Methods and compositions for enzymatic wound debridement

Abstract

Disclosed herein are methods and compositions for enzymatic wound debridement. In some aspects, provided are methods for debriding a wound, comprising applying a composition including a poloxamer and a composition including a collagenase to the wound. In some aspects, the composition including a poloxamer and the composition including a collagenase are applied sequentially. Also provided herein are related kits and uses thereof.

Description

DESCRIPTIONMETHODS AND COMPOSITIONS FOR ENZYMATIC WOUND DEBRIDEMENTCROSS-REFERENCE WITH RELATED APPLICATIONS

[0001] This application claims priority to and the benefit of priority of U.S. Provisional Application No. 63 / 604,638, filed November 30, 2023, the contents of which is incorporated into the present application by reference.FIELD

[0002] The present disclosure relates generally to topical enzymatic wound debriding compositions and methods of treating wounds in need of debridement.BACKGROUND

[0003] A wound is a disruption of the structure and function of tissue, such as cuts, tears, bums, breaks, or other damage to living tissue. A dermal wound involves the disruption of the skin and associated soft tissue architecture. Dermal wounds can be partial or full thickness wounds. They can also be acute wounds, chronic wounds, or burns (which can be acute or chronic). Wounds may also include, without limitation, internal organ wounds; mucous membrane wounds; vascular tissue wounds; soft tissue wounds including ligaments, tendons, and cartilage; and bone wounds. Wounds can result from various conditions and injuries and may be classified in a variety of ways.

[0004] Topical compositions containing proteolytic enzymes such as trypsin, papain, bromelain, subtilisin, sutilains, and collagenase have been used for enzymatic wound debridement. Generally, the standard of care is to apply the composition to the wound in need of debridement once daily (once every 24 hours) or more often if the composition becomes soiled. Because many proteolytic enzymes are susceptible to degradation in water-based compositions, many wound debriding compositions are made with anhydrous, hydrophobic bases such as petrolatum, mineral oil and / or vegetable oil. However, enzymatic wound debriding compositions based on hydrophobic bases are generally not miscible in the aqueous environment of a wound bed, and thus contact of the proteolytic enzyme with the wound bed is generally hindered. Other compositions are made with anhydrous, hydrophilic bases such as propylene glycol or poloxamers.

[0005] Promoting rapid and effective wound healing is an essential component of medical care for individuals with wounds. Treatment for promoting wound healing can includedebridement, the removal of dead, damaged, or infected tissue, which can improve the healing potential of the remaining healthy tissue. Debridement is the process of removing non-viable tissue and other foreign debris from a wound to prevent or diminish infection and facilitate healing. The clinical significance of wound debridement should not be underestimated, and debridement for most wounds is considered a standard in wound management. It can provide the benefits of removal of necrotic tissue and bacteria and senescent cells, as well as the stimulating activity of growth factors. The mode of debridement can be tailored to the particular wound presentation, considering factors such as comorbidities, other lower-risk options, and the subject’s comfort and desires.

[0006] Debridement can occur by a variety of means, including surgical, mechanical, chemical, enzymatic, and autolytic (self-digestion). Autolytic debridement involves the use of the body's own enzymes and moisture to re-hydrate, soften and finally liquefy hard eschar and slough. Mechanical and surgical debridement both use instruments or physical tools for the mechanical removal of necrotic tissue from the wound. Enzymatic debridement employs enzymes that degrade proteinaceous materials, such as collagen matrix, in necrotic tissue. An example of enzymatic debridement is the use of Collagenase SANTYL® Ointment, which is an FDA-approved ointment prescribed for the removal of dead tissues from wounds. The SANTYL® Ointment, which is described in U.S. Patent Nos. 3,705,083 and 3,821,364 (both of which are incorporated herein by reference), includes as its active ingredient a protease mixture derived from the fermentation by Clostridium histolyticum. The mixture includes unseparated collagenase, in that collagenases and other non-specific proteases (e.g., a neutral protease) are present in the Ointment. The ointment possesses the ability to digest collagen in necrotic tissue, thereby debriding the wound.

[0007] In some aspects, following debridement, a wound can be more effectively treated with wound healing agents to promote wound repair. During the wound repair stage, cells begin to grow and rebuild missing and damaged tissues. Small blood vessels develop to deliver a blood supply to the wound. Skin cells then migrate, and scabs form within hours of the initial wound. These skin and epithelial cells can cover a properly closed surgical incision within 48 hours. In an open wound, the creation of granulation tissue can take longer. Examples of wound repair can be found, for example, in U.S. Patent No. 5,851,522 and in Herman IM, Stimulation of Human Keratinocyte Migration and Proliferation In Vitro: Insights into the Cellular Responses to Injury and Wound Healing, WOUNDS: A Compendium of Clinical Research and Practice, 1996, Volume 8, No. 2, Pages 33-41, both of which are incorporated herein by reference. U.S. Patent No. 5,851,522 discloses a purified form of collagenase (i.e.,Clostridiopeptidase A collagenase (EC 3.4.24.3), obtained by fermentation of Clostridium histolyicum) that was purified to be substantially free from other proteinases. Similarly, Herman (1996) discusses the use of purified Clostridium collagenase for wound healing.

[0008] Methods of enzymatic debridement require that the enzymes retain activity following application to achieve the desired effect. Identifying compositions and methods that provide a suitable environment in which activity of the enzymes can be maintained while also providing access to substrate in the wound has been challenging.

[0009] Given the importance of effectively debriding wounds, pursuit of superior compositions for and methods of wound debridement that can (1) provide a suitable environment in which activity of the enzymes can be maintained, and (2) provide access to substrate in the wound, are warranted. Such compositions and methods would have numerous applications, such as clinical and / or commercial uses similar to uses of fresh intact tissues, while providing an extended shelf-life for tissues compared to fresh tissue.SUMMARY

[0010] A discovery has been made that provides a solution to at least one or more of the aforementioned problems associated with methods for wound debridement. In some aspects, inclusion of a poloxamer in a composition for enzymatic wound debridement can help preserve enzymatic activity (e.g., collagenase activity) while providing the enzyme access to the wound substrate. In some aspects, the inventors discovered that the sequential application of a first composition including the poloxamer, and the application of a second composition including the collagenase, to a wound, results in a surprising improvement in collagenase activity, for example in comparison to the application of a single composition that includes both the poloxamer and the collagenase.

[0011] In some aspects, the methods and compositions provided herein provide several advantages. In some aspects, the methods provide a surprising, yet simple and economical solution for improving enzyme (e.g., collagenase) activity for enzymatic debridement. The separate application of the poloxamer and collagenase also provides flexibility and opportunities for the separate optimization of each composition and / or application thereof. For example, modifications to either composition can be made without affecting the other, as needed. It may be that different formulations of the first composition may be appropriate for different wound types, whereas the second composition can remain the same or different, or vice versa. It may also be that the separate compositions may be more readily or cost-effectively produced, shipped, stored, and / or applied, in comparison to a single composition includingboth the poloxamer and collagenase. Such flexibility may also improve the ability to tailor wound treatment for different wounds and individuals as needed, in a cost-effective manner. In some embodiments, the first composition including the poloxamer can be formulated and / or comprised in a cleanser or wound dressing, while the second composition can be formulated and / or comprised in a wound dressing or spray.

[0012] In some aspects, provided herein is a method for debriding a wound, comprising applying a first composition including a poloxamer to the wound, and applying a second composition including a collagenase to the wound. In some embodiments, the first composition can be applied to the wound before and / or after the second composition. In some embodiments, the collagenase activity is maintained or increased compared to a method comprising applying a composition that includes both a poloxamer and a collagenase. In some embodiments, collagenase activity is maintained or increased compared to a method comprising applying a composition that includes a collagenase but does not include a poloxamer (e.g., applying a single composition that includes a collagenase but not a poloxamer). In some embodiments, collagenase activity is maintained or increased compared to a method that does not comprise applying a poloxamer to the wound.

[0013] In some aspects, provided herein is a method for debriding a wound. In some aspects, the method comprises enzymatic wound debridement. In some aspects, the method comprises: (a) applying a first composition including a poloxamer to the wound; and (b) applying a second composition including a collagenase to the wound. In some aspects, the method is different than, and provides advantages in comparison to, a method in which a single composition including both the poloxamer and collagenase is applied to the wound. Thus, in some embodiments, the second composition does not include a poloxamer.

[0014] In some embodiments, collagenase activity (e.g., the activity of the collagenase in the second composition) is maintained or increased compared to a method comprising applying a composition that includes a poloxamer and a collagenase, such as a single composition including both the poloxamer and the collagenase. In some embodiments, the collagenase activity is maintained or increased compared to a method comprising applying a composition that includes a collagenase but does not include a poloxamer. In some embodiments, the collagenase activity is maintained or increased compared to a method that does not comprise applying a poloxamer to the wound. In some embodiments, the collagenase activity is maintained or increased compared to a method comprising applying a collagenase but not a poloxamer to the wound.

[0015] In some aspects, the maintained or increased collagenase activity is the activity of the collagenase after a period of time following application of the collagenase to the wound, such at least, or at or about, 1 minute, 10 minutes, 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5, hours, 6 hours, 12 hours, 18 hours, 24 hours, or more. In some aspects, the collagenase activity can be determined and / or measured by any suitable means, such as by any assay known to one skilled in the art for measuring collagenase activity (e.g., as described herein). In some aspects, the collagenase activity is the ability of the collagenase to digest (e.g., cleave) collagen at one or more sites.

[0016] In some aspects, the first composition including the poloxamer and the second composition including the collagenase can be applied to the wound in any suitable order. In some embodiments, the first composition is applied to the wound before the second composition. In some embodiments, the first composition is applied to the wound after the second composition. In some aspects, the application of the first and / or second composition can be repeated any suitable number of times, and at any suitable interval. In some aspects, the method for debridement can be repeated at suitable intervals, such as daily or weekly.

[0017] The first and / or second composition can be, can be formulated as, or can be comprised in, any suitable composition. Accordingly, first and / or second composition can include any other suitable components in addition to those described herein. In some aspects, the first composition comprises: (a) 1-15 wt. % poloxamer; and (b) 1-40 wt. % water soluble solvent. In some aspects, the poloxamer is poloxamer 407 and the water soluble solvent is propylene glycol. In some aspects, the first composition further comprises 0.01-5 wt. % of one or more preservatives. In some aspects, the one or more preservatives comprise one or more parabens, benzyl alcohol, phenoxyethanol, or a combination thereof. In some aspects, the first composition further comprises one or more buffering or osmolarity agents, and wherein the first composition has a pH of 6-8. In some aspects, the buffering or osmolarity agents comprise sodium acetate, Tris buffer, or sodium chloride. In some aspects, the first composition further comprises water. In specific aspects, the first composition comprises: (a) 2.5-7.5 wt. % poloxamer; (b) 2-6 wt. % propylene glycol; (c) 0.01-1 wt. % methyl paraben; (d) 0.01-1 wt. % propyl paraben; (e) 0.01-2 wt. % benzyl alcohol; (f) sodium acetate; and (g) sodium chloride.

[0018] In some embodiments, the collagenase is comprised in a protease mixture. In some embodiments, the protease mixture includes collagenase ColG and collagenase ColH. In some embodiments, the protease mixture includes collagenase ColG, collagenase ColH, and one or more additional proteases. In some embodiments, the protease mixture includes collagenase ColG, collagenase ColH, and a non-specific neutral protease. In some embodiments, the non-specific neutral protease is Clostripain. In some aspects, Clostripain is a proteinase originally isolated from Clostridium histolyticum that cleaves proteins on the carboxyl peptide bond of arginine. In some embodiments, the protease mixture includes collagenase ColG, collagenase ColH, and Clostripain. In some embodiments, the protease mixture is the active pharmaceutical ingredient (API) in SANTYL® Ointment. SANTYL® Ointment is described, for example, in U.S. Patent Application No. 15 / 534,547, and in U.S. Patent Nos. 3,705,083 and 3,821,364 (each of which are incorporated herein by reference). SANTYL® Ointment includes as its active ingredient a protease mixture derived from the fermentation by Clostridium histolyticum. The mixture includes unseparated collagenase, in that collagenases and other non-specific proteases (e.g., a neutral protease) are present in the Ointment. The Ointment possesses the ability to digest collagen in necrotic tissue, thereby debriding the wound.

