Methods for measurement of endotoxin contaminants in enzymatic compositions

Abstract

Disclosed herein are methods for measuring endotoxin contaminants in a composition comprising an enzymatic debriding agent that may be used for enzymatic wound debridement. In some aspects, the methods comprise heating the composition, contacting the composition with a broad spectrum metalloprotease inhibitor, and measuring a concentration of endotoxin contaminants in the composition. Also provided herein are compositions comprising an enzymatic debriding agent for which a concentration of endotoxin contaminants is determined according to the disclosed methods and methods of manufacturing formulations including such compositions.

Description

METHODS FOR MEASUREMENT OF ENDOTOXIN CONTAMINANTS INENZYMATIC COMPOSITIONSCROSS-REFERENCE WITH RELATED APPLICATIONS

[0001] This application claims priority to and the benefit of U.S. Provisional Application No. 63 / 557,739, filed February 26, 2024, which is incorporated herein by reference.FIELD

[0002] The present disclosure relates generally to compositions comprising enzymatic debriding agents and methods for measurement of endotoxin contaminants in such compositions.BACKGROUND

[0003] The presence of eschar and other necrotic tissue in a wound can impede the healing process, causing the wound to become a slow-healing or “chronic” wound. A wound is a disruption of the structure and function of tissue, such as cuts, tears, burns, 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. Diabetic foot ulcers, venous leg ulcers, arterial leg ulcers, decubitus ulcers, stasis ulcers, dermal ulcers, burns, and pressure ulcers are non-limiting examples of wounds.

[0004] Promoting rapid and effective wound healing is an essential component of medical care for individuals with wounds. Treatment for promoting wound healing can include debridement, 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. In some aspects, following debridement, a wound can bemore effectively treated with wound healing agents to promote wound repair, during which cells begin to grow and rebuild missing and damaged tissues, small blood vessels develop to deliver a blood supply to the wound, and skin cells migrate to form scabs.

[0005] 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.

[0006] Enzymatic debridement employs enzymes that degrade proteinaceous materials, such as collagen matrix, in necrotic tissue. Enzymatic wound debridement agents can be used to digest eschar and other necrotic tissue, thereby facilitating the healing process of wounds. Topical compositions containing proteolytic enzymes such as thermolysin, trypsin, papain, bromelain, subtilisin, sutilains, and collagenase have been used for enzymatic wound debridement. Many of these proteolytic enzymes are produced by bacteria. For example, thermolysin is a thermostable neutral metalloproteinase enzyme produced by the Grampositive bacteria Bacillus thermoproteolyticus . In addition to producing desired proteolytic enzymes, however, bacteria also produce endotoxins.

[0007] Endotoxins are small bacterially-derived hydrophobic lipopolysaccharide (LPS) molecules that can easily contaminate proteolytic enzyme compositions. Bacteria shed endotoxin in large amounts upon cell death and when they are actively growing and dividing. Endotoxins have a high heat stability, making it difficult to eliminate them under regular sterilizing conditions. They are also amphipathic molecules that carry a net negative charge in solution. Because of their hydrophobicity, they are likely to have strong affinities for other hydrophobic materials. In vivo, many proteins in plasma, saliva, lungs and in granules of neutrophils bind endotoxins, and endotoxins are potent activators of the innate immune system, even at very low doses. The presence of endotoxin in products for administration to humans can result in pyrogenic responses ranging from fever and chills to irreversible and fatal septic shock.

[0008] Methods of enzymatic debridement require that the measured levels of endotoxin contaminants of enzyme compositions used in such methods do not exceed safety thresholds for human use. Existing methods for measuring endotoxin contaminants in enzyme compositions include, for example, gel-clot, limulus amebocyte lysate, turbidimetric, or photometric assays. However, these methods may not efficiently detect endotoxin contaminants or may not be sensitive enough to detect low levels of endotoxin contaminants,where proteases, enzymes that degrade proteins, can interfere with each of these methods. Additionally, some heat-stable enzymes (e.g., metallopeptidases such as therm oly sin and collagenases, or serine proteases like alcalase) pose a challenge to accurate endotoxin analyses as they are inherently difficult to inactivate, and temperatures required for heat inactivation can result in loss of endotoxin. The proteolytic activity of these enzymes has also been shown to interfere with endotoxin testing methods. Avoiding such interference can require dilution of the enzymatic compositions, which can result in an increase in the amount of endotoxin contaminants that need to be present in the compositions for detection by the assays. Accordingly, identifying improvements to such methods to provide more accurate endotoxin measurements has therefore been challenging.

[0009] Given the importance of safely and effectively debriding wounds, pursuit of superior methods for accurately measuring endotoxin contaminants in enzymatic wound debridement compositions are warranted. Such compositions and methods would have numerous applications, such as clinical and / or commercial uses, by providing for more efficient and sensitive endotoxin values using existing measurement methods to help ensure the safety of enzymatic wound debridement compositions.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 and measuring endotoxin contaminants in enzymatic wound debridement compositions. In some aspects, utilizing heat inactivation in combination with metalloproteinase inhibition can resolve enzymatic interference to increase the efficiency and sensitivity of assays to measure endotoxin contaminants. In some aspects, the methods and compositions provided herein provide several advantages. In some aspects, the inventors discovered that utilizing heat inactivation in combination with metalloproteinase inhibition permits accurate measurement of endotoxin contaminants 1) without enzymatic interference, and 2) without significant dilution of the enzymatic compositions, which can avoid an increase in the limit of detection for endotoxin contaminants in the enzymatic compositions. In this way, the method can increase provide for detection of reduced amounts of endotoxin contaminants by increasing the amount of enzyme that can be more accurately be measured. Accordingly, in some aspects, the methods provide a surprising, yet simple and economical, solution for improving detection of endotoxin contaminants in enzymatic wound debridement compositions.

[0011] Disclosed herein, in some aspects, is a method for measuring endotoxin contaminants in a composition comprising an enzymatic debriding agent. In some aspects, the method comprises heating the composition, contacting the composition with a broad spectrum metalloprotease inhibitor, and measuring a concentration of endotoxin contaminants in the composition. In specific aspects, the enzymatic debriding agent is thermolysin.

[0012] Also disclosed herein, in some aspects, is a method for manufacturing a formulation including a composition comprising an enzymatic debriding agent and endotoxin contaminants. In specific aspects, the enzymatic debriding agent is thermolysin. In some aspects, the method comprises obtaining the formulation including the composition, and measuring the endotoxin contaminants in the composition. In some aspects, endotoxin contaminants in the composition are measured by heating the composition, mixing the composition with a broad spectrum metalloprotease inhibitor, and measuring a concentration of endotoxin contaminants in the composition. In some aspects, the formulation is a hydrogel. In some aspects, the composition is solubilized or suspended in the formulation. In some aspects, the endotoxin contaminants are measured in the composition prior to obtaining the formulation including the composition. In some aspects, the endotoxin contaminants are measured in the composition after obtaining the formulation including the composition.

[0013] Also disclosed herein is a composition comprising an enzymatic debriding agent, and a concentration of endotoxin contaminants in the composition is determined by a method comprising heating the composition, contacting the composition with a broad spectrum metalloprotease inhibitor, and measuring a concentration of endotoxin contaminants in the composition. In specific aspects, the enzymatic debriding agent is thermolysin. In some aspects, the composition is solubilized or suspended in a hydrogel.

[0014] The composition can be heated to 90 °C to 100 °C. In specific aspects, the composition is heated to about 95 °C. The composition can be heated for 5 minutes to 15 minutes. In some aspects, the composition is heated for about 10 minutes.

[0015] In some aspects, the method further comprises cooling the composition after heating the composition. The composition can be cooled to 20 °C to 25 °C. In some aspects, the composition is not further diluted.

