derivative is DPPC.
  • the reporter molecule is comprised in a detectable fragment that gets cleaved from the wound dressing material upon contact with an enzyme. In some embodiments, the reporter molecule is not comprised in the fragment that gets cleaved from the wound dressing material upon contact with an enzyme. In some embodiments, the reporter molecule is visualized by the naked eye. In some embodiments, the reporter molecule is visualized under UV light. In some embodiments, the reporter molecule is visualized using a fluorescent lamp.
  • R optionally comprises a quencher fragment.
  • the quencher fragment prevents the reporter molecule from fluorescing.
  • the quencher fragment is a protecting group.
  • the quencher fragment is an acetate group.
  • modified wound dressing materials containing a target sequence for one or more enzymes.
  • enzyme-catalyzed cleavage releases a detectable fragment.
  • the detectable fragment may comprise a reporter molecule. Qualitative or quantitative measurement of the levels of detectable fragments enables a determination of the presence or absence of infection in the wound.
  • the enzyme-catalyzed cleavage releases a non-detectable fragment.
  • enzyme interaction with the wound dressing material cleaves a quencher fragment, and allows for a reporter molecule bound to the wound dressing material to fluoresce. Qualitative or quantitative measurement of the fluorescence enables a determination of the presence or absence of infection in the wound.
  • peptide-modified wound dressing materials containing a target sequence for one or more proteases.
  • Protease-catalyzed cleavage releases a detectable peptide fragment.
  • the detectable peptide fragment comprises a reporter molecule.
  • Qualitative or quantitative measurement of the levels of detectable peptide fragments enables a determination of the presence or absence of elevated proteases in the wound.
  • the enzyme-catalyzed cleavage releases a non-detectable fragment.
  • enzyme interaction with the wound dressing material cleaves a quencher fragment, and allows for a reporter molecule bound to the wound dressing material to fluoresce. Qualitative or quantitative measurement of the fluorescence enables a determination of the presence or absence of infection in the wound.
  • R comprises a quencher fragment. In some embodiments, the quencher fragment is preventing the reporter molecule from fluorescing. In some embodiments, the quencher fragment is a protecting group. In some embodiments, the quencher fragment is an acetate group. In some embodiments of a wound dressing material of Formula I, R comprises a chemical moiety that is nucleophilic reaction product. In some embodiments, R comprises a maleimide-thiol adduct. In some embodiments, R comprises a haloacetamide-thiol conjugation product. In some embodiments, R comprises a haloacetamide conjugation product.
  • the wound dressing material has the structure of Formula Ia:
  • R is a region comprising a reporter molecule
  • m is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, or more
  • n is an integer selected from 200 to 4000, e.g., 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3100, 3200, 3300, 3400, 3500, 3600, 3700, 3800, 3900, 4000, including all unitary values in between, for example, 201, 202, 203, etc.
  • n is an integer selected from 300 to 3500.
  • n is an integer selected from 400 to 3200.
  • R is a peptide
  • the wound dressing material has the structure of Formula Ib:
  • R is a region comprising a reporter molecule
  • n is an integer selected from 200 to 4000, e.g., 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3100, 3200, 3300, 3400, 3500, 3600, 3700, 3800, 3900, 4000, including all unitary values in between, for example, 201, 202, 203, etc.
  • n is an integer selected from 300 to 3500. In still further embodiments, n is an integer selected from 400 to 3200.
  • R is a peptide region comprising a reporter molecule and at least one amino acid. In some embodiments, R is a peptide region comprising a reporter molecule and one amino acid.
  • the wound dressing material has the structure of Formula IIa:
  • M is a gel-forming polymer selected from cellulose, chemically modified cellulose, pectin, alginate, chitosan, hyaluronic acid, polysaccharide, or gum-derived polymer, or any combination thereof;
  • PEP is a peptide region comprising a reporter molecule and at least one amino acid;
  • L is a linker that connects M and PEP, wherein L comprises one or more polyethylene glycol subunits or polypropylene subunits.
  • the wound dressing material has the structure of Formula IIb:
  • PEP is a peptide region comprising a reporter molecule and at least one amino acid
  • m is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, or more
  • n is an integer selected from 200 to 4000.
  • n is an integer selected from 300 to 3500.
  • n is an integer selected from 400 to 3200.
  • the wound dressing material has the structure of Formula IIc:
  • PEP is a peptide region comprising a reporter molecule and at least one amino acid
  • m is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, or more
  • n is an integer selected from 200 to 4000, e.g., 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3100, 3200, 3300, 3400, 3500, 3600, 3700, 3800, 3900, 4000, including all unitary values in between, for example, 201, 202, 203, etc.
  • n is an integer selected from 300 to 3500.
  • n is an integer selected from 400 to 3200.
  • compositions are Compositions:
  • Embodiments described herein further relate to compositions containing the compounds of Formula I or Formula II. Such compositions may be prepared using conventional methods.
  • the resulting stock composition of compounds of Formula I or Formula II may be further modified into desired form, e.g., gels, balms, lotions, cream, paste, ointments, etc. using conventional methods, e.g., using carriers, gelling agents, emollients, surfactants, humectants, viscosity enhancers, emulsifiers, etc. See, e.g., WO 2011/126384 and WO 2013/004953, which are incorporated by reference.
  • Carriers for use in the composition may include, but are not limited to, water, glycerin, diglycerin, glycerin derivatives, glycols, glycol derivatives, sugars, ethoxylated and/or propoxylated esters and ethers, urea, sodium PCA, alcohols, ethanol, isopropyl alcohol, and combinations thereof.
  • the carrier is propylene glycol.
  • the composition contains a carrier in an amount from about 1% by weight of the composition to about 99.9% by weight of the composition, more typically from about 2% by weight of the composition to about 95% by weight of the composition, and more typically from about 5% by weight of the composition to about 90% by weight of the composition.
  • Thermo-reversible gelling agents are defined as ingredients that are soluble, partially soluble, or miscible in a hydrophilic carrier at elevated temperatures, such as 50° C., wherein the agents have the ability to thicken the carrier when cooled to 25° C., but will be less viscous at 50° C. when application to a substrate is necessary.
  • Suitable hydrophilic carriers include water, glycols, e.g., propylene glycol.
  • Thermo-reversible gelling agents for use in the composition may include salts of fatty acids such as sodium stearate, sodium palmitate, potassium stearate. These salts can be added to the composition or can be created in-situ by addition of the fatty acid and neutralizing with appropriate base.
  • compositions are to provide stearic acid and sodium hydroxide to produce sodium stearate.
  • Other common hermos-reversible gelling agents could include, e.g., polyethylene glycols and derivatives such as PEG-20, PEG-150 distearate, PEG-150 pentaerythrityl tetrastearate, disteareth-75 IPDI, disteareth-100 IPDI, fatty alcohols, e.g., cetyl alcohol, fatty acids such as stearic acid, hydroxystearic acid and its derivatives, and combinations thereof.
  • the composition can contain various other ingredients and components.
  • other ingredients that may be included within the composition are emollients, sterols or sterol derivatives, natural and synthetic fats or oils, viscosity enhancers, rheology modifiers, polyols, surfactants, alcohols, esters, silicones, clays, starch, cellulose, particulates, moisturizers, film formers, slip modifiers, surface modifiers, skin protectants, humectants, sunscreens, and the like.
  • Embodiments described herein further relate to pharmaceutical compositions and/or preparations comprising one or more of the aforementioned compounds of Formula I or Formula II and a carrier.
  • pharmaceutically acceptable is employed herein to refer to those compounds, salts, compositions, dosage forms, etc., which are—within the scope of sound medical judgment—suitable for use in contact with the tissues of human beings and/or other mammals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.
  • “pharmaceutically acceptable” means approved by a regulatory agency of the federal or a state government, or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in mammals (e.g., animals), and more particularly, in humans.
  • compositions may be prepared by any suitable means known in the art.
  • suitable means known in the art.
  • examples of such compositions include those adapted for: (a) topical application, e.g., articles (e.g., gauzes, pads, swabs, dressings), creams, ointments, gels, lotions, etc.; (b) parenteral administration, e.g., subcutaneous, intramuscular or intravenous injection as a sterile solution or suspension; (c) oral administration, external application (e.g. drenches including aqueous and non-aqueous solutions or suspensions), tablets, boluses, powders, granules, pellets for admixture with feedstuffs, pastes for application to the tongue, etc.
  • the pharmaceutical compositions may comprise one or more antibiotic agents.
  • antibiotic or “antimicrobial agent” refers to a substance that inhibits the growth of or destroys microorganisms.
  • the antibiotic is useful in curbing the virulence of an infectious agent and/or treating an infectious disease.
  • Antibiotic also refers to semi-synthetic substances wherein a natural form produced by a microorganism, e.g., yeast or fungus is structurally modified.
  • the antibiotic is selected from the group consisting of ⁇ -lactams (including, ⁇ -lactamase inhibitors and cephalosporins), fluoroquinolones, aminoglycosides, tetracyclines and/or glycylcyclines and/or polymyxins.
  • ⁇ -lactams including, ⁇ -lactamase inhibitors and cephalosporins
  • fluoroquinolones aminoglycosides
  • tetracyclines and/or glycylcyclines and/or polymyxins Any combination of antimicrobial agents may also be employed, e.g., at least one ⁇ -lactam and at least one fluoroquinolone; at least one aminoglycoside and one cephalosporin; at least one ⁇ -lactam and one ⁇ -lactamase inhibitor, optionally together with an aminoglycoside, etc.
  • ⁇ -lactam includes natural and semi-synthetic penicillins and penicillin derivatives, e.g., benzathine penicillin, benzylpenicillin (penicillin G), phenoxymethylpenicillin (penicillin V), procaine penicillin and oxacillin; methicillin, dicloxacillin and flucloxacillin; temocillin; amoxicillin and ampicillin; azlocillin, carbenicillin, ticarcillin, mezlocillin and piperacillin; biapenem, doripenem, ertapenem, imipenem, meropenem, panipenem and PZ-601; cephalexin, cephalothin, cefazolin, cefaclor, cefuroxime, cefamandole, cefotetan, cefoxitin, cefotaxime, and cefpodoxime; cefepime and cefpirome; cefadroxil
  • Fluoroquinolones include, ciprofloxacin, garenoxacin, gatifloxacin, gemifloxacin, levofloxacin, and moxifloxacin.
  • Aminoglycosides include, for e.g., kanamycin, amikacin, tobramycin, dibekacin, gentamicin, sisomicin, netilmicin, neomycin B, neomycin C, neomycin E (paromomycin) and streptomycin, including, synthetic derivatives clarithromycin and azithromycin.