[0019] In some embodiments, the second composition is comprised in a wound dressing or a spray. In some embodiments, the wound dressing is a patch, wrap, or bandage. In some embodiments, the second composition further comprises petrolatum. For example, in some embodiments, the second composition comprises collagenase ColG, collagenase ColH, a nonspecific neutral protease, and petrolatum. In some embodiments, the second composition further includes an antimicrobial agent. Any suitable microbial agents may be included in the second composition, and appropriate antimicrobial agents for inclusion could be readily selected by one of skill in the art.

[0020] In some embodiments, the first composition is comprised in a wound dressing or a cleanser. In some embodiments, the cleanser is aqueous. In some embodiments, the wound dressing is a patch, wrap, or bandage.

[0021] In some aspects, the composition including the poloxamer (e.g., the first composition) can facilitate improved activity of collagenase (and other enzymes) when the collagenase is applied to the wound. For example, without wishing to be bound by theory, the composition including the poloxamer can accelerate the extraction and solubilization of enzymes from the composition including the collagenase, which may be provided in a hydrophobic matrix (such as in white petrolatum, as in SANTYL® Ointment). In this way, the poloxamer can allow the collagenase and / or other enzymes to more effectively access wound substrates, thereby increasing enzymatic activity (e.g., the rate at which wound collagen is digested) upon application.

[0022] In some aspects, provided herein is a method for preparing a wound for debridement with a composition comprising collagenase, the method comprising, prior to debriding the wound with the composition comprising collagenase, applying a composition comprising apoloxamer to the wound. In some embodiments, the collagenase activity is maintained or increased compared to a method comprising applying a composition that includes a poloxamer and a collagenase. In some embodiments, the collagenase activity is maintained or increased compared to a method comprising applying a composition that includes a collagenase but does not include a poloxamer. In some embodiments, the collagenase activity is maintained or increased compared to a method comprising applying a collagenase but not a poloxamer to the wound.

[0023] In some aspects, provided herein is a kit, such as a kit for enzymatic wound debridement. In some aspects, provided herein is a kit comprising a first composition including a poloxamer and a second composition including a collagenase. In some embodiments, the first composition and the second composition are comprised in the same container. In some embodiments, the first composition and the second composition are comprised in different containers. In some embodiments, the first composition is comprised in a cleanser, and the second composition is comprised in a wound dressing or spray.

[0024] Accordingly, provided herein are aspects 1-45. Aspect 1 is a method for debriding a wound, the method comprising: (a) applying a first composition including a poloxamer to the wound; and (b) applying a second composition including a collagenase to the wound. Aspect 2 is the method of Aspect 1, wherein the first composition comprises: (a) 1-15 wt. % poloxamer; and (b) 1-40 wt. % water soluble solvent. Aspect 3 is the method of Aspect 1 or 2, wherein the poloxamer is poloxamer 407 and the water soluble solvent is propylene glycol. Aspect 4 is the method of any one of Aspects 1-3, wherein the first composition further comprises 0.01-5 wt. % of one or more preservatives. Aspect 5 is the method of any one of Aspects 1-4, wherein the one or more preservatives comprise one or more parabens, benzyl alcohol, phenoxyethanol, or a combination thereof. Aspect 6 is the method of any one of Aspects 1-5, wherein the first composition further comprises one or more buffering or osmolarity agents, and wherein the first composition has a pH of 6-8. Aspect 7 is the method of any one of Aspects 1-6, wherein the buffering or osmolarity agents comprise sodium acetate, Tris buffer, or sodium chloride. Aspect 8 is the method of any one of Aspects 1-7, wherein the first composition further comprises water. Aspect 9 is the method of any one of Aspects 1-8, wherein the first composition comprises: (a) 2.5-7.5 wt. % poloxamer; (b) 2-6 wt. % propylene glycol; (c) 0.01-1 wt. % methyl paraben; (d) 0.01-1 wt. % propyl paraben; (e) 0.01-2 wt. % benzyl alcohol; (f) sodium acetate; and (g) sodium chloride. Aspect 10 is the method of any one of Aspects 1-9, wherein collagenase activity is maintained or increased compared to a method comprising applying a composition that includes a poloxamer and a collagenase.Aspect 11 is the method of any one of Aspects 1-9, wherein collagenase activity is maintained or increased compared to a method comprising applying a collagenase but not a poloxamer to the wound. Aspect 12 is the method of any one of Aspects 1-11, wherein the first composition is applied to the wound before the second composition. Aspect 13 is the method of any one of Aspects 1-11, wherein the first composition is applied to the wound after the second composition. Aspect 14 is the method of any one of Aspects 1-13, wherein the collagenase is comprised in a protease mixture. Aspect 15 is the method of any one of Aspects 1-14, wherein the protease mixture includes collagenase ColG, collagenase ColH, and a non-specific neutral protease. Aspect 16 is the method of any one of Aspects 1-15, wherein the protease mixture is the active pharmaceutical ingredient (API) in SANTYL® Ointment. Aspect 17 is the method of any one of Aspects 1-16, wherein the second composition is comprised in a wound dressing or a spray. Aspect 18 is the method of any one of Aspects 1-17, wherein the wound dressing is a patch, wrap, or bandage. In some embodiments, the second composition comprises collagenase ColG, collagenase ColH, a non-specific neutral protease, and petrolatum. Aspect 19 is the method of any one of Aspects 1-18, wherein the second composition further includes an antimicrobial agent. Aspect 20 is the method of any one of Aspects 1-19, wherein the first composition is comprised in a wound dressing or a cleanser. Aspect 21 is the method of any one of Aspects 1-20, wherein the cleanser is aqueous. Aspect 22 is the method of any one of Aspects 1-21, wherein the wound dressing is a patch, wrap, or bandage.

[0025] Aspect 23 is a method for preparing a wound for debridement with a composition comprising collagenase, the method comprising, prior to debriding the wound with the composition comprising collagenase, applying a composition comprising a poloxamer to the wound. Aspect 24 is the method of Aspect 23, wherein collagenase activity is maintained or increased compared to a method comprising applying a composition that includes a poloxamer and a collagenase. Aspect 25 is the method of Aspect 23, wherein collagenase activity is maintained or increased compared to a method comprising applying a collagenase but not a poloxamer to the wound. Aspect 26 is the method of any one of Aspects 23-25, wherein the first composition comprises: (a) 1-15 wt. % poloxamer; and (b) 1-40 wt. % water soluble solvent. Aspect 27 is the method of any one of Aspects 23-26, wherein the poloxamer is poloxamer 407 and the water soluble solvent is propylene glycol. Aspect 28 is the method of any one of Aspects 26-27, wherein the first composition further comprises 0.01-5 wt. % of one or more preservatives. Aspect 29 is the method of any one of Aspects 26-28, wherein the one or more preservatives comprise one or more parabens, benzyl alcohol, phenoxyethanol, or a combination thereof. Aspect 30 is the method of any one of Aspects 26-29, wherein the firstcomposition further comprises one or more buffering or osmolarity agents, and wherein the first composition has a pH of 6-8. Aspect 31 is the method of any one of Aspects 26-30, wherein the buffering or osmolarity agents comprise sodium acetate, Tris buffer, or sodium chloride. Aspect 32 is the method of any one of Aspects 26-31, wherein the first composition further comprises water. Aspect 33 is the method of any one of Aspects 26-32, wherein the first composition comprises: (a) 2.5-7.5 wt. % poloxamer; (b) 2-6 wt. % propylene glycol; (c) 0.01- 1 wt. % methyl paraben; (d) 0.01-1 wt. % propyl paraben; (e) 0.01-2 wt. % benzyl alcohol; (f) sodium acetate; and (g) sodium chloride.

[0026] Aspect 34 is a kit comprising a first composition including a poloxamer and a second composition including a collagenase. Aspect 35 is the kit of Aspect 34, wherein the first composition and the second composition are comprised in the same container. Aspect 36 is the kit of Aspect 34, wherein the first composition and the second composition are comprised in different containers. Aspect 37 is the kit of any one of Aspects 34-36, wherein the first composition is comprised in a cleanser, and wherein the second composition is comprised in a wound dressing or spray. Aspect 38 is the method of any one of Aspects 34-37, wherein the first composition comprises: (a) 1-15 wt. % poloxamer; and (b) 1-40 wt. % water soluble solvent. Aspect 39 is the method of any one of Aspects 34-38, wherein the poloxamer is poloxamer 407 and the water soluble solvent is propylene glycol. Aspect 40 is the method of any one of Aspects 34-39, wherein the first composition further comprises 0.01-5 wt. % of one or more preservatives. Aspect 41 is the method of any one of Aspects 34-40, wherein the one or more preservatives comprise one or more parabens, benzyl alcohol, phenoxyethanol, or a combination thereof. Aspect 42 is the method of any one of Aspects 34-41, wherein the first composition further comprises one or more buffering or osmolarity agents, and wherein the first composition has a pH of 6-8. Aspect 43 is the method of any one of Aspects 34-42, wherein the buffering or osmolarity agents comprise sodium acetate, Tris buffer, or sodium chloride. Aspect 44 is the method of any one of Aspects 34-43, wherein the first composition further comprises water. Aspect 45 is the method of any one of Aspects 34-44, wherein the first composition comprises: (a) 2.5-7.5 wt. % poloxamer; (b) 2-6 wt. % propylene glycol; (c) 0.01- 1 wt. % methyl paraben; (d) 0.01-1 wt. % propyl paraben; (e) 0.01-2 wt. % benzyl alcohol; (f) sodium acetate; and (g) sodium chloride.

[0027] The disclosed materials, compositions, and components may be used for, may be used in conjunction with, may be used in preparation for, or are products of the disclosed method and compositions. These and other materials are disclosed herein, and it is understood that when combinations, subsets, interactions, groups, etc. of these materials are disclosed that,while specific reference of each various individual and collective combinations and permutation of these materials may not be explicitly disclosed, each is specifically contemplated and described herein. Thus, for example, if a class of components A, B, and C are disclosed as well as a class of components D, E, and F and an example of a combination molecule, A-D is disclosed, then even if each is not individually recited, each is individually and collectively contemplated. Thus, is this example, each of the combinations A-E, A-F, B- D, B-E, B-F, C-D, C-E, and C-F are specifically contemplated and should be considered disclosed from disclosure of A, B, and C; D, E, and F; and the example combination A-D. Likewise, any subset or combination of these is also specifically contemplated and disclosed. Thus, for example, the sub-group of A-E, B-F, and C-E are specifically contemplated and should be considered disclosed from disclosure of A, B, and C; D, E, and F; and the example combination A-D. This concept applies to all aspects of this application including, but not limited to, steps in methods of making and using the disclosed compositions. Thus, if there are a variety of additional steps that can be performed it is understood that each of these additional steps can be performed with any specific aspect or combination of aspects of the disclosed methods, and that each such combination is specifically contemplated and should be considered disclosed.

[0028] It is contemplated that any aspect discussed in this specification can be implemented with respect to any method or composition of the present disclosure, and vice versa. Furthermore, compositions of the present disclosure can be used to achieve methods of the present disclosure.

[0029] Other objects, features and advantages of the present disclosure will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating specific aspects of the present disclosure, are given by way of illustration only, since various changes and modifications within the spirit and scope of the present disclosure will become apparent to those skilled in the art from this detailed description.BRIEF DESCRIPTION OF THE DRAWINGS

[0030] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present disclosure. The present disclosure may be better understood by reference to one or more of these drawings in combination with the detailed description of specific aspects presented herein.