[0016] In some aspects, the composition is contacted with an effective amount of the broad spectrum metalloprotease inhibitor to deactivate the enzymatic debriding agent. In some aspects, the effective amount comprises greater than 10 nanomolar broad spectrum metalloprotease inhibitor for up to 3 milligrams of enzymatic debriding agent per milliliter of enzymatic debriding agent solution or up to 15,000 Protease Units (PU) per milliliter ofenzymatic debriding agent solution. In some aspects, the broad spectrum metalloprotease inhibitor is a hydroxamate-based inhibitor, a thiol-based inhibitor, or an endogenous inhibitor. In some aspects, the broad spectrum metalloprotease inhibitor is ilomastat, batimastat, marimastat, MMI-270, cipemastat, MMI-166, ABT-770, prinomastat, rebimastat, tanomastat, a2-macroglobulin, GM 1489, or a combination thereof.

[0017] In some aspects, the concentration of endotoxin contaminants is measured by a gelclot, limulus amebocyte lysate, turbidimetric or photometric assay. In some aspects, the concentration of endotoxin contaminants is greater than 0.05 endotoxin units per mg of the composition.

[0018] Also disclosed herein are the following Aspects 1-22. Aspect 1 is a method for measuring endotoxin contaminants in a composition comprising an enzymatic debriding agent, the method comprising: heating the composition; contacting the composition with a broad spectrum metalloprotease inhibitor; and measuring a concentration of endotoxin contaminants in the composition. Aspect 2 is the method of Aspect 1, wherein the composition is heated to 90 °C to 100 °C for 5 minutes to 15 minutes. Aspect 3 is the method of Aspect 1 or 2, wherein the composition is heated to about 95 °C. Aspect 4 is the method of any one of Aspects 1-3, wherein the composition is heated for about 10 minutes. Aspect 5 is the method of any one of Aspects 1-4, further comprising cooling the composition after heating the composition. Aspect 6 is the method of Aspect 5, wherein the composition is cooled to 20 °C to 25 °C. Aspect 7 is the method of any one of any one of Aspects 1-6, wherein the composition is contacted with an effective amount of the broad spectrum metalloprotease inhibitor to deactivate the enzymatic debriding agent. Aspect 8 is the method of any one of Aspects 1-7, wherein the effective amount comprises greater than 10 nanomolar for up to 3 milligrams of enzymatic debriding agent per milliliter or up to 15,000 Protease Units (PU) per milliliter. Aspect 9 is the method of any one of Aspects 1-8, wherein the broad spectrum metalloprotease inhibitor is a hydroxamate-based inhibitor, a thiol -based inhibitor, or an endogenous inhibitor. Aspect 10 is the method of any one of Aspects 1-9, wherein the broad spectrum metalloprotease inhibitor is ilomastat, batimastat, marimastat, MMI-270, cipemastat, MMI-166, ABT-770, prinomastat, rebimastat, tanomastat, a2 -macroglobulin, GM 1489, or a combination thereof. Aspect 11 is the method of any one of Aspects 1-10, wherein the composition is not further diluted. Aspect 12 is the method of any one of Aspects 1-11, wherein the concentration of endotoxin contaminants is measured by a gel-clot, limulus amebocyte lysate, turbidimetric or photometric assay. Aspect 13 is the method of any one of Aspects 1-12, wherein the concentration of endotoxin contaminants is greater than 0.05 endotoxin units per mg of the composition.

[0019] Aspect 14 is a method for manufacturing a formulation including a composition comprising an enzymatic debriding agent and endotoxin contaminants, the method comprising: obtaining the formulation including the composition; and measuring the endotoxin contaminants in the composition by: heating the composition; mixing the composition with a broad spectrum metalloprotease inhibitor; and measuring a concentration of endotoxin contaminants in the composition. Aspect 15 is the method of Aspect 14, wherein the formulation is a hydrogel. Aspect 16 is the method of Aspect 14 or 15, wherein the composition is solubilized or suspended in the formulation. Aspect 17 is the method of any one of Aspects 14-16, wherein the endotoxin contaminants are measured in the composition prior to obtaining the formulation including the composition. Aspect 18 is the method of any one of Aspects 14- 17, wherein the endotoxin contaminants are measured in the composition after obtaining the formulation including the composition. Aspect 19 is the method of any one of Aspects 1-18, wherein the enzymatic debriding agent is thermolysin.

[0020] Aspect 20 is a composition comprising an enzymatic debriding agent, wherein a concentration of endotoxin contaminants in the composition is determined by a method comprising: heating the composition; contacting the composition with a broad spectrum metalloprotease inhibitor; and measuring a concentration of endotoxin contaminants in the composition. Aspect 21 is the composition of Aspect 20, wherein the composition is solubilized or suspended in a hydrogel. Aspect 22 is the composition of 20 or 21, wherein the enzymatic debriding agent is thermolysin.

[0021] 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 andshould 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.

[0022] 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.

[0023] 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

[0024] 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.

[0025] FIG. 1 shows an example of the effect of sample preparation on endotoxin amounts from various thermolysin lots. In the first experimental preparation, samples were heated at 95 °C for 10 minutes. In the second experimental preparation, samples were heated at 95 °C for 10 minutes, then combined with a metalloprotease inhibitor, GM6001. For each lot, thermolysin enzymatic interference was not sufficiently inactivated, and the endotoxin test results were falsely higher and had a great degree of variation under the first sample preparation (Heat) compared to the second sample preparation (Heat + Protease Inhibitor).DETAILED DESCRIPTION

[0026] The present disclosure relates to methods and compositions (e.g., for measurement of endotoxin contaminants), methods of processing and / or making the compositions disclosed herein, and uses of compositions for the treatment of wounds (e.g., dermal wounds). Themethods and compositions provided herein can provide more efficient and sensitive measurement of endotoxin contaminants compared to existing methods for measuring endotoxin contaminants in enzymatic wound debridement compositions. For example, methods for measuring endotoxin contaminants that utilize heat inactivation in combination with metalloproteinase inhibition can better avoid enzymatic interference without significant dilution of the enzymatic compositions, which reduce or prevent undesirable increases in the limit of detection for endotoxin contaminants in enzymatic wound debridement compositions. Accordingly, the methods and compositions provided herein can provide safer and more effective enzymatic wound debridement compositions that can provide greater beneficial effects for wound healing as compared to commercially available products (e.g., products for wound debridement). These and other non-limiting aspects of the present invention are described in the following sections.I. Exemplary Definitions

[0027] 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.

[0028] 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.

[0029] 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 within0.5% of an associated disclosed value. The terms may be removed from the associated disclosed value and the exact value may be used instead.

[0030] The term “substantially” and its variations are defined as being largely but not necessarily wholly what is specified as understood by one of ordinary skill in the art, and in one non-limiting embodiment substantially refers to ranges within 10%, within 5%, within 1%, or within 0.5%.

[0031] 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 of values 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.

[0032] 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, efc.), 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.”

[0033] The terms “wt.%”, “w / w”, “vol.%”, “v / v”, “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.

[0034] 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) maymean “one,” but it is also consistent with the meaning of “one or more,” “at least one,” and“one or more than one.”

[0035] 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.

[0036] 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, / .< ., 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.

[0037] 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.

[0038] 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 one non-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.

[0039] The terms “inhibiting” or “reducing” or any variation of these terms includes any measurable decrease or complete inhibition to achieve a desired result, such as a measurable decrease in or a complete inhibition of the activity of an enzymatic wound debridement agent (e.g., thermolysin). The terms “promote” or “increase” or any variation of these terms includes any measurable increase to achieve a desired result.

