Method of debridement of chronic wounds

Abstract

This invention relates to a method for wound debridement. In particular, this invention relates to a method for debridement of chronic wounds, the method comprising applying a debridement preparation in the form of a hydrogel to the wound site, the debridement preparation comprising a mixture of proteolytic enzymes obtained from bromelain and a water-soluble gelling agent, the debridement preparation being applied to the wound site up to 10 times over a period of up to 4 weeks, thereby achieving debridement of the chronic wound.

Description

Invention Field

[0001] This invention relates to a method for wound debridement. In particular, this invention relates to a method for debridement of chronic wounds, comprising applying a debridement preparation to the wound site, the debridement preparation comprising a mixture of proteolytic enzymes obtained from bromelain and a water-soluble gelling agent, the debridement preparation being applied to the wound site up to 10 times over a period of up to 4 weeks, thereby achieving debridement of chronic wounds. Background of the Invention

[0003] Chronic wounds, or wounds that are difficult to heal, are a common condition that causes suffering for millions of people each year. Most chronic wounds are caused by local or generalized vascular insufficiency, which reduces blood flow to the skin and subcutaneous tissues. The most common types of chronic wounds, or wounds that are difficult to heal, include: pressure ulcers (decubiti or “bed sores”), diabetic ulcers, arterial ulcers; venous ulcers; and postoperative / post-traumatic ulcers, or combinations thereof.

[0004] Chronic wounds result in severe damage to the skin. This damage can affect the entire thickness of the skin and often includes deeper tissues. Damaged skin loses the anatomical structure of healthy skin; the stratum corneum is at least partially damaged, and therefore the inner layers of the skin are no longer protected from external environmental damage. Furthermore, damaged skin often contains eschar, diseased cells, and / or abnormal cells that must be removed to promote healing. Eschar remaining in situ allows the wound to extend and deepen into adjacent undamaged tissue. This eschar also acts as a medium for bacterial growth and a source of potentially life-threatening infections, contamination, and sepsis.

[0005] Studies investigating the composition and structure of eschar and necrotic tissue in chronic wounds have shown that chronic wounds comprise multiple protein substances representing extracellular matrix proteins and autolytic debridement degradation products. These studies suggest that chronic wound debridement may require more than one enzyme-specific approach to degrade the various components of inactive and necrotic tissue within the wound.

[0006] The removal of eschar, diseased cells, and / or abnormal cells, also known as "debridement," can be performed surgically, mechanically (dressing changes, bathing), through autolysis procedures (promoting impregnation with dressings), or enzymatically. Surgery is one of the most common debridement procedures, in which small areas of necrotic tissue are removed from the entire damaged skin. This method is limited to small, non-tangential surfaces. Surgery also involves removing most of the healthy tissue, which, if preserved, can serve as a source of the natural healing process. Surgical procedures are also more expensive and require more medical resources.

[0007] Enzymatic debridement offers advantages over mechanical and surgical debridement, primarily due to its less painful nature, greater selectivity, and the fact that it does not require the assistance of well-trained medical personnel. The use of proteolytic enzymes for debridement is well-known in the art. These enzymes include those isolated from bacteria and those commonly found in plant sources, such as papain (papaya), fig (figase), and bromelain (bromelain). Bromelain-derived hydrolytic enzymes, particularly those derived from pineapple plants, are specifically described in U.S. Patents 4,197,291, 4,226,854, 4,307,081, 4,329,430, and 5,830,739, for the dissection and isolation of nonviable tissue, especially eschar, from viable tissue in mammalian hosts.

[0008] The therapeutic activity derived from topical proteolytic enzymes is particularly controlled by the inherent catalytic properties of the enzymes. A major problem associated with the topical use of compositions containing proteolytic enzymes is that the catalytic activity of the enzyme is rapidly diminished due to the typically low pH of the lesion area, the adsorption of enzyme molecules to the surface of the wound bed and / or dressing, and the partial inhibition of enzymatic activity within wound exudate. Therefore, obtaining stable enzymatic formulations is complex.

[0009] Several ointments are currently marketed for removing eschar, such as Ointments. These ointments are typically applied daily for several months to achieve the desired wound debridement.

[0010] U.S. Patent No. 4,668,228 to Bolton et al. discloses a debridement bandage containing dried, powdered proteolytic enzymes, such as subtilisin and bromelain, on the adhesive mass surface of a closed or semi-closed surgical tape. According to U.S. Patent No. 4,668,228, when the debridement bandage is applied to a burn surface, water from the wound that is impermeable to the backing of the closed bandage activates the debridement enzymes.

[0011] U.S. Patent No. 4,784,653 to Bolton et al. discloses an absorbent adhesive dressing for use in treating ulcer and burn wounds, comprising a three-layer sandwich structure having a sealing membrane as the outer layer, an absorbent fiber layer as the middle layer, and a wet-stick adhesive as the inner wound-facing adhesive layer, the adhesive layer being made of an acrylic polymer having both hydrophilic and hydrophobic properties. According to U.S. Patent No. 4,784,653, a debridement enzyme may be added to the adhesive mass if desired.

[0012] U.S. Patent No. 5,271,943 to Bogart et al. discloses a therapeutic gel having a minimum yield point of about 800 poise and a maximum apparent viscosity of about 100,000 cp, the gel comprising water, sodium chloride and a gelling agent.

[0013] U.S. Patent No. 5,514,370 to Stern et al. discloses a pharmaceutical composition for topical application comprising a high concentration of collagenase in a non-aqueous excipient. U.S. Patent No. 5,514,370 also discloses a method of treating a wound comprising applying to the wound a composition consisting primarily of a non-aqueous excipient and collagenase.

[0014] Rolf's U.S. Patent No. 5,804,213 discloses a pre-packaged wound dressing comprising a natural or synthetic hydrocolloid in the form of dried particles. According to U.S. Patent No. 5,804,213, the hydrocolloid in the form of dried particles is contained in a compartment of a sealed container separated from water. After mixing with water, the mixture is fluid enough to allow it to be poured or applied to the wound. Upon application to the wound, the hydrated hydrocolloid dispersion begins to solidify to form a solid, self-supporting, flexible dressing composed of water, hydrocolloid, and a bioactive ingredient.

[0015] U.S. Patent No. 6,548,556 to Hobson et al. discloses an enzymatically catalytic anhydrous hydrophilic debridement agent that uses a combination of a proteolytic enzyme and an anhydrous hydrophilic poloxamer carrier.

[0016] U.S. Patent No. 8,062,661 to Caldwell et al. discloses a method for debridement of a skin wound, which includes contacting the skin wound with a hydrogel patch debridement composition and removing the hydrogel patch debridement composition from the skin wound to remove foreign matter from the skin wound.

[0017] International application publication No. WO 2006 / 054309, belonging to the applicant of this invention, discloses a debridement composition obtained from bromelain that can be used to remove eschar tissue and for wound healing.

[0018] International application publication No. WO 2013 / 011514, belonging to the applicant of this invention, discloses a proteolytic extract from bromelain for the treatment of connective tissue diseases, including Dupuytren's disease and Peyronie's disease, associated with excessive collagen deposition.

[0019] U.S. Provisional Patent Application No. 62 / 289,246, filed January 31, 2016, discloses a debridement composition comprising a mixture of proteolytic enzymes in dry form obtained from bromelain and an aqueous gel carrier, wherein the proteolytic enzyme mixture is mixed with the aqueous gel carrier prior to use to form a debridement composition suitable for the debridement and treatment of chronic wounds.

[0020] There is a long-standing and unmet need for improved methods for enzymatic debridement of chronic wounds that achieve complete wound debridement and contribute to the closure and healing of chronic wounds. Invention Overview

[0022] This invention provides a method for wound debridement and / or treatment of wounds, particularly chronic wounds, the method comprising applying a debridement preparation topically to the wound site in a regimen of up to 10 applications over a period of up to 4 weeks, wherein the debridement preparation is formulated as a hydrogel comprising: (i) a proteolytic enzyme mixture obtained from bromelain, the proteolytic enzyme mixture comprising stem bromelain (EC 3.4.22.32) and ananain (EC 3.4.22.31); and (ii) a water-soluble gelling agent, wherein the water-soluble gelling agent is not a cross-linked polymer of acrylic acid, and wherein the debridement preparation remains in contact with the wound site for at least 4 hours, thereby achieving debridement of eschar / necrotic tissue and various forms of devitalized necrotic tissue.

[0023] Wound debridement is a key process in wound bed preparation (WBP) and is considered an essential intervention in chronic wound management that promotes wound healing and complete wound closure.

[0024] It is known that currently available enzymatic debridements for treating chronic wounds include, for example Ointments are applied daily for extended periods, such as three, six, or even twelve months, to remove eschar.