  • Tetracyclines include naturally-occurring compounds (e.g., tetracycline, chlortetracycline, oxytetracycline, demeclocycline) or semi-synthetic agents (e.g., lymecycline, meclocycline, methacycline, minocycline, rolitetracycline).
  • Glycylcyclines e.g., minocycline/tigecycline
  • Polymyxins include, e.g., polymyxin B and polymyxin E (colistin).
  • the compositions may contain an antibiotic at a concentration of 0.1 mg/mL, 0.5 mg/L, 1 mg/mL, 2 mg/mL, 3 mg/mL, 4 mg/mL, 5 mg/mL, 6 mg/mL, 7 mg/mL, 8 mg/mL, 9 mg/mL, 10 mg/mL, 11 mg/mL, 12 mg/mL, 13 mg/mL, 14 mg/mL, 15 mg/mL, 16 mg/mL, 17 mg/mL, 18 mg/mL, 19 mg/mL, 20 mg/mL, 21 mg/mL, 22 mg/mL, 23 mg/mL, 24 mg/mL, 25 mg/mL, 26 mg/mL, 27 mg/mL, 28 mg/mL, 29 mg/mL, 30 mg/mL, 31 mg/mL, 32 mg/mL, 33 mg/mL, 34 mg/mL, 35 mg/mL, 36 mg/mL, 37 mg/mL, 38 mg/mL, 10
  • wound dressings comprising wound dressing materials as described herein, e.g., compounds of Formula I or Formula II.
  • the wound dressings consist essentially of the wound dressing materials as described herein, e.g., a compound of Formula I or Formula II.
  • wound dressing disclosed herein are biocompatible, biodegradable, non-immunogenic and readily commercially available.
  • the compounds of Formula I or Formula II are provided in the form of particles, such as fiber particles or powder particles, optionally containing a medicament.
  • the materials preferably contain CMC fibers.
  • compositions may preferably comprise an intimate mixture of the dressing material and other compounds.
  • the intimate mixture comprises a mixed solution or dispersion of the dressing material and a suitable vehicle, such as a solvent, or a solid composition produced by removing solvent from such a solution or dispersion.
  • the dressing material makes up at least 5%, more preferably at least 10%, 20%, 30%, 50%, 75%, 90% or greater % by weight of the material.
  • the material consists essentially of the dressing material.
  • Other components of the material may include 0-25% by weight, for example from about 1 to about 20% by weight, of one or more other biocompatible polysaccharides, for example alginates such as sodium alginate or calcium alginate, starch derivatives such as sodium starch glycolate, cellulose derivatives such as methyl cellulose or carboxymethyl cellulose, or glycosaminoglycans such as hyaluronic acid or its salts, chondroitin sulfate or heparan sulfate.
  • the materials may also comprise up to about 25% by weight, for example from about 1 to about 20% by weight, of one or more structural proteins selected from the group consisting of fibronectin, fibrin, laminin, elastin, collagen and mixtures thereof.
  • the protein comprises collagen, and more preferably it consists essentially of collagen.
  • the materials may also comprise up to about 20% by weight, preferably from about 2% to about 10% by weight of water.
  • the materials may also contain 0-40% by weight, for example from about 5 to about 25% by weight, of a plasticizer, preferably a polyhydric alcohol such as glycerol or sorbitol.
  • the materials may also comprise up to about 10% by weight, for example from about 0.01 to about 5% by weight, typically from about 0.1 to about 2% by weight of one or more therapeutic wound healing agents, such as non-steroidal anti-inflammatory drugs (e.g., acetaminophen), steroids, local anesthetics, antimicrobial agents, or growth factors (e.g., fibroblast growth factor or platelet derived growth factor).
  • therapeutic wound healing agents such as non-steroidal anti-inflammatory drugs (e.g., acetaminophen), steroids, local anesthetics, antimicrobial agents, or growth factors (e.g., fibroblast growth factor or platelet derived growth factor).
  • the antimicrobial agent may, for example, comprise an antiseptic, an antibiotic, or mixtures thereof.
  • Preferred antibiotics include tetracycline, penicillins, terramycins, erythromycin, bacitracin, neomycin, polymycin B, mupirocin, clindamycin and mixtures thereof.
  • Preferred antiseptics include silver, including colloidal silver, silver salts including salts of one or more of the anionic polymers making up the material, silver sulfadiazine, chlorhexidine, povidone iodine, triclosan, sucralfate, quaternary ammonium salts and mixtures thereof.
  • the weight ratio of the wound dressing material to other auxiliary agents and materials is from about 1:99 to about 99:1. More preferably, the weight ratio is in the range about 1:9 to about 9:1, more preferably it is in the range about 4:1 to about 1:4, still more preferably in the range about 2:1 to about 1:2.
  • the material may be in any convenient form, such as a powder, microspheres, flakes, a mat or a film.
  • the material is in the form of a semisolid or gel ointment for topical application.
  • the material is in the form of a freeze-dried or solvent-dried bioabsorbable sponge for application to a chronic wound.
  • the average pore size of the sponge is in the region of 10-500 ⁇ m, more preferably about 100-300 ⁇ m.
  • a suitable sponge has been made by freeze-drying or solvent drying an aqueous dispersion comprising compounds of Formula I or Formula II, together with suitable therapeutic agents.
  • the material is in the form of a flexible film, which may be continuous or interrupted (e.g. perforated).
  • the flexible film preferably comprises a plasticizer to render it flexible, such as glycerol.
  • wound dressing materials in sheet form comprising an active layer of a composition comprising compounds of Formula I or Formula II.
  • the active layer would normally be the wound contacting layer in use, but in some embodiments it could be separated from the wound by a liquid-permeable top sheet.
  • the area of the active layer is from about 1 cm 2 to about 400 cm 2 , particularly from about 4 cm 2 to about 100 cm 2 .
  • the wound dressing material further comprises a backing sheet extending over the active layer opposite to the wound facing side of the active layer.
  • the backing sheet is larger than the active layer such that a marginal region of width 1 mm to 50 mm, preferably 5 mm to 20 mm extends around the active layer to form a so-called island dressing.
  • the backing sheet is preferably coated with a pressure sensitive medical grade adhesive in at least its marginal region.
  • the back sheet is substantially liquid-impermeable.
  • the backing sheet is semipermeable, e.g., the backing sheet is preferably permeable to water vapor, but not permeable to liquid water or wound exudate.
  • the backing sheet is also microorganism-impermeable.
  • Suitable continuous conformable backing sheets will preferably have a moisture vapor transmission rate (MVTR) of the backing sheet alone of 300 to 5000 g/m 2 /24 hrs., preferably 500 to 2000 g/m 2 /24 hrs. at 37.5° C. at 100% to 10% relative humidity difference.
  • the backing sheet thickness is preferably in the range of 10 to 1000 micrometers, more preferably 100 to 500 micrometers.
  • the MVTR of the dressing as a whole is lower than that of the backing sheet alone because the apertured sheet partially obstructs moisture transfer through the dressing.
  • Suitable polymers for forming the backing sheet include polyurethanes and poly alkoxyalkyl acrylates and methacrylates.
  • the backing sheet comprises a continuous layer of a high density blocked polyurethane foam that is predominantly closed-cell.
  • a suitable backing sheet material is a polyurethane film.
  • the adhesive layer should be moisture vapor transmitting and/or patterned to allow passage of water vapor.
  • the adhesive layer is preferably a continuous moisture vapor transmitting, pressure-sensitive adhesive layer of the type conventionally used for island-type wound dressings, for example, a pressure sensitive adhesive based on acrylate ester copolymers, polyvinyl ethyl ether and polyurethane. Polyurethane-based pressure sensitive adhesives may be selectively used.
  • the dressing may comprise further layers of a multilayer absorbent article may be built up between the active layer and the protective sheet.
  • these layers may comprise an apertured plastic film to provide support for the active layer in use, in which case the apertures in the film are preferably aligned in register with the apertures in the hydrogel layer.
  • the dressing may comprise an absorbent layer between the active layer and the protective sheet, especially if the dressing is for use on exuding wounds.
  • the optional absorbent layer may be any of the layers conventionally used for absorbing wound fluids, serum or blood in the wound healing art, including gauzes, nonwoven fabrics, superabsorbents, hydrogels and mixtures thereof.
  • the absorbent layer comprises a layer of absorbent foam, such as an open celled hydrophilic polyurethane foam.
  • the absorbent layer may be a nonwoven fibrous web, for example a carded web of viscose staple fibers.
  • the wound dressing may be protected by a removable cover sheet.
  • the cover sheet is normally formed from flexible thermoplastic material. Suitable materials include polyesters and polyolefins.
  • the adhesive-facing surface of the cover sheet is a release surface. That is to say, a surface that is only weakly adherent to the active layer and the adhesive on the backing sheet to assist peeling of the hydrogel layer from the cover sheet.
  • the cover sheet may be formed from a non-adherent plastic such as a fluoropolymer, or it may be provided with a release coating such as a silicone or fluoropolymer release coating.
  • the wound dressing is sterile and packaged in a microorganism-impermeable container.
  • kits comprising, in one or separate compartments, the compounds of Formula I or Formula II, optionally together with an excipient, carrier or oil.
  • the kits may further comprise additional ingredients, e.g., gelling agents, emollients, surfactants, humectants, viscosity enhancers, emulsifiers, etc., in one or more compartments.
  • additional ingredients e.g., gelling agents, emollients, surfactants, humectants, viscosity enhancers, emulsifiers, etc.
  • the kits may optionally comprise instructions for formulating an article for diagnosing, detecting or treating wounds, e.g., chronic or infected wounds.
  • the kits may also comprise instructions for using the components, either individually or together, in the treatment of wounds.
  • kits comprising a package and at least one absorbent article (described above) comprising the aforementioned compositions.
  • the kits may comprise the individual components separately, optionally together with secondary information, useable in or with the package.
  • the wound is a chronic wound, for example a wound selected from the group consisting of venous ulcers, decubitis ulcers and diabetic ulcers.
  • Embodiments of the disclosed technology further provide for surfaces comprising the aforementioned compounds of Formula I or Formula II, wherein the reporter or peptide is oriented to permit binding to a partner, e.g., an enzyme.
  • a partner e.g., an enzyme.
  • the surface is a surface of a solid support. Numerous and varied solid supports are known to those in the art.