[0040] The term “effective,” as that term is used in the specification and / or claims, means adequate to accomplish a desired, expected, or intended result. For example, an “effective amount” of an inhibitor may be an amount sufficient to achieve a measurable decrease in or complete inhibition of the activity of an enzymatic wound debridement agent (e.g., thermolysin).

[0041] 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.

[0042] The term “body” as used herein means the body of a subject.

[0043] 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 burns, wounds, ulcerations, and lacerations, ablations (including laser, freezing, cryo-surgery, heat and electrical ablations), and surgical incisions.

[0044] 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 bums, 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 bum, second-degree burn (partial thickness burns), third degree burn (full thickness burns), infection of bum wound, infection of excised and unexcised burn wound, loss of epithelium from a previously grafted or healed burn, or bum wound impetigo.

[0045] 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.II. Methods for Measuring Endotoxin Contaminants

[0046] Disclosed herein are methods of measuring endotoxin contaminants in a composition comprising an enzymatic wound debriding agent (e.g., thermolysin, collagenase(s), and the like). Also disclosed are compositions comprising an enzymatic wound debriding agent (e.g., thermolysin, collagenase(s), and the like) for which a concentration of endotoxin contaminants in the composition is determined according to the described methods. Also disclosed are methods for manufacturing a formulation including a composition comprising an enzymatic wound debriding agent (e.g., thermolysin, collagenase(s), and the like) and endotoxin contaminants, where the formulation including the composition is obtained, and a concentration of endotoxin contaminants in the composition is determined according to the described methods. In some aspects, the endotoxin contaminants are measured in the composition prior to obtaining the formulation including the composition. In some aspects, the endotoxin contaminants are measured in the composition after obtaining the formulation including the composition.

[0047] In some aspects, the methods of measuring endotoxin contaminants comprise improvements to existing methods of measuring endotoxin contaminants to increase the efficiency or sensitivity of the existing methods. For example, methods for measuring endotoxin contaminants disclosed herein can better avoid enzymatic interference without significant dilution of the enzymatic compositions, which can reduce or prevent undesirable increases in the limit of detection for endotoxin contaminants in enzymatic wound debridement compositions. Accurate measurement of endotoxin contaminants in enzymatic wound debridement compositions can provide safer and more effective compositions for wound healing as compared to commercially available products (e.g., products for wound debridement).

[0048] In some aspects, the enzymatic wound debriding agent (e.g., thermolysin, collagenase(s), etc.} is supplied as a stock solution. The stock solution may or may not be a dilution of a more concentrated stock solution. In some aspects, the stock solution of the enzymatic wound debriding agent (e.g., thermolysin, collagenase(s), etc. is diluted into a composition comprising the enzymatic wound debriding agent (e.g., thermolysin, collagenase(s), etc.}. In some aspects, the method does not require dilution of the composition comprising an enzymatic wound debriding agent (e.g., thermolysin, collagenase(s), etc.}. Inother words, in some aspects, the composition comprising the enzymatic wound debriding agent (e.g., thermolysin, collagenase(s), etc.) is not diluted to avoid further dilution of the enzymatic wound debriding agent (e.g., thermolysin, collagenase(s), etc.).

[0049] In some aspects, disclosed is a method of measuring endotoxin contaminants in a composition comprising an enzymatic wound debriding agent (e.g., thermolysin, collagenase(s), etc.). In some aspects, the method comprises heating the composition comprising an enzymatic wound debriding agent (e.g., thermolysin, collagenase(s), etc.), contacting the composition comprising an enzymatic wound debriding agent (e.g., thermolysin, collagenase(s), etc.) with a broad spectrum metalloprotease inhibitor, and measuring a concentration of endotoxin contaminants in the composition comprising an enzymatic wound debriding agent (e.g., thermolysin, collagenase(s), etc.).A. Heating

[0050] In some aspects, the composition is heated to a temperature sufficient to inactivate the enzymatic wound debriding agent (e.g., thermolysin, collagenase(s), etc.). For example, the composition can be heated to 90 °C to 100 °C, e.g., at least, at most, exactly, or between any two of 90 °C, 91 °C, 92 °C, 93 °C, 94 °C, 95 °C, 96 °C, 97 °C, 98 °C, 99 °C, or 100 °C. In some aspects, the composition is heated to 90 °C to 100 °C, 92 °C to 98 °C, or 94 °C to 96 °C. In specific aspects, the composition is heated to about 95 °C.

[0051] The composition can be heated for 5 minutes to 15 minutes, e.g., at least, at most, exactly, or between any two of 5 min, 6 min, 7 min, 8 min, 9 min, 10 min, 11 min, 12 min, 13 min, 14 min, or 15 min. In some aspects, the composition is heated for 5 minutes to 15 minutes, 7 minutes to 13 minutes, or 9 minutes to 11 minutes. In specific aspects, the composition is heated for about 10 minutes.

[0052] In some aspects, the composition is cooled after it is heated. For example, the composition can be cooled to room temperature after heating the composition to 90 °C to 100 °C. In some aspects, the composition is cooled to 20 °C to 25 °C, e.g., at least, at most, exactly, or between any two of 20 °C, 21 °C, 22 °C, 23 °C, 24 °C, or 25 °C, after heating the composition to 90 °C to 100 °C.B. Metalloprotease Inhibitors

[0053] In some aspects, the method further comprises, after heating the composition comprising an enzymatic wound debriding agent (e.g., thermolysin, collagenase(s), etc.), contacting the composition with a broad spectrum metalloprotease inhibitor. The composition can be contacted with an effective amount of the broad spectrum metalloprotease inhibitor toinhibit or deactivate the enzymatic wound debriding agent (e.g., thermolysin, collagenase(s), etc. ). In some aspects, the effective amount is greater than 10 nM e.g. , at least, at most, exactly, or between any two of 10 nM, 20 nM, 30 nM, 40 nM, 50 nM, 60 nM, 70 nM, 80 nM, 90 nM, 100 nM, 200 nM, 300 nM, 400 nM, 500 nM, 600 nM, 700 nM, 800 nM, 900 nM, or 1000 nM) of broad spectrum metalloprotease inhibitor for up to 3 mg of enzyme per mL of enzyme solution (e.g., at least, at most, exactly or between any two of 10 ng or pg, 20 ng or pg, 30 ng or pg, 40 ng or pg, 50 ng or pg, 60 ng or pg, 70 ng or pg, 80 ng or pg, 90 ng or pg, 100 ng or pg, 200 ng or pg, 300 ng or pg, 400 ng or pg, 500 ng or pg, 600 ng or pg, 700 ng or pg, 800 ng or pg, 900 ng or pg, 1000 ng or pg, 1250 ng or pg, 1500 ng or pg, 1750 ng or pg, 2000 ng or pg, 2250 ng or pg, 2500 ng or pg, 2750 ng or pg, or 3000 ng or pg). In some aspects, the effective amount is up to 15,000 Protease Units (PU) per mL of enzyme solution (e.g., at least, at most, exactly, or between any two of 1 PU / mL, 500 PU / mL, 1000 PU / mL, 1500 PU / mL, 2000 PU / mL, 2500 PU / mL, 3000 PU / mL, 3500 PU / mL, 4000 PU / mL, 4500 PU / mL, 5000 PU / mL, 5500 PU / mL, 6000 PU / mL, 6500 PU / mL, 7000 PU / mL, 7500 PU / mL, 8000 PU / mL, 8500 PU / mL, 9000 PU / mL, 9500 PU / mL, 10000 PU / mL, 10500 PU / mL, 11000 PU / mL, 11500 PU / mL, 12000 PU / mL, 12500 PU / mL, 13000 PU / mL, 13500 PU / mL, 14000 PU / mL, 14500 PU / mL, 15000 PU / mL,), where one PU is defined as the quantity of enzyme in grams that liberates the equivalent of 1.5 pg per mL of L-tyrosine per minute from casein under the conditions of the assay.