[0031] FIGs. 1A-1B show results from a first (FIG. 1A) and second (FIG. IB) assay assessing collagenase activity in a saline solution or exemplary wound cleansers containing a poloxamer.

[0032] FIG. 2 shows results from an assay assessing collagenase activity in different solutions containing a poloxamer.

[0033] FIGs. 3A-3B shows results from a first (FIG. 3A) and second (FIG. 3B) assay assessing collagenase activity in different exemplary cleanser formulations containing a poloxamer.

[0034] FIG. 4 shows results from an assay assessing collagenase activity in different solutions containing various surfactants.

[0035] FIGs. 5A-5B show results from an assay assessing collagenase activity in different solutions containing various metal chloride salts (FIG. 5A) or sodium salts (FIG. 5B).

[0036] FIG. 6 shows results from an Artificial Wound Eschar (AWE) model assay assessing collagen digestion following application of mixed or separate compositions comprising SANTYL and a poloxamer.DETAILED DESCRIPTION

[0037] The present disclosure relates to methods and compositions (e.g., for enzymatic wound debridement), methods of processing and / or making the compositions disclosed herein, and uses of the compositions for the treatment of wounds (e.g., dermal wounds). The compositions can further comprise a carrier, such as a pharmaceutically acceptable carrier. The compositions provided herein can provide greater beneficial effects for wound healing as compared to commercially available products (e.g., products for wound debridement). As one non-limiting example, the compositions provided herein can provide a suitable environment in which activity of debriding enzymes can be maintained. As another non-limiting example of the beneficial effects of the compositions provided herein, the compositions may provide improved access to substrate in the wound. Thus, the methods and compositions described in this disclosure, including the methods and compositions for enzymatic wound debridement, may provide more favorable conditions to support the wound healing process in comparison to existing methods and compositions.I. Exemplary Definitions

[0038] In various aspects, the subject of the herein disclosed methods is a vertebrate, e.g., a mammal. Thus, the subject of the herein disclosed methods can be a human, non-human primate, horse, pig, rabbit, dog, sheep, goat, cow, cat, guinea pig or rodent. In some aspects,the subject is a human. The term does not denote a particular age or sex. Thus, adult and newborn subjects, as well as fetuses, whether male or female, are intended to be covered. A patient refers to a subject afflicted with a disease or disorder. The term “patient” includes human and veterinary subjects.

[0039] The term “body” as used herein means the body of a subject.

[0040] The term “tissue injury” means an injury of any tissue such as skin or the outer layer of any organ. By injury, it is meant a pathology that involves or results from a mechanical, metabolic, or other insult. Examples of such tissue injuries are bums, wounds, ulcerations, and lacerations, ablations (including laser, freezing, cryo-surgery, heat and electrical ablations), and surgical incisions.

[0041] The term “wound” as used herein means tendon repair, cartilage repair (e.g., femoral condyle, tibial plateau), ACL replacement at the tunnel / bone interface, dental tissue augmentation, fistulas (e.g., Crohn's disease, G-tube, tracheoesophogeal), missing tissue at adhesion barriers (e.g., nasal septum repair, vaginal wall repair, abdominal wall repair, tumor resection), dermal wounds (e.g., partial thickness burns, toxic epidermal necrolysis, epidermolysis bullosa, pyoderma gangrenosum, ulcers e.g., diabetic ulcers (e.g., foot), venous leg ulcers), surgical wounds, hernia repair, tendon repair, bladder repair, periosteum replacement, keloids, organ lacerations, epithelial defects, and repair or replacement of a tympanic membrane. Optionally, the wound is a laceration, scrape, thermal or chemical burn, incision, puncture, or wound caused by a projectile. Optionally, the wound is an epidermal wound, skin wound, chronic wound, acute wound, external wound, internal wounds, congenital wound, ulcer, or pressure ulcer. Such wounds may be accidental or deliberate, e.g., wounds caused during or as an adjunct to a surgical procedure. Optionally, the wound is closed surgically prior to administration. Optionally, the burn is a first-degree burn, second-degree bum (partial thickness burns), third degree burn (full thickness burns), infection of burn wound, infection of excised and unexcised burn wound, loss of epithelium from a previously grafted or healed bum, or burn wound impetigo.

[0042] The compositions disclosed herein are useful in treating wounds. Non-limiting examples of wound sites to which the compositions can be applied include those that are surgically induced or associated with surgery involving the spine, laminectomy, knee, shoulder, or child birth, trauma related wounds or injuries, cardiovascular procedures, angiogenesis stimulation, brain / neurological procedures, burn and wound care, and ophthalmic procedures. Direction for such procedures, including the selection of wound sites and / ormethodologies, can be found, for example, in WO 2009 / 132186 and US 2010 / 0098743, which are hereby incorporated by reference.

[0043] Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of skill in the art to which the disclosed method and compositions belong. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present method and compositions, the particularly useful methods, devices, and materials are as described. Publications cited herein and the material for which they are cited are hereby specifically incorporated by reference. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such disclosure by virtue of prior invention. No admission is made that any reference constitutes prior art. The discussion of references states what their authors assert, and applicants reserve the right to challenge the accuracy and pertinence of the cited documents. It will be clearly understood that, although a number of publications are referred to herein, such reference does not constitute an admission that any of these documents forms part of the common general knowledge in the art.

[0044] The terms “optional” or “optionally” as used herein mean that the subsequently described event, circumstance, or material may or may not occur or be present, and that the description includes instances where the event, circumstance, or material occurs or is present and instances where it does not occur or is not present.

[0045] The terms “about” or “approximately” as used herein are defined as being close to as understood by one of skill in the art, and in one non-limiting aspect the terms are defined to be within 10%, preferably within 5%, more preferably within 1%, and most preferably within 0.5% of an associated disclosed value. The terms may be removed from the associated disclosed value and the exact value may be used instead.

[0046] Ranges may be expressed herein as from “about” one particular value, and / or to “about” another particular value. When such a range is expressed, also specifically contemplated and considered disclosed is the range from the one particular value and / or to the other particular value unless the context specifically indicates otherwise. Similarly, when values are expressed as approximations, by use of the antecedent “about,” it will be understood that the particular value forms another, specifically contemplated aspect that should be considered disclosed unless the context specifically indicates otherwise. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint unless the context specifically indicates otherwise. Finally, it should be understood that all of the individual values and sub-ranges ofvalues contained within an explicitly disclosed range are also specifically contemplated and should be considered disclosed unless the context specifically indicates otherwise. The foregoing applies regardless of whether in particular cases some or all of these aspects are explicitly disclosed.

[0047] When the lower limit value of a given percentage range does not include the % symbol and / or the percentage type (e.g., w / w, v / v, etc.), then the percentage type for the lower limit value is the same as for the upper limit value of the given percentage range. For example, the percentage range of “0.01 to 0.5% w / w” means “0.01% w / w to 0.5% w / w.”

[0048] The terms “wt.%”, “w / w”, “vol.%”, “N / N”, “w / v”, or “mol.%” refers to a weight percentage of a component, a volume percentage of a component, or molar percentage of a component, respectively, based on the total weight, the total volume of material, or total moles, which includes the component. In non-limiting examples, 10 grams of component in 100 grams of a material is 10 wt.% or 10% w / w of component, 10 mL of component in 100 mL of a material is 10 vol.% or 10% v / v of component, and 10 grams of component in 100 mL of a material is 10 w / v of component.

[0049] The use of the word “a” or “an” when used in conjunction with the terms “comprising”, “having”, “including”, or “containing” (or any variations of these words) may mean “one,” but it is also consistent with the meaning of “one or more,” “at least one,” and “one or more than one.”

[0050] The phrase “and / or” means “and” or “or”. To illustrate, A, B, and / or C includes: A alone, B alone, C alone, a combination of A and B, a combination of A and C, a combination of B and C, or a combination of A, B, and C.

[0051] For purposes of this application, a number value with one or more decimal places can be rounded to the nearest whole number using standard rounding guidelines, i.e., round up if the number being rounded is 5, 6, 7, 8, or 9; and round down if the number being rounded is 0, 1, 2, 3, or 4. For example, 0.42 can be rounded to 0.4.

[0052] The words “comprising” (and any form of comprising, such as “comprise” and “comprises”), “having” (and any form of having, such as “have” and “has”), “including” (and any form of including, such as “includes” and “include”) or “containing” (and any form of containing, such as “contains” and “contain”) as used herein are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.

[0053] The compositions and methods for their use can “comprise,” “consist essentially of,” or “consist of’ any of the ingredients or steps disclosed throughout the specification. With respect to the transitional phrases “consist essentially of’ or “consisting essentially of,” in onenon-limiting aspect, a basic and novel characteristic of the compositions and methods of the present disclosure are their abilities to treat wounds and / or repair tissue in a subject with composition or dehydrated compositions comprising placental tissue, preferably with compositions comprising dehydrated placental tissue.II. Collagenase and protease mixtures

[0054] In some aspects, provided herein are compositions comprising collagenase, such as the second composition. In some embodiments, the composition comprising collagenase further comprises one or more additional proteolytic enzymes (i.e., proteases). In some embodiments, the collagenase is comprised in a protease mixture, which can be any suitable protease mixture, such as any provided herein. The protease mixture can include collagenase ColG, collagenase ColH, and a non-specific neutral protease. In some embodiments, the protease mixture is the active pharmaceutical ingredient (API) in SANTYL® Ointment. Collagenase, exemplary proteases, and protease mixtures are described below, which may be used in connection with the methods and compositions provided herein.

[0055] Any proteolytic enzyme useful for wound debridement is suitable for compositions of the present disclosure. Proteolytic enzymes (proteases) break down protein by hydrolysis of the peptide bonds that link amino acids together in the polypeptide chain of a protein. In some aspects, proteases can be divided into four major groups on the basis of catalytic mechanism: serine proteases, cysteine proteases, metalloproteases, and aspartic proteases. Some proteases have been identified with other catalytic amino acids in the active site, such as threonine and glutamic acid; however, they do not form major groups.A. Collagenases

[0056] In some aspects, provided herein is a composition comprising collagenase, and related methods, for wound debridement. A suitable proteolytic enzyme for wound debridement is the metalloprotease collagenase. The collagenase can be substantially pure or it may contain detectable levels of other proteases.

[0057] The amount (potency or concentration) of collagenase in the compositions of the present disclosure is at an effective level to debride the wound. Generally, the potency of collagenase in the compositions can vary from about 1 to about 10,000 collagenase units per gram of product, based on the activity of the collagenase used in the product. In various embodiments, the potency, expressed as collagenase units per gram of product, is from about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240,250, 260, 270, 280, 290, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500 to about 10000, or any range or numerical amount derivable therein.

[0058] The compositions provided herein can comprise any suitable concentration of collagenase. The concentration of collagenase in the compositions generally can vary from about 0.001% w / w to about 8% w / w. In various embodiments, the concentration, expressed as percentage weight by weight, is from about 0.001, 0.002, 0.003, 0.004, 0.005, 0.006, 0.007, 0.008, 0.009, 0.0100.015, 0.020, 0.025, 0.030, 0.035, 0.040, 0.050, 0.055, 0.060, 0.065, 0.070, 0.075, 0.080, 0.085, 0.090, 0.095, 0.100, 0.125, 0.150, 0.175, 0.20, 0.25, 0.30 ,0.35, 0.40, 0.45, 0.50, 0.55, 0.60, 0.65, 0.70, 0.75, 0.80, 0.85, 0.90, 0.95, 1, 2, 3, 4, 5, 6, 7 to about 8 or any range or numerical amount derivable therein.