[0025] It is now disclosed that the debridement preparation of the present invention can be applied to chronic wounds up to 10 times, with each application keeping the debridement preparation at the wound site for approximately 24 hours, resulting in substantially complete removal of eschar from the chronic wound. The method of the present invention requires a short treatment regimen, improves patient compliance, and achieves debridement of chronic or difficult-to-heal wounds within a few days, at a faster rate than any currently known enzymatic debridement method. Therefore, the method of the present invention is highly advantageous compared to currently known enzymatic wound debridement techniques.

[0026] According to some embodiments, the debridement preparation used to carry out the method of the present invention comprises the following components:

[0027] (a) A composition in a dried or lyophilized form, said composition comprising:

[0028] (i) A mixture of proteolytic enzymes obtained from bromelain, the mixture of proteolytic enzymes comprising stem bromelain (EC 3.4.22.32) and ananain (EC 3.4.22.31);

[0029] (ii) A water-soluble gelling agent, wherein the water-soluble gelling agent is not a cross-linked polymer of acrylic acid;

[0030] (iii) Anti-aggregating agents or anti-coagulation agents;

[0031] (iv) pH adjusters; and

[0032] (b) Water,

[0033] Prior to use, the composition (a) is mixed with water (b) to form a debridement preparation, characterized in that it is a homogeneous hydrogel having a viscosity in the range of about 2,000,000 cP to about 8,500,000 cP and a pH in the range of about 6.0 to about 8.0, wherein the amount of protein in the debridement preparation ranges from about 0.5% (w / w) to about 7% (w / w) of the total weight of the debridement preparation.

[0034] In some embodiments, the debridement formulations of the present invention have a pH ranging from about 6.4 to about 8.0, for example, about 7.0. Within this pH range, the activity of proteolytic enzymes is substantially maximum. To achieve these pH values, the debridement formulations of the present invention contain a pH adjuster, thus enabling the production of highly effective enzymatic debridements.

[0035] It is also disclosed that, due to the protein properties of the active agent in the debridement preparation, namely a mixture of proteolytic enzymes obtained from bromelain, this mixture tends to form aggregates or clumps. To prevent the formation of aggregates or clumps, the debridement preparation also contains an anti-aggregating agent or anti-coagulant, thereby enabling the formation of a homogeneous hydrogel.

[0036] The invention also discloses that the composition of the debridement preparation is sufficiently viscous to allow proteolytic enzymes to penetrate into the eschar tissue of a chronic wound to effectively remove inactive tissue, and to be positioned at the wound site without leakage from the wound site.

[0037] The invention also discloses that, due to the composition of the debridement agent, its preparation process is simple, easy, and rapid, requiring only a few minutes, for example less than two minutes, to mix the components and obtain a homogeneous hydrogel. Because it is easy to mix, the debridement agent of the present invention can be prepared by the patient without the assistance of medical personnel. Therefore, the method of the present invention is particularly advantageous for elderly patients with chronic wounds or wounds that are difficult to heal.

[0038] According to a first aspect, the present invention provides a method for wound debridement, the method comprising applying a therapeutically effective amount of a debridement preparation to a wound site of a subject requiring such treatment in a regimen of up to 10 applications over a period of up to 4 weeks, wherein the debridement preparation is in the form of a hydrogel comprising: (i) a proteolytic enzyme mixture obtained from bromelain, the proteolytic enzyme mixture comprising stem bromelain (EC 3.4.22.32) and ananain (EC 3.4.22.31); and (ii) a water-soluble gelling agent, wherein the water-soluble gelling agent is not a cross-linked polymer of acrylic acid, and wherein with each application, the debridement preparation remains in contact with the wound site for at least 4 hours.

[0039] According to some embodiments, the wound to be debrided is a chronic wound. According to other embodiments, the chronic wound is selected from the group consisting of diabetic ulcers, venous stasis ulcers, arterial insufficiency ulcers, pressure ulcers, postoperative wounds, and post-traumatic wounds. Each possibility represents a separate embodiment of the invention. According to another embodiment, the chronic wound is a diabetic lower extremity ulcer or a venous leg ulcer.

[0040] According to another embodiment, the debridement agent is applied in a regimen of up to 10 applications, wherein each application is made daily, and the debridement agent remains in contact with the wound site for approximately 4-24 hours, such as approximately 6 hours, approximately 8 hours, approximately 10 hours, approximately 12 hours, approximately 16 hours, approximately 24 hours, or any integer number of hours therebetween. Each possibility represents a separate embodiment of the invention. According to one embodiment, for up to 10 applications, optionally for up to 8 applications, each application maintains contact with the wound site for approximately 24 hours. According to an exemplary embodiment, each application is made daily, with the debridement agent applied for approximately 24 hours for 10 consecutive days.

[0041] According to another embodiment, the debridement agent is applied up to 10 times every other day, with each application maintaining contact between the debridement agent and the wound site for approximately 48 hours. According to yet another embodiment, the debridement agent is applied up to 8 times every other day, with each application maintaining contact between the debridement agent and the wound site for approximately 48 hours.

[0042] According to another implementation plan, the debridement agent is applied three times a week, up to 10 times, or up to 8 times, wherein the debridement agent is kept in contact with the wound site for a period of time consisting of two groups: one group of approximately 48 hours per application and the other group of approximately 72 hours per application.

[0043] According to another implementation scheme, the application of the debridement agent as defined above may be repeated once, twice, or until the wound is completely debrided. Alternatively or additionally, if the eschar recurs and wound closure has not been achieved, the scheme may be repeated once, twice, or more until the eschar is completely removed.

[0044] According to yet another embodiment, the application of the debridement agent is followed by a cessation of application for at least one day, such as two, three, four, five, six days, one week, two weeks, three weeks, four weeks or longer, or any integer period thereof. Each possibility represents a separate embodiment of the invention.

[0045] According to another embodiment, the method further includes the step of cleaning the wound site after contact with the debridement agent for at least 4 hours, such as, for example, after contact with the debridement agent for about 6 hours, about 8 hours, about 10 hours, about 12 hours, about 24 hours, about 48 hours, or about 72 hours.

[0046] According to another embodiment, the method may further include the step of administering an active agent to the subject, said active agent being selected from the group consisting of anesthetics, antibacterial agents, antifungal agents, and anti-inflammatory agents. The active agent, such as, for example, an anesthetic, may be applied topically to the wound site or may be administered orally or parenterally before, simultaneously with, or after the application of a debridement preparation.

[0047] According to some embodiments, the wound cleaning agent used in the wound cleaning method of the present invention comprises the following components:

[0048] (a) A composition in dry or powder form, said composition comprising:

[0049] (i) A proteolytic enzyme mixture obtained from bromelain, the proteolytic enzyme mixture comprising stem bromelain (EC 3.4.22.32) and ananain (EC 3.4.22.31);

[0050] (ii) Water-soluble gelling agents;

[0051] (iii) Anti-aggregation agent;

[0052] (iv) pH adjusters; and

[0053] (b) Water,

[0054] Prior to use, the composition (a) is mixed with water (b) to form the debridement preparation, characterized in that it is a homogeneous hydrogel having a viscosity in the range of about 2,000,000 cP to about 8,500,000 cP and a pH in the range of about 6.0 to about 8.0, and wherein the amount of protein in the debridement preparation ranges from about 0.5% (w / w) to about 7% (w / w) of the total weight of the debridement preparation.

[0055] According to another embodiment, the amount of protein in the debridement preparation ranges from about 2% (w / w) to about 7% (w / w) of the total weight of the debridement preparation, optionally from about 1% (w / w) to about 5% (w / w) of the total weight of the debridement preparation, and more preferably about 1%, 2%, 2.5%, 3%, 4%, 5%, 6%, or about 7% of the total weight of the debridement preparation. Each possibility represents a separate embodiment of the invention. According to one embodiment, the amount of protein in the debridement preparation is about 2% (w / w) of the total weight of the debridement composition.

[0056] According to other embodiments, the water-soluble gelling agent is selected from the group consisting of naturally occurring gelling agents, semi-synthetic gelling agents, and synthetic gelling agents. According to another embodiment, the naturally occurring gelling agent is a naturally occurring polysaccharide, such as, for example, galactomannan, glucomannan, natural gums, starch, agar, and pectin. Each possibility represents a separate embodiment of the invention. According to one embodiment, the water-soluble naturally occurring gelling agent is guar gum present in an amount ranging from about 0.25% (w / w) to about 5% (w / w) of the total weight of the debridement preparation.

[0057] According to another embodiment, the anti-aggregating agent of the debridement preparation is an oligosaccharide selected from the group consisting of lactose, sucrose, mannitol, and glucose. Each possibility represents a separate embodiment of the invention. According to one embodiment, the anti-aggregating agent is lactose present in an amount ranging from about 10% (w / w) to about 25% (w / w) of the total weight of the debridement preparation.