[0054] Inhibitors contemplated to be useful in the methods disclosed herein are those that comprise a functional group (e.g., hydroxamates, carboxylates, thiols and phosphorous-based) that block protein cleavage by binding or chelating catalytic Ca2+or Zn2+or that dislodge the enzymes from their receptors. Classes of metalloprotease inhibitors contemplated for use according to the methods disclosed herein include but are not limited to: EDTA, 1,10- phenanthroline, and other chelating compounds that lower the concentration of metal to the point where the metal is removed from the enzyme active site; classical lock and key inhibitors that bind tightly by approximating the transition state of the hydrolysis of the peptide, preventing it from acting on other substrates; and protein inhibitors.

[0055] Non-limiting examples of these metalloprotease inhibitors are hydroxymates, thiols, carbamoylphosphonates, hydroxyureas, hydrazines, P-lactams, squaric acids, nitrogenous ligands, endogenous inhibitors, or a combination thereof. In some aspects, the metalloprotease inhibitor is ilomastat, batimastat, marimastat, MMI-270, cipemastat, MML 166, ABT-770, prinomastat, rebimastat, tanomastat, a2-macroglobulin, GM 1489, or a combination thereof. In specific aspects, the metalloprotease inhibitor is ilomastat, batimastat,marimastat, or a combination thereof. In specific aspects, the metalloprotease inhibitor is ilomastat, also known as GM6001 MMP inhibitor or N-[(2R)-2- (hydroxamidocarbonylmethyl)-4-methylpentanoyl]-L-tryptophan methylamide. In specific aspects, the metalloprotease inhibitor is batimastat, or (4-N-Hydroxyamino)-2 R-isobutyl-3 S- (thienylthiomethyl)succinyl)-L-phenylalanine-N-methylamide. In specific aspects, the metalloprotease inhibitor is marimastat, or (2S,3R)-N4-[(lS)-2,2-Dimethyl-l- [(methylamino)carbonyl] propyl]-Nl,2-dihydroxy-3-(2-methylpropyl)butanediamide.C. Endotoxin Contaminant Measurement

[0056] In some aspects, the method further comprises, after contacting the composition with a broad spectrum metalloprotease inhibitor, measuring a concentration of endotoxin contaminants in the composition comprising the enzymatic wound debriding agent (e.g., thermolysin, collagenase(s), etc.). In some aspects, the measured concentration of endotoxin contaminants in the composition comprising the enzymatic wound debriding agent (e.g., thermolysin, collagenase(s), etc.) corresponds to the limit of detection of the assay used to measure the endotoxin contaminant concentration, i.e., the lowest concentration of endotoxin contaminants capable of being detected by the assay. In some aspects, the measured concentration of endotoxin contaminants in the composition comprising the enzymatic wound debriding agent (e.g., thermolysin, collagenase(s), etc.) is improved after heat inactivation and broad spectrum metalloprotease inhibition of the enzyme(s), i.e., the assay is more sensitive and accurate for measuring endotoxin contaminants) compared to existing methods for measuring endotoxin contaminants that do not including enzyme heating and inhibition steps. In some aspects, the measured concentration of endotoxin contaminants in the composition comprising the enzymatic wound debriding agent (e.g., thermolysin, collagenase(s), etc.) is greater than 0.05 endotoxin units per mg of the composition, i.e., the method is sufficiently sensitive and accurate to measure levels of endotoxin as low as 0.05 endotoxin units per mg of the composition.

[0057] The concentration of endotoxin contaminants in the composition comprising the enzymatic wound debriding agent (e.g., thermolysin, collagenase(s), etc.) may be measured using any assay for measuring endotoxin contaminants known in the art. Non-limiting examples of assays for measuring a concentration of endotoxin contaminants in a composition include the bacterial endotoxin test (BET) described in US Pharmacopoeia <85> to detect or quantify endotoxins from bacteria using amoebocyte lysate from horseshoe crab (Limulus polyphemus or Tachypleus tridentatus). Techniques to this test include, e.g., the gel-clottechnique, which is based on gel formation, and photometric techniques including, e.g., the turbidimetric technique, which is based on the development of turbidity after cleavage of endogenous substrate, and the chromogenic or photometric technique, which is based on development of color after cleavage of a synthetic peptide-chromagen complex.

[0058] To conduct a BET, all glassware and other heat-stable materials can be depyrogenated in a hot air oven using a validated process. A commonly used minimum time and temperature is 30 min at 250 °C. Plastic materials should be shown to be free of detectable endotoxin so as not to interfere in the test.

[0059] Test reagents and solutions can include amoebocyte lysate, water, standard endotoxin stock solution and dilutions thereof, and sample solution and dilutions thereof. Amoebocyte lystate is a lyophilized product obtained from the lysate of amoebocytes (white blood cells) from horseshoe crab (Limulus polyphemus or Tachypleus tridentatus). Alternative, non animal-based reagents may be produced using the recombinant DNA technology and may use one or more cloned zymogen proteases that comprise all of or part of the natural lysate reaction cascade. Two types of recombinant reagents are currently available: Recombinant Factor C (rFC) and Cascade Reagents (rCR). Recombinant Factor C (rFC) reagents contain the recombinant Factor C constituent of the clotting cascade. Recombinant Cascade Reagents (rCR) include Factor C, Factor B, and the proclotting enzyme in their formulations. The test methodology for using rFC and rCR are endpoint chromogenic or photometric techniques described in USP <85>, but may require validation and demonstration of comparability with the official method.

[0060] Standard endotoxin stock solution is prepared from a USP endotoxin reference standard that has been calibrated to the current World Health Organization International Standard for endotoxin. The USP endotoxin reference standard (RSE) has a defined potency of 10,000 USP Endotoxin Units (EU) per vial. One USP EU is equal to one International Unit (IU) of endotoxin. The entire contents of one vial of the RSE can be reconstituted with 5 mL of water for BET that is produced by procedures that show no reaction with the lysate employed, at the detection limit of the reagent. The reconstituted RSE can be mixed vigorously for 30 minutes and used for making appropriate serial dilutions.

[0061] Sample solutions can be prepared by dissolving or diluting drugs or extracting medical devices using water for BET. Some substances or preparations may be more appropriately dissolved, diluted, or extracted in other aqueous solutions. If necessary, the pH of the solution (or dilution thereof) can be adjusted so that the pH of the mixture of the water for BET and sample falls within the pH range specified by the amoebocyte lysate manufacturer,e.g., a pH of 6.0 to 8.0. The pH may be adjusted using an acid, base, or suitable buffer as recommended by the amoebocyte lysate manufacturer. Acids and bases may be prepared from concentrates or solids with water for BET in containers free of detectable endotoxin. Buffers may be validated to be free of detectable endotoxin and interfering factors.

[0062] The maximum valid dilution (MVD) can then be determined. The MVD is the maximum allowable dilution of a specimen at which the endotoxin limit can be determined. It applies to injections or to solutions for parenteral administration in the form constituted or diluted for administration, or, where applicable, to the amount of drug by weight if the volume of the dosage form for administration could be varied. The general equation to determine MVD is: MVD = (Endotoxin limit x Concentration of sample solution) / (X), where the concentration of sample solution and are as defined herein. Where the endotoxin limit concentration is specified in the individual monograph in terms of volume (in EU per mL), the limit should be divided by X, which is the labeled sensitivity (in EU per mL) of the amoebocyte lysate, to obtain the MVD factor. Where the endotoxin limit concentration is specified in the individual monograph in terms of weight or units of active drug (in EU per mg or in EU per unit), the limit should be multiplied by the concentration (in mg per mL or in units per mL) of the drug in the solution tested or of the drug constituted according to the label instructions, whichever is applicable, and the product of the multiplication should be divided by X to obtain the MVD factor. The MVD factor so obtained is the limit dilution factor for the preparation for the test to be valid.