[0059] In one embodiment, the collagenase is derived from Clostridium histolyticum however, in other embodiments the collagenase can be derived from other sources. Methods for producing a suitable collagenase are disclosed in US patents 3,705,083; 3,821,364; 5,422,261; 5,332,503; 5,422,103; 5,514,370; 5,851,522; 5,718,897; and 6,146,626 all of which are herein incorporated by reference.B. Thermolysin and Other Metalloproteases

[0060] Metalloproteases are among the proteases in which the nucleophilic attach on a peptide bond is mediated by a water molecule, while a divalent metal cation, usually zinc but sometimes cobalt, manganese, nickel or copper, activates the water molecule. The metal ions are extremely important for the activity. Any compounds that have potential to interact with the metal ion, chelating or oxidation, will affect the enzymatic activity. Non-limiting examples of metalloproteases in this family include thermolysin, collagenases, matrix metallo proteinases (MMPs), bacillolysin, dispase, vibriolysin, pseudolysin, stromelysin, and various bacterial derived neutral metalloproteases.

[0061] In some embodiments, the metalloprotease is thermolysin. In some embodiments, the one or more additional proteolytic enzymes comprise thermolysin. In some embodiments, any composition provided herein can comprise thermolysin. In some aspects, provided herein are compositions comprising thermolysin, such as the second composition. In some embodiments, any composition comprising collagenase herein may also comprise thermolysin. Alternatively, thermolysin may be used instead of collagenase in any of the compositions provided herein. In some embodiments, thermolysin may be substituted for collagenase in any of the compositions provided herein, such as the second composition. Thus, in some embodiments, provided herein is a method for debriding a wound, the method comprising: (a)applying a first composition including a poloxamer to the wound; and (b) applying a second composition including a thermolysin to the wound. In some aspects, the method provides similar benefits with respect to enzymatic activity of thermolysin as for collagenase. For example, in some aspects, thermolysin activity is maintained or increased compared to a method comprising applying a thermolysin but not a poloxamer to the wound.

[0062] In some embodiments, the amount (potency or concentration) of thermolysin in the compositions of the present disclosure is at an effective level to debride the wound. Generally, the potency of thermolysin in the compositions can vary from about 1 to about 10,000 thermolysin units per gram of product, based on the activity of the thermolysin used in the product. In various embodiments, the potency, expressed as thermolysin units per gram of product, is from about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500 to about 10000, or any range or numerical amount derivable therein.

[0063] The compositions provided herein can comprise any suitable concentration of thermolysin. The concentration of thermolysin in the compositions generally can vary from about 0.001% w / w to about 8% w / w. In various embodiments, the concentration, expressed as percentage weight by weight, is from about 0.001, 0.002, 0.003, 0.004, 0.005, 0.006, 0.007, 0.008, 0.009, 0.0100.015, 0.020, 0.025, 0.030, 0.035, 0.040, 0.050, 0.055, 0.060, 0.065, 0.070, 0.075, 0.080, 0.085, 0.090, 0.095, 0.100, 0.125, 0.150, 0.175, 0.20, 0.25, 0.30 ,0.35, 0.40, 0.45, 0.50, 0.55, 0.60, 0.65, 0.70, 0.75, 0.80, 0.85, 0.90, 0.95, 1, 2, 3, 4, 5, 6, 7 to about 8 or any range or numerical amount derivable therein.

[0064] The thermolysin can be from any suitable source, such as a bacterial species that produces thermolysin (e.g., Bacillus ihermoproleolylicus). or can be obtained by synthetic methods.C. Other Proteases

[0065] Serine proteases depend upon the hydroxyl group of a serine residue acting as the nucleophile that attacks the peptide bond. The major clans found in humans include the chymotrypsin-like, the subtilisin-like, the alpha / beta hydrolase, and signal peptidase clans. In evolutionary history, serine proteases were originally digestive enzymes. In mammals, they evolved by gene duplication to serve functions in blood clotting, the immune system, and inflammation. These proteases have a broad substrate specificity and work in a wide pH range.Non-limiting examples of serine proteases include trypsin, chymotrypsin, subtilisin, sutilains, plasmin, and elastases. Trypsin is a serine protease that can be derived from the pancreas of healthy bovine or porcine animals, or both. Trypsin can also be derived from recombinant sources. The pharmaceutical grade (USP / NF) of trypsin is known as Crystallized Trypsin. It contains not less than 2500 USP Trypsin Units per mg, calculated on the dried basis, and not less than 90.0% and not more than 110.0% of the labeled potency. The potency assay of trypsin as well as the definition of a USP Trypsin Unit are found in the Crystallized Trypsin monograph of the USP 31 (Official August 1, 2008) herein incorporated by reference.

[0066] Peptidases in which the nucleophile that attach the scissile peptide bond in the sulfhydryl group of a cysteine residue are known as cysteine proteases. Cysteine proteases are commonly encountered in fruits including papaya, pineapple, and kiwifruit. Cysteine proteases have a broad specificity and are widely used under physiological conditions. In this family, papain has been used extensively for wound debridement for a long time. Other cysteine proteases, such as bromelain and analain, have also been investigated for the applications in wound debridement. Other non-limiting examples of cysteine proteases include calpain, caspases, chymopapain, and clostripain.

[0067] Aspartic peptidases are so named because aspartic acid residues are the ligands of the activated water molecule. In most enzymes in this family, a pair of aspartic residues act together to bind and activate the catalytic water molecule. All or most aspartic peptidases are endopeptidases. Most aspartic peptidases have a broad specificity. However, the optimum pH of most aspartic peptidases is in the acidic range. Non-limiting examples of aspartic peptidases are pepsin, chymosin, beta-secretase, plasmepsin, plant acid proteases and retroviral proteases.C. Protease mixtures

[0068] In some aspects, the protease mixture of compositions of the present disclosure can be produced by fermenting Clostridium histolyticum and can include collagenases and a neutral protease. In particular, the mixture can include two collagenases, ColG (MW about 114 kDa) and ColH (MW about 110 kDa), and a non-specific neutral protease (a metalloproteinase with a MW about 35 kDa). It can have a very limited amount of clostripain (cysteine protease). In some embodiments, the activity of clostripain in the mixture is not detected by casein zymogram. In some embodiments, the mixture includes 1 wt. % to 10 wt. % or 1 wt. % to 5 wt. % or 2 wt. % to 4 wt. % or about 3 wt.% of a neutral protease (molecule weight (MW) about 35 kDa), which is a non-specific metalloprotease. In some embodiments, the protease mixture of compositions of the present disclosure does not include or contains limited amounts(e.g., less than 1 wt. % or less than 0.5 wt. % or less than 0.1 wt. % or less than 0.01 wt.%) of clostripain.

[0069] A non-limiting example of a protease mixture that can be used in connection with the compositions of the present disclosure is the active ingredient used in the aforementioned SANTYL® Ointment (called PK collagenase), which is an FDA-approved prescription medicine that debrides wounds to prepare the wound bed for further administration of wound healing agents. The Ointment is a sterile composition that contains 250 collagenase units per gram of white petrolatum USP.III. Poloxamers and Related Compositions

[0070] In some aspects, provided herein are compositions comprising a poloxamer, such as the first composition including a poloxamer. Any suitable poloxamer can be used in connection with the provided compositions and methods for wound debridement, such as any poloxamer provided herein or known to one skilled in the art. The composition including a poloxamer can be any composition including a poloxamer provided herein. In some embodiments, the composition including a poloxamer can be any of the compositions provided in Table 1.

[0071] Poloxamers are synthetic block copolymers of ethylene oxide and propylene oxide represented by the formula:HO(C2H4OMC3H6O) / ?(C2H4O)flH, in which a and b represent the number of repeat units. Generally, a is from 2 to 150 and b is from 15 to 70 depending on the particular poloxamer. Poloxamers can be either liquid or solid at 25 °C depending on their molecular weights.

[0072] The following suitable non-limiting examples of liquid poloxamers are described using CTFA / INCI nomenclature: poloxamer 101, poloxamer 105, poloxamer 122, poloxamer 123, poloxamer 124, poloxamer 181, poloxamer 182, poloxamer 183, poloxamer 184, poloxamer 212, poloxamer 231, poloxamer 282, poloxamer 331, poloxamer 401, and poloxamer 402.

[0073] The following suitable non-limiting examples of solid poloxamers are described using CTFA / INCI nomenclature: poloxamer 108, poloxamer 188, poloxamer 217, poloxamer 237, poloxamer 238, poloxamer 288, poloxamer 338, poloxamer 407, poloxamer 185, poloxamer 215, poloxamer 234, poloxamer 235, poloxamer 284, poloxamer 333, poloxamer 334, poloxamer 335, and poloxamer 403.

[0074] The liquid and solid poloxamers are available commercially from the BASF Corporation under the PLURONIC® and LUTROL® tradenames and from the UNIQEMA Corporation under the SYNPERONIC® trademark. Pharmaceutical grade (USP / NF) poloxamers are poloxamer 124, poloxamer 188, poloxamer 237, poloxamer 338, and poloxamer 407. Both pharmaceutical grade and cosmetic grade poloxamers are suitable for the compositions of the present disclosure.

[0075] The amount of such poloxamer in the composition can range from 1 to 25% w / w, e.g., at least, at most, exactly, or between any two of 1 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6,6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15, 15.5, 16, 16.5, 17,17.5, 18, 18.5, 19, 19.5, 20, 20.5, 21, 21.5, 22, 22.5, 23, 23.5, 24, 24.5, or 25 wt. %; or 2.5, 3, 5, 7.5, 10, 12.5, or 15 wt. %; or 2, 2.05, 2.1, 2.15, 2.2, 2.25, 2.3, 2.35, 2.4, 2.45, 2.5, 2.55, 2.6, 2.65, 2.7, 2.75, 2.8, 2.85, 2.9, 2.95, 3, 3.05, 3.1, 3.15, 3.2, 3.25, 3.3, 3.35, 3.4, 3.45, 3.5, 3.55, 3.6, 3.65, 3.7, 3.75, 3.8, 3.85, 3.9, 3.95, 4, 4.05, 4.1, 4.15, 4.2, 4.25, 4.3, 4.35, 4.4, 4.45, 4.5, 4.55, 4.6, 4.65, 4.7, 4.75, 4.8, 4.85, 4.9, 4.95, or 5 wt. %, depending on the poloxamer included in the composition, although more or less can be added to achieve a desired result for the composition. For example, in one aspect, the first composition comprises poloxamer 407, and the poloxamer 407 is included in the composition at 1 to 15% w / w, e.g., at least, at most, exactly, or between any two of 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5, or 15 wt. %. Without wishing to be bound by theory, >15 wt. % poloxamer 407 can form a thermoreversible gel in aqueous systems such as saline, blood, wound fluid etc. The formation of such structures could inhibit metalloproteinase and / or slow the solubilization process of the second composition, which could disadvantageously slow the speed of necrotic tissue digestion. In another aspect, the first composition comprises poloxamer 407, and the poloxamer 407 is included in the composition at 1 to 15% w / w, e.g., at least, at most, exactly, or between any two of 1, 1.5, 2, 2.5, 3, 3.5, 4,4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5, or 15 wt. %. For other poloxamer-type surfactants (e.g., poloxamer 124, poloxamer 188) that do not form thermoreversible hydrogels, poloxamer can be included in the compositions at concentrations higher than concentrations of poloxamer 407, e.g., at least, at most, exactly, or between any two of 1 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12,12.5, 13, 13.5, 14, 14.5, 15, 15.5, 16, 16.5, 17, 17.5, 18, 18.5, 19, 19.5, 20, 20.5, 21, 21.5, 22,22.5, 23, 23.5, 24, 24.5, or 25 wt. %.

[0076] Certain compositions of the present disclosure (e.g., the first or second composition, or compositions comprising the first or second composition) can include hydrophilic polyols,which are water-soluble, polar aliphatic alcohols with at least two hydroxyl groups, and include polymeric polyols, e.g., polyethylene glycols and poloxamers. In some embodiments, the liquid hydrophilic polyol is a liquid polyethylene glycol or a liquid poloxamer, or mixtures thereof. Other examples of liquid hydrophilic polyols include but are not limited to propylene glycol, butylene glycol, pentylene glycol, hexylene glycol, glycerin, hexylene glycol, methoxy polyethylene glycol, propylene carbonate, and ethoxydiglycol.