  • Useful solid supports include natural polymeric carbohydrates and their synthetically modified, cross-linked or substituted derivatives, such as agar, agarose, cross-linked alginic acid, substituted and cross-linked guar gums, cellulose esters, especially with nitric acid and carboxylic acids, mixed cellulose esters, and cellulose ethers; natural polymers containing nitrogen, such as proteins and derivatives, including cross-linked or modified gelatins; natural hydrocarbon polymers, such as latex and rubber; synthetic polymers which may be prepared with suitably porous structures, such as vinyl polymers, including polyethylene, polypropylene, polystyrene, polyvinylchloride, polyvinylacetate and its partially hydrolyzed derivatives, polyacrylamides, polymethacrylates, copolymers and terpolymers of the above polycondensates, such as polyesters, polyamides, and other polymers, such as polyurethanes or polyepoxides; porous inorganic materials such as sulfates
  • the support is a well of an array plate, e.g., a microarray.
  • array plate e.g., a microarray.
  • Methods for constructing such arrays are known in the art, e.g., Cao et al., Appl Environ Microbiol., 77(23): 8219-8225, 2011.
  • Each compound of Formula I or Formula II (or the reporter alone) may be spotted in triplicate to eliminate irregular data due to physical defects in the array.
  • Embodiments of the disclosed technology further provide for diagnostic systems comprising the aforementioned compositions and/or kits.
  • the various components of the diagnostic systems may be provided in a variety of forms.
  • the compounds of Formula I or Formula II e.g., compounds containing peptide reporters
  • these lyophilized reagents may be pre-mixed before lyophilization so that when reconstituted they form a complete mixture with the proper ratio of each of the components ready for use in the assay.
  • the diagnostic systems of the present invention may contain a reconstitution reagent for reconstituting the lyophilized reagents of the kit.
  • Embodiments described herein further relate to LC detection systems.
  • An LC detection system utilizes monitoring of a change in alignment of 5CB liquid crystals (LCs) as the detection method.
  • LCs liquid crystals
  • a lipid is added to the part of the peptide sequence that would be cleaved.
  • the lipid once released would cause an alignment change in 5CB.
  • the alignment of 5CB can be detected through crossed polarizing lenses and shows a change from dark to bright; the system would comprise containment of the LC at the correct alignment until the point of use and may also involve the use of crossed polarized lenses and a microscope for detection and/or visualization.
  • the wound dressing comprises a wound dressing material having the structure of Formula Ia:
  • R is a region comprising a reporter molecule; m is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; and n is an integer selected from 200 to 4000. In further embodiments, n is an integer selected from 300 to 3500. In still further embodiments, n is an integer selected from 400 to 3200. In some embodiments, R is a peptide region comprising a reporter molecule and at least one amino acid.
  • the wound dressing comprises a wound dressing material having the structure of Formula Ib:
  • R is a region comprising a reporter molecule; and n is an integer selected from 200 to 4000. In further embodiments, n is an integer selected from 300 to 3500. In still further embodiments, n is an integer selected from 400 to 3200.
  • R is a peptide region comprising a reporter molecule and at least one amino acid. In some embodiments, R is a peptide region comprising a reporter molecule and one amino acid.
  • wound dressings comprising a wound dressing material having the structure of Formula II:
  • M is a gel-forming polymer
  • PEP is a peptide region comprising a reporter molecule and at least one amino acid
  • L is a linker that connects M and PEP.
  • the wound dressing comprises a wound dressing material having the structure of Formula IIa:
  • M is a gel-forming polymer selected from cellulose, chemically modified cellulose, pectin, alginate, chitosan, modified chitosan, hyaluronic acid, polysaccharide, or gum-derived polymer, CES, oxidized cellulose (or a derivative thereof), or any combination thereof;
  • PEP is a peptide region comprising a reporter molecule and at least one amino acid;
  • L is a linker that connects M and PEP, wherein L comprises one or more polyethylene glycol subunits or polypropylene subunits.
  • the wound dressing comprises a wound dressing material having the structure of Formula IIb:
  • PEP is a peptide region comprising a reporter molecule and at least one amino acid
  • m is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10
  • n is an integer selected from 200 to 4000.
  • n is an integer selected from 300 to 3500.
  • n is an integer selected from 400 to 3200.
  • the wound dressing comprises a wound dressing material having the structure of Formula IIc:
  • PEP is a peptide region comprising a reporter molecule and at least one amino acid
  • m is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10
  • n is an integer selected from 200 to 4000.
  • n is an integer selected from 300 to 3500.
  • n is an integer selected from 400 to 3200.
  • Embodiments provided herein further relate to methods of making compounds of Formula I or Formula II, including precursors thereof.
  • precursor includes any compound which is employed as a reactant to generate an intermediary or a final product.
  • M is a gel-forming polymer comprising a plurality of monomers selected from the group consisting of cellulose, carboxymethylcellulose (CMC), oxidized cellulose (or a derivative thereof), cellulose ethyl sulfonate (CES), pectin, alginate, chitosan, modified chitosan, hyaluronic acid, polysaccharide, or gum-derived polymer, or any combination or mixture thereof and R is a reporter region, comprising, conjugating the gel-forming polymer with the reporter molecule, e.g., via covalent bond.
  • CMC carboxymethylcellulose
  • CES cellulose ethyl sulfonate
  • the reporter R is a substrate for a wound-specific marker, e.g., a wound-specific enzyme such as a hydrolase, and more specifically a protease, as described before.
  • the substrate for the wound-specific marker comprises, for example, a hydrolysable substrate, e.g., an amino acid, a sugar, a peptide, a polysaccharide, a nucleic acid, a lipid, or a combination thereof.
  • the gel-forming polymer is conjugated to the reporter molecule via a peptide, a glycosidic, an amide, an ester, an ether, an anhydride or a similar linkage.
  • a “peptide bond” is formed by the condensation reaction between two amino acids, wherein the acid moiety of one reacts with the amino moiety of the other to produce a peptide bond (—CO—NH—) between the two amino acids.
  • a “glycosidic bond” is formed between the hemiacetal or hemiketal group of a saccharide (or a molecule derived from a saccharide) and the hydroxyl group of some compound such as an alcohol.
  • glycoside A substance containing a glycosidic bond is a glycoside.
  • glycoside is now extended to also cover compounds with bonds formed between hemiacetal (or hemiketal) groups of sugars and several chemical groups other than hydroxyls, such as —SR (thioglycosides), —SeR (selenoglycosides), —NR1R2 (N-glycosides), or even —CR1R2R3 (C-glycosides).
  • amide refers to refers to either —N(R 1 )—C( ⁇ O)— or —C( ⁇ O)—N(R 1 )— wherein R 1 is defined herein to include hydrogen as well as other groups.
  • substituted amide refers to the situation where R1 is not hydrogen, while the term “unsubstituted amide” refers to the situation where R1 is hydrogen.
  • ester refers to a chemical compound derived from an acid (organic or inorganic) in which at least one hydroxyl group is replaced by an alkoxy group. Esters have a generic formula —C( ⁇ O)—OR 1 or R 1 —C( ⁇ O)—O— wherein R 1 is defined herein to include hydrogen as well as other groups.
  • esters include, but are not limited to, alkyl, alkenyl, alkynyl, aryl, heteroaryl, aralkyl, heteroaralkyl, cycloalkyl and heterocyclyl esters of acidic groups, including, but not limited to, carboxylic acids, phosphoric acids, phosphinic acids, sulfonic acids, sulfinic acids and boronic acids.
  • sulfonyl represents a group of the formula —SO2-alkyl or —SO2-aryl wherein “alkyl” includes saturated monovalent hydrocarbon radicals having straight, branched or cyclic moieties or combinations thereof and contains 1-20 carbon atoms, preferably 1-5 carbon atoms and “aryl” includes an organic radical derived from an aromatic hydrocarbon by removal of one hydrogen, such as phenyl or naphthyl, optionally substituted by 1 to 5 substituents independently selected from the group halogen, hydroxy, thiol, amino, nitro, cyano, acyl, acyloxy, sulfonyl, sulfinyl, alkylamino, carboxy, ester, ether, amido, sulfonic acid, sulfonamide, alkylthio, oxyester.
  • sulfinyl represents a group of the formula —SO-alkyl or —SO-aryl wherein “alkyl” and “aryl” are defined above.
  • sulfonamide represents a group of formula —SO2NH2.
  • oxyester means a group of formula —O—COO-alkyl, or —O—COO-aryl wherein “alkyl” and “aryl” are defined above.
  • ether means a group of formula alkyl-O-alkyl or alkyl-O-aryl or aryl-O-aryl wherein “alkyl” and “aryl” are defined above.
  • amido means a group of formula —CONRR′ wherein R and R′ are independently selected from hydrogen, “alkyl” or “aryl”.
  • oxyamido means a group of formula —O—CONRR′ wherein R and R′ are independently selected from hydrogen, “alkyl” or “aryl”.
  • alkoxy as used herein includes —O-alkyl groups wherein “alkyl” is defined above.
  • alkylthio as used herein, includes alkyl groups wherein “alkyl” is defined above.
  • alkylamino as used herein, includes —NHalkyl or —N(alkyl)2 groups wherein “alkyl” is defined above.
  • a method of making a compound of Formula I comprising the structure M-L-R, wherein, M and R are each, as described previously and L is a linker which is a monomer or a polymer of a neutral polymer, e.g., a polymer selected from the group consisting of an ethoxylated polyhydric alcohol, a polyvinyl pyrrolidone polymer, a polypropylene, a polyalkylene glycol, a polyamine, including, ethers, amides, and esters thereof.
  • a neutral polymer e.g., a polymer selected from the group consisting of an ethoxylated polyhydric alcohol, a polyvinyl pyrrolidone polymer, a polypropylene, a polyalkylene glycol, a polyamine, including, ethers, amides, and esters thereof.
  • the M is conjugated to the L via a first ester, oxyester, amide, amido, oxyamido, ether, sulfonyl, sulfinyl, sulfonamide, alkoxy, alkylthio, alkylamino, or a similar linkage.
  • the linker L is conjugated to the reporter region R via a second ester, oxyester, amide, amido, oxyamido, ether, sulfonyl, sulfinyl, sulfonamide, alkoxy, alkylthio, alkylamino, or a similar linkage.
  • the two linkages may be identical or different, e.g., M may be conjugated to the L via an ester linkage while L may be conjugated to the R via a peptide linkage.
  • the compound of Formula I having the structure M-L-R is synthesized by first conjugating the gel-forming polymer M with the linker L to generate a precursor M-L and then conjugating the precursor M-L with the reporter region R to generate the compound of Formula I.
  • the compound of Formula I having the structure M-L-R is synthesized by first conjugating the linker L with the reporter region R to generate the precursor L-R, which is then conjugated to the gel-forming polymer M to generate the compound of Formula I.
  • the compound of Formula I having the structure M-L-R may be synthesized in a single reaction chamber or multiple reaction chambers.
  • the compositions, dressing materials, articles, kits and systems described herein are useful in diagnosing or treating wounds, particularly chronic or infected wounds.