[0063] The endotoxin limit for parenteral drugs, defined on the basis of dose, is equal to KIM, where K is the threshold human pyrogenic dose of endotoxin per kg of bodyweight, and M is equal to the maximum recommended human dose of product per kg of body weight in a single hour period. The endotoxin limit for parenteral drugs is specified in individual monographs in units such as EU / mL, EU / mg, or EU / Unit of biological activity.1. Gel-Clot Techniques

[0064] The gel-clot techniques (e.g., gel-clot limit test, gel-clot assay) detect or quantify endotoxins based on clotting of amoebocyte lysate in the presence of endotoxin. The concentration of endotoxin required to cause the lysate to clot under standard conditions is the labeled sensitivity of the amoebocyte lysate. To ensure both the precision and validity of the test, the labeled amoebocyte lysate sensitivity and interfering factors can be tested as described in USP <85>. Once the precision and validity of the test is verified, a gel-clot limit test may be conducted by preparing Solutions A, B, C, and D as shown in Table 1. Solution A and positiveproduct control Solution B are prepared using a dilution not greater than the MVD and as directed in the interfering factors test. Positive control Solutions B and C contain the standard endotoxin preparation at a concentration corresponding to twice the labeled amoebocyte lysate sensitivity. The negative control Solution D is water for BET.Table 1Preparation of Solutions for Gel-Clot Limit Test

[0065] The gel-clot limit test is performed on these solutions according to the following procedure. First, a volume of the amoebocyte lysate is mixed with an equal volume (such as 0.1 mL aliquots) of one of the standard solutions in each test tube. The reaction mixture is incubated for a constant period according to directions of the amoebocyte lysate manufacturer (usually at 37 ± 1 for 60 ± 2 minutes), avoiding vibration. To test the integrity of the gel, each tube is taken in turn directly from the incubator and inverted through about 180 degrees in one smooth motion. If a firm gel has formed that remains in place upon inversion, the result is recorded as positive. A result is negative if an intact gel is not formed. The endpoint is the last positive test in the series of decreasing concentrations of endotoxin. The mean value of the logarithms of the endpoint concentration is calculated, and then the anti -logarithm of the mean value is calculated using the following equation: Geometric Mean Endpoint Concentration = antilog (Xc7 / ), where Xc is the sum of the log endpoint concentrations of the dilution series used, and / is the number of replicate test tubes. The geometric mean endpoint concentration is the measured sensitivity of the amoebocyte lysate (in EU / mL). The test is not valid unless both replicates of positive control Solutions B and C are positive and those of negative control Solution D are negative. The preparation being tested complies with the test when a negative result is found for both tubes containing Solution A. The preparation being tested does not comply with the test when a positive result is found for both tubes containing Solution A. The test is repeated when a positive result is found for 1 tube containing Solution A and a negative result for the other one. The preparation being tested complies with the test when a negative result is found for both tubes containing Solution A in the repeat result. If the test is positivefor the preparation being tested at a dilution less than the MVD, the test may be repeated at a dilution not greater than the MVD.

[0066] Additionally, or alternatively, once the precision and validity of the test is verified, a gel-clot assay may be conducted by preparing Solutions A, B, C, and D as shown in Table 2. Solution A is a sample solution being tested at the dilution, not to exceed the MVD, and subsequent dilution of the sample solution should not exceed the MVD. Water for BET is used to make a dilution series of four tubes containing the sample solution being tested at concentrations of 1, ’A, 14, and 1 / 8 relative to the dilution with which the test for interfering factors was completed. Other dilutions may be used as appropriate. Solution B is Solution A containing standard endotoxin at a concentration of 2k (positive product control). Solution C is two series of 4 tubes of water for BET containing the standard endotoxin at a concentration of 2k, k, 0.5k, and 0.25k, respectively. Solution D is water for BET (negative control).Table 2Preparation of Solutions for Gel-Clot Assay

[0067] The gel-clot assay is performed on these solutions according to the procedure described for the gel-clot limit test. The test is not valid unless the following conditions are met: (1) both replicates of negative control Solution D are negative; (2) both replicates of positive product control Solution B are positive; and (3) the geometric mean endpoint concentration of Solution C is in the range of 0.5 to 2. To determine the endotoxin concentration of Solution A, the endpoint concentration for each replicate series of dilutions is calculated by multiplying each endpoint dilution factor by X. The endotoxin concentration in the sample is the geometric mean endpoint concentration of the replicates calculated by the Geometric Mean Endpoint Concentration formula. If the test is conducted with a diluted sample solution, the concentration of endotoxin in the original sample solution is calculated by multiplying by the dilution factor. If none of the dilutions of the sample solution is positive in a valid assay, report the endotoxin concentration is reported as less than X (if the diluted sample was tested, less than X times the lowest dilution factor of the sample). If all dilutions are positive, the endotoxin concentration is reported as equal to or greater than the greatest dilution factor multiplied by X (e.g., initial dilution factor times 8 times X in Table 2). The article meets the requirements of the test if the concentration of endotoxin is less than that specified in the individual monograph.2. Photometric Techniques

[0068] The turbidimetric method measures increases in turbidity. Depending on the test principle used, this technique is classified as either endpoint-turbidimetric or kinetic- turbidimetric. The endpoint-turbidimetric technique is based on the quantitative relationshipbetween the concentration of endotoxins and the turbidity (absorbance or transmission) of the reaction mixture at the end of an incubation period. The kinetic-turbidimetric technique is a method to measure either the onset time needed to reach a predetermined absorbance of the reaction mixture or the rate of turbidity development.

[0069] The chromogenic method measures the chromophore released from a suitable chromogenic peptide by the reaction of endotoxins with amoebocyte lysate. Depending on the test principle employed, this technique is classified as either endpoint-chromogenic or kinetic- chromogenic. The endpoint-chromogenic technique is based on the quantitative relationship between the concentration of endotoxins and the release of chromophore at the end of an incubation period. The kinetic-chromogenic technique is a method to measure either the onset time needed to reach a predetermined absorbance of the reaction mixture or the rate of color development.

[0070] All photometric tests are carried out at the incubation temperature recommended by the amoebocyte lysate manufacturer, which is usually 37 °C ± 1 °C. To assure the precision or validity of the turbidimetric and chromogenic techniques, preparatory tests such as those described in USP <85> may be conducted to verify that the criteria for the standard curve are valid and that the sample solution does not inhibit or enhance the reaction. Revalidation for the test method is required when conditions that are likely to influence the test result change.

[0071] The procedure for photometric techniques include preparing a standard curve. Using the Standard Endotoxin Solution, at least three endotoxin concentrations are prepared to generate the standard curve. Testing is performed using at least three replicates of each standard endotoxin concentration according to the manufacturer’s instructions for the amoebocyte lysate (with regard to volume ratios, incubation time, temperature, pH, etc. . If the desired range in the kinetic methods is greater than two logs, additional standards should be included to bracket each log increase within the range of the standard curve. The absolute value of the correlation coefficient must be greater than or equal to 0.980 for the range of endotoxin concentrations indicated by the manufacturer of the amoebocyte lysate.