[0077] Polyethylene glycols are homo-polymers of ethylene glycol and water represented by the formula:H(OCH2CH2)„OH, in which n represents the average number of oxyethylene groups. Polyethylene glycols can be either liquid or solid at 25 °C depending on their molecular weights.

[0078] The following suitable non-limiting examples of liquid polyethylene glycols are described using USP nomenclature: polyethylene glycol 200, polyethylene glycol 300, polyethylene glycol 400, polyethylene glycol 500, and polyethylene glycol 600.

[0079] The following suitable non-limiting examples of solid polyethylene glycols are described using USP nomenclature: polyethylene glycol 700, polyethylene glycol 800, polyethylene glycol 900, polyethylene glycol 1000, polyethylene glycol 1100, polyethylene glycol 1200, polyethylene glycol 1300, polyethylene glycol 1400, polyethylene glycol 1450, polyethylene glycol 1500, polyethylene glycol 1600, polyethylene glycol 1700, polyethylene glycol 1800, polyethylene glycol 1900, polyethylene glycol 2000, polyethylene glycol 2100, polyethylene glycol 2200, polyethylene glycol 2300, polyethylene glycol 2400, polyethylene glycol 2500, polyethylene glycol 2600, polyethylene glycol 2700, polyethylene glycol 2800, polyethylene glycol 2900, polyethylene glycol 3000, polyethylene glycol 3250, polyethylene glycol 3350, polyethylene glycol 3750, polyethylene glycol 4000, polyethylene glycol 4250, polyethylene glycol 4500, polyethylene glycol 4750, polyethylene glycol 5000, polyethylene glycol 5500, polyethylene glycol 6000, polyethylene glycol 6500, polyethylene glycol 7000, polyethylene glycol 7500, and polyethylene glycol 8000.

[0080] The liquid and solid polyethylene glycols are available commercially from the DOW Chemical Company under the CARBOWAX™ tradename and from the BASF Corporation under LUTROL® E and PLURACARE® E tradenames. Both pharmaceutical grade (USP / NF) and cosmetic grade polyethylene glycols are suitable for the compositions of the present disclosure.A. Additives

[0081] Compositions comprising a poloxamer, such as the first composition including a poloxamer, can include additional ingredients. For instance, in some aspects, the first composition including the poloxamer can further include one or more water soluble solvents, one or more preservatives, and one or more metal salts. In some aspects, any one or more of the foregoing further components may be excluded from the first composition including the poloxamer disclosed herein.

[0082] The water-soluble solvents can include propylene glycol or other diols. The total amount of such solvents can range from 1 to 40% w / w, e.g., at least, at most, exactly, or between any two of 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11,11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15, 15.5, 16, 16.5, 17, 17.5, 18, 18.5, 19, 19.5, 20, 20.5, 21,21.5, 22, 22.5, 23, 23.5, 24, 24.5, 25, 25.5, 26, 26.5, 27, 27.5, 28, 28.5, 29, 29.5, 30, 30.5, 31,31.5, 32, 32.5, 33, 33.5, 34, 34.5, 35, 35.5, 36, 36.5, 37, 37.5, 38, 38.5, 39, 39.5, or 40 wt. %, although more or less can be added to achieve a desired result for the first composition. For example, in some aspects, the first composition comprises propylene glycol, and the propylene glycol is included in the composition at, e.g., at least, at most, exactly, or between any two of 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, or 6 wt. %. In specific aspects, propylene glycol is included in the composition at about 2, 2.2, or 4 wt. %.

[0083] The one or more preservatives can be used to preserve the first composition and can include one or more parabens, benzyl alcohol, and / or phenoxyethanol. The one or more parabens can include methyl paraben and / or propyl paraben. The total amount of such preservatives can range from 0.01 to 5% w / w, e.g., at least, at most, exactly, or between any two of 0.01, 0.05, 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 0.55, 0.6, 0.65, 0.7, 0.75, 0.8, 0.85, 0.9, 0.95, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, or 5 wt. %, although more or less can be added to achieve a desired result for the first composition.

[0084] For example, in some aspects, the first composition comprises methyl paraben, and the methyl paraben is included in the composition at 0.01 to 1% w / w, e.g., at least, at most, exactly, or between any two of 0.01, 0.05, 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 0.55, 0.6, 0.65, 0.7, 0.75, 0.8, 0.85, 0.9, 0.95, or 1 wt. %. In some aspects, the first composition comprises methyl paraben, and the methyl paraben is included in the composition at, e.g., at least, at most, exactly, or between any two of 0.15, 0.155, 0.16, 0.165, 0.17, 0.175, 0.18, 0.185, 0.19, 0.195, 0.2, 0.205, 0.21, 0.215, 0.22, 0.225, 0.23, 0.235, 0.24, 0.245, 0.25, 0.255, 0.26,0.265, 0.27, 0.275, 0.28, 0.285, 0.29, 0.295, or 0.3 wt. %. In specific aspects, methyl paraben is included in the composition at about 0.15, 0.18, 0.20, 0.23, 0.25, or 0.30 wt. %.

[0085] Additionally or alternatively, the first composition can comprise propyl paraben, and the propyl paraben is included in the composition at 0.01 to 1% w / w, e.g., at least, at most, exactly, or between any two of 0.01, 0.05, 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 0.55, 0.6, 0.65, 0.7, 0.75, 0.8, 0.85, 0.9, 0.95, or 1 wt. %. In some aspects, the first composition comprises propyl paraben, and the propyl paraben is included in the composition at, e.g., at least, at most, exactly, or between any two of 0.03, 0.035, 0.04, 0.045, 0.05, 0.055, 0.06, 0.065, 0.07, 0.075, 0.08, 0.085, 0.09, 0.095, 0.1, 0.105, 0.11, 0.115, 0.12, 0.125, 0.13, 0.135, 0.14, 0.145, or 0.15 wt. %. In specific aspects, propyl paraben is included in the composition at about 0.03, 0.04, 0.06, 0.15, or 0.6 wt. %.

[0086] Additionally or alternatively, the first composition can comprise benzyl alcohol, and the benzyl alcohol is included in the composition at 0.01 to 2% w / w, e.g., at least, at most, exactly, or between any two of 0.01, 0.05, 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 0.55, 0.6, 0.65, 0.7, 0.75, 0.8, 0.85, 0.9, 0.95, 1, 1.05, 1.1, 1.15, 1.2, 1.25, 1.3, 1.35, 1.4, 1.45, 1.5, 1.55, 1.6, 1.65, 1.7, 1.75, 1.8, 1.85, 1.9, 1.95, or 2 wt. %. In some aspects, the first composition comprises benzyl alcohol, and the benzyl alcohol is included in the composition at, e.g., at least, at most, exactly, or between any two of 0.15, 0.155, 0.16, 0.165, 0.17, 0.175, 0.18, 0.185, 0.19, 0.195, or 1 wt. %. In specific aspects, benzyl alcohol is included in the composition at about 0.5 or 1 wt. %.

[0087] Additionally or alternatively, the first composition can comprise phenoxyethanol, and the phenoxyethanol is included in the composition at 0.01 to 2% w / w, e.g., at least, at most, exactly, or between any two of 0.01, 0.05, 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 0.55, 0.6, 0.65, 0.7, 0.75, 0.8, 0.85, 0.9, 0.95, 1, 1.05, 1.1, 1.15, 1.2, 1.25, 1.3, 1.35, 1.4, 1.45, 1.5, 1.55, 1.6, 1.65, 1.7, 1.75, 1.8, 1.85, 1.9, 1.95, or 2 wt. %. In some aspects, the first composition comprises phenoxyethanol, and the phenoxyethanol is included in the composition at, e.g., at least, at most, exactly, or between any two of 0.15, 0.155, 0.16, 0.165, 0.17, 0.175, 0.18, 0.185, 0.19, 0.195, or 1 wt. %. In specific aspects, phenoxyethanol is included in the composition at about 0.5, 0.8, or 1 wt. %.

[0088] The one or more metal salts can be used as buffering or stabilization agents and can include sodium acetate, sodium bicarbonate, sodium ascorbate, sodium chloride, potassium chloride, magnesium chloride, calcium chloride, and / or barium chloride. The total amounts of salts can range from 0.01 to 1% w / w, e.g., at least, at most, exactly, or between any two of 0.01, 0.05, 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 0.55, 0.6, 0.65, 0.7, 0.75, 0.8, 0.85, 0.9,0.95, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, or 10 wt. %, although more or less can be added to achieve a desired result for the first composition. Without wishing to be bound by theory, the amount of the one or more metal salts included in the first composition can be adjusted to provide an optimal pH for metalloproteinase activity, e.g., to provide a pH of at least, at most, exactly, or between any two of 6, 6.05, 6.1, 6.15, 6.2, 6.25, 6.3, 6.35, 6.4, 6.45, 6.5, 6.55, 6.6, 6.65, 6.7, 6.75, 6.8, 6.85, 6.9, 6.95, 7, 7.05, 7.1, 7.15, 7.2, 7.25, 7.3, 7.35,7.4, 7.45, 7.5, 7.55, 7.6, 7.65, 7.7, 7.75, 7.8, 7.85, 7.9, 7.95, or 8.

[0089] For example, in some aspects, the first composition comprises sodium acetate, and the sodium acetate is included in the first composition to provide a pH of at least, at most, exactly, or between any two of 6, 6.05, 6.1, 6.15, 6.2, 6.25, 6.3, 6.35, 6.4, 6.45, 6.5, 6.55, 6.6, 6.65, 6.7, 6.75, 6.8, 6.85, 6.9, 6.95, 7, 7.05, 7.1, 7.15, 7.2, 7.25, 7.3, 7.35, 7.4, 7.45, 7.5, 7.55, 7.6, 7.65, 7.7, 7.75, 7.8, 7.85, 7.9, 7.95, or 8. In some aspects, the first composition comprises sodium acetate, and the sodium acetate is included in the composition at, e.g., at least, at most, exactly, or between any two of about 0.06, 0.15, 0.23, 0.24, 0.25, 0.27, 0.30, 0.50, or 0.51 wt. % .

[0090] Additionally or alternatively, the first composition can comprise sodium chloride, and the sodium chloride can be included in the first composition as an osmolarity agent at a concentration of, e.g., at least, at most, exactly, or between any two of 1, 1.5, 2, 2.5, 3, 3.5, 4,4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, or 10 wt. %. In specific aspects, sodium chloride is included in the composition at about 0.50, 0.51, 0.52, 0.53, 0.54, 0.55, 0.55, 0.56, 0.57, 0.58, 0.59, or 0.60 wt. %.

[0091] In some aspects, the first composition including the poloxamer further includes water. The amount of water that can be added can be an amount that fills out the formulation (e.g., q.s. to 100%).

[0092] Buffers having an appropriate pH range (e.g., 6.5 to 9 or 7 to 8 or about 7.5) for collagenase can also be used. An example of such a buffer is Tris buffer (10 mM, pH=7.5).

[0093] In some aspects, the first composition includes the poloxamer, propylene glycol, methyl paraben, propyl paraben, sodium acetate, sodium chloride, benzyl alcohol, and water.IV. Compositions, Carriers, and Other Ingredients

[0094] The compositions of the present disclosure (e.g., the first composition and / or the second composition, or any composition comprising a collagenase or poloxamer) can include additional components such as carriers or diluents, which can be non-active components. Insome aspects, the additional components do not substantially interfere with or inhibit the intended purpose of the composition.

[0095] The compositions may, for example, take the form of solutions, suspensions, instillations, sprays, salves, creams, gels, foams, ointments, emulsions, lotions, paints, sustained release formulations, dissolvable gel-forming films, or powders, and can contain any suitable concentration of active ingredient. For example, the compositions can contain about 0.01% to about 1% of active ingredient(s), about l%-50% or active ingredient(s), about 2%- 60% of active ingredient(s), about 2%-70% of active ingredient(s), or up to about 90%) of active ingredient(s). Other suitable formulations include poloxamer gel-based formulations, carboxymethylcellulose (CMC)-based formulations, hydroxylethylcellulose (HEC)-based formulations, hydroxypropycellulose (HPC)-based formulations, and hyroxypropylmethylcellulose (HPMC)-based formulations. Other useful formulations include slow or delayed release preparations. In some specific embodiments, the composition is formulated as an ointment.