  • any type of wound may be diagnosed and/or treated, the embodiments are particularly suitable for diagnosing and treating wounds that exude wound fluid.
  • the wound may be a chronic or acute wound.
  • Representative examples of chronic wounds include, e.g., venous ulcers, pressure sores, decubitis ulcers, diabetic ulcers and chronic ulcers of unknown aetiology.
  • Representative examples of acute wounds include, e.g., acute traumatic laceration, perhaps resulting from an intentional operative incision.
  • a wound fluid refers to any wound exudate or other fluid (suitably substantially not including blood) that is present at the surface of the wound, or that is removed from the wound surface by aspiration, absorption or washing.
  • the determining, measuring or quantifying is suitably carried out on wound fluid that has been removed from the body of the patient, but can also be performed on wound fluid in situ.
  • wound fluid does not normally refer to blood or tissue plasma remote from the wound site.
  • the wound fluid is mammalian wound fluid, suitably human wound fluid.
  • the diagnostic method comprises contacting a wound with at least one composition comprising a compound of Formula I or Formula II, a dressing material comprising such compounds, article comprising such materials or compounds, kits comprising such materials or compounds, or a system comprising such materials or compounds described herein; and measuring a parameter associated with the wound.
  • the parameter being measured is a level or activity of a wound-specific hydrolase.
  • the parameter being measured is the activity of the hydrolase.
  • the measurement may either be made in situ or ex situ.
  • in situ refers to processes, events, objects, or components that are present or take place within the context of the system or device, including, the surrounding environment, for example, the biological material with which the composition, article, system or device is in contact with.
  • an in situ reaction may refer to the reaction of the various components present in the device (e.g., compound of Formula I or Formula II), including, components provided by the human skin tissue (e.g., wound exudate containing the enzyme).
  • ex situ refers to outside of the environment.
  • the measurement is performed ex situ, e.g., removing the fluid from the wound for analysis in the apparatus or device of the invention.
  • the measurement is made in situ.
  • the method comprising determining a level of a reporter, e.g., a product of a substrate acted upon by a wound-specific enzyme. More specifically, the method comprises determining a level of a hydrolase enzyme product.
  • determining includes measuring a numerical value of the activity or level of said hydrolase; establishing if the activity or level falls above or below a predetermined range; and/or comparing the numerical value of activity or level with a control standard.
  • the control standard may comprise determining a level or activity of the hydrolase in a biopsy material obtained from an unwounded site or from a healthy subject.
  • the term “determining” comprises measuring the parameter (e.g., activity or level) of at least one wound specific protease is selected from the group consisting of MMP-1 (collagenase), MMP-2 (gelatinase A), MMP-3 (stomelysin 1), MMP-8 (neutrophil collagenase), MMP-9 (gelatinase B), human neutrophil elastase (HNE), cathepsin G, urokinase-type plasminogen activator (uPA), and lysozyme, or a combination thereof; establishing if said parameter exceeds a first predetermined threshold; and/or comparing the numerical value of parameter with a control standard.
  • MMP-1 collagenase
  • MMP-2 gelatinase A
  • MMP-3 stomelysin 1
  • MMP-8 neutral collagenase
  • MMP-9 gelatinase B
  • HNE human neutrophil elastase
  • cathepsin G cathe
  • the control standard may comprise determining a parameter of the protease in a biopsy material obtained from an unwounded site or from a healthy subject.
  • the term “determining” comprises establishing whether a weighted average (weighted sum) of the parameters associated with a plurality of the aforementioned proteases exceeds a predetermined threshold value for said weighted average.
  • the parameter is activity level of the analyte (e.g. a protease) in a wound fluid.
  • the activity of an individual analyte is expressed in terms units/mL.
  • the parameter is the level of the analyte (e.g., protease) in a wound fluid.
  • the term amount is also indicative of the activity of a particular analyte.
  • the term “combined amount” or “combined activity” refers to a single numerical value that results from the application of a mathematical function to a plurality of values, for example those amounts obtained for a number of individual analytes.
  • the term “combined amount” or “combined activity” may refer to the sum or product of a group of individual values.
  • the term “combined amount” or “combined activity” relates to the sum of a group of individual values.
  • the amount of elastase refers to elastase-like activity (e.g., U/mL) and the amount of metalloproteinase (MMP) refers to total concentration of the respective analyte (e.g., in ng/mL).
  • MMP metalloproteinase
  • the term “quantifying” refers to measuring an absolute numerical quantity of a particular analyte(s) or substrate(s) in a sample, within the margins of experimental error.
  • markers or analytes refers to any chemical entity that is identified or determined using the apparatus, devices, kits or methods defined herein.
  • the markers or analytes determined or identified by the apparatus, devices, kits or methods of the present invention are cleaved products of the aforementioned enzymes.
  • the term “predetermined range” refers to a data range or profile that the skilled person would understand is indicative of a particular sub-class of patient.
  • the predetermined range may be a data range or profile that is typical of a wound that would respond well to a particular wound treatment, such as antibiotic therapy.
  • the predetermined range may suitably refer to a data range that is typical of a wound that would not respond well to a particular wound treatment, such as antibiotic therapy.
  • the term “predetermined threshold” refers to a minimum level that the skilled person would determine is indicative of a non-healing wound based on statistical analysis of levels determined for known healing and non-healing wounds, for example as explained further above.
  • the threshold should be set at an appropriate level so that non-healing wounds with high protease activity are correctly identified. Increasing the threshold will increase the chance of only non-healing wounds being over the threshold. However, if the threshold is too high, wounds that are non-healing due to a high level of proteases would not be identified and clinically this would mean they would not receive the required protease modulating treatment.
  • control standard refers to a data set or profile that can be used as a reference or comparison in order to define or normalize another data point or set of data.
  • control or “control standard” may be data set or profile that is indicative of a particular sub-class of patient.
  • control standard may be a data set or profile indicative of healing or non-healing wound status.
  • control or “control standard” can be a data set or profile that can be used as a comparative tool to allow a skilled person to determine whether a wound is likely to be responsive or non-responsive to a wound treatment, such as antibiotic therapy.
  • control standard is a data set or profile indicative of a patient that does not respond well to wound treatment.
  • control standard is a data set or profile indicative of a patient that responds well to wound treatment. Patients that tend to respond well to wound treatment as disclosed herein exhibit lower combined amount or activity of hydrolases than patients that tend not to respond well to the treatment. For example, patients that tend to respond well to wound treatment as disclosed herein exhibit lower combined amounts of at least one wound-specific hydrolase.
  • the threshold matrix metalloproteinase (MMP) activity is about 5 U/mL to about 30 U/mL, including all values in between, e.g., about 6 U/mL, about 7 U/mL, about 8 U/mL, about 9 U/mL, about 10 U/mL, about 11 U/mL, about 12 U/mL, about 13 U/mL, about 14 U/mL, about 15 U/mL, about 16 U/mL, about 17 U/mL, about 18 U/mL, about 19 U/mL, about 20 U/mL, about 21 U/mL, about 22 U/mL, about 23 U/mL, about 24 U/mL, about 25 U/mL, or more, indicate chronic wound infection.
  • MMP matrix metalloproteinase
  • Units of activity (U) are typically used to describe enzyme catalytic activity, where a unit (U) refers to the amount of enzyme that catalyzes the conversion of 1 micromole ( ⁇ mole) of substrate per minute.
  • 1 enzyme unit (U) 1 ⁇ mol/min, where ⁇ mol refers to the amount of substrate converted.
  • the threshold human neutrophil elastase activity is about 5 U/mL to about 30 U/mL, including all values in between, e.g., about 6 U/mL, about 7 U/mL, about 8 U/mL, about 9 U/mL, about 10 U/mL, about 11 U/mL, about 12 U/mL, about 13 U/mL, about 14 U/mL, about 15 U/mL, about 16 U/mL, about 17 U/mL, about 18 U/mL, about 19 U/mL, about 20 U/mL, about 21 U/mL, about 22 U/mL, about 23 U/mL, about 24 U/mL, about 25 U/mL, or more, indicate chronic wound infection.
  • the threshold human neutrophil elastase activity levels of at least 9.6 indicate chronic wound infection. In some embodiments, human neutrophil elastase activity levels of at least 22.9 U/mL indicate chronic wound infection.
  • the threshold cathepsin G activity levels of about 10 U/mL to about 100 U/mL including all values in between, e.g., about 15 U/mL, about 20 U/mL, about 25 U/mL, about 30 U/mL, about 35 U/mL, about 40 U/mL, about 45 U/mL, about 50 U/mL, about 55 U/mL, about 60 U/mL, about 65 U/mL, about 70 U/mL, about 75 U/mL, about 80 U/mL, about 85 U/mL, about 90 U/mL, about 95 U/mL, about 100 U/mL, about 110 U/mL, about 120 U/mL, or more, indicate chronic wound infection.
  • cathepsin G activity levels of at least 50 U/mL, at least 40 U/mL, at least 30 U/mL, at least 20 U/mL, at least 15 U/mL or at least 10 U/mL indicates chronic wound infection.
  • Embodiments disclosed herein further relate to treatment of chronic or infected wounds using the compositions, materials, articles, dressings, kits and/or systems described herein.
  • the therapeutic embodiment includes, contacting a composition, material, article, dressing, kit, system or devices of the invention with a subject in need thereof.
  • the method may include determination of whether the subject is responding to the treatment.
  • wounds are “responsive to treatment” or not.
  • the skilled person will readily be able to determine the levels of the proteases identified in the present claims that are predictive or indicative of a good response or poor response to wound treatment, particularly to treatment with wound dressings comprising oxidized cellulose.
  • responsive and responder(s) refer to wounds that are considered to respond well to wound treatment, particularly to treatment with a pharmacological agent, e.g., antibiotics.
  • non-responsive and non-responder(s) refers to wounds that are not considered to respond well to wound treatment, particularly to treatment with the pharmacological agent, e.g., antibiotics. For instance, patients who exhibit better than 50% wound closure after 4 weeks of wound treatment are considered to be responsive to said treatment.
  • a patient may be simultaneously diagnosed and treated with the compositions, articles, systems, or devices described herein.
  • the term “simultaneously” means performing the stated objectives, e.g., diagnosis and treatment, together.
  • a patient may be sequentially diagnosed and treated with the compositions, articles, systems, or devices described herein.
  • Embodiments described herein further enable a care giver or a patient to determine quickly and reliably whether a wound is likely to be non-healing, and to select an appropriate therapy based on this determination.
  • non-healing wounds may require the application of special wound dressings such as wound dressings comprising specific therapeutic agents, to promote healing.
  • embodiments described herein further provide methods of treatment of a wound, e.g., chronic or infected wounds, comprising determining whether a wound is healing or non-healing, followed by applying a wound dressing comprising a therapeutic agent to the wound if it is non-healing.