[0072] Once a standard curve is generated, an endotoxin concentration at or near the middle of the endotoxin standard curve is selected, and Solutions A, B, C, and D are prepared as shown in Table 3. Solution A is the sample solution that may be diluted not to exceed MVD. Solution B is the preparation being tested at the same dilution as Solution A, containing added endotoxin at a concentration equal to or near the middle of the standard curve. Solution C is the standard endotoxin at the concentrations used in the validation of the method described in USP <85>. Solution D is water for BET (negative control).Table 3Preparation of Solutions for Photometric Assays

[0073] The photometric assay is performed on these solutions at least in duplicate following the instructions for the amoebocyte lysate used (with regard to volume of sample and amoebocyte lysate, volume ratio of sample to amoebocyte lysate, incubation time, etc.). The endotoxin concentration of each of the replicates of test Solution A is calculated using the standard curve generated by positive control Solution C series. The test is not valid unless the following conditions are met: (1) the results of control Solution C series comply with the requirements for validation defined by USP <85>; (2) the endotoxin recovery, calculated from the concentration found in Solution B after subtracting the endotoxin concentration found in Solution A is within 50 to 200%; and (3) the result of negative control series D does not exceed the limit of the blank value required in the description of the amoebocyte lysate used. In photometric assays, the preparation being tested complies with the test if the mean endotoxin concentration of the replicates of Solution A, after correction for dilution and concentration, is less than the endotoxin limit for the product.III. Enzymatic Wound Debriding Agents & Related Compositions

[0074] In some aspects, provided herein are compositions including an enzymatic wound debriding agent (e.g., thermolysin, collagenase(s), and the like), for which a concentration of endotoxin contaminants is determined according to the disclosed methods. In some aspects, the compositions including an enzymatic wound debriding agent (e.g., thermolysin, collagenase(s), and the like) are comprised in a formulation (e.g., a hydrogel, an ointment, etc.).

[0075] The compositions of the present disclosure (e.g., compositions including an enzymatic wound debriding agent (e.g., thermolysin, collagenase(s), and the like)), or formulations including such compositions, can include additional components such as carriers or diluents, which can be non-active components. In some aspects, the additional components do not substantially interfere with or inhibit the intended purpose of the composition or formulation. For example, the compositions or formulations can contain about 0.01% to about1% 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).

[0076] 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. 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 aspects, the composition is formulated as a hydrogel. In some specific embodiments, the composition is formulated as an ointment.

[0077] The compositions or formulations can be packaged in any package suitable for dispensing a wound debrider. The compositions or formulations 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. The compositions or formulations can be prepared by techniques and methods known by one of ordinary skill in the art using processing equipment known by one of ordinary skill in the art, e.g., blenders, mixers, mills, homogenizers, dispersers, dissolvers, etc.

[0078] The composition or formulation may be present in a vehicle or administered in any effective amount. In some aspects, the composition may be formulated as 100, 150, 200, 250, 300, 350, 400, or 500 units (or any derivable range therein) of enzyme per gram of composition. In some embodiments, the composition or formulation is applied every 2, 6, or 8 hours or every 1, 2, 4, 7, or 10 days (or any derivable range thereof).

[0079] In some aspects, the compositions or formulations described herein can be provided in the form of a wound dressing or spray. The term “wound dressing” used herein is taken to include any medically or pharmaceutically acceptable wound covering or support matrix. Examples of suitable wound dressing 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 anadhesive 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. Specific, nonlimiting examples of wound dressings are patches, wraps, and bandages.A. Enzymatic Wound Debriding Agents

[0080] Any enzyme useful for wound debridement is suitable for compositions, formulations, and methods of the present disclosure. In some aspects, the enzymatic wound debriding agents comprise a protease or a mixture of proteases. 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: metalloproteases, serine proteases, cysteine proteases, 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.

[0081] In some aspects, the enzymatic wound debriding agent can include a protease or mixture of proteases, which can be any suitable protease mixture, such as any provided herein. For example, in some aspects, the enzymatic wound debriding agent can include therm oly sin. In some aspects, the enzymatic wound debriding agent can include collagenase ColG, collagenase ColH, and a non-specific neutral protease. In some aspects, the enzymatic wound debriding agent is the active pharmaceutical ingredient (API) in SANTYL® Ointment. Exemplary proteases are described below, which may be used in connection with the methods and compositions provided herein.1. Metalloproteases

[0082] In some aspects, provided herein is a composition comprising one or more metalloproteases, and related methods, for wound debridement. Metalloproteases are among the proteases in which nucleophilic attack 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 important for the activity, and compounds that have potential to interact with the metal ion, chelating or oxidation, may affect the enzymatic activity. Non-limiting examples of metalloproteases in this family include thermolysin, collagenases, matrix metalloproteinases (MMPs), bacillolysin, dispase, vibriolysin, pseudolysin, stromelysin, and various bacterial derived neutral metalloproteases. i. Thermolysin

[0083] In some aspects, the metalloprotease is thermolysin. In some aspects, the enzymatic wound debriding agent comprises thermolysin. In some aspects, provided herein is a composition comprising thermolysin, and related methods, for wound debridement. In some aspects, any composition provided herein can comprise thermolysin. In some aspects, any composition comprising thermolysin herein may also comprise collagenase. Alternatively, thermolysin may be used instead of collagenase in any of the compositions provided herein. In some aspects, thermolysin may be substituted for collagenase in any of the compositions provided herein. Thus, in some aspects, provided herein is a method for debriding a wound by applying a composition including a thermolysin to the wound.

[0084] In some aspects, 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.

[0085] 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 aspects, 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, 0.010 0.015, 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.

[0086] The thermolysin can be from any suitable source, such as a bacterial species that produces thermolysin (e.g., Bacillus thermoproteolyticus), or can be obtained by synthetic methods. ii. Collagenase

[0087] In some aspects, the metalloprotease is collagenase. In some aspects, the enzymatic wound debriding agent comprises one or more collagenases. In some aspects, provided herein is a composition comprising one or more collagenases, and related methods, for wound debridement. The collagenase(s) can be substantially pure or may contain detectable levels of other proteases. For example, the collagenase(s) may be derived from the fermentation by Clostridium histolyticum and may include unseparated collagenase, in that collagenases and other non-specific proteases (e.g., a neutral protease) are present. In some aspects, any composition provided herein can comprise collagenase(s). In some aspects, any composition comprising collagenase(s) herein may also comprise thermolysin. Alternatively, collagenase(s) may be used instead of thermolysin in any of the compositions provided herein. In some aspects, collagenase(s) may be substituted for thermolysin in any of the compositions provided herein. Thus, in some aspects, provided herein is a method for debriding a wound by applying a composition including one or more collagenases to the wound.

[0088] 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.

[0089] 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, 0.010 0.015, 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.

[0090] In one embodiment, the collagenase is derived from Clostridium hislolylicum 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.2. Serine Proteases

[0091] 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 subtili sin-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.3. Cysteine Proteases

[0092] 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.4. Aspartic Proteases

[0093] Aspartic proteases 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 acttogether 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.5. Protease Mixtures

[0094] In some aspects, protease mixtures 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 protease 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 aspects, 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 aspects, 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.

[0095] A non-limiting example of a protease mixture that can be used in connection with the compositions and methods 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.B. Hydrogel Compositions

[0096] In some aspects, the enzymatic wound debriding agent(s) are stabilized in an aqueous environment by using a nonionic cellulose ether to create a hydrogel that contains the enzymatic wound debriding agent(s). The compositions can be formulated as hydrogels in which the continuous medium is an aqueous environment that has been gelled with a nonionic cellulose ether. Hydrogels are typically in a semi-solid dosage form. The enzymatic wound debriding agent(s) can be suspended or solubilized within the gel. The enzymatic wound debriding agent(s) can also be partially solubilized and partially suspended within the gel. Further, other additives can also be added to the hydrogels. In some aspects, a portion of the enzyme within the hydrogel can be solubilized (e.g., less than 50, 40, 30, 20, 10, 5, 1, or 0.5% w / w can be solubilized).