[0096] Gels or jellies may be produced using a suitable gelling agent including, but not limited to, gelatin, tragacanth, or a cellulose derivative and may include glycerol as a humectant, emollient, and preservative. Ointments are semi-solid preparations that consist of the active ingredient incorporated into a fatty, waxy, or synthetic base. Examples of suitable creams include, but are not limited to, water-in-oil and oil-in-water emulsions. Water-in-oil creams may be formulated by using a suitable emulsifying agent with properties similar, but not limited, to those of the fatty alcohols such as cetyl alcohol or cetostearyl alcohol and to emulsifying wax. Oil-in-water creams may be formulated using an emulsifying agent such as cetomacrogol emulsifying wax. Suitable properties include the ability to modify the viscosity of the emulsion and both physical and chemical stability over a wide range of pH. The water soluble or miscible cream base may contain a preservative system and may also be buffered to maintain an acceptable physiological pH. In some embodiments, the compositions comprise petrolatum. In some embodiments, the compositions comprise white petrolatum.

[0097] Foam preparations may be formulated to be delivered from a pressurized aerosol canister, via a suitable applicator, using inert propellants. Suitable excipients for the formulation of the foam base include, but are not limited to, propylene glycol, emulsifying wax, cetyl alcohol, and glyceryl stearate. Potential preservatives include methylparaben and propylparaben.

[0098] Suitable carriers and diluents include isotonic saline solutions, for example phosphate-buffered saline. Suitable diluents and excipients also include, for example, water,saline, dextrose, glycerol, or the like, and combinations thereof. In addition, if desired, substances such as wetting or emulsifying agents, stabilizing or pH buffering agents may also be present.

[0099] The term “pharmaceutically acceptable carrier” can refer to any pharmaceutical carrier that does not itself induce the production of antibodies harmful to the individual receiving the composition, and which can be administered without undue toxicity. Suitable carriers can be large, slowly metabolized macromolecules such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, and amino acid copolymers.

[0100] Pharmaceutically acceptable salts can also be present, e.g., mineral acid salts such as hydrochlorides, hydrobromides, phosphates, sulfates, and the like; and the salts of organic acids such as acetates, propionates, malonates, benzoates, and the like.

[0101] Suitable carrier materials include any carrier or vehicle commonly used as a base for creams, lotions, sprays, foams, gels, emulsions, lotions or paints for topical administration. Examples include emulsifying agents, inert carriers including hydrocarbon bases, emulsifying bases, non-toxic solvents or water-soluble bases. Examples include poloxamers, petrolatum, white petrolatum, HPMC, CMC and other cellulose-based ingredients, lanolin, hard paraffin, liquid paraffin, soft yellow paraffin or soft white paraffin, white beeswax, yellow beeswax, cetostearyl alcohol, cetyl alcohol, dimethicones, emulsifying waxes, isopropyl myristate, microcrystalline wax, oleyl alcohol and stearyl alcohol. In some embodiments, the composition (e.g., the composition including collagenase) comprises petrolatum. In some embodiments, the composition comprises white petrolatum.

[0102] Auxiliary agents such as casein, gelatin, albumin, glue, sodium alginate, carboxymethylcellulose, methylcellulose, hydroxyethylcellulose or polyvinyl alcohol may also be included in the compositions described herein.

[0103] The compositions provided herein may include hydrophobic bases. Hydrophobic bases can comprise, but are not limited to, plant, animal, paraffinic, and synthetic derived fats, butters, greases, waxes, solvents, and oils; mineral oils, vegetable oils, petrolatum, water insoluble organic esters and triglycerides, silicones, or fluorinated compounds; or mixtures thereof. In some embodiments, the hydrophobic phase comprises petrolatum.

[0104] Plant derived materials include, but are not limited to, arachis (peanut) oil, balsam Peru oil, carnauba wax, candelilla wax, castor oil, hydrogenated castor oil, cocoa butter, coconut oil, corn oil, cotton seed oil, jojoba oil, macadamia seed oil, olive oil, orange oil, orange wax, palm kernel oil, rapeseed oil, safflower oil, sesame seed oil, shea butter, soybean oil, sunflower seed oil, tea tree oil, vegetable oil, and hydrogenated vegetable oil.

[0105] Non-limiting examples of animal derived materials include beeswax, cod liver oil, emu oil, lard, mink oil, shark liver oil, squalane, squalene, and tallow.

[0106] Non-limiting examples of paraffinic materials include isoparaffin, microcrystalline wax, heavy mineral oil, light mineral oil, ozokerite, petrolatum, and paraffin.

[0107] Suitable non-limiting examples of organic esters and triglycerides include C12-15 alkyl benzoate, isopropyl myristate, isopropyl palmitate, medium chain triglycerides, trilaurin, and trihydroxystearin.

[0108] Non-limiting examples of silicones are dimethicone and cyclomethicone. A nonlimiting example of a fluorinated compound is polytetrafluoroethylene (PTFE).

[0109] In some embodiments, the composition (e.g., the composition including collagenase) comprises petrolatum. In some aspects, petrolatum is a purified mixture of semisolid hydrocarbons obtained from petroleum and varies from dark amber to light yellow in color. White petrolatum is wholly or nearly decolorized petrolatum and varies from cream to snow white in color. Petrolatum and White Petrolatum can also vary in melting point, viscosity, and consistency.

[0110] Various grades are available commercially from the PENRECO Corporation under the tradenames: PENRECO®ULTIMA, PENRECO®SUPER, PENRECO®SNOW, PENRECO®REGENT, PENRECO®LILY, PENRECO®CREAM, PENRECO® ROY AL, PENRECO® BLOND, and PENRECO® AMBER. Various grades are also available commercially from the SONNEBORN Corporation under the tradenames: ALBA®, SUPER WHITE PROTOPET®, SUPER WHITE FONOLINE®, WHITE PROTOPET IS®, WHITE PROTOPET 2L®, WHITE PROTOPET 3C®, WHITE FONOLINE®, PERFECT A®, YELLOW PROTOPET 2A®, YELLOW FONOLINE®, PROTOLINE®, SONOJELL #4®, SONOJELL #9®, MINERAL JELLY #10®, MINERAL JELLY #14®, MINERAL JELLY #17®, AND CARNATION TROUGH GREASE®.

[0111] Petrolatum and White Petrolatum are available in cosmetic grade and pharmaceutical (USP / NF) grade and both are suitable for the compositions of the present disclosure.

[0112] The compositions can be anhydrous as defined herein. The compositions can be semisolid or liquid. The composition can be impregnated on a pad, gauze, or sponge. The compositions can also be sterile.

[0113] The compositions can be topical. The compositions can include additional materials known in the art that are suitable for topical compositions, e.g., absorbents, deodorizers,surfactants, solvents, rheology modifiers, film formers, stabilizers, emollients, moisturizers, preservatives, antimicrobials, antioxidants, chelating agents, fragrances, and colorants.

[0114] The compositions can also include additional pharmaceutical active ingredients known in the art that are suitable for topical compositions of this nature, e.g., antimicrobial agents, wound healing agents, anesthetic agents, vulnerary agents, and haemostatic agents. A non-limiting example of a vulnerary agent is balsam Peru.

[0115] The compositions can be packaged in any package suitable for dispensing a wound debrider. The compositions can be packaged in multi-use, single-dose, or metered dose packages. Non-limiting examples include a tube, bottle, jar, pump container, pressurized container, bladder container, aerosol container, aerosol spray container, non-aerosol spray container, syringe, pouch, or sachet.

[0116] The compositions of the present disclosure can be prepared by techniques and methods known by one of ordinary skill in the art by dissolving or suspending the proteolytic enzyme in part or all of the available hydrophilic polyol. The resulting solution or suspension can be mixed with a hydrophobic base to form a dispersion, wherein the hydrophobic base becomes the continuous phase and the hydrophilic polyol / enzyme phase becomes the dispersed phase. These compositions can be prepared using processing equipment known by one of ordinary skill in the art, e.g., blenders, mixers, mills, homogenizers, dispersers, dissolvers, etc.

[0117] The composition may be present in a vehicle or administered in any effective amount. In some embodiments, the composition including collagenase is formulated as 250 units of collagenase per gram of composition (Unit definition: The potency assay of collagenase in SANTYL® Ointment is based on the digestion of undenatured collagen from bovine Achilles tendon at pH 7.2 and 37 °C for 24 hours. The number of peptide bonds cleaved is measured by reaction with ninhydrin. Amino groups released from the collagen by a trypsin digestion control are subtracted. One net collagenase unit will hydrolyze undenatured collagen to produce ninhydrin reactive material equivalent to 1.09 nmol of leucine equivalents per minute.) In some embodiments composition may be formulated as 100, 150, 200, 250, 300, 350, 400, or 500 units (or any derivable range therein) of collagenase per gram of composition. In some embodiments, the composition is applied every 2, 6, or 8 hours or every 1, 2, 4, 7, or 10 days (or any derivable range thereof).

[0118] In some aspects, the compositions described herein can be provided in the form of a wound dressing. The wound dressing may contain one or more agents specific to a desired biomarker. The term “wound dressing” used herein is taken to include any medically or pharmaceutically acceptable wound covering or support matrix. Examples of suitable wounddressing materials include, but are not limited to, a) films, including those of a semipermeable or a semi-occlusive nature such as polyurethane copolymers, polyurethane film, acrylamides, acrylates, paraffin, polysaccharides, cellophane and lanolin; b) hydrocolloids including carboxymethylcellulose protein constituents of gelatin, pectin, and complex polysaccharides including Acacia gum, guar gum and karaya, which may be utilized in the form of a flexible foam, formulated in polyurethane, or formulated as an adhesive mass such as polyisobutylene; c) polymers such as agar, starch or propylene glycol, which typically contain about 80% to about 90% water and are conventionally formulated as sheets, powders, pastes and gels in conjunction with cross-linked polymers such as polyethylene oxide, polyvinyl pyrrolidone, acrylamide, propylene glycol; d) foams such as polysaccharide which consist of a hydrophilic open-celled contact surface and hydrophobic closed-cell polyurethane; e) impregnates including pine mesh gauze, paraffin and lanolin-coated gauze, polyethylene glycol-coated gauze, knitted viscose, rayon, and polyester; and f) cellulose-like polysaccharide such as alginates, including calcium alginate, which may be formulated as non-woven composites of fibers or spun into woven composites.

[0119] The compositions described herein may also include additional therapeutic components that are known to treat skin conditions and / or wounds. Such therapeutic components include antimicrobials such as, for example, antiseptics and antibiotics.

[0120] Antiseptics are disinfectants that can be used on intact skin and some open wounds to kill or inhibit microorganisms. They often have multiple microbial targets, a broad antimicrobial spectrum, and residual anti-infective activity but are often toxic to host tissues (e.g., fibroblasts, keratinocytes, and possibly leukocytes). Commonly used antiseptics include hydrogen peroxide, which has limited bactericidal and debriding activity; and chlorhexidine, which has long-acting activity against a wide range of both gram-negative and gram-positive bacteria.

[0121] Antibiotics are chemicals produced either naturally (by a microorganism) or synthetically that in dilute solution inhibit or kill other microorganisms. They usually act on one specific cell target, have a narrower spectrum of activity, are relatively nontoxic, and are more susceptible to losing their effectiveness to bacterial resistance. The first topical antibiotics were derived from agents developed for systemic use (i.e., sulfonamides in the mid- 19308), followed in the next decade by topical penicillins, bacitracin, gramicidin, aminoglycosides (including neomycin), polymixin, tetracyclines, and chloramphenicol. Agents introduced later include fusidic acid, clindamycin, mupirocin and retapamulin. Antibiotics that may be used in the compositions described in the disclosure include bacitracin, fisidic acid,gentamicin, mafenide acetate, mupirocin and mupirocin calcium, neomycin sulfate, nitrofurazone, polymixin B, retapumulin, and sulfacetamide.