  • Embodiments described herein provide methods and assays for diagnosis or detection of infected wounds.
  • the methods are suitable for the detection of bacterial infectious agents.
  • the wounds are infected with gram-negative bacteria.
  • Typical gram-negative bacteria include proteobacteria such as E. coli, Salmonella, Pseudomonas , and Helicobacter , and cyanobacteria .
  • the wounds are infected with gram-positive bacteria.
  • gram-positive bacteria is meant a bacterium or bacteria that contain(s) teichoic acid (e.g., lipoteichoic acid and/or wall teichoic acid), or a functionally equivalent glycopolymer (e.g., a rhamnopolysaccharide, teichuronic acid, arabinogalactan, lipomannan, and lipoarabinomannan) in its cell wall.
  • teichoic acid e.g., lipoteichoic acid and/or wall teichoic acid
  • a functionally equivalent glycopolymer e.g., a rhamnopolysaccharide, teichuronic acid, arabinogalactan, lipomannan, and lipoarabinomannan
  • the bacteria include pathogenic bacteria that infect mammalian hosts (e.g., bovine, murine, equine, primate, feline, canine, and human hosts).
  • pathogenic bacteria include, e.g., members of a bacterial species such as Bacteroides, Clostridium, Streptococcus, Staphylococcus, Pseudomonas, Haemophilus, Legionella, Mycobacterium, Escherichia, Salmonella, Shigella, Vibrio , or Listeria .
  • the infectious bacteria is selected from the group consisting of Clostridium difficile , Carbapenem-Resistant Enterobacteriaceae (CR- Klebsiella spp; CR- E. coli ), and Neisseria gonorrhoeae .
  • the infectious bacteria is selected from the group consisting of multidrug-resistant Acinetobacter , drug-resistant Campylobacter , extended spectrum ⁇ -Lactamase (ESBL)-producing enterobacteriaceae, vancomycin-resistant enterococcus , multidrug-resistant Pseudomonas aeruginosa , drug-resistant non-typhoidal Salmonella , drug-resistant Salmonella enterica serovar Typhi , drug-resistant Shigella , methicillin-resistant Staphylococcus aureus (MRSA), drug-resistant Streptococcus pneumoniae , and drug-resistant Tuberculosis.
  • multidrug-resistant Acinetobacter drug-resistant Campylobacter
  • ESBL extended spectrum ⁇ -Lactamase
  • the infectious bacteria is selected from the group consisting of vancomycin-resistant Staphylococcus aureus , erythromycin-resistant Group A Streptococcus , clindamycin-Resistant Group B Streptococcus.
  • the chronic or infected wounds are found in host subjects.
  • the hosts are mammals, e.g., a rodent, a human, a livestock animal, a companion animal, or a non-domesticated or wild animal.
  • the subject may be a rodent, e.g. a mouse, a rat, a guinea pig, etc.
  • the subject may be a livestock animal.
  • suitable livestock animals may include pigs, cows, horses, goats, sheep, llamas and alpacas.
  • the subject may be a companion animal.
  • companion animals may include pets such as dogs, cats, rabbits, and birds.
  • the subject may be a zoo animal.
  • a “zoo animal” refers to an animal that may be found in a zoo. Such animals may include non-human primates, large cats, wolves, and bears.
  • the subject is a human.
  • kits for detecting levels of one or more enzymes in a mammalian wound comprising the steps of: (a) placing the wound dressing material described herein in contact with the mammalian wound; (b) visually comparing the wound dressing material in contact with the mammalian wound with one or more reference samples; and (c) obtaining a qualitative determination of the concentration of reporter molecules in the wound dressing material in contact with the mammalian wound.
  • the method of detecting the level of one or more enzymes in a mammalian wound consists essentially of the steps of: (a) placing the wound dressing material described herein in contact with the mammalian wound; (b) visually comparing the wound dressing material in contact with the mammalian wound with one or more reference samples; and (c) obtaining a qualitative determination of the concentration of reporter molecules in the wound dressing material in contact with the mammalian wound.
  • kits for detecting levels of one or more enzymes in a mammalian wound comprising the steps of: (a) placing the wound dressing material described herein in contact with the mammalian wound; (b) obtaining a quantitative determination of the concentration of reporter molecules in the wound dressing material in contact with the mammalian wound; and (c) comparing the quantitative determination with one or more reference samples.
  • the method of detecting the level of one or more enzymes in a mammalian wound consists essentially of the steps of: (a) placing the wound dressing material described herein in contact with the mammalian wound; (b) obtaining a quantitative determination of the concentration of reporter molecules in the wound dressing material in contact with the mammalian wound; and (c) comparing the quantitative determination with one or more reference samples.
  • kits for detecting levels of one or more proteases in a mammalian wound comprising the steps of: (a) placing the wound dressing material described herein in contact with the mammalian wound; (b) visually comparing the wound dressing material in contact with the mammalian wound with one or more reference samples; and (c) obtaining a qualitative determination of the concentration of reporter molecules in the wound dressing material in contact with the mammalian wound.
  • the method of detecting the level of one or more proteases in a mammalian wound consists essentially of the steps of: (a) placing the wound dressing material described herein in contact with the mammalian wound; (b) visually comparing the wound dressing material in contact with the mammalian wound with one or more reference samples; and (c) obtaining a qualitative determination of the concentration of reporter molecules in the wound dressing material in contact with the mammalian wound.
  • kits for detecting levels of one or more proteases in a mammalian wound comprising the steps of: (a) placing the wound dressing material described herein in contact with the mammalian wound; (b) obtaining a quantitative determination of the concentration of reporter molecules in the wound dressing material in contact with the mammalian wound; and (c) comparing the quantitative determination with one or more reference samples.
  • the method of detecting the level of one or more proteases in a mammalian wound consists essentially of the steps of: (a) placing the wound dressing material described herein in contact with the mammalian wound; (b) obtaining a quantitative determination of the concentration of reporter molecules in the wound dressing material in contact with the mammalian wound; and (c) comparing the quantitative determination with one or more reference samples.
  • kits to diagnose a chronic wound in a mammal comprising the steps of: (a) placing the wound dressing material described herein in contact with the mammalian wound; (b) visually comparing the wound dressing material in contact with the mammalian wound with one or more reference samples; and (c) obtaining a qualitative determination of the concentration of reporter molecules in the wound dressing material in contact with the mammalian wound.
  • the method to diagnose a chronic wound in a mammal consists essentially of the steps of: (a) placing the wound dressing material described herein in contact with the mammalian wound; (b) visually comparing the wound dressing material in contact with the mammalian wound with one or more reference samples; and (c) obtaining a qualitative determination of the concentration of reporter molecules in the wound dressing material in contact with the mammalian wound.
  • kits to diagnose a chronic wound in a mammal comprising the steps of: (a) placing the wound dressing material described herein in contact with the mammalian wound; (b) obtaining a quantitative determination of the concentration of reporter molecules in the wound dressing material in contact with the mammalian wound; and (c) comparing the quantitative determination with one or more reference samples.
  • the method to diagnose a chronic wound in a mammal consists essentially of the steps of: (a) placing the wound dressing material described herein in contact with the mammalian wound; (b) obtaining a quantitative determination of the concentration of reporter molecules in the wound dressing material in contact with the mammalian wound; and (c) comparing the quantitative determination with one or more reference samples.
  • kits to treat a wound in a mammal comprising the steps of: (a) placing the wound dressing material described herein in contact with the mammalian wound; (b) visually comparing the wound dressing material in contact with the mammalian wound with one or more reference samples; (c) obtaining a qualitative determination of the concentration of reporter molecules in the wound dressing material in contact with the mammalian wound; and (d) administering medical treatment to the mammal; wherein the medical treatment comprises antibiotic therapy only when the concentration of reporter molecules indicates that the mammalian wound is chronic.
  • the method to treat a wound in a mammal consists essentially of the steps of: (a) placing the wound dressing material described herein in contact with the mammalian wound; (b) visually comparing the wound dressing material in contact with the mammalian wound with one or more reference samples; (c) obtaining a qualitative determination of the concentration of reporter molecules in the wound dressing material in contact with the mammalian wound; and (d) administering medical treatment to the mammal; wherein the medical treatment comprises antibiotic therapy only when the concentration of reporter molecules indicates that the mammalian wound is chronic.
  • kits to treat a wound in a mammal comprising the steps of: (a) placing the wound dressing material described herein in contact with the mammalian wound; (b) obtaining a quantitative determination of the concentration of reporter molecules in the wound dressing material in contact with the mammalian wound; (c) comparing the wound dressing material in contact with the mammalian wound with one or more reference samples; and (d) administering medical treatment to the mammal; wherein the medical treatment comprises antibiotic therapy only when the concentration of reporter molecules indicates that the mammalian wound is chronic.
  • the method to treat a wound in a mammal consists essentially of the steps of: (a) placing the wound dressing material described herein in contact with the mammalian wound; (b) obtaining a quantitative determination of the concentration of reporter molecules in the wound dressing material in contact with the mammalian wound; (c) comparing the wound dressing material in contact with the mammalian wound with one or more reference samples; and (d) administering medical treatment to the mammal; wherein the medical treatment comprises antibiotic therapy only when the concentration of reporter molecules indicates that the mammalian wound is chronic.
  • the diagnosis and treatment is conducted in situ.
  • Embodiments described herein therefore allow diagnosis and treatment of wounds in an easy, non-invasive manner.
  • the diagnosis may be made in real time and the treatment may be applied to the infected wound or to the patient (systemically) and the progress of wound treatment be monitored over real-time, e.g., dissipation of the signal generated by the reporter molecule due to wound-healing.
  • n is an integer selected from 400 to 3200.
  • Sodium carboxymethylcellulose (NaCMC) fiber (443 mg, 1.08 mmol) was dissolved in deionized water (44 ml) to give a 1% solution.
  • Dowex 650C monosphere ion exchange resin was added in order to provide the acidified CMC (CMC-H). The monospheres were removed by filtration and then tetrabutylammonium hydroxide (TBAH) 40% (aq) was added until the pH was 8-9. The resulting solution was stirred for 30 min before being lyophilized overnight.
  • the lyophilized material (0.38 g) was dissolved in 40 ml dry DMF under nitrogen with stirring and gentle heating over a period of approximately 1 h. The resulting solution was cloudy and off-white.
  • FTIR 3360, 3320 (N—H/O—H), 2875 (C—H), 1650 with shoulder (amidic C ⁇ O of BOC), 1590 (C ⁇ O of CMC).
  • Solubility of Polymer 1 was determined in a similar manner as for compound 1-c. Data is shown below: x indicates insoluble material.