[0097] The combination of the cellulose ether / water / enzyme produces a surprising stable formulation that can be used to treat wounds. For instance, data confirms that such a hydrogel can be stored at room temperature (about 20-25 °C) for 24 months (e.g., at least, at most, exactly, or between any two of 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, or 24 months) and still retain at least 80% or 90% of its original enzymatic activity (i.e., the enzymatic activity does not decrease by more than 30%, e.g., at least, at most, exactly, or between any two of 1%, 5%, 10%, 15%, 20%, 25%, 30%,). This stability is surprising because proteases (e.g., thermolysin, collagenase(s), etc.) are expected to be highly unstable and quickly degraded in an aqueous environment such as a hydrogel.

[0098] Nonionic cellulose ethers are high-molecular-weight compounds that can be made by replacing the hydrogen atoms of hydroxyl groups in the glucose units of cellulose with alkyl or hydroxylalkyl groups. Non-limiting examples of nonionic alkyl cellulose ethers include methyl cellulose (MC), ethyl cellulose (EC), and ethyl methyl cellulose (EMC). Non-limiting examples of nonionic hydroxyalkyl cellulose ethers include hydroxyethyl cellulose (ELEC), hydroxylpropyl cellulose (HPC), hydroxymethyl cellulose (HMC), hydroxypropylmethyl cellulose (HPMC), ethylhydroxyethyl cellulose (EHEC), hydroxyethylmethy cellulose (HEMC), methylhydroxyethyl cellulose (MHEC), methylhydroxypropylcellulose (MHPC), and hydroxyethylcarboxymethyl cellulose (HECMC). Any one or more of the foregoing nonionic cellulose ethers may be included in or excluded from the compositions of the disclosure. There are a wide range of commercial sources for each of these cellulose ethers (e.g., Dow Chemical Company (USA), Ashland (USA), Samsung Fine Chemicals (USA)). Additional commercial sources of these nonionic cellulose ethers can be found in the International Cosmetic Ingredient Dictionary and Handbook, 12th Edition (2008), volumes 1- 3.

[0099] The amount of nonionic cellulose ether within the hydrogel can vary as needed to achieve a particular viscosity. In certain instances, the amount can range from 0.1 to 30% w / w, or 0.1 to 20% w / w, or 0.1 to 10% w / w, or 0.1 to 5% w / w of the nonionic cellulose ether or can include at least, at most, exactly, or between any two of 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, or 30% w / w. In one aspect, the amount of hydroxy ethylcellulose within the hydrogel can range from 2.5 to 4.5% w / w (e.g., at least, at most, exactly, or between any two of 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, or 4.5% w / w). In another aspect, the amount of hydroxypropylcellulose within the hydrogel can range from 0.01 to 10% w / w (e.g., at least, at most, exactly, or between any two of 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1,0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10% w / w). In still another aspect, the amount of hydroxypropylmethylcellulose within the hydrogel can range from 1.5 to 2.5% w / w (e.g., at least, at most, exactly, or between any two of 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, or 2.5% w / w).

[0100] In addition to enzyme, nonionic cellulose ether, and water, the hydrogels can include additional ingredients. For instance, water-soluble solvents such as propylene glycol or other diols can be used. Triols and polyols can also be used in the hydrogels. A non-limiting example of a triol is glycerin. The amount of such solvents can range from 1 to 20% w / w (e.g., at least, at most, exactly, or between any two of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20% w / w), although more or less can be added to achieve a desired result for the hydrogel. The hydrogel can also include metal salts. Non-limiting examples of metal salts include sodium chloride and calcium chloride. Metal salts such as NaCh or CaCh can be used as stabilization agents. The amounts of salt can range from 0.01 to 1% w / w (e.g., at least, at most, exactly, or between any two of 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1% w / w), although more or less can be added to achieve a desired result for the hydrogel.

[0101] Buffers having an appropriate pH range for enzymes can be used. The buffer, in certain aspects, can have a pH range of about 7.0 to 8.0 or 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9 or 8. In some aspects, the buffer can have a pH range of about 7.5. An example of such a buffer is Tris buffer (10 mM, pH=7.5). The water source for the hydrogel can come from the buffer solution. Therefore, the amount of buffer that can be added is an amount that fills out the formulation (e.g., q.s. to 100%). The hydrogel can be sterile or it can be preserved by the inclusion of preservatives. Non-limiting examples of preservatives include methylparaben, propylparaben, and phenoxyethanol.

[0102] Further additives can be added to achieve a given tactile property or to add an additional functional aspect to the hydrogel (e.g., agents that can further aid in the wound healing process such as vulnerary agents, antimicrobial agents, anti-inflammatory agents, pain- relieving agents, etc. . Preservatives such as methylparaben, propylparaben, and phenoxyethanol can be used to preserve the hydrogel. The amounts of such preservatives can range from 0.01 to 1% w / w (e.g., at least, at most, exactly, or between any two of 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1% w / w), although more or less can be added to achieve a desired result for the hydrogel.

[0103] The viscosity of the hydrogel can be modified to meet a desired consistency. In some aspects, the viscosity of the hydrogel can range from 5,000 to 100,000 cps, or 10,000 to100,000 cps, or 15,000 to 100,000 cps, 30,000 to 80,000 cps, or can be about 5,000, 10,000, 15,000, 20,000, 30,000, 40,000, 50,000, 60,000, 70,000, 80,000, 90,000, or 100,000 or any range therein, as measured with a Brookfield RV Viscometer (spindle 14 with small sample adapter) at 10 rpm at room temperature read at 30 seconds.

[0104] Hydrogels can be prepared by mixing propylene glycol and preservatives (e.g., methylparaben and propylparaben) in de-ionized (DI) water or Tris buffer (pH=7.5) at 70 °C. Upon solubilization ( / .< ., clear solution is obtained), the solution can be cooled to room temperature (RT) and a gelling agent e.g., Hydroxy ethylcellulose-HEC) can be added. The mixture can then be stirred until homogeneous (i.e., no visible particles of gelling agent, HEC), thereby forming a hydrogel. The active phase can be made by mixing therm oly sin, NaCl and CaCh in DI water or Tris buffer (pH=7.5) at appropriate concentrations. Upon homogenization (a white slurry will be obtained), the active phase can then be added to a hydrogel (at appropriate concentration). The final mixture can then be stirred at RT to obtain a hydrogel.C. Other Compositions

[0105] 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.

[0106] 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.D. Additional Composition Ingredients

[0107] The composition and formulation disclosed herein may include additional ingredients. For example, 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.

[0108] 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.

[0109] 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.

[0110] 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.

[0111] Auxiliary agents such as casein, gelatin, albumin, glue, sodium alginate, carboxymethylcellulose, methylcellulose, hydroxy ethylcellulose or polyvinyl alcohol may also be included in the compositions described herein.

[0112] 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.

[0113] 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, com 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.

[0114] Non-limiting examples of animal derived materials include beeswax, cod liver oil, emu oil, lard, mink oil, shark liver oil, squalane, squalene, and tallow.

[0115] Non-limiting examples of paraffinic materials include isoparaffin, microcrystalline wax, heavy mineral oil, light mineral oil, ozokerite, petrolatum, and paraffin.

[0116] Suitable non-limiting examples of organic esters and triglycerides include Cl 2- 15 alkyl benzoate, isopropyl myristate, isopropyl palmitate, medium chain triglycerides, trilaurin, and trihydroxystearin.

[0117] Non-limiting examples of silicones are dimethicone and cyclomethicone. A nonlimiting example of a fluorinated compound is polytetrafluoroethylene (PTFE).