[0058] According to another embodiment, the pH adjuster of the debridement preparation is selected from the group consisting of potassium phosphate, potassium carbonate, sodium phosphate, and sodium carbonate. Each possibility is a separate embodiment of the invention. According to one embodiment, the pH adjuster is a combination of dipotassium hydrogen phosphate and potassium dihydrogen phosphate present in an amount ranging from about 2% (w / w) to about 10% (w / w) of the total weight of the debridement preparation.

[0059] According to another embodiment, the viscosity of the debridement agent to be used in the method of the present invention ranges from about 2,000,000 cP to about 7,000,000 cP, and optionally from about 2,400,000 cP to about 6,200,000 cP. Each possibility is a separate embodiment of the present invention.

[0060] According to another embodiment, the pH range of the debridement agent to be used in the method of the present invention is from about 6.0 to about 7.0. According to one embodiment, the pH is about 7.0.

[0061] According to some embodiments, water is present in an amount ranging from about 55% (w / w) to about 90% (w / w) of the total weight of the debridement composition.

[0062] According to another embodiment, the debridement preparation used in the method of the present invention further comprises an agent selected from the group consisting of defoamers such as, for example, polyethylene glycol (PEG), antioxidants, and preservatives. According to an exemplary embodiment, the debridement preparation further comprises PEG.

[0063] According to another implementation plan, the debridement preparation also includes an active agent selected from the group consisting of anesthetics, analgesics, anti-inflammatory agents, antibiotics, antifungals, growth factors, and healing promoters.

[0064] According to some embodiments, the wound to be treated by the method of the present invention is a chronic wound, and the debridement preparation comprises:

[0065] (i) A mixture of proteolytic enzymes obtained from bromelain, which is named the active main ingredient (API) throughout the specification and claims;

[0066] (ii) Guar gum in amounts ranging from about 0.25% (w / w) to about 5% (w / w) of the total weight of the debridement preparation;

[0067] (iii) Lactose in amounts ranging from about 10% (w / w) to about 25% (w / w) of the total weight of the debridement preparation;

[0068] (iv) Potassium phosphate in amounts ranging from about 2% (w / w) to about 10% (w / w) of the total weight of the debridement composition; and

[0069] (v) Add water to a volume equal to 100% (w / w) of the total weight of the debridement preparation.

[0070] The amount of protein in the debridement preparation ranges from about 0.5% (w / w) to about 7% (w / w) of the total weight of the debridement preparation, preferably from about 1% (w / w) to about 5% (w / w).

[0071] According to another embodiment, the wound to be treated by the method of the present invention is a chronic wound, and the debridement preparation comprises:

[0072] (i) A mixture of proteolytic enzymes obtained from bromelain, which is named the active main ingredient (API) throughout the specification and claims;

[0073] (ii) Guar gum in amounts ranging from about 0.25% (w / w) to about 5% (w / w) of the total weight of the debridement preparation;

[0074] (iii) Lactose in amounts ranging from about 10% (w / w) to about 25% (w / w) of the total weight of the debridement preparation;

[0075] (iv) Potassium phosphate in amounts ranging from about 2% (w / w) to about 10% (w / w) of the total weight of the debridement composition;

[0076] (v) PEG in amounts ranging from about 0.5% (w / w) to about 10% (w / w) of the total weight of the debridement composition; and

[0077] (vi) Add water to a volume equal to 100% (w / w) of the total weight of the debridement preparation.

[0078] The amount of protein in the debridement preparation ranges from about 0.5% (w / w) to about 7% (w / w) of the total weight of the debridement preparation, preferably from about 1% (w / w) to about 5% (w / w).

[0079] According to one embodiment, the debridement agent to be used in the method of the present invention comprises:

[0080] Element Formulation (%) w / w API 2 Guar gum 3.5 lactose 18.05 dipotassium hydrogen phosphate 2.5 Potassium dihydrogen phosphate 0.8 PEG-3350 2 Water for Injection 71.15 .

[0081] According to another embodiment, the debridement agent to be used in the wound debridement method of the present invention is prepared by the following steps:

[0082] (a) Obtaining a composition in dry or powder form, said composition comprising:

[0083] (i) A mixture of proteolytic enzymes obtained from bromelain;

[0084] (ii) Water-soluble gelling agents;

[0085] (iii) Anti-aggregation agent;

[0086] (iv) pH adjusters; and

[0087] (b) Prior to use, the composition (a) is mixed with water to form the debridement preparation, wherein the debridement preparation is characterized as a homogeneous hydrogel having a viscosity in the range of about 2,000,000 cP to about 8,500,000 cP and a pH in the range of about 6.0 to about 8.0.

[0088] According to another aspect, the present invention provides a method for treating wounds and / or promoting wound closure and / or wound healing, the method comprising the step of topically applying a therapeutically effective amount of a debridement preparation to a wound site of a subject requiring such treatment in a regimen of up to 10 applications over a period of up to 4 weeks, wherein the debridement preparation is present in the form of a hydrogel comprising: (i) a proteolytic enzyme mixture obtained from bromelain, the proteolytic enzyme mixture comprising stem bromelain (EC 3.4.22.32) and ananain (EC 3.4.22.31); and (ii) a water-soluble gelling agent, wherein the water-soluble gelling agent is not a cross-linked polymer of acrylic acid, and wherein, as defined in any of the above regimens, the debridement preparation remains in contact with the wound site for at least 4 hours with each application.

[0089] According to another aspect, the present invention provides a debridement preparation comprising:

[0090] (a) A composition in dry or powder form, said composition comprising:

[0091] (i) A proteolytic enzyme mixture obtained from bromelain, the proteolytic enzyme mixture comprising stem bromelain (EC 3.4.22.32) and ananain (EC 3.4.22.31);

[0092] (ii) A water-soluble gelling agent, wherein the water-soluble gelling agent is not a cross-linked polymer of acrylic acid;

[0093] (iii) Anti-aggregation agent;

[0094] (iv) pH adjusters; and

[0095] (b) Water,

[0096] The composition (a) is mixed with water (b) to form a debridement preparation, the debridement preparation being a homogeneous hydrogel having a viscosity in the range of about 2,000,000 cP to about 8,500,000 cP and a pH in the range of about 6.0 to about 8.0, and wherein the amount of protein in the debridement preparation ranges from about 0.5% (w / w) to about 7% (w / w) of the total weight of the debridement preparation.

[0097] According to some implementation plans, the debridement preparation includes:

[0098] (a) A composition in dry or powder form, said composition comprising:

[0099] (i) A proteolytic enzyme mixture obtained from bromelain, the proteolytic enzyme mixture comprising stem bromelain (EC 3.4.22.32) and ananain (EC 3.4.22.31);

[0100] (ii) Guar gum in an amount ranging from about 0.25% (w / w) to about 5% (w / w) of the total weight of the debridement preparation;

[0101] (iii) Lactose in an amount ranging from about 10% (w / w) to about 25% (w / w) of the total weight of the debridement preparation;

[0102] (iv) pH adjusters; and

[0103] (b) Water in amounts ranging from about 55% (w / w) to about 90% (w / w),

[0104] The composition (a) is mixed with water (b) to form a debridement preparation, the debridement preparation being a homogeneous hydrogel having a viscosity in the range of about 2,000,000 cP to about 8,500,000 cP and a pH in the range of about 6.0 to about 8.0, and wherein the amount of protein in the debridement preparation ranges from about 0.5% (w / w) to about 7% (w / w) of the total weight of the debridement preparation, preferably from about 1% (w / w) to about 5% (w / w).

[0105] According to one implementation plan, the debridement preparation includes:

[0106] Element Formulation (%) w / w API 2 Guar gum 3.5 lactose 18.05 dipotassium hydrogen phosphate 2.5 Potassium dihydrogen phosphate 0.8 PEG-3350 2 Water for Injection 71.15 .

[0107] According to another aspect, a debridement preparation is provided for use in wound debridement and / or treatment of wounds and / or promotion of wound closure and / or wound healing, the debridement preparation comprising: (i) a proteolytic enzyme mixture obtained from bromelain, said proteolytic enzyme mixture comprising stem bromelain (EC 3.4.22.32) and ananain (EC 3.4.22.31); and (ii) a water-soluble gelling agent, wherein the water-soluble gelling agent is not a cross-linked polymer of acrylic acid, wherein, according to the principles of the invention, the debridement preparation is applied topically to the wound site in a manner of up to 10 applications over a period of up to 4 weeks, and wherein with each application, the debridement preparation remains in contact with the wound site for at least 4 hours.

[0108] These and other embodiments of the invention will be better understood in conjunction with the following drawings, description, embodiments, and claims. Brief description of the attached diagram

[0110] Figure 1A-1F These are photographs of chronic wounds induced in pigs. Figure 1A It shows the chronic wound before treatment, and Figure 1B and 1C The images show chronic wounds treated with the debridement formulation of the present invention on days 7 and 10, respectively. As a control, the chronic wounds before treatment are shown. Figure 1D ) or treatment with the medium only on days 7 and 10 (respectively) Figure 1E and 1F Chronic wounds during treatment.