[0122] The compositions described herein may also comprise additional agents that reduce skin inflammation such as, for example, antihistamines, corticosteroids (e.g., hydrocortisone or clobetasol propionate), and immunosuppressants (e.g., pimecrolimus and tacrolimus).

[0123] The compositions described herein may also be combined with other treatments known in the art to promote wound repair.

[0124] The compositions provided herein may also comprise any suitable agents for reducing pain, such as topical analgesics. A variety of topical analgesics may be used in connection with the compositions of the present disclosure. The most common topical analgesics are local anesthetics and anti-inflammatories such as salicylates or NSAIDS, and counter-irritants including capsaicin and aromatic compounds. Anesthetics such as lidocaine, that act on local sensory afferents, are intended to totally block pain receptors and numb the area of application. Salicylates and NSAIDS such as ibuprofen, are anti-inflammatory compounds that inhibit pain and inflammation and are generally taken internally. Counter- irritants and aromatics, especially terpenes, are substances such as menthol, oil of wintergreen, camphor, eucalyptus, mustard plasters and turpentine oil, that mask sensations of pain by stimulating local pain afferents and thereby creating a feeling of cold or heat over the affected area. Capsaicin is a natural ingredient found in cayenne peppers. Capsaicin is believed to operate in an anesthetic fashion by depleting Substance-P from sensory afferents and thereby suppressing transmission of pain to the brain. Menthol is a compound obtained from peppermint oils, or other mint oils, or made synthetically. Menthol has local anesthetic and counterirritant qualities. Topical analgesics can include menthol / menthyl derivatives, such as 1-menthol or menthyl lactate. Capsaicin, or other capsaicinoids, vanilloids, or vanillyl butyl ether, may also be used.V. Methods of Use

[0125] Disclosed are methods of treating a wound (e.g., methods for wound debridement) comprising applying a first composition including a poloxamer to the wound and applying a second composition including a collagenase to the wound. In some aspects, the methods for wound debridement can facilitate removal of necrotic tissue and other debris from the wound. In some aspects, the wound debridement can promote wound healing, and improve outcome of subsequent treatments to facilitate wound healing.

[0126] The compositions and methods disclosed herein can, in some aspects, be useful for the treatment of wounds of a subject by applying the compositions to or on the wound. A wound can include a disruption of the structure and function of tissue. In addition to the other nonlimiting examples disclosed elsewhere herein, wounds can include: internal organ wounds; mucous membrane wounds; vascular tissue wounds; soft tissue wounds including ligaments, tendons, and cartilage; bone wounds; and dermal wounds. In some aspects, the wound is a dermal wound. In some aspects, the compositions can be applied topically to a dermal wound. In some aspects, the compositions can be applied so as to be in direct contact with at least a portion of the wound surface.

[0127] A dermal wound may involve the disruption of the skin and associated soft tissue architecture. Dermal wounds may be partial or full thickness wounds. They may also be acute wounds, chronic wounds, or bums, which may be acute or chronic. Non-limiting examples of a bum wound include a superficial (first degree) bum, a partial thickness (second degree) bum, a full thickness (third degree) burn, or a radiation bum. Non-limiting examples of a chronic wound include a dermal ulcer, a diabetic ulcer, a diabetic foot ulcer, a venous ulcer, a venous leg ulcer, an arterial ulcer, an arterial leg ulcer, a decubitus ulcer, a stasis ulcer, an ischemic ulcer, a vascular ulcer, a pressure ulcer (stage I-IV), a podiatric wound, a draining wound, a tunneling wound, or an undermining wound. Non-limiting examples of an acute wound include a trauma wound, a laceration, an abrasion, a skin tear, a skin lesion, a blister, a surgical incision, a donor skin site, a skin graft, a laser surgery wound, a Mohs surgery wound, or a dehisced wound. In some aspects, the dermal wound includes necrotic tissue. In some aspects, removal of the necrotic tissue is facilitated by the method of debridement.

[0128] In some aspects, compositions disclosed herein may provide a topical wound covering which functions as a protective barrier for the wound.

[0129] In some aspects, compositions disclosed herein may be used as a dressing to provide a physical barrier for the management of chronic wounds (such as diabetic foot ulcers, venous leg ulcers, pressure ulcers), acute wounds, and in the post-operative care of surgical incisions. In some aspects, compositions disclosed herein may be sutured onto the wound bed, which either independently or in combination with its physical barrier function, may protect the innate wound healing response. In some aspects, the subject device may also act as a biodegradable scaffold that supports the body’s own wound healing processes.

[0130] In some aspects, the compositions disclosed herein may be used, optionally as a wound dressing, for management of chronic, acute and post-surgical exuding wounds including partial- and full-thickness wounds, pressure ulcers, venous ulcers, diabetic ulcers, chronicvascular ulcers, tunneled, surgical wounds (e.g., donor sites / grafts, post-Mohs surgery, postlaser surgery, podiatric, wound dehiscence), trauma wounds (e.g., abrasions, lacerations, second-degree bums and skin tears), and draining wounds. In some aspects, the compositions disclosed herein may be used as a dressing to provide a protective cover to chronic wounds, acute wounds, and in post-operative care of surgical incisions. In some aspects, the dressing may promote an environment that helps with wound management by serving as protective barrier. In some aspects, the dressing’s thickness and tensile strength may enable the dressing to be sutured on the wound.

[0131] In some aspects, the compositions disclosed herein may provide benefits, including barrier that protects the wound environment, ability to suture the subject device on the wound, terminal sterilization and viral inactivation reducing the risk of microbial and viral contamination of the device, ability of biodegrading within 10-21 days (e.g., at least, at most, exactly, or between any two of 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, or 21 days), elasticity properties of subject device which may allow it to conform to complex wound anatomy, and / or availability in multiple sizes.

[0132] In another aspect, the compositions or methods disclosed herein may be applied to the wound periodically, for example, daily. The compositions and methods may be applied in conjunction with the application of other wound dressings including but not limited to gauze bandages, sponge wound dressings, foam wound dressings (e.g., ALLEVYN™ foam dressing), antimicrobial wound dressings, ECM based wound dressings, placental tissue wound dressings, wound debriding dressings, calcium alginate dressings, hydrogels, and wound dressings with vulnerary agents. For example, after the debridement, the wound may be covered with another wound dressing. The method may be performed before or after the application of another wound dressing.

[0133] In some aspects, the compositions and methods for wound debridement disclosed herein may support and promote subsequent wound healing processes, such as cellular migration, vascular ingrowth, and / or the formation of granulation tissues.VI. Kits and Packaging

[0134] The compositions of the present disclosure may be packaged in any package configuration suitable, for example, for use in storing, shipping, and / or using the compositions of the present disclosure. Non-limiting examples of packaging configurations may include containers, such as plastic packages, foil packages, pouches, packets, and / or boxes. In certain aspects where the composition is flowable (e.g., in liquid or hydrogel form), the compositionsbottles, jars, bottles with pumps, toddles, tubes (e.g., aluminum, plastic, or laminated), jars, non-aerosol pump sprayers, and / or aerosol containers could be used. The packages may be configured for single-dose or multiple-dose administration.

[0135] Containers such as kits that have multiple compartments may also be used. For instance, the composition including a poloxamer and the composition including a collagenase can be provided in different compartments. Kits may also include 3, 4, 5, or more additional compartments or containers.

[0136] In various aspects, the compositions described herein can be provided in a kit. The kit can include a first composition including a poloxamer and a second composition including a collagenase.

[0137] Packaging may also include informational material relating to the compositions of the present disclosure. In various aspects, the informational material can be descriptive, instructional, marketing or other material that relates to the methods described herein and / or to the use of the placental tissue for the methods described herein. Instructions may include an explanation of how to apply, use, and maintain the products or compositions, for example in accordance with the methods for wound debridement provided herein.EXAMPLES

[0138] The following examples are included to demonstrate aspects of the present disclosure. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the present disclosure, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific aspects which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the present disclosure.I. Example 1 - Wound Cleanser Formulations

[0139] Wound cleansers with varying 0 wt. % to 15 wt. % concentrations of Poloxamer- 407 (an exemplary Poloxamer) were formulated, as shown in Table 1. The wound cleansers were made by dissolving parabens (methyl paraben (MP), propyl paraben (PP)), propylene glycol (PG), Poloxamer-407 (Pol), sodium acetate buffer (SA), sodium chloride (SC), and optionally, preservative (benzyl alcohol (BA), phenoxyethanol (PE)), in water at room temperature. Formulation # 53 also included TRIS buffer and IN HC1. Clear and transparent water-like formulations were obtained.Table 1. Wound cleanser formulations

[0140] Collagenase activity was assessed in exemplary wound cleansers having 0, 2.5, 5, 7.5, 10, 12.5, or 15 wt. % Poloxamer from Table 1.II. Example 2 - Collagenase activity is compatible with the presence of poloxamers

[0141] The enzymatic activity of C. collagenase when formulated in various compositions was assessed by Collagen Fluorescein Conjugate (Coll-FITC) or by FALGPA (2- furanacryloyl-l-leucylglycyl-l-prolyl-l-alanine) assay (described in Jovanovic et al., Wounds 2012;24(9):242-253, incorporated by reference herein in its entirety).

[0142] For the Coll-FITC assay (Invitrogen, DQ Collagen, type I from bovine skin, fluorescein conjugate), briefly, 0.3 g of Collagenase SANTYL Ointment (CSO) was measured into a conical tube, followed by 1 ml of 1 mg / ml Coll-FITC solution in tricine buffer. The reaction was performed in 5 ml of tricine buffer, 5 ml of normal saline, or 5 ml of the exemplary wound cleansers from Table 1 having 0, 2.5, 5, 7.5, 10, 12.5, or 15 wt. % Poloxamer, with all other formulation ingredients and concentrations being constant. The resulting enzymatic digestion was measured as FITC fluorescence at 520 nm. As shown in FIGs. 1A-1B, collagenase activity was improved in the wound cleansers containing poloxamer, in comparison to the saline solution.

[0143] The collagenolytic activity (i.e., collagenase activity) of C. collagenase in the presence poloxamer cleanser formulations having 0, 2.5, 5, 7.5, 10, 12.5, or 15 wt. % Poloxamer was also measured using N-(3[2-Furyl]acryloyl)-Leu-Gly-Pro-Ala (FALGPA) substrate. The concentrations of enzyme, substrate, and salts were the following: C. collagenase 0.1 mg / ml, FALGPA substrate 1 mg / ml; tricine buffer (positive control) or poloxamer cleanser formulations were used as solubilization media for enzyme and substrate. The enzyme activity was measured as decreased absorbance at 345 nm for the first 35 minutes at room temperature. The slope of the linear curve is used as the activity rate (Vmax). Using a 96-well microplate, 100 pL of the enzyme solution was mixed with 150 pL of the FALGPA solution. The kinetic reaction was monitored for 35 minutes, and kinetic rates (for enzymatic activity) were determined by recording the absorbance change at 345 nm. Rates were reportedas Vmax (milli-OD per minute). The results are shown as percent enzymatic activity and are given by the following formula:Vmax tested article% Activity = - - — - - X100Vmax enzyme control solution

[0144] Collagenase enzymatic activity was assessed when formulated in various solutions containing an exemplary poloxamer, Poloxamer-407, and comprising only commonly used GRAS (Generally Recognized as Safe by the Food and Drug Administration) ingredients. Collagenase activity was compared to a control condition in which the collagenase was suspended in a Tris-buffered saline (TBS) solution. FIG. 2 shows results from collagenase in TBS or collagenase in any of four different solutions containing Poloxamer-407. In particular, the 026-0128A02 formulation included -12.5% Poloxamer-407; the 026-0129 A01 formulation included -11.4% Poloxamer-407, the 026-0202A02 formulation included -18% Poloxamer- 407, and the 026-0209 AO 1 formulation included -15.5% Poloxamer-407. The results show that collagenase activity was maintained or improved in the solutions containing Poloxamer-407, in comparison to the control condition.