[0118] 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.

[0119] 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®ROYAL, 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®, PERFECTA®, YELLOW PROTOPET 2A®, YELLOW FONOLINE®, PROTOLINE®, SONOJELL #4®, SONOJELL #9®, MINERAL JELLY #10®, MINERAL JELLY #14®, MINERAL JELLY #17®, AND CARNATION TROUGH GREASE®.

[0120] 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.

[0121] 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.

[0122] 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.

[0123] 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.

[0124] 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.

[0125] 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.

[0126] 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.

[0127] 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).

[0128] The compositions described herein may also be combined with other treatments known in the art to promote wound repair.

[0129] 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.IV. Methods of Use

[0130] Disclosed are methods of treating a wound (e.g., methods for wound debridement) comprising applying to the wound a composition or formulation including an enzymatic wound debriding agent (e.g., thermolysin, collagenase(s), and the like), for which a concentration of endotoxin contaminants was determined according to the disclosed methods. 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.

[0131] The compositions and formulations including such compositions disclosed herein can, in some aspects, be useful for the treatment of wounds of a subject by applying thecompositions to or on the wound. A wound can include a disruption of the structure and function of tissue. In addition to the other non-limiting 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.

[0132] 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 burn 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.

[0133] In some aspects, compositions and formulations including such compositions disclosed herein may provide a topical wound covering which functions as a protective barrier for the wound.

[0134] In some aspects, compositions and formulations including such 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 and formulations including such 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.

[0135] In some aspects, the compositions and formulations including such 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, pressureulcers, venous ulcers, diabetic ulcers, chronic vascular ulcers, tunneled, surgical wounds (e.g., donor sites / grafts, post-Mohs surgery, post-laser surgery, podiatric, wound dehiscence), trauma wounds (e.g., abrasions, lacerations, second-degree burns and skin tears), and draining wounds. In some aspects, the compositions and formulations including such 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.

[0136] In some aspects, the compositions and formulations including such 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.

[0137] In another aspect, the compositions and formulations including such compositions disclosed herein may be applied to the wound periodically, for example, daily. The compositions and formulations including such compositions 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.

[0138] In some aspects, the compositions and formulations including such compositions 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.V. Kits and Packaging

[0139] The compositions or formulations of the present disclosure, for which a concentration of endotoxin contaminants is or was determined according to the disclosed methods, 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 compositions bottles, 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.

[0140] 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.

[0141] In various aspects, the compositions or formulations described herein can be provided in a kit. The kit can include the composition or formulation and one or more broad spectrum metalloprotease inhibitors, for example.

[0142] Packaging may also include informational material relating to the compositions and formulations 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 compositions and formulations 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 measuring endotoxin contaminants and / or wound debridement provided herein.EXAMPLES

[0143] 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 stillobtain a like or similar result without departing from the spirit and scope of the present disclosure.I. Example 1 - Endotoxin Measurement In Thermolysin Samples After Heat Inactivation & GM6001 Inhibitor Treatment

[0144] Thermolysin lyophilized sample was dissolved in water in accordance with the USP <85> for the Limulus Amebocyte Lysate method. Once the thermolysin powder was completely dissolved to achieve 1 milligram of thermolysin per milliliter of solution, 1 milliliter of the sample solution was transferred to a polystyrene tube suitable for heating at 95 °C. The thermolysin solution was heated at 95 °C for 10 minutes. The sample was cooled to room temperature, then vortexed to ensure reincorporation of condensation. The cooled thermolysin solution was combined with a metalloprotease inhibitor, GM6001 (ilomastat; N- [(2R)-2-(hydroxamidocarbonylmethyl)-4-methylpentanoyl]-L-tryptophan methylamide) to provide a 10 nanomolar GM6001 solution, and the solution was vortexed briefly to evenly incorporate the inhibitor. The thermolysin sample was then analyzed in accordance with the USP <85> for the Limulus Amebocyte Lysate method, as described elsewhere herein. A positive endotoxin control, control standard endotoxin derived from Escherichia coh. was used to ensure that endotoxin would be detected if it were present in the sample.

[0145] While GM6001 was selected as a representative broad spectrum protease inhibitor for testing, it is expected that other broad spectrum protease inhibitors, including but not limited to batimastat, marimastat, MMI-270, cipemastat, MMI-166, ABT-770, prinomastat, rebimastat, tanomastat, a2-macroglobulin, GM 1489, would produce similar results according to the Limulus Amebocyte Lysate method.* * *

[0146] 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 measuring endotoxin contaminants in a composition comprising an enzymatic debriding agent, the method comprising: heating the composition; contacting the composition with a broad spectrum metalloprotease inhibitor; and measuring a concentration of endotoxin contaminants in the composition.

2. The method of claim 1, wherein the composition is heated to 90 °C to 100 °C for 5 minutes to 15 minutes.

3. The method of claim 2, wherein the composition is heated to about 95 °C.

4. The method of claim 2, wherein the composition is heated for about 10 minutes.

5. The method of any one of claims 1-4, further comprising cooling the composition after heating the composition.

6. The method of claim 5, wherein the composition is cooled to 20 °C to 25 °C.

7. The method of any one of claims 1-6, wherein the composition is contacted with an effective amount of the broad spectrum metalloprotease inhibitor to deactivate the enzymatic debriding agent.

8. The method of claim 7, wherein the effective amount comprises greater than 10 nanomolar for up to 3 milligrams of enzymatic debriding agent per milliliter or up to 15,000 Protease Units (PU) per milliliter.

9. The method of any one of claims 1-8, wherein the broad spectrum metalloprotease inhibitor is a hydroxamate-based inhibitor, a thiol-based inhibitor, or an endogenous inhibitor.

10. The method of any one of claims 1-9, wherein the broad spectrum metalloprotease inhibitor is ilomastat, batimastat, marimastat, MMI-270, cipemastat, MMI-166, ABT-770, prinomastat, rebimastat, tanomastat, a2 -macroglobulin, GM 1489, or a combination thereof.

11. The method of any one of claims 1-10, wherein the composition is not further diluted.

12. The method of any one of claims 1-11, wherein the concentration of endotoxin contaminants is measured by a gel-clot, limulus amebocyte lysate, turbidimetric or photometric assay.

13. The method of any one of claims 1-12, wherein the concentration of endotoxin contaminants is greater than 0.05 endotoxin units per mg of the composition.

14. A method for manufacturing a formulation including a composition comprising an enzymatic debriding agent and endotoxin contaminants, the method comprising: obtaining the formulation including the composition; and measuring the endotoxin contaminants in the composition by: heating the composition; mixing the composition with a broad spectrum metalloprotease inhibitor; and measuring a concentration of endotoxin contaminants in the composition.

15. The method of claim 14, wherein the formulation is a hydrogel.

16. The method of claim 14 or 15, wherein the composition is solubilized or suspended in the formulation.

17. The method of any one of claims 14-16, wherein the endotoxin contaminants are measured in the composition prior to obtaining the formulation including the composition.

18. The method of any one of claims 14-17, wherein the endotoxin contaminants are measured in the composition after obtaining the formulation including the composition.

19. The method of any one of claims 1-18, wherein the enzymatic debriding agent is thermolysin.

20. A composition comprising an enzymatic debriding agent, wherein a concentration of endotoxin contaminants in the composition is determined by a method comprising: heating the composition; contacting the composition with a broad spectrum metalloprotease inhibitor; and measuring a concentration of endotoxin contaminants in the composition.

21. The composition of claim 20, wherein the composition is solubilized or suspended in a hydrogel.

22. The composition of claim 20 or 21, wherein the enzymatic debriding agent is thermolysin.

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