[0111] Figure 2 The percentage of cleared area of ​​induced chronic wounds in pigs after the 10th treatment is shown as a function of the concentration of the active pharmaceutical ingredient (API) applied to the wound over a continuous 24-hour period. The broad dashed line indicates the confidence interval (95%) of the model.

[0112] Figure 3 The area under the curve (AUC) for cleansing is shown as a function of the concentration of the API applied continuously for 24 hours to induced chronic wounds in pigs. The broad dashed line indicates the confidence interval (95%) of the model.

[0113] Figure 4 The area under the curve (AUC) of eschar is shown as a function of the concentration of the API applied continuously for 24 hours to induced chronic wounds in pigs. The broad dashed line indicates the confidence interval (95%) of the model.

[0114] Detailed description of the invention

[0115] This invention provides a method for wound debridement and / or wound treatment and / or wound closure and / or wound healing, the method comprising the step of topically applying a therapeutically effective amount of a debridement preparation to a wound site of a subject requiring such treatment in a regimen of up to 10 applications over a period of up to 4 weeks, wherein the debridement preparation is in the form of a hydrogel comprising: (i) a proteolytic enzyme mixture obtained from bromelain, the proteolytic enzyme mixture comprising stem bromelain (EC 3.4.22.32) and ananain (EC 3.4.22.31); and (ii) a water-soluble gelling agent, wherein the water-soluble gelling agent is not a cross-linked polymer of acrylic acid, and wherein with each application, the debridement preparation remains in contact with the wound site for at least 4 hours. This invention also provides a debridement preparation in the form of a hydrogel comprising a proteolytic enzyme mixture obtained from bromelain and a water-soluble gelling agent other than a cross-linked polymer of acrylic acid.

[0116] It is now disclosed that applying the debridement preparation of the present invention to induced chronic wounds in pigs up to 10 times, with each application maintaining contact between the debridement preparation and the wound site for 24 hours, results in substantially complete removal of the eschar from the chronic wound. Similar removal can be achieved by applying the debridement preparation to the chronic wound three times a week for up to 10 times, with the debridement composition maintaining contact with the wound site twice for 48 hours each time, and by maintaining contact with the wound site once for 72 hours each time.

[0117] Debridement agents

[0118] The present invention provides a wound debridement preparation comprising a mixture of proteolytic enzymes obtained from bromelain as the active ingredient and a variety of excipients.

[0119] As used throughout the specification and claims, the term "a mixture of proteolytic enzymes obtained from bromelain" refers to an enzyme product partially purified from bromelain.

[0120] The term "bromelain" refers to a protein extract derived from the stem of the pineapple plant, which may be commercially available.

[0121] A mixture of proteolytic enzymes obtained from bromelain (also known as...) or Bromelain and its products are disclosed in WO 2006 / 054309 and WO 2013 / 011514, the contents of which are incorporated herein by reference as if fully set forth herein. The proteolytic enzyme mixture obtained from bromelain contains at least two cysteine ​​proteases present in bromelain: stem bromelain (EC 3.4.22.32) and ananain (EC 3.4.22.31). The proteolytic enzyme mixture may also contain one or more cysteine ​​protease precursors of bromelain, such as, for example, ananain (EC 3.4.22.31) precursor, fruit bromelain (EC 3.4.22.33) precursor, and stem bromelain (EC 3.4.22.32) precursor. The proteolytic enzyme mixture may also contain cysteine ​​protease fragments (see, for example, WO 2006 / 054309), bromelain-like lectins, and / or bromelain inhibitors. According to one implementation, the proteolytic enzyme mixture obtained from bromelain contains stem bromelain (EC 3.4.22.32), ananain (EC 3.4.22.31), a cysteine ​​protease precursor of bromelain, and bromelain-like lectin.

[0122] The proteolytic enzyme mixture can be obtained by the procedure disclosed in WO 2013 / 011514. As a final step in the preparation, the proteolytic enzyme mixture is lyophilized and stored as a lyophilized powder until use.

[0123] The proteolytic enzyme mixture is highly stable and can be stored at 2°C–8°C for extended periods, such as up to three years. After this period, the proteolytic enzyme mixture retains at least 90% of the original debridement activity determined immediately after the preparation process.

[0124] The proteolytic enzyme mixture is referred to as the main active ingredient (API) throughout the specification and claims. According to the invention, the amount of protein or optionally API in the debridement preparation ranges from about 0.5% (w / w) to about 7% (w / w) of the total weight of the debridement preparation. According to another embodiment, the amount of protein or API ranges from about 1% (w / w) to about 5% (w / w) of the total weight of the debridement preparation, such as about 1%, 2%, 3%, 4%, 5%, 6%, 7%, or optionally about 2% (w / w) of the total weight of the debridement preparation.

[0125] As used interchangeably throughout the specification and claims, the terms "dried," "dry," "freeze-dried," or "powdered" mean a composition containing water in an amount not exceeding 5% (w / w) of the total weight of the composition, optionally in an amount not exceeding about 3%, 2%, 1%, 0.5%, or even optionally not exceeding about 0.1% (w / w) of the total weight of the composition. According to one embodiment, the composition is water-free.

[0126] As used herein, the term "hydrogel" refers to an aqueous composition capable of maintaining a gel-like form.

[0127] The term "homogeneous" hydrogel means a hydrogel with a uniform viscosity (e.g., fully mixed throughout).

[0128] The excipients in debridement preparations are all pharmaceutically acceptable. The term "pharmaceuticalally acceptable" means that they are approved by federal or state regulatory agencies or listed in the United States Pharmacopeia or other generally recognized pharmacopoeias for use in humans.

[0129] The term “about” refers to a value that is 10% higher or lower than the value shown.

[0130] According to some implementation schemes, the excipients of the debridement preparation are water-soluble. The term "water-soluble" refers to an agent that typically has a solubility in water at room temperature in the range of 1 gr / ml to 1 gr / 30 ml.

[0131] Water-soluble gelling agents can be naturally occurring gelling agents, semi-synthetic gelling agents, and synthetic gelling agents. The gelling agents according to the present invention do not include cross-linked polymers of acrylic acid.

[0132] Water-soluble naturally occurring gelling agents include, but are not limited to, water-soluble naturally occurring polysaccharides, such as, for example, galactomannan, glucomannan, starch, agar, pectin, alginate, carrageenan, or combinations thereof. Each possibility represents a separate embodiment. Non-limiting examples of galactomannan and glucomannan are guar gum, locust bean gum, xanthan gum, gum arabic, tragacanth gum, gellan gum, and mixtures thereof. Each possibility represents a separate embodiment. According to one embodiment, the water-soluble naturally occurring gelling agent is guar gum.

[0133] Other biopolymers include, for example, chitin, chitosan, collagen, gelatin, glycosaminoglycans such as heparin, chondroitin sulfate, dermatan sulfate, and heparan sulfate, proteoglycans, fibronectin, and laminin.

[0134] Semi-synthetic gelling agents include, but are not limited to, cellulose ethers (such as hydroxyethyl cellulose, methyl cellulose, carboxymethyl cellulose, hydroxypropyl methyl cellulose), polyvinylpyrrolidone, polyvinyl alcohol, hydroxypropyl guar gum, etc.

[0135] Synthetic gelling agents include, but are not limited to, carboxyvinyl polymers, polyvinylpyrrolidone, polyvinyl acetate polymers, polyvinyl chloride polymers, and polyvinylidene chloride polymers.

[0136] The debridement composition may also contain at least one excipient selected from the group consisting of anti-aggregating agents and pH adjusters.

[0137] The anti-agglomeration agent or anti-coagulation agent suitable for carrying out the present invention is any known anti-agglomeration agent, such as water-soluble oligosaccharides, for example, lactose, sucrose, mannitol, sorbitol, and glucose. Each possibility represents a separate embodiment. According to one embodiment, the anti-agglomeration agent is lactose.

[0138] The pH adjuster preferably has a pKa higher than 6.0. In some embodiments, the pH adjuster can be any known pH adjuster, such as, for example, potassium phosphate, potassium carbonate, sodium carbonate, and sodium phosphate. According to some embodiments, the pH adjuster is a combination of potassium dihydrogen phosphate and dipotassium hydrogen phosphate present in an amount ranging from about 2% (w / w) to about 10% (w / w) of the total weight of the debridement preparation. It is now disclosed that higher amounts of potassium dihydrogen phosphate and dipotassium hydrogen phosphate in the debridement preparation can cause bleeding at the application site. Therefore, it is disclosed that if the pH adjuster is a combination of potassium dihydrogen phosphate and dipotassium hydrogen phosphate, their total amount is preferably no more than about 10% of the total weight of the debridement preparation to achieve effective debridement without undesirable bleeding.

