METHODS OF DEBRIDEMENT OF CHRONIC WOUNDS

Abstract

Methods for wound debridement and specifically methods of debridement of chronic wounds. These methods provide topically applying to a wound site a debriding formulation in the form of a hydrogel that includes a proteolytic enzyme mixture obtained from bromelain and a water-soluble gelling agent, with the debriding formulation being applied to the wound site up to ten times over a period of up to four weeks, thereby achieving debridement of chronic wounds.

Claims

1. to 35. (canceled)

36. A method for debridement of a wound comprising topically applying to the wound site of a subject in need of such treatment a therapeutically effective amount of a debriding formulation in a regimen of up to ten applications during a time period of up to four weeks, wherein the debriding formulation formulated in the form of a hydrogel comprising: (i) a proteolytic enzyme mixture obtained from bromelain 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 other than a cross-linked polymer of acrylic acid, and wherein said debriding formulation is maintained in contact with the wound site for at least four hours per application.

37. The method according to claim 36, wherein the wound is a chronic wound.

38. The method according to claim 37, wherein the chronic wound is selected from the group consisting of a diabetic ulcer, a venous stasis ulcer, an arterial insufficiency ulcer, a pressure ulcer, a post-operative wound, and a post-trauma wound.

39. The method according to claim 36, wherein applying the debriding formulation is performed in a regimen of up to ten applications, and wherein the debriding formulation is maintained in contact with the wound site for about 24 hours per application.

40. The method according to claim 36, wherein applying the debriding formulation is performed in a regimen of up to 10 applications, and wherein the debriding formulation is maintained in contact with the wound site for about 48 hours per application.

41. The method according to claim 36, wherein applying the debriding formulation is performed in a regimen of up to 10 applications, three applications per week, and wherein the debriding formulation is maintained in contact with the wound site for a duration selected from the group consisting of 48 hours per application and 72 hours per application.

42. The method according to claim 36, wherein the regimen is repeated once, twice, or as needed until the wound is completely debrided.

43. The method according to claim 36, wherein the debriding formulation comprises the following ingredients: (a) a composition in a dry or powdered form comprising: (i) the 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); (ii) the water-soluble gelling agent; (iii) an anti-aggregation agent; (iv) a pH adjusting agent; and (b) water, wherein, prior to use, the composition (a) is admixed with the water (b) to form said debriding formulation characterized by being a homogenous hydrogel having a viscosity in the range of about 2,000,000 centipoise (cP) to about 8,500,000 cP and a pH ranging from about 6.0 to about 8.0, and wherein the amount of proteins in the debriding formulation ranges from about 0.5% (w/w) to about 7% (w/w) of the total weight of said debriding formulation.

44. The method according to claim 43, wherein the water-soluble gelling agent is selected from the group consisting of naturally occurring gelling agents, semi-synthetic gelling agents and synthetic gelling agents.

45. The method according to claim 44, wherein the water-soluble naturally occurring gelling agent is a polysaccharide.

46. The method according to claim 45, wherein the polysaccharide is a galactomannan or glucomannan.

47. The method according to claim 46, wherein the galactomannan 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 debriding formulation.

48. The method according to claim 43, wherein the anti-aggregation agent is an oligosaccharide.

49. The method according to claim 48, wherein the oligosaccharide is selected from the group consisting of lactose, sucrose, mannitol, and glucose.

50. The method according to claim 49, wherein the oligosaccharide is lactose present in an amount ranging from about 10% (w/w) to about 25% (w/w) of the total weight of the debriding formulation.

51. The method according to claim 43, wherein the pH adjusting agent is potassium phosphate present in an amount ranging from about 2% (w/w) to about 10% (w/w) of the total weight of the debriding formulation.

52. The method according to claim 36, wherein the debriding formulation comprises: (i) the proteolytic enzyme mixture, wherein the amount of proteins in the debriding formulation ranges from about 0.5% (w/w) to about 7% (w/w), preferably from about 1% (w/w) to about 5% (w/w) of the total weight of the debriding formulation; (ii) guar gum in an amount ranging from about 0.25% (w/w) to about 5% (w/w) of the total weight of the debriding formulation; (iii) lactose in an amount ranging from about 10% (w/w) to about 25% (w/w) of the total weight of the debriding formulation; (iv) potassium phosphate in an amount ranging from about 2% (w/w) to about 10% (w/w) of the total weight of the debriding formulation; and (v) water in an amount to complete to 100% (w/w) of the total weight of the debriding formulation.

53. The method according to claim 52, wherein the debriding formulation comprises: TABLE-US-00005 Ingredient (%) w/w of formulation API 2 Guar gum 3.5 Lactose 18.05 Potassium phosphate dibasic 2.5 Potassium phosphate monobasic 0.8 PEG-3350 2 Water for injection 71.15

54. The method according to claim 36, wherein the debriding formulation is prepared by the following steps: (a) obtaining a composition in a dry or powdered form which comprises: (i) the proteolytic enzyme mixture obtained from bromelain; (ii) the water-soluble gelling agent; (iii) an anti-aggregation agent; (iv) a pH adjusting agent; and (b) admixing, prior to use, the composition (a) with water to form the debriding formulation characterized by being a homogenous hydrogel having a viscosity in the range of about 2,000,000 centipoise (cP) to about 8,500,000 cP and a pH ranging from about 6.0 to about 8.0.