  • Solubility of Polymer 2 was determined in a similar manner as for compound 1-c. Data is shown below: x indicates insoluble material.
  • Polymer 2 was placed in a solution of piperidine: DMF (1:4 v/v, 6 ml) for approximately 2 h. The resulting de-protected product was then washed with EtOH before conducting a Kaiser test to confirm presence of free amine. This de-protected product (0.080 g, 0.15 mmol) was added to a 15-ml plastic separating column set up with a 10 ⁇ m polyethylene frit and a luer tip. A solution of Fmoc-Phe-OH (0.063 g, 0.16 mmol), HBTU (0.062 g, 0.16 mmol), DIPEA (0.23 ml, 1.8 mmol) and dry DMF (6 ml) was prepared and added to the column.
  • Fmoc-Lys-OH.HCl (0.0405 g, 0.100 mmol) was placed in a round bottomed flask, to which a 5:2 mixture of 1,4-dioxane: 10% K 2 CO 3 (aq) (3 ml) was added dropwise. The mixture was stirred and Dabsyl chloride (0.033 g, 0.10 mmol) was added to the mixture which was then stirred at RT overnight open to the atmosphere. The solution was diluted with 150 ml water. The layers were separated, and the aqueous layer was extracted with diethyl ether (10 ml ⁇ 3). The combined organic layers were dried and concentrated.
  • Sodium carboxymethylcellulose (NaCMC) of DoS 0.7 (2.0 g, 8.33 mmol) was dissolved in de-ionized water (180 mL) to give a 1% solution.
  • Dowex 650C monosphere ion exchange resin was added with stirring for 10 minutes. The monospheres were removed by filtration before tetrabutylammonium hydroxide (TBAH) 40% (aq) was added in 0.1 mL aliquots until the pH was 8-9 (3.5 mL, 36.14 mmol). The resulting solution was stirred for 30 minutes before being lyophilized over seven days.
  • TBAH tetrabutylammonium hydroxide
  • the lyophilized material was dissolved in dry DMF (240 mL) under a nitrogen atmosphere, stirring and gentle heating was required over a period of approximately 2 hours.
  • the solution was cooled to approx. 4° C. before 2-chloro-N-methylpyridinium iodide (CMP-I) (1.5 g, 5.8 mmol) was added with vigorous stirring.
  • CMP-I 2-chloro-N-methylpyridinium iodide
  • 2,2′-(ethylenedioxy)bis(ethylamine) (1.3567 g, 9.17 mmol) was added to the reaction, along with dry triethylamine (5 mL). The reaction was kept at 4° C.
  • Polymer 5 was soaked in dry DMF (6 mL) for 20 min in a filtration tube.
  • Compound 4-c (0.0416 g, 0.063 mmol) and HBTU (0.2893 g, 0.8571 mmol) were added along with DIPEA (0.2 mL, 0.8571 mmol).
  • the tube was secured and rotated on a lab mixer overnight at RT.
  • a Kaiser test was used to confirm presence of free amine.
  • Solubility of polymer 6 was examined in the following manner. A small amount of polymer 6 was placed into two separate glass-bottom petri dishes. The material was saturated with either pH 4 general lab buffer or pH 9 K 2 HPO 4 /MgCl 2 buffer (20 ⁇ L each). Each was viewed under the Zeiss Axioimager light microscope ⁇ 10 magnification optical lens plus ⁇ 10 magnification on the eye piece.
  • 1-Ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) (1.92 g, 10.01 mmol) was added to the solution with stirring.
  • L-cysteine (0.50 g, 4.1 mmol) was added to the reaction with stirring.
  • Solubility of Polymer 7 was determined in a similar manner as for compound 1-c. Polymer 7 was found to be insoluble in water, DMF, acetone, MeOH, EtOH, and DCM.
  • Polymer 5 (1.00 g, 2.86 mmol) was ground into a fine powder and mixed with DMF (25 ml) for 20 min.
  • Fmoc-Cys(StBu)-OH (3.7 g, 8.6 mmol), HBTU (2.87 g, 8.57 mmol) and DIPEA (1.10 ml, 8.57 mmol) were added with stirring at RT, open to air (a centrifuge tube and lab rotor were used to facilitate mixing overnight). The mixture was sonicated for 30 sec before being allowed to stir overnight.
  • the solid was filtered and washed with DCM (20 mL ⁇ 5) and MeOH (20 mL ⁇ 5). The resulting product was dried under in vacuo to provide compound 8-b as an off-white solid.
  • Polymer 5 (1.0 g, 2.714 mmol) was dispersed in PBS (25 mL) with gentle heating, stirring and sonication. The reaction vessel was purged with nitrogen gas. To a solution of Traut's reagent (0.600 g, 4.304 mmol) in PBS (50 mL), was added EDTA (0.044 g, 0.15 mmol). Once fully dissolved, this solution was added to the reaction mixture, which was stirred at RT under nitrogen atmosphere for 1 hour. The resulting product was separated by filtration and washed with PBS (3 ⁇ 10 mL) and methanol (3 ⁇ 10 mL). The white/off-white powdered solid product was then dried in vacuo and stored under nitrogen.
  • ⁇ C 10,000 Hz, CP MAS: 222.37 (spinning side band), shoulder of 172.5 (C8, C ⁇ O of CMC), 172.5 (C8, C ⁇ O of amide, possibly also some of the acid group on the Ellman's reagent), 156.0 (C15, small peak) 123.7 (spinning side band), 144.5 (aromatic region of Ellman's Reagent), 103.0 (C1), 96.79, 82.25 (C7, shoulder), 74.5 (C2, C3, C4, C5), 61.4 (C6), 39.3 plus shoulder (C9, C10, C11, C12, C13, C14), 32.2 sharp peak (possible contaminant).
  • the intermediate disulfide product 10-a (0.1 g, 0.1422 mmol), mercaptobenzoic acid (0.1 g, 0.7112 mmol) and methanol: water (4:1 ratio, 5 mL) were combined and stirred for 2 h at RT under nitrogen atmosphere.
  • the resulting product was filtered and washed with methanol (3 ⁇ 10 mL), DCM (3 ⁇ 10 mL) and further washed with methanol (3 ⁇ 10 mL) before being dried in vacuo to produce polymer 10 as a yellow solid powder.
  • Weight 0.0821 g (87% yield). Strong yellow color from an Ellman test indicates that some free thiol was still present, possibly in unreacted remaining starting materials.
  • Aminophenyl fluorescein (0.005 g, 0.0180 mmol), HBTU (0.0136 g, 0.036 mmol) and polymer 5 (0.013 g, 0.036 mmol) were added to a 25 mL RBF with a stir bar and purged with N 2 (g). Dry DMF (5 mL) was added and the reaction was stirred at RT overnight covered with foil to protect from the light. The reaction solution was filtered through a filtration tube and washed several times with DMF. The supernatant maintained an orange color; it was kept along with the washings and dried in vacuo. The solid product was recovered and dried under vacuum.
  • Enzyme efficacy was examined with esterase ( ⁇ 50 units/mL achieved by mixing neat esterase (0.01 mL) in PBS (0.99 mL).
  • Fluorescence microscopy indicated a distinct observable difference in fluorescence between the sample of polymer 12 and control sample (polymer 12 in PBS without enzyme), demonstrating that there was activity by the enzyme on the polymer to release the ester groups from the attached fluorescein.
  • N 2 wells had 40 ⁇ L of PBS added (control) into the test dispersion only.
  • N 2 wells had 40 ⁇ L of weak enzyme solution pipetted into the test dispersion.
  • N 2 wells had 40 ⁇ L of strong enzyme solution pipetted into the test dispersion.
  • N 2 wells had 40 ⁇ L of 1 M NaOH solution pipetted into the test dispersion.
  • FIG. 1 shows the plot for the strong esterase up to 30 min and plots the trend line for this and the weak esterase over the whole 60 min.
  • NaCMC fiber (2 ⁇ AQUACEL Dressings 10 ⁇ 10 cm) with DoS approx. 0.2-0.3 (1.97 g, 8.33 mmol) was broken apart into small, open fibers by hand and dispersed in a solution of ethanol/deionized water (80:20 v/v) (180 mL).
  • Dowex 650C monosphere ion exchange resin was added in order to provide the acidified CMC (CMC-H) and was mixed for approximately 30 min. The monospheres were removed carefully by filtration and tetrabutylammonium hydroxide (TBAH) 40% (aq) was added until the pH was 8-9. The resulting solution was stirred for 30 min before being concentrated in vacuo.
  • the solid was filtered and then washed with acetone (3 ⁇ 100 mL) then DMF (3 ⁇ 100 mL), sonication was performed during the wash steps in order to encourage the fibers to disperse in the wash solution.
  • a Kaiser test was used to confirm presence of terminal amines (a reading at 570 nm equating to 3.08 ⁇ mol amine).
  • Solubility of Polymer 13 was determined in a similar manner as for compound 1-c. Data is shown below: x indicates insoluble material.
  • Polymer 13 (0.250 g, 1.396 mmol) was placed in a RB flask under a nitrogen atmosphere.
  • Compound 14-a (90 mg, 0.679 mmol) was dissolved in dry DMF (5 mL) under nitrogen atmosphere and added to polymer 13 with stirring.
  • the reaction mixture was stirred for 90 min at RT under a nitrogen atmosphere before being filtered and washed with DMF (5 ⁇ 5 mL) and methanol (5 ⁇ 5 mL).
  • a Kaiser test was used to confirm presence of terminal amines (a reading at 570 nm equating to 1.86 ⁇ mol amine).
  • Solubility of Polymer 14 was determined in a similar manner as for compound 1-c. Data is shown below: x indicates insoluble material.
  • Polymer 5-a (prepared as in Example 5 using 2.0 g of NaCMC) was dissolved in dry DMF (150 mL) under nitrogen. Stirring and heating to ⁇ 50° C. was required over a period of ⁇ 2 h in addition to multiple cycles of sonication for 1 min to provide a clear solution. The solution was cooled to ⁇ 4° C. and stirred before 2-chloro-N-methylpyridinium iodide (CMP-I) (1.4875 g, 5.8 mmol) was added. The solution became a viscose “jelly” and was mixed vigorously and a further 20 mL dry DMF was added which broke down and diluted the gel.
  • CMP-I 2-chloro-N-methylpyridinium iodide
  • Polymer 15 (1.000 g, 2.273 mmol) was ground into a fine powder and mixed with DMF (15 mL) for 20 min.
  • Compound 8-a (1.14 g, 2.639 mmol), HBTU (2.59 g, 6.82 mmol) and DIPEA (1.19 mL, 6.82 mmol) were added with stirring at RT, open to air (a centrifuge tube and lab rotor were used to facilitate mixing overnight). The mixture was sonicated for 30 s before being allowed to stir overnight.