[0139] The composition may also contain an antifoaming agent. Antifoaming agents are known in the art and include, but are not limited to, polyethylene glycols, such as PEG-1450, PEG-3350, etc. The composition may also contain preservatives, such as benzyl alcohol, parabens, methylparaben, or propylparaben; and / or antioxidants, such as ascorbic acid, dihydroquinone, butylated hydroxytoluene, and dithiothreitol.

[0140] The composition may also contain anesthetics, antibacterial agents, antifungal agents, anti-inflammatory agents, analgesics, growth factors and / or healing promoters.

[0141] Anesthetics include, but are not limited to, lidocaine, linocaine, celecoxib, bupivacaine, prilocaine, ropivacaine, benzocaine, mepivacaine, and cocaine. Each possibility represents a separate implementation scheme.

[0142] Antibacterial agents include, but are not limited to, amanfadine hydrochloride, amanfadine sulfate, amikacin, amikacin sulfate, aminoglycosides, amoxicillin, ampicillin, anesamcinolone, bacitracin, β-lactams, clotrimazole, capreomycin, carbenicillin, cephalexin, cefotaxime, cefotaxime, cefazolin, cefepime, cefadroxil, cefotaxime, chloramphenicol, chlorhexidine, chlorhexidine gluconate, chlorhexidine hydrochloride, dihydrochloroquine, chloroquine, and chlortetracycline. Chlortetracycline hydrochloride, Ciprofloxacin, Cyclobazin, Clindamycin, Clindamycin hydrochloride, Clotrimazole, Cloxacillin, Demeclocycline, Diclofenac, Diiodohydroxyquinoline, Doxycycline, Ethambutol, Ethambutol hydrochloride, Erythromycin, Erythromycin etoritate, Erythromycin stearate, Farnesol, Flucloxacillin, Gentamicin, Gentamicin sulfate, Bacitracin, Griseofulvin, Haloprothin, Haloquinone alcohol, Hexachlorophene, Minocycline, Clodohydroxyquinoline, Kanamycin, Kanamycin sulfate, Lincomycin, Lincomycin hydrochloride Macrolides, meclocycline, methacin, methacin hydrochloride, urotropine, urotropine hippurate, urotropine mandelate, methicillin, metronidazole, miconazole, miconazole hydrochloride, minocycline, minocycline hydrochloride, mupirocin, nafcillin, neomycin, neomycin sulfate, netilmicin, netilmicin sulfate, nitrofurazone, norfloxacin, nystatin, octopirox, tylosin, cephalosporins, oxacillin, oxytetracycline, oxytetracycline hydrochloride, parachlorometracycline Cresol, paromomycin, paromomycin sulfate, penicillins, penicillin G, penicillin V, pentamicillin, pentamicillin hydrochloride, feneccillin, polymyxin, quinolones, streptomycin sulfate, tetracycline, tobramycin, tonaphalate, triclosan, rifampin, rifamycin, rolicycline, silver salts, spectinomycin, spiramycin, streptomycin, sulfonamides, tetracyclines, tetracycline, tobramycin, tobramycin sulfate, triclocarban, triclosan, trimethoprim-sulfamethoxazole, tylosin, vancomycin, and breviculin. Each possibility represents a separate implementation scheme.

[0143] Antifungal agents include, but are not limited to, nystatin, clotrimazole, miconazole, ketoconazole, fluconazole, thiabendazole, econazole, clomidazole, isoconazole, thiabendazole, thiabendazole, thioconazole, bifonazole, oxiconazole, fenteconazole, oxomoconazole, sertaconazole, and flutriazole. Each possibility represents a separate implementation scheme.

[0144] Anti-inflammatory agents can be nonsteroidal anti-inflammatory agents, steroidal anti-inflammatory agents, or combinations thereof. Non-limiting examples of nonsteroidal anti-inflammatory agents include oxacins, such as piroxicam, isoxicam, tenoxicam, and sudoxicam; salicylates / salts, such as aspirin, disalcid, benorilate, trilisate, safapryn, solprin, diflunisal, and fendusal; and acetic acid derivatives, such as diclofenac, fenclofenac, indomethacin, sulindac, tometidine, isocolic acid, furofenac, thiophene, zidomecin, asimexin, fentiac, zolpidem, and clindamycin. NAC), oxifene, biphenylacetic acid, and ketorolac; fenamates, such as mefenamic acid, meclofenamic acid, flufenamic acid, niflufenic acid, and tofenamic acid; propionic acid derivatives, such as ibuprofen, naproxen, benzoxalofen, flurbiprofen, ketoprofen, fenofibfen, fenbufen, indoprofen, pirprofen, carprofen, oxaprozin, pranoprofen, imidofen, thioprofen, sulprofen, amineprofen, and tiaprofenic acid; pyrazoles, such as phenylbutazone, hydroxybutazine, fepranoprofen, azaprofen, and trimethoprim. Extracts of these nonsteroidal anti-inflammatory drugs may also be used. Each possibility represents a separate implementation scheme.

[0145] Non-limiting examples of steroidal anti-inflammatory drugs include corticosteroids such as hydrocortisone, hydroxytriamcione, alpha-methyldexamethasone, dexamethasone phosphate, beclomethasone dipropionate, clobetasol valerate, desonide, deoxymethasone, desoxycorticosterone acetate, dexamethasone, diclomethasone, difluralasone diacetate, diflucortisone valerate, fluadrenolone, fluclosonide, flucortisone, dexamethasone neovalerate, fluocinolone, fluocinolone acetate, foctocin butyl ester, flucortisone, fluprednisolone acetate, fluprednisolone, fluprednisolone acetate, flucortisone, halcinonide, hydrocortisone acetate, hydrocortisone butyrate, methylprednisolone, triamcinolone, cortisone, todoxason, fluocinolone acetate, flucortisone, difluralasone diacetate, flucortisone, diflucortisone diacetate, acetone-fluorohydrocephalosporin, methylhydroxysone, ansilafal, ansilafate, betamethasone and its ester balances, chlorprednisolone, chlorprednisolone acetate, chlorcotropene, disilone, diclofenac, difluprednisolone, fludiclofenac, flumexone, fluperazine, fluprednisolone, hydrocortisone valerate, hydrocortisone cyclopropionate, hydrocortisone ester, methylprednisolone, peramisone, prednisolone, prednisolone, beclomethasone dipropionate, and triamcinolone. Each possibility represents a separate implementation.

[0146] Analgesics include, but are not limited to, codeine, hydrocodone, oxycodone, fentanyl, and propoxyphene. Each possibility represents a separate implementation scheme.

[0147] Growth factors include, but are not limited to, epidermal growth factor, fibroblast growth factor, and insulin-like growth factor.

[0148] Healing promoters include, but are not limited to, hyaluronic acid.

[0149] The viscosity of the gel formulations of this invention can be measured by any known means. According to some embodiments, an absolute viscometer with a plate geometry can be used to calculate the viscosity of the gel formulations described herein. All viscosity ranges mentioned herein are measured at room temperature.

[0150] According to the principles of the invention, the composition (a) and water (b), which are present in dry or powder form, can be placed in the first and second compartments of a single container, respectively, or they can be placed in two separate containers. Before use, the composition (a) and water (b) are mixed to form a wound-cleansing preparation.

[0151] The debridement preparation of the present invention has a low bacterial bioload, and therefore reduces the risk of further contamination of the wound site. According to some embodiments, the debridement preparation is sterile.

[0152] According to some implementation plans, the debridement preparation includes:

[0153] (a) A composition in dry or powder form, said composition comprising:

[0154] (i) A proteolytic enzyme mixture obtained from bromelain, the proteolytic enzyme mixture comprising stem bromelain (EC 3.4.22.32) and ananain (EC 3.4.22.31);

[0155] (ii) A water-soluble gelling agent, wherein the water-soluble gelling agent is not a cross-linked polymer of acrylic acid;

[0156] (iii) Anti-aggregation agent;

[0157] (iv) pH adjusters; and

[0158] (b) Water,

[0159] The composition (a) is mixed with water (b) to form a debridement preparation, the debridement preparation being a homogeneous hydrogel having a viscosity in the range of about 2,000,000 cP to about 8,500,000 cP and a pH in the range of about 6.0 to about 8.0, and wherein the amount of protein in the debridement preparation ranges from about 0.5% (w / w) to about 7% (w / w) of the total weight of the debridement preparation.

[0160] According to some implementation plans, the debridement preparation includes:

[0161] (a) A composition in dry or powder form, present in a first compartment of a container or in a first container, said composition comprising:

[0162] (i) A proteolytic enzyme mixture obtained from bromelain, the proteolytic enzyme mixture comprising stem bromelain (EC 3.4.22.32) and ananain (EC 3.4.22.31);

[0163] (ii) Guar gum in amounts ranging from about 0.25% (w / w) to about 5% (w / w) of the total weight of the debridement preparation;

[0164] (iii) Lactose in amounts ranging from about 10% (w / w) to about 25% (w / w) of the total weight of the debridement preparation;

[0165] (iv) pH adjusters; and

[0166] (b) The portion present in the second compartment of the container or the portion present in the second container is approximately 55%.