[0145] Next, collagenase enzymatic activity was assessed when formulated in various exemplary wound cleanser solutions having 0, 2.5, 5, 7.5, 10, 12.5, or 15 wt. % Poloxamer, with all other formulation ingredients and concentrations being constant. The ingredients of the exemplary wound cleansers were: Poloxamer-407 (principal surfactant), glycerin or propylene glycol (moisturizers), acetate buffer at pH=7.40, NaCl (osmolality ~300mGsm / kg), methyl paraben / propyl paraben, and optionally, benzyl alcohol or phenoxyethanol (preservatives). Collagenase activity was compared to a control condition in which the cleanser did not contain the poloxamer. As shown in FIGs. 3A-3B, collagenase activity was maintained or improved in the solutions containing Poloxamer-407, in comparison to the control condition.III. Example 3 - Collagenase activity is incompatible with other surfactants

[0146] The collagenolytic activity (i.e., collagenase activity) of C. collagenase in the presence poloxamer cleanser formulations containing various surfactants was also measured using N-(3[2-Furyl]acryloyl)-Leu-Gly-Pro-Ala (FALGPA) substrate. The concentrations of enzyme, substrate, and surfactants were the following: C. collagenase 0.1 mg / ml, FALGPA substrate 1 mg / ml; tricine buffer (positive control) or solution containing 10 mg / mL (1 wt. %) surfactant were used as solubilization media for enzyme and substrate. The enzyme activity was measured as decreased absorbance at 345 nm for the first 35 minutes at room temperature. The slope of the linear curve is used as the activity rate (Vmax). Using a 96-well microplate,100 pL of the enzyme solution was mixed with 150 |aL of the FALGPA solution. The kinetic reaction was monitored for 35 minutes, and kinetic rates (for enzymatic activity) were determined by recording the absorbance change at 345 nm. Rates were reported as Vmax (milli- OD per minute). The results are shown as percent enzymatic activity and are given by the following formula:Vmax tested article% Activity = — - ■, — ■, — — X100Vmax enzyme control solution

[0147] The surfactants that were tested were: Poloxamer-124, Poloxamer-188, Cetylpyridium Chloride (CTC), Cetylethyldimethyl Bromide (CEDB), Ariasilk CDM, Ammonyx C, and Sodium Dodecyl Sulfate (SDS). Collagenase activity was compared to a control condition in which the solution did not contain one of the surfactants. As shown in FIG. 4, collagenase activity was maintained or improved in the presence of the exemplary poloxamers (Poloxamer-124 or Poloxamer-188), suggesting that poloxamers in general are compatible with collagenase activity. In contrast, collagenase activity was eliminated or drastically reduced in the presence of each of the other surfactants, showing that poloxamers are somewhat unique among surfactants in their ability to maintain or increase collagenase activity.

[0148] Taken together, the results demonstrate that compositions such as solutions and wound cleansers that a poloxamer can maintain or improve collagenase activity. The results support the utility of poloxamers in compositions for wound treatments, such as enzymatic wound debridement.IV. Example 4 - Collagenase activity is compatible with up to 500 mM metal salts

[0149] The collagenolytic activity (i.e., collagenase activity) of C. collagenase in the presence poloxamer cleanser formulations having up to 500 mM metal salts was also measured using N-(3[2-Furyl]acryloyl)-Leu-Gly-Pro-Ala (FALGPA) substrate. The concentrations of enzyme, substrate, and salts were the following: C. collagenase 0.1 mg / ml, FALGPA substrate 1 mg / ml; tricine buffer (positive control) or solution containing up to 500 mM salt were used as solubilization media for enzyme and substrate. The enzyme activity was measured as decreased absorbance at 345 nm for the first 35 minutes at room temperature. The slope of the linear curve is used as the activity rate (Vmax). Using a 96-well microplate, 100 pL of the enzyme solution was mixed with 150 pL of the FALGPA solution. The kinetic reaction was monitored for 35 minutes, and kinetic rates (for enzymatic activity) were determined by recording the absorbance change at 345 nm. Rates were reported as Vmax (milli-OD perminute). The results are shown as percent enzymatic activity and are given by the following formula:Vmax tested article% Activity = - - — - - X100Vmax enzyme control solution

[0150] The metal chloride salts that were tested were: sodium chloride, potassium chloride, magnesium chloride, calcium chloride, barium chloride. Collagenase activity was compared for 100 mM and 500 mM of each metal chloride salt. As shown in FIG. 5A, collagenase activity was maintained or improved in the presence of 100 mM of all metal chloride salts and at 500 mM potassium chloride and calcium chloride.

[0151] Also tested were various sodium salts, including: sodium bicarbonate, sodium ascorbate, and sodium acetate. Collagenase activity was compared for 100 mM to 500 mM of each sodium salt. As shown in FIG. 5B, collagenase activity was maintained or improved in the presence of 100 mM to 500 mM sodium salts.

[0152] Taken together, the results demonstrate that compositions such as solutions and wound cleansers that include up to 500 mM metal salts can maintain or improve collagenase activity. The results support the utility of metal salts in compositions for wound treatments, such as enzymatic wound debridement.V. Example 5 - Sequential application of poloxamer and collagenase promotes collagenase activity in a wound model

[0153] Digestion of collagen (i.e., collagenase activity) was assessed in an Artificial Wound Eschar (AWE) model under conditions in which collagenase and a poloxamer were applied to the artificial wound sequentially in separate compositions, or in a single pre-mixed composition. The Artificial Wound Eschar model assay was performed as described, for example, in “ Study on the debridement efficacy of formulated enzymatic wound debriding agents by in vitro assessment using artificial wound eschar and by an in vivo pig model”, Shi et. al.. Wound Repair Regen, 2009, 17(6) : 853 , and in U.S. Patent No. 9,694,100, both of which are herein incorporated by reference. Briefly, bovine fibrinogen was polymerized using bovine thrombin (CalBiochem, La Jolla, CA) to form fibrin. Before the preparation of the fibrin, fibrinogen was labeled with 7-amino-4-methyl-coumarin. Labeling was accomplished by mixing fibrinogen in a coumarin solution (0.02 mg / mL in Tris buffer) with a final fibrinogen concentration of 10 mg / mL (in Tris buffer). The mixture was incubated at room temperature, with rotary shaking, for 1 hour. The coumarin-labeled fibrin was derived by adding a thrombin solution (2.5 U / mL) to the fibrinogen-coumarin solution and allowed to clot for 1 hour. Thefibrin clot was gently removed and repeatedly washed with water to remove any residual dye. The washed fibrin-coumarin was allowed to stand, overnight, in distilled water and methanol to remove excess dye. The fibrin-coumarin was subsequently dried and ground into a fine powder using a mortar and pestle. Because the conjugation is performed in a fibrinogen solution, the dried fibrin-coumarin is uniform in dye concentration. The resulting structure is referred to as “Artificial Wound Eschar (AWE).”

[0154] Collagenase was provided in SANTYL® Ointment (as described herein). The artificial wound was treated according to three conditions: 1) application of SANTYL alone, 2) application of a mixture of SANTYL and a composition comprising Poloxamer-407, and 3) sequential application of SANTYL followed by application of the composition comprising Poloxamer-407. Cumulative digestion of collagen was assessed and quantified at various time points up to 24 hours following application. As shown in FIG. 6, application of a mixture of SANTYL and a composition comprising Poloxamer-407 resulted in essentially identical levels of collagen digestion at all time points. Surprisingly, the sequential application of SANTYL and the composition comprising Poloxamer-407 resulted in increased collagen digestion (i.e., collagenase activity) in comparison to the application of the same two compositions applied simultaneously, including at timepoints from 12 to 24 hours.

[0155] The results suggest that the application of a composition including a collagenase and a separate composition including a poloxamer to a wound can improve collagenase activity in comparison to the application of a single composition that includes both the collagenase and the poloxamer. The results further support the therapeutic utility of applying the separate compositions to wounds, for example in methods of enzymatic wound debridement as described herein.* * *

[0156] All of the methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this disclosure have been described in terms of preferred aspects, it will be apparent to those of skill in the art that variations may be applied to the methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the disclosure. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the disclosure as defined by the appended claims.

Claims

CLAIMS1. A method for debriding a wound, the method comprising:(a) applying a first composition including a poloxamer to the wound; and(b) applying a second composition including a collagenase to the wound.

2. The method of claim 1, wherein the first composition comprises:(a) 1-15 wt. % poloxamer; and(b) 1-40 wt. % water soluble solvent.

3. The method of claim 2, wherein the poloxamer is poloxamer 407 and the water soluble solvent is propylene glycol.

4. The method of claim 2, wherein the first composition further comprises 0.01-5 wt. % of one or more preservatives.

5. The method claim 4, wherein the one or more preservatives comprise one or more parabens, benzyl alcohol, phenoxyethanol, or a combination thereof.

6. The method of 2, wherein the first composition further comprises one or more buffering or osmolarity agents, and wherein the first composition has a pH of 6-8.

7. The method of claim 6, wherein the buffering agents comprise sodium acetate or Tris buffer and the osmolarity agents comprise sodium chloride.

8. The method of claim 1, wherein the first composition comprises:(a) 2.5-7.5 wt. % poloxamer;(b) 2-6 wt. % propylene glycol;(c) 0.01-1 wt. % methyl paraben;(d) 0.01-1 wt. % propyl paraben;(e) 0.01-2 wt. % benzyl alcohol;(f) sodium acetate; and(g) sodium chloride.

9. The method of claim 1, wherein collagenase activity is maintained or increased compared to a method comprising applying a composition that includes a poloxamer and a collagenase.

10. The method of claim 1, wherein collagenase activity is maintained or increased compared to a method comprising applying a collagenase but not a poloxamer to the wound.

11. The method of claim 1, wherein the first composition is applied to the wound before the second composition.

12. The method of claim 1, wherein the first composition is applied to the wound after the second composition.

13. The method of claim 1, wherein the collagenase is comprised in a protease mixture.

14. The method of claim 13, wherein the protease mixture includes collagenase ColG, collagenase ColH, and a non-specific neutral protease.

15. The method of claim 13, wherein the protease mixture is the active pharmaceutical ingredient (API) in SANTYL® Ointment.

16. The method of claim 1, wherein the second composition is comprised in a wound dressing or a spray.

17. The method of claim 16, wherein the wound dressing is a patch, wrap, or bandage.

18. The method of claim 1, wherein the second composition comprises collagenase ColG, collagenase ColH, a non-specific neutral protease, and petrolatum.

19. The method of claim 18, wherein the second composition further includes an antimicrobial agent.

20. The method of claim 1, wherein the first composition is comprised in a wound dressing or a cleanser.

21. The method of claim 20, wherein the cleanser is aqueous.

22. The method of claim 20, wherein the wound dressing is a patch, wrap, or bandage.

23. A method for preparing a wound for debridement with a composition comprising collagenase, the method comprising, prior to debriding the wound with the composition comprising collagenase, applying a composition comprising a poloxamer to the wound.

24. The method of claim 23, wherein collagenase activity is maintained or increased compared to a method comprising applying a composition that includes a poloxamer and a collagenase.

25. The method of claim 23, wherein collagenase activity is maintained or increased compared to a method comprising applying a collagenase but not a poloxamer to the wound.

26. A kit comprising a first composition including a poloxamer and a second composition including a collagenase.

27. The kit of claim 26, wherein the first composition and the second composition are comprised in the same container.

28. The kit of claim 26, wherein the first composition and the second composition are comprised in different containers.

29. The kit of claim 26, wherein the first composition is comprised in a cleanser, and wherein the second composition is comprised in a wound dressing or spray.

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