  • the solid was filtered and washed with DCM (5 ⁇ 20 mL) and methanol (5 ⁇ 20 mL). The resulting coupled intermediate was dried in vacuo.
  • This intermediate (850 mg) was added to a solution of piperidine: DMF (20:80 v/v), (20 mL) and bubbled with nitrogen gas in order to stir the mixture for 1 hour, after which the solid was filtered and washed with ethanol (3 ⁇ 50 mL) and DCM (3 ⁇ 50 mL). This process was then repeated and the amine intermediate was dried in vacuo. This amine intermediate was added to a RB flask and purged with nitrogen gas. TCEP (428 mg) was dissolved in deionized water (2.6 mL) and added to the amine intermediate with stirring, followed by the addition of methanol (5.2 mL).
  • Solubility assessment indicates that polymer 16 forms a hydrogel in pH 9 buffer (K 2 HPO 4 /MgCl 2 ).
  • polymer 16 was treated with TCEP in order to de-couple any disulfide bonds that had formed.
  • TCEP (0.1685 g, 0.196 mmol, 4 equiv) was dissolved in 1 mL water and added to polymer 16 (0.0754 g, 0.147 mmol) and was stirred at RT for 30 min. The solid was filtered and washed with water (3 ⁇ 10 mL) and freeze-dried yielding solid off-white/cream colored powder which was then washed with the DMF (3 ⁇ 10 mL).
  • Enzyme efficacy was examined with esterase ( ⁇ 58 units/mL achieved by mixing neat esterase (0.01 mL) in PBS (0.99 mL).
  • N 4 wells had 40 ⁇ L of PBS added (control) into the test dispersion only.
  • N 4 wells had 40 ⁇ L of weak enzyme solution pipetted into the test dispersion.
  • N 4 wells had 40 ⁇ L of strong enzyme solution pipetted into the test dispersion.
  • FIG. 3 shows the fluorescence reading at each time point minus the baseline reading (the particle suspension only). The instrument did not reach the saturation point during this experiment as it had previously. As expected, fluorescence for the weak and strong esterase solutions increased over time.
  • Polymer 5 (300 mg, 0.81 mmol) was ground into a fine powder and mixed with DMF (20 mL) for 20 minutes.
  • Fmoc-Ala-OH (1.00 g, 2.31 mmol
  • HBTU (1.84 g, 4.86 mmol
  • EDC 100 mg, 0.52 mmol
  • DIPEA 6.2 mL, 4.86 mmol
  • FTIR 3300 (N—H/O—H), 2850 (C—H), 1748 (amide), 1648 (C ⁇ O, amide of coupled product), 1589 (C ⁇ O of CMC), 1403/1022, 896.
  • Polymer 19 (200 mg, 0.470 mmol) was ground into a fine powder and mixed with DMF (20 mL) for 20 minutes.
  • Fmoc-Ala-OH (0.439 g, 1.41 mmol)
  • HBTU 0.539 g, 1.41 mmol
  • EDC 270 mg, 1.41 mmol
  • DIPEA 0.018 mL, 1.41 mmol
  • FTIR Peaks at 3300 (N—H/O—H), 2900/2850 (C—H), 1748 (amide), 1648 (C ⁇ O, amide of coupled product), 1584 (C ⁇ O of CMC), 1403/1025, 900.
  • Polymer 20 (150 mg, 0.2785 mmol) was ground into a fine powder and mixed with DMF (20 mL) for 20 minutes.
  • Fmoc-Pro-OH (0.282 g, 0.836 mmol), HBTU (0.317 g, 0.8356 mmol), EDC (160 mg, 0.836 mmol) and DIPEA (0.20 mL, 0.836 mmol) were added to a filtration column tube and sealed.
  • a lab rotor was used to facilitate mixing overnight.
  • the solid was filtered and washed with DCM (5 ⁇ 20 mL) and methanol (5 ⁇ 20 mL).
  • FTIR Peaks at 3300 (N—H/O—H), 2850 (C—H), 1748 (amide), 1648 (C ⁇ O, amide of coupled product), 1585 (C ⁇ O of CMC), 1453/1403/1024/1095/1058, 961, 841.
  • Polymer 22 (50 mg, 0.707 mmol) was ground into a fine powder and mixed with DMF (10 mL) for 20 minutes.
  • Fmoc-Cys(StBu)-OH (716 mg, 2.122 mmol), HBTU (805 mg, 2.122 mmol), EDC (407 mg, 2.122 mmol) and DIPEA (0.274 mL, 2.122 mmol) were added to a filtration column tube and sealed.
  • a lab rotor was used to facilitate mixing overnight.
  • the solid was filtered and washed with DCM (5 ⁇ 20 mL) and methanol (5 ⁇ 20 mL).
  • Polymer 23-a (25 mg) was placed in a solution of TCEP in water (5 mL) and stirred for 2 hours at RT, after which the solid was filtered and washed with ethanol (3 ⁇ 50 mL) and DCM (3 ⁇ 50 mL). This process was then repeated, and the product was dried to provide Polymer 23 as off-white powder. A positive Kaiser test result indicated presence of free amine. This material was used in Example 24 without additional characterization.
  • TCEP (0.0357 g, 0.125 mmol, 4 equiv) was dissolved in 1 mL water and added to polymer 23 (25.3 mg, 0.0312 mmol), and the mixture was stirred at RT for 30 min. The solid was filtered and washed with water (10 mL ⁇ 3) and freeze-dried yielding solid off-white/cream colored powder which was then washed with the DMF (10 mL ⁇ 3).
  • Polymer 13 was mixed with DMF (30 mL) for 20 min. Fmoc-C(StBu)-OH (1.5 g, 3.5 mmol), HBTU (3.09 g, 8.14 mmol) and DIPEA (1.05 mL, 8.14 mmol) were added with stirring at RT, open to air (a centrifuge tube and lab rotor were used to facilitate mixing overnight). After rotating overnight, the solid was filtered and washed with DCM (5 ⁇ 50 mL), methanol (5 ⁇ 50 mL) and again with DCM (5 ⁇ 50 mL). The resulting product was dried to provide Polymer 25 and used directly in the next example (Example 26).
  • CMC-Cys (7) (10 mg, 0.029 mmol 1 eq.) was stirred at RT for 1 hour with TCEP (16.8 mg, 0.058 mmol, 4 eq.) in DI water (1 mL) in order to de-couple any unwanted disulphide bonds.
  • the solid was filtered and washed with water (10 mL ⁇ 3), freeze-dried and then washed again with DMF (10 mL ⁇ 3).
  • Fluorescein diacetate-5-maleimide (12-a) (11 mg, 0.029 mmol, 1 eq.) was dissolved in dry DMF (3 mL) and added to the CMC-CYS under a nitrogen atmosphere. The reaction was stirred at RT overnight and was protected from the light.
  • NaCMC fibers of DoS 0.3 (2.0 g, 8.33 mmol) were dispersed in a solution of ethanol: de-ionized water (80:20 v/v) (180 mL) to give a 1% suspension.
  • Dowex 650C monosphere ion exchange resin was added with stirring for 30 minutes. The monospheres were removed by filtration before tetrabutylammonium hydroxide (TBAH) 40% (aq) was added in 0.1 mL aliquots until the pH was 8-9 (3.0 mL, 1.16 mmol). The resulting solution was stirred for 30 minutes before being reduced in vacuo.
  • TBAH tetrabutylammonium hydroxide
  • the film-like material was dissolved in dry DMF (100 mL) under a nitrogen atmosphere, stirring and gentle heating was required overnight resulting in a viscous gel-like solution with a uniform texture.
  • the solution was cooled to approx. 4° C. and CMP-I (1.48 g, 5.8 mmol) was added with vigorous stirring.
  • 2,2′-(ethylenedioxy)bis(ethylamine) (1.36 g, 9.17 mmol) was added to the reaction, along with dry triethylamine (3 mL). The reaction was kept at 4° C.
  • Fluorescein diacetate-5-maleimide (12-a) (0.125 g, 0.240 mmol, 1 eq.) was dissolved in dry DMF (15 mL) and added to the CMC-PEG-NH-CYS under a nitrogen atmosphere. The reaction was stirred at RT overnight and was protected from the light. The reaction mixture was reduced in vacuo, the product was washed with DCM (3 ⁇ 50 mL), Methanol (3 ⁇ 50 mL) and reduced in vacuo. The off-white solid fibers were then washed with water (5 ⁇ 75 mL) with gentle heating, stirring and sonication before being filtered and lyophilised yielding off-white solid fibers (30) (1.10 g, 72%).
  • Fluorescein-5-maleimide (25 mg, 59 mmol, 0.1 eq.) was dissolved in dry DMF (70 mL) and added to the CMC-PEG-NH-AAPVC(Fmoc) under a nitrogen atmosphere. The reaction was stirred at RT for 3 hours and was protected from the light. The reaction mixture was reduced in vacuo, the product was washed with DCM (3 ⁇ 50 mL), methanol (3 ⁇ 50 mL), DI water (3 ⁇ 50 mL), again with DCM 3 ⁇ 50 mL) and again with methanol (3 ⁇ 50 mL) and methanol (3 ⁇ 50 mL) and reduced in vacuo yielding an off-white solid (31) (436 mg, 68%).
  • N-Bromophenyl Maleimide (37 mg, 0146 mmol, 1 eq.) was dissolved in dry DMF (20 mL) and added to the CMC-PEG-NH-AAPVC(Fmoc) under a nitrogen atmosphere. The reaction was stirred at RT for 3 hours and was protected from the light. The reaction mixture was reduced in vacuo, the product was washed with DCM (3 ⁇ 50 mL), methanol (3 ⁇ 50 mL), DI water (3 ⁇ 50 mL), again with DCM 3 ⁇ 50 mL) and again with methanol (3 ⁇ 50 mL) and methanol (3 ⁇ 50 mL) and reduced in vacuo yielding an off-white solid (32) (129 mg, 69%).
  • wound specific proteases including other proteases such as trypsin, chymotrypsin and thermolysin was assayed.
  • Trypsin cleaves P1-P1′ wherein P1 is Lys or Arg, and P1′ is non-specific (except when followed by proline).
  • Chymotrypsin cleaves P1-P1′ wherein P1 is any aromatic amino residue, Trp, Tyr or Phe, and P1′ is non-specific.
  • Thermolysin is a metalloendopeptidase which cleaves P2-P1-P1′-P2′ wherein P1 is non-specific, P1′ is Leu, Phe, Ile, Val, Met, Ala and P2′ is not Pro.