[0167] (w / w) to about 90% (w / w) of water.

[0168] It should be understood that the wound-cleansing preparation of the present invention is formulated as a gel, i.e., a hydrogel, and is thus applied to the wound site. Preferably, the wound-cleansing preparation is not a patch preparation. According to some embodiments, the preparation does not contain adhesives and is therefore non-adhesive.

[0169] Uses of wound cleaning agents

[0170] This invention provides a method for debridement and / or treatment of skin wounds, the method comprising the step of topically applying a therapeutically effective amount of a debridement preparation to a wound site of a subject requiring such treatment in a regimen of up to 10 applications over a period of up to 4 weeks, wherein the debridement preparation is present in the form of a hydrogel comprising: (i) a proteolytic enzyme mixture obtained from bromelain, the proteolytic enzyme mixture comprising stem bromelain (EC 3.4.22.32) and ananain (EC 3.4.22.31); and (ii) a water-soluble gelling agent, wherein the water-soluble gelling agent is not a cross-linked polymer of acrylic acid, and wherein with each application, the debridement preparation remains in contact with the wound site for at least 4 hours.

[0171] According to some implementation plans, the wound is a chronic wound or a wound that is difficult to heal.

[0172] As used interchangeably throughout this specification and claims, the terms "chronic wound," "chronic skin wound," or "wound that is difficult to heal" refer to a wound that fails to undergo a series of orderly and timely events to achieve a durable structural, functional, and / or aesthetic closure as a wound should. A wound that fails to heal within one month is considered a chronic wound.

[0173] According to some implementation schemes, chronic wounds are selected from the group consisting of diabetic ulcers, venous stasis ulcers, arterial insufficiency ulcers, pressure ulcers, postoperative wounds, and post-traumatic wounds. Each possibility represents a separate implementation scheme. According to another implementation scheme, chronic wounds are diabetic lower extremity ulcers or venous leg ulcers.

[0174] As used herein, “wound debridement” refers to the removal of non-viable tissue from the wound: necrotic eschar, slough or fibrin, foreign bodies, and bacteria / biofilm. Necrotic eschar is thin or thick, leathery, dead, black, brown, or tan tissue, while slough and biofilm are exudative, white or yellowish-green patchy, fine tissues on the wound bed. Necrotic tissue, foreign bodies, and bacteria hinder the body’s healing process by producing or stimulating the production of metalloproteinases that interfere with the local wound healing process. This harsh environment allows bacteria to proliferate and further colonize the wound within the exudate, debris, and purulent discharge (“slough”) covering the wound bed. Additionally, bacteria secrete structural products that, along with slough, form biofilms, thus protecting their colonies from potential damage. Bacteria produce their respective wound-inhibiting enzymes and, more importantly, consume most of the scarce, available local resources necessary for wound healing.

[0175] According to some embodiments, wound debridement refers to the removal of at least 50%, optionally at least 75%, of the non-viable tissue present before treatment. Each possibility represents a separate embodiment of the invention. According to some embodiments, wound debridement refers to the removal of at least 90%, or at least 95%, and preferably 100% of the non-viable tissue present before treatment; such debridement, i.e., the removal of 90% or more of the non-viable tissue present before treatment, is referred to throughout the specification and claims as “complete wound debridement.”

[0176] In chronic or slow-healing wounds, several different factors can play a significant role. Exposed surfaces such as bone, tendons, fascia, or even fat do not support cell proliferation, and they dry out and become foreign bodies, such as synthetic implants. Any interference with the local blood supply (arteries, veins, lymph, pressure, etc.) can cause the wound to become difficult to heal and chronic. Granulation tissue may become intractable, atrophy, lose its rich vascular matrix, become darker and less transparent in color, and will play no role in the wound healing and closure process.

[0177] As used in this article, “wound bed preparation” refers to the creation of a wound bed through appropriate debridement to accelerate intrinsic healing or promote the effectiveness of other treatments. It is a process of debridement, removing various “burdens” within the wound and the patient that hinder healing. Burdens within the wound include exudate, bacteria, biofilms, and necrotic debris / cellular debris. The patient’s overall health is important for the healing process. In chronic or difficult-to-heal wounds, complete removal of unsightly eschar, necrotic tissue, or biofilm may create a clean wound bed; however, if such a wound bed is not supported by the patient’s overall or limb condition, it may still be insufficient for future healing.

[0178] A wound bed prepared for healing is a wound bed free of eschar, necrotic tissue, fibrin, or biofilm. This wound bed is also a viable bed of healthy tissue and / or healthy granulation tissue (level > 7 on a particle size scale), which will allow the wound to close spontaneously on a viable, clean bed through scar formation and contracture-epidermal formation (optionally using methods such as biological dressings, wound healing-enhancing dressings, synthetic wound dressings, vacuum or ozone wound healing systems) or will support autologous STSG (split-thickness skin graft) or skin allogeneic graft.

[0179] The term "wound closure" refers to the process of regeneration of the cellular covering layer of tissue. Therefore, promoting wound closure means having a positive effect on the regeneration of the cellular covering layer. A positive effect can be to accelerate the regeneration process or to reduce the damaged area of ​​the wound. Wound closure is also defined as complete epithelialization, no drainage, and no need for additional dressings, and is confirmed during two consecutive study visits two weeks apart.

[0180] The term "therapeutic effective amount" is the amount of a mixture of proteolytic enzymes sufficient to provide a beneficial effect to a subject to whom the composition is administered.

[0181] According to some implementation plans, the debridement agent can be applied to the wound site up to 10 times, with each application lasting 4 hours and the debridement agent remaining in contact with the wound site.

[0182] According to another implementation scheme, the debridement agent can be applied to the wound site in up to 10 applications, with each application maintaining contact with the wound site for approximately 24 hours. Therefore, the debridement agent can be applied daily for up to 10 consecutive days, maintaining contact with the wound site for approximately 24 hours each time, or it can be applied 1, 2, 3, 4, 5, 6, 7, 8, or 9 times consecutively, with each application lasting approximately 24 hours, with applications discontinued for a day or longer as needed.

[0183] According to another implementation scheme, the debridement agent can be applied to the wound site in up to 10 applications, with each application maintaining contact with the wound site for approximately 48 hours. Therefore, the debridement agent can be applied every other day for up to 20 consecutive days, or it can be applied once, twice, three times, four times, five times, six times, seven times, eight times, or nine times every other day, with applications discontinued as needed for a day or longer interval.

[0184] According to another implementation scheme, the debridement agent can be applied to chronic wounds three times a week for up to 10 times, wherein the duration of contact between the debridement agent and the wound site is selected from the group consisting of 48 hours per application and 72 hours per application.

[0185] According to another implementation scheme, the debridement agent can be applied to the wound site in up to 10 applications, with each application maintaining contact between the debridement agent and the wound site for approximately 72 hours.

[0186] According to some implementation schemes, the debridement agent remains in contact with the wound site for up to approximately 72 hours with each application.

[0187] The wound site can be cleaned after a specified period of application following contact with the debridement agent, such as at least 4 hours, 24 hours, 48 ​​hours, or 72 hours after treatment. Therefore, the method of the present invention may further include the step of cleaning the wound site after said contact and before subsequent application of the debridement agent. If application is stopped, the wound site can be covered with a wet dressing such as moist saline gauze.

[0188] According to some embodiments, the method of the present invention may further include the step of covering the debridement preparation with a closure layer or closure dressing to keep or retain the composition at the wound site.

[0189] According to another embodiment, the method of the present invention may also include the step of protecting the wound edges and the skin around the wound during debridement.

[0190] The range of numerical values ​​indicated throughout the specification and claims includes any integers therein.

[0191] It should be understood that any of the above-defined regimens may be repeated once, twice, three times, or more (optionally with application cessation) until the eschar / necrotic tissue is completely removed. Application cessation may take days, weeks, or months. The debridement regimen may be repeated as needed to remove the eschar. If the eschar recurs, the debridement regimen may be repeated as needed to remove the eschar.

[0192] This invention encompasses combination therapies, wherein the methods of this invention can be combined with known debridement methods, such as surgical debridement or sharp debridement. According to some embodiments, the methods of this invention can be performed prior to surgical debridement or sharp debridement. Optionally, the methods of this invention can be performed after surgical debridement or sharp debridement.

[0193] According to some implementation schemes, the amount of API applied ranges from 100cm. 2 Approximately 0.1 to 2 g of a sterile lyophilized proteolytic enzyme mixture was applied to the wound surface. According to another embodiment, the amount of hydrogel applied to the wound site was per 100 cm². 2 The wound surface is approximately 20g.

[0194] Example 1

[0195] Gel formulation

[0196] The following debridement agents have been developed:

[0197]

[0198] *Other quantities (w / w) of the API being evaluated: 0.1%; 0.5%; 1%; and 2%.

[0199] **The amount of lactose should be adjusted according to the amount of API.

[0200] The debridement preparation is prepared by mixing a dry or powdered composition containing API, guar gum, lactose, dipotassium hydrogen phosphate and potassium dihydrogen phosphate, and PEG-3350 with water to form a hydrogel with a uniform appearance and a viscosity ranging from 2,40,000 cP to 6,200,000 cP.

[0201] Example 2

[0202] Remove eschar with gel preparation

[0203] The aim of this study was to determine the dosage of the active ingredient in a gel formulation that provides maximum efficacy for eschar removal in chronic wounds.

[0204] A chronic wound model was established in hybrid domestic pigs.

[0205] Before applying the gel, the wound edges were protected with a thick layer of petroleum jelly. Approximately 2 g of gel was applied to each wound site to cover it for 24 hours, followed by a non-absorbent dressing. Each wound was photographed before and after each application. The following dosages were examined: placebo (0%), 0.1%, 0.5%, 1%, 2%, and 5%.

[0206] This procedure is followed for up to 11 consecutive days of treatment, or until a clean wound bed is achieved. This period is called the "treatment period." Following the treatment period is a two-week "recovery period" without treatment. During the recovery period, the wound is photographed three times a week.

[0207] Wound area, cleaned area, and eschar volume were visually evaluated, measured using ImageJ software (NIH, MD, USA), and analyzed using JMP statistical software (SAS Inc., NC, USA).

[0208] On the first day of treatment, all wounds were covered with a thick layer of eschar. The eschar comprised two distinct areas:

[0209] a. The center of the wound, which is covered by a thin layer of eschar;

[0210] b. The edge of the wound, characterized by completely necrotic tissue.

[0211] Representative photographs of wounds before and 7 or 10 days after treatment with gel preparations or gel media are shown. Figure 1A-1F middle.

[0212] At the start of treatment, the chronic wound had already formed an eschar. Figure 1A and Figure 1D This is a representative photograph showing a chronic wound before treatment. The eschar comprises two distinct areas: the center of the wound where the skin has been completely removed and the wound periphery. In the center, a thin layer of eschar forms, while at the periphery, the eschar contains necrotic skin. In chronic wounds treated with gel formulations containing APIs, the eschar softens and gradually dissolves with each treatment, until the dissolution at the periphery causes the eschar to detach completely from the viable tissue. Figure 1B and 1C In contrast, chronic wounds treated with gel-mediated treatment show almost no change in appearance and consistency during the treatment period. Figure 1E and 1F ).

[0213] The cleaned area is calculated as a percentage of the total wound size. Taking into account both the area and thickness of the eschar, the eschar volume is calculated as a percentage of the eschar volume prior to the first treatment.

[0214] Treatment efficacy is evaluated by measuring the area under the curve (AUC), where the x-axis represents the number of treatment days and the y-axis represents the percentage of cleared area or eschar volume. More effective treatment results in a larger AUC for the percentage of cleared area and a smaller AUC for eschar volume.

[0215] To evaluate the irritation caused by the formulation, five unsuspecting evaluators scored each wound based on photographs taken throughout the experiment.

[0216] Table 1. Summary of debridement results

[0217] Parameter (Y) Gel formulation placebo % of cleaned area 82 52 % of eschar from day 0 7 31 AUC cleanup 454 274 AUC eschar 531 850

[0218] At the end of the treatment period, the percentage of the wound area cleaned was:

[0219] The percentage of the initial wound area cleared at the end of treatment was found to be significantly dependent on the amount of API administered. The dependence on the amount of API was linear. Figure 2 ).

[0220] When the API dosage in the gel formulation is 5%, an average of 82% of wounds are cleaned.

[0221] At the end of the treatment period, the percentage of eschar relative to the initial wound volume. :

[0222] The percentage of eschar remaining at the initial amount at the end of treatment was found to be significantly dependent on the amount of API. The dependence on the amount of API was linear.

[0223] When the API dosage in the gel formulation is 5%, an average of 93% of the eschar is removed.

[0224] The area under the curve (AUC) is calculated, where the x-axis represents the number of treatment days and the y-axis represents the percentage of cleared area. :

[0225] This parameter indicates the effectiveness of the treatment in clearing the area: the more effective the treatment, the higher the AUC. The AUC of the percentage of area cleared during treatment is significantly dependent on the amount of API. Figure 3 As shown, the AUC percentage of the cleaned area is linearly dependent on the amount of API.

[0226] The area under the curve (AUC) is calculated, where the x-axis represents the number of treatment days and the y-axis represents the percentage of eschar to the initial amount of eschar. :

[0227] This parameter indicates the efficacy of the treatment in removing eschar: the more effective the treatment, the smaller the AUC. This parameter was found to be significantly dependent on the amount of API. The dependence on the amount of API is linear. Figure 4 ).

[0228] Taken together, these results indicate that the effects of the API are dose- and time-dependent.

[0229] Stimulate

[0230] Five evaluators, unaware of the treatments, scored each wound based on photographs taken throughout the experiment. They found that the placebo-induced stimulation was significantly lower than that in the treatment group. Stimulation was dose-dependent and completely disappeared within one or two days of the follow-up period in all treatments.

[0231] Example 3

[0232] Efficacy and safety of APIs in gel formulations - clinical studies

[0233] The aim of this study was to evaluate the safety and efficacy of two doses, 2% (w / w) and 5% (w / w) of the gel formulation (also known as EX-02) disclosed in Example 1 above in the debridement of chronic venous leg ulcers and diabetic lower extremity ulcers, compared with placebo.

[0234] This study is a multicenter, prospective, randomized, placebo-controlled, double-blind international study.

[0235] At 3cm 2 With 200cm 2 Adults with >50% necrotic / necrotic / fibrinous inactive tissue on chronic wounds (venous leg ulcers, diabetic lower extremity ulcers) between (surface area) were included in the study.

[0236] Patients were randomly assigned to receive either a low-dose EX-02 treatment (2% w / w), a high-dose EX-02 treatment (5% w / w), or a placebo treatment. Treatment was administered three times a week for up to 10 applications (up to 10 visits), or until complete debridement was achieved, whichever came first. Each application lasted 24 ± 2 hours, or three times a week for 48 ± 4 hours and 72 ± 4 hours, respectively. After each application, the wound was cleaned, photographed, and wound size and removal of non-viable tissue (using digital planimetry software) and changes in granulation tissue, wound status, and safety parameters were evaluated. Treatment was administered continuously throughout the weekdays. During weekends, the wound was covered with completely moistened (moist-to-moist) saline gauze.

[0237] After the debridement period, patients were treated according to standard procedures, with weekly assessments (wound assessments) until complete wound closure, continuing for up to 12 weeks from the last application (up to 12 visits). For patients who achieved wound closure, three additional monthly visits were conducted to confirm closure; the first monthly visit was conducted two weeks after wound closure was achieved. For patients who did not achieve wound closure during the 12-week visitation period, only three monthly visits were conducted (week 30). The placebo was prepared using only excipient powder and water for gel preparation.

[0238] The following endpoints were evaluated for all wounds and compared between EX-02 and placebo:

[0239] Primary endpoint

[0240] The incidence of complete debridement (removal of non-viable tissue) at the end of the debridement period (up to 8 days of treatment)

[0241] Secondary endpoint

[0242] 1. Time to achieve complete debridement (within up to 8 treatments);

[0243] 2. Number of application applications / treatment days to achieve complete debridement;

[0244] 3. Evaluate changes in wound debridement status during the treatment period: percentage reduction in non-viable tissue (per day during the 8 treatment sessions);

[0245] 4. Time required to achieve complete granulation (up to 12 weeks);

[0246] 5. Incidence of complete granulation (week 12);

[0247] 6. Percentage change in granulation tissue over time (each week during the baseline-12-week period);

[0248] 7. Incidence of complete wound closure (up to 12 weeks). Wound closure was defined as complete epithelial formation, no discharge, and no need for additional dressings, as confirmed in two consecutive study visits 2 weeks apart;

[0249] 8. Time required for wound closure (up to 12 weeks);

[0250] 9. Wound size reduction: percentage reduction in wound size over time (every week from baseline up to 12 weeks);

[0251] 10. Infection rate.

[0252] Those skilled in the art will understand that this invention is not limited to what has been specifically shown and described above. Rather, the scope of the invention is defined by the following claims.