  • Esterases hydrolyze the ester linkages of carboxylic acid esters and it therefore behaves differently to proteases, which specifically hydrolyze amino acid sequences.
  • the substrate used for enzymatic assays is CMC-PEG-NH-Phe-Phe-Lys (Dabsyl) and the enzymatic activity was determined using a UV-visible based method.
  • the Phe-Phe-Lys (Dabsyl) sequence should be hydrolyzed by chymotrypsin. Trypsin, was used as a negative control since it should not cleave this sequence.
  • the activity of the enzymes on the substrate CMC-PEG-NH-Cys-maleimide fluorescein diacetate was analyzed using confocal fluorescence microscopy.
  • the solid particles were first viewed alone and then after the addition of esterase solution ( ⁇ 50 units/mL).
  • the microscopy images demonstrate the increase in fluorescence observed when the enzyme solution was added to the sample.
  • the advantage of using this method is that one can visualize the particles and observe a difference in the fluorescence intensity on addition of an enzyme solution, which confirmed the coupling of the fluorescein diacetate to the CMC polymer.
  • multi-plate readers may be used to assess the cleavage of the substrates and the results quantified with Michaelis-Menton kinetics.
  • CMC-PEG-NH-Cys-maleimide-fluorescein diacetate powder (0.2 mg) was dispersed in PBS (320 ⁇ L). Sonication, vortex mixing and gentle heating were required in order to disperse the compound. 40 ⁇ L of substrate suspension was added to the wells of a 96-well plate, then either weak esterase solution (58 U/mL), strong esterase solution (116 U/mL) or PBS (negative control) was added (40 ⁇ L per well). A cycle period of 5 minutes over 2 hours was selected for fluorescence measurements.
  • the rate of the 116 U/mL esterase reaction was twice that of the 58 U/mL sample, therefore doubling the enzyme concentration caused a doubling of the rate.
  • the rates of reaction were significantly lower than that of the shorter PEG linker equivalent. This reduction was expected since the loading of the peptide onto the CMC was lower.
  • the AAPV peptide sequence is a substrate for elastase, with predicted cleavage sites at P1 of Ala and Val.
  • an elastase assay was performed in a similar manner to the esterase assays. Three forms of substrates were tested: (a) powder form; (b) wet spun alginate with the compound in fiber format, and (c) hydrocolloid gel containing either 0.8% or 8% of the compound.
  • An important aspect to consider when designing a medical device is its biological safety and other factors such as cytotoxicity, sensitization, hemocompatibility, pyrogenicity, implantation, genotoxicity, carcinogenicity, reproductive and developmental toxicity, biodegradation, etc.
  • the study of the interactions between new materials and cells grown in-vitro can give a good indicator of the toxicity of these materials, and therefore an array of these methods are used (Eisenbrand et al., Food Chem. Toxicol. 2002, 40, 193-236).
  • a suitable cell type must be selected in order to make the test appropriate for the area of the body and function related to the device's end use.
  • the method may be quantitative or purely visual, and modem techniques allow a sophisticated viewpoint of cell interactions, such as the use of time-lapse video imaging of cells via a microscope.
  • fibroblast cells are important in the wound healing process. They begin to migrate towards the wound bed approximately 24 hours after the injury during the late stages of the inflammatory phase. They modify the wound environment throughout the proliferation and epithelialisation phases by production of mediators including proteases like the MMPs. Finally fibroblast levels reduce back to normal levels at the remodeling stage and once the new wound extracellular matrix has achieved sufficient strength (Bainbridge et al., J. Wound Care 2013, 22, 407-408). Fibroblasts are therefore a suitable cell line to use when mimicking the wound healing process.
  • Cultures of normal fibroblasts had been obtained with informed consent from patients. Patients with diabetes, systemic immunosuppression or with evidence of local infection were excluded from the study. Three patient cell lines were available for this study: Patients A, F and G.
  • a 6 mm biopsy was taken from the patient's thigh. Cultures were established by a single-cell suspension technique following enzymatic degradation of the specimens. Briefly, tissue was incubated overnight with Dispase (2 mg/mL; Boehringer Mannheim, Lewes, UK) to separate epidermal tissue from the dermal tissue. Dermal tissue specimens were then disaggregated overnight utilizing bacterial Clostridium histolyticum A collagenase (1 mg/mL; Boehringer Mannheim).
  • Fibroblast cultures were maintained in Fibroblast-Serum Containing Medium (F-SCM) containing Dulbecco's Modified Eagle's Medium (DMEM) supplemented with L-glutamine (2 mM), non-essential amino acids (lx), antibiotics (100 U/mL penicillin G; 100 mg/mL streptomycin sulphate; 0.25 mg/mL amphotericin B) and 1% (v/v) foetal calf serum (FCS).
  • F-SCM Fibroblast-Serum Containing Medium
  • DMEM Dulbecco's Modified Eagle's Medium
  • lx non-essential amino acids
  • antibiotics 100 U/mL penicillin G
  • streptomycin sulphate 100 mg/mL streptomycin sulphate
  • FCS 1% foetal calf serum
  • the scratch assay is a technique in which a confluent mono-layer of fibroblasts is grown on a flat surface, then this surface is ‘scratched’ or ‘wounded’ to create a channel separating two areas of fibroblast cells.
  • the sample solution is added onto the top of the cells and the channel is monitored over time using confocal microscopy in order to see how the cells respond (Liang et al., Nat. Protocols 2007, 2, 329-333). If the surrounding area containing the sample is amenable to cells, the cells will proliferate and migrate to fill the channel during the time-period; if the sample is not amenable then the cells will not migrate and will die.
  • Human dermal fibroblast cells were seeded into 24-well tissue culture plates (2 ⁇ 104 cells per well), cultured to 80-90% confluence and the monolayers were wounded by scratching along the surface of the tissue culture plastic with a 200 ⁇ L pipette tip. Monolayers were washed with PBS and the compounds added at 0.66 mg/mL or 0.066 mg/mL in DMEM. The cells were then re-fed with F-SCM and incubated under standard culture conditions on the motorized, heated and gassed stage of a confocal microscope with Cell-IQ system.
  • a collagen matrix model is an in vitro tool which represents the dermis during the reorganization phase of healing.
  • FPCLs fibroblast populated collagen lattices
  • Fibroblasts derived from culture by trypsinisation were utilized to construct the fibroblast populated collagen lattices (FPCLs).
  • Type I rat-tail collagen was purchased from First Link. 1.5 ⁇ 105 fibroblasts (in 750 ⁇ L F-SCM) were added to 60 mm bacteriological plates containing 2 ⁇ DMEM (40 parts 10 ⁇ DMEM, 10 parts NaHCO 3 (7.5% (w/v)), 4 parts L-glutamine (200 mM), 4 parts non-essential amino acids (100 ⁇ ), 140 parts H20 and 5 parts NaOH (1 M); 3 mL), 0.1M NaOH (750 ⁇ L), FCS (750 ⁇ L), 2.25 mL of type I collagen (1.7 mg/mL) and the test compounds (0.66 mg/mL).
  • the plates were incubated at 37° C. for 60 minutes to allow collagen polymerization. They were then detached from the edge of the plate and 2 mL of F-SCM was added. FPCLs were maintained at 37° C. in a 5% CO2 humidified atmosphere.
  • the fibroblasts remained viable and proliferated to fill the scratch channel during the test period.
  • the results show the time taken for the fibroblasts to completely close the scratch channel. As shown in FIG. 5 , all samples closed the channel within 70 hours.
  • the CMC powder sample was used as a reference as this is known to be safe for use in wound contact applications.
  • the CMC-PEG-NH 2 fiber was the only sample in which the scratch closed in a shorter time period than the control.
  • the CMC-PEG-NH 2 powder scratch closure time was roughly equivalent to the other modified CMC samples ( ⁇ 40-60 hours), suggesting that the cells may have some preference for the physical structure of the sample in fiber form.
  • FIG. 6 The scratches for Patient A containing 0.066 mg/mL of 12, CMC-PEG-NH2 powder ( FIG. 6 ) and Patient A containing 0.66 mg/mL of 12, CMC-PEG-NH2 powder ( FIG. 7 ) both closed over time as the fibroblasts replicated and migrated into the channel.
  • FIG. 6 and FIG. 7 are representative of the observations of nearly all of the tests performed across all samples and cell lines from patients A, F and G.
  • CMC-longer PEG-NH2 powder (#83) generated anomalous results in one study, when the test was repeated with samples obtained from three different patients, the fibroblasts did proliferate and migrate.
  • the glass slide, used as a base is preferably free from impurities such as grease. Cleaning was performed using Piranha solution, a strong oxidizing agent which removes all organic matter from the glass. Due to the strong oxidizing nature of the piranha solution, appropriate safety precautions were taken to avoid contact with skin and also avoid explosion.
  • OTS octadecyltrichlorosilane
  • TEM grids were placed onto the OTS coated glass to make the grid for the LC solutions to be held within.
  • 5CB was added carefully to the TEM grids using a capillary tube to ensure that each grid is sufficiently filled, but not over-filled so as to create a dome of solution on top of the grid.
  • 5CB alignment was checked using the crossed polarized lens of a light microscope. 5CB is homeotropic at this stage, which is due to the alignment of the LCs with the OTS coated glass slide.
  • FIG. 14 shows micrographs showing 5CB filled TEM grids upon application of CMC gel.

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IL262011A (en) 2018-10-31
BR112018070191A2 (pt) 2019-01-29
ES2968013T3 (es) 2024-05-06
AU2017244116B2 (en) 2023-03-02
JP7395630B6 (ja) 2024-01-23
CL2018002782A1 (es) 2019-03-15
AU2017244116C1 (en) 2023-06-01
EP3436012B1 (de) 2023-09-20
CO2018011720A2 (es) 2018-11-13
WO2017173026A1 (en) 2017-10-05
PL3436012T3 (pl) 2024-04-08
JP2019509853A (ja) 2019-04-11
JP7394526B2 (ja) 2023-12-08
KR20180132093A (ko) 2018-12-11
CN109219436A (zh) 2019-01-15
SG11201808528VA (en) 2018-10-30
US20240123091A1 (en) 2024-04-18
US20210275693A1 (en) 2021-09-09
TW201806625A (zh) 2018-03-01
JP2022065128A (ja) 2022-04-26
EP3436012C0 (de) 2023-09-20
EP3436012A4 (de) 2019-10-23
ECSP18081922A (es) 2019-02-28
CA3019548A1 (en) 2017-10-05
JP7395630B2 (ja) 2023-12-11
MX2018011811A (es) 2019-08-21
AR108054A1 (es) 2018-07-11
AU2017244116A1 (en) 2018-11-15
EP3436012A1 (de) 2019-02-06

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