Claims

1. Use of the dry form of the composition in the preparation of a debridement formulation for chronic wound debridement, said debridement formulation comprising the dry form of the composition and water, said dry form of the composition comprising: (i) A mixture of proteolytic enzymes obtained from bromelain, i.e., API, said proteolytic enzyme mixture comprising stem bromelain (EC 3.4.22.32) and ananain (EC 3.4.22.31); (ii) A water-soluble gelling agent, wherein the water-soluble gelling agent is a naturally occurring polysaccharide, and the naturally occurring polysaccharide is guar gum; (iii) Anti-aggregating agents; (iv) pH adjusters; and in, Prior to use, the composition is mixed with water to form the wound-cleansing preparation, characterized in that it is a homogeneous hydrogel having a viscosity in the range of 2,400,000 centipoise (cP) to 6,200,000 cP and a pH in the range of 6.0 to 8.0, and wherein the amount of protein in the wound-cleansing preparation ranges from 0.5% (w / w) to 5% (w / w) of the total weight of the wound-cleansing preparation. The aforementioned debridement preparation is suitable for topical application to the wound site in a regimen of up to 10 applications over a period of up to 4 weeks. Furthermore, the debridement agent is suitable for maintaining contact with the wound site for at least 4 hours with each application.

2. The use according to claim 1, wherein the chronic wound is selected from the group consisting of diabetic ulcers, venous stasis ulcers, arterial insufficiency ulcers, pressure ulcers, and post-traumatic wounds.

3. The use according to any one of claims 1 and 2, wherein the debridement preparation is suitable for application in a regimen of up to 10 applications, and wherein the debridement preparation is suitable for maintaining contact with the wound site for 24 hours during each application.

4. The use according to any one of claims 1 and 2, wherein the debridement preparation is suitable for application in a regimen of up to 8 applications, and wherein the debridement preparation is suitable for maintaining contact with the wound site for 24 hours during each application.

5. The use according to any one of claims 1 and 2, wherein the debridement preparation is suitable for application in a regimen of up to 10 applications, and wherein the debridement preparation is suitable for maintaining contact with the wound site for 48 hours with each application.

6. The use according to any one of claims 1 and 2, wherein the debridement preparation is suitable for application in a regimen of up to 8 applications, and wherein the debridement preparation is suitable for maintaining contact with the wound site for 48 hours with each application.

7. The use according to any one of claims 1 and 2, wherein the debridement preparation is suitable for application in a regimen of three times a week for up to 10 applications, and wherein the debridement preparation is suitable for maintaining contact with the wound site for a duration selected from the group consisting of 48 hours per application and 72 hours per application.

8. The use according to any one of claims 1 and 2, wherein the debridement preparation is suitable for application in a regimen of three times a week for up to eight times, and wherein the debridement preparation is suitable for maintaining contact with the wound site for a duration selected from the group consisting of 48 hours per application and 72 hours per application.

9. The use according to claim 3, wherein the procedure is repeated once, twice, or as needed until the wound is completely cleaned.

10. The use according to claim 4, wherein the procedure is repeated once, twice, or as needed until the wound is completely cleaned.

11. The use according to claim 5, wherein the procedure is repeated once, twice, or as needed until the wound is completely cleaned.

12. The use according to claim 6, wherein the procedure is repeated once, twice, or as needed until the wound is completely cleaned.

13. The use according to claim 7, wherein the procedure is repeated once, twice, or as needed until the wound is completely cleaned.

14. The use according to claim 8, wherein the procedure is repeated once, twice, or as needed until the wound is completely cleaned.

15. The use according to any one of claims 9-14, wherein the application is stopped between repetitive processes.

16. The use according to claim 1, wherein the wound site is suitable for cleaning before subsequent application, at least 4 hours after the debridement preparation has been in contact with the wound site, at least 24 hours after contact, at least 48 hours after contact, or at least 72 hours after contact.

17. The use according to claim 1, wherein the amount of protein in the debridement preparation ranges from 1% (w / w) to 5% (w / w) of the total weight of the debridement preparation.

18. The use according to claim 17, wherein the amount of protein in the debridement preparation is 2% (w / w) of the total weight of the debridement preparation.

19. The use according to claim 1, wherein the guar gum is present in an amount ranging from 0.25% (w / w) to 5% (w / w) of the total weight of the debridement preparation.

20. The use according to claim 1, wherein the anti-aggregating agent is an oligosaccharide.

21. The use according to claim 20, wherein the oligosaccharide is selected from the group consisting of lactose, sucrose, mannitol and glucose.

22. The use according to claim 21, wherein the oligosaccharide is lactose present in an amount ranging from 10% (w / w) to 25% (w / w) of the total weight of the debridement preparation.

23. The use according to claim 1, wherein the pH adjuster is potassium phosphate present in an amount ranging from 2% (w / w) to 10% (w / w) of the total weight of the debridement preparation.

24. The use according to claim 23, wherein the potassium phosphate is a combination of dipotassium hydrogen phosphate and potassium dihydrogen phosphate.

25. The use according to claim 1, wherein the pH range of the debridement preparation is from 6.0 to 7.

0.

26. The use according to claim 1, wherein water is present in an amount ranging from 55% (w / w) to 90% (w / w) of the total weight of the debridement preparation.

27. The use according to claim 1, wherein the debridement preparation further comprises an agent selected from the group consisting of an antifoaming agent, an antioxidant, and a preservative.

28. The use according to claim 1, wherein the debridement preparation further comprises an active agent selected from the group consisting of anesthetics, anti-inflammatory agents, antibiotics, antifungals, analgesics, and growth factors.

29. The use according to claim 1, wherein the debridement agent comprises: (i) the protein hydrolase mixture, wherein the amount of protein in the debridement preparation ranges from 1% (w / w) to 5% (w / w) of the total weight of the debridement preparation; (ii) Guar gum in an amount ranging from 0.25% (w / w) to 5% (w / w) of the total weight of the debridement preparation; (iii) Lactose in an amount ranging from 10% (w / w) to 25% (w / w) of the total weight of the debridement preparation; (iv) Potassium phosphate in an amount ranging from 2% (w / w) to 10% (w / w) of the total weight of the debridement preparation; and (v) Water to a volume of 100% (w / w) of the total weight of the debridement preparation.

30. The use according to claim 29, wherein the debridement preparation comprises: 。 31. The use according to claim 1, wherein the debridement preparation is prepared by the following steps: (a) Obtaining a composition in a dried form, said composition comprising: (i) A mixture of proteolytic enzymes obtained from bromelain; (ii) Water-soluble gelling agents; (iii) Anti-aggregating agents; (iv) pH adjusters; and (b) Prior to use, the composition (a) is mixed with water to form the debridement preparation, the debridement preparation being a homogeneous hydrogel having a viscosity in the range of 2,000,000 centipoise (cP) to 8,500,000 cP and a pH range of 6.0 to 8.

0.

32. A wound-cleansing preparation, said wound-cleansing preparation comprising: (a) A composition in dried form, said composition comprising: (i) A mixture of proteolytic enzymes obtained from bromelain, i.e., API, said proteolytic enzyme mixture comprising stem bromelain (EC 3.4.22.32) and ananain (EC 3.4.22.31); (ii) A water-soluble gelling agent, wherein the water-soluble gelling agent is a naturally occurring polysaccharide, and the naturally occurring polypeptide is guar gum; (iii) Anti-aggregating agents; (iv) pH adjusters; and (b) Water, in, Prior to use, the composition (a) is mixed with water (b) to form a wound-cleansing preparation, characterized as a homogeneous hydrogel having a viscosity in the range of 2,400,000 centipoise (cP) to 6,200,000 cP and a pH in the range of 6.0 to 8.0, and wherein the amount of protein in the wound-cleansing preparation ranges from 0.5% (w / w) to 5% (w / w) of the total weight of the wound-cleansing preparation.

33. The debridement preparation according to claim 32, wherein the debridement preparation comprises: (a) A composition present in the first compartment of a container or in the dry form of a first container, said composition comprising: (i) A proteolytic enzyme mixture obtained from bromelain, the proteolytic enzyme mixture comprising stem bromelain (EC 3.4.22.32) and ananain (EC 3.4.22.31), wherein the amount of protein in the debridement preparation ranges from 1% (w / w) to 5% (w / w) of the total weight of the debridement preparation; (ii) Guar gum in an amount ranging from 0.25% (w / w) to 5% (w / w) of the total weight of the debridement preparation; (iii) Lactose in an amount ranging from 10% (w / w) to 25% (w / w) of the total weight of the debridement preparation; (iv) pH adjusters; and (b) Water present in the second compartment of the container or in an amount of 55% (w / w) to 90% (w / w) in the second container. in, Prior to use, the composition (a) is mixed with water (b) to form a wound-cleansing preparation, characterized as a homogeneous hydrogel having a viscosity in the range of 2,400,000 centipoise (cP) to 6,200,000 cP and a pH in the range of 6.0 to 8.

0.

34. The debridement preparation according to claim 33, wherein the debridement preparation comprises: 。

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