55. A method of treating a wound comprising a step of topically applying to the wound site of a subject in need of such treatment a therapeutically effective amount of a debriding formulation in a regimen of up to ten applications during a period of up to four weeks, wherein the debriding formulation formulated in the form of a hydrogel comprising: (i) a proteolytic enzyme mixture obtained from bromelain 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 other than a cross-linked polymer of acrylic acid, and wherein said debriding formulation is contacted with the wound site for at least four hours per application.

56. A debriding formulation comprising: (a) a composition in a dried or lyophilized form comprising: (i) a proteolytic enzyme mixture obtained from bromelain 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 other than a cross-linked polymer of acrylic acid; (iii) an anti-aggregation agent; (iv) a pH adjusting agent; and (b) water, wherein, prior to use, the composition (a) being admixed with the water (b) to form a debriding formulation characterized by being a homogenous hydrogel having a viscosity in the range of about 2,000,000 centipoise (cP) to about 8,500,000 cP and a pH ranging from about 6.0 to about 8.0, and wherein the amount of proteins in the debriding formulation ranges from about 0.5% (w/w) to about 7% (w/w) of the total weight of the debriding formulation.

57. The debriding formulation according to claim 56 comprising: (a) a composition in a dried or lyophilized form, present in a first compartment of a container or in a first container, the composition comprising: (i) a proteolytic enzyme mixture obtained from bromelain comprising stem bromelain (EC 3.4.22.32) and ananain (EC 3.4.22.31), wherein the amount of proteins in the debriding formulation ranges from about 0.5% (w/w) to about 7% (w/w), preferably from about 1% (w/w) to about 5% (w/w) of the total weight of the debriding formulation; (ii) guar gum in an amount ranging from about 0.25% (w/w) to about 5% (w/w) of the total weight of the debriding formulation; (iii) lactose in an amount ranging from about 10% (w/w) to about 25% (w/w) of the total weight of the debriding formulation; (iv) a pH adjusting agent; and (b) water in an amount of about 55% (w/w) to about 90% (w/w) present in a second compartment of the container or in a second container, wherein, prior to use, the composition (a) being admixed with the water (b) to form a debriding formulation characterized by being a homogenous hydrogel having a viscosity in the range of about 2,000,000 centipoise (cP) to about 8,500,000 cP and a pH ranging from about 6.0 to about 8.0.

58. The debriding formulation according to claim 57 comprising: TABLE-US-00006 Ingredient (%) w/w of formulation API 2 Guar gum 3.5 Lactose 18.05 Potassium phosphate dibasic 2.5 Potassium phosphate monobasic 0.8 PEG-3350 2 Water for injection 71.15

Description

BRIEF DESCRIPTION OF THE FIGURES

[0104] FIGS. 1A-F are photographs of chronic wounds induced in pigs. FIG. 1A shows the chronic wound before treatment and FIGS. 1B and 1C show the chronic wound at the 7th and 10.sup.th day of treatment, respectively, with the debriding formulation of the present invention. As a control, a chronic wound before treatment (FIG. 1D) or after treatment with vehicle only at the 7.sup.th and 10.sup.th day (FIGS. 1E and 1F, respectively) are shown.

[0105] FIG. 2 shows the percent clean area after the 10.sup.th treatment of chronic wounds induced in pigs as a function of the concentration of the active pharmaceutical ingredient (API) applied to the wound for 24 hours. Wide dash lines indicate confidence intervals (95%) of the model.

[0106] FIG. 3 shows the clean area under the curves (AUC) as a function of the concentration of API applied to chronic wounds induced in pigs for 24 hours. Wide dash lines indicate confidence intervals (95%) of the model.

[0107] FIG. 4 shows the eschar area under the curves (AUC) as a function of the concentration of API applied to chronic wounds induced in pigs for 24 hours. Wide dash lines indicate confidence intervals (95%) of the model.

DETAILED DESCRIPTION OF THE INVENTION

[0108] The present invention provides methods for debridement of a wound and/or for treating a wound and/or for promoting closure of a wound and/or for healing a wound comprising a step of topically applying to a wound site of a subject in need of such treatment a therapeutically effective amount of a debriding formulation in a regimen of up to ten applications during a time period of up to four weeks, wherein the debriding formulation present in the form of a hydrogel comprising: (i) a proteolytic enzyme mixture obtained from bromelain 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 other than a cross-linked polymer of acrylic acid, and wherein said debriding formulation is maintained in contact with the wound site for at least four hours per application. The present invention further provides a debriding formulation in the form of a hydrogel which comprises a proteolytic enzyme mixture obtained from bromelain and a water-soluble gelling agent other than a cross-linked polymer of acrylic acid.

[0109] It is now disclosed that applying the debriding formulation of the present invention on a chronic wound induced in pigs for up to 10 applications, when the debriding formulation is maintained in contact with the wound site for 24 hours per application, resulted in essentially complete eschar debridement of the chronic wounds. Similar debridement can be achieved if the debriding formulation is applied to a chronic wound three times a week for up to 10 applications when the debriding composition is maintained in contact with the wound site twice for 48 hours per application and once for 72 hours per application.

Debriding Formulation

[0110] The present invention provides a debriding formulation comprising a proteolytic enzyme mixture obtained from bromelain as an active ingredient and various excipients.

[0111] The term proteolytic enzyme mixture obtained from bromelain as used throughout the specification and claims refers to an enzymatic preparation partially purified from bromelain.

[0112] The term bromelain refers to a protein extract derived from the stems of pineapple plants which can be purchased commercially.

[0113] The proteolytic enzyme mixture obtained from bromelain (also termed Debrase or NexoBrid) and the preparation thereof are disclosed in WO 2006/054309 and WO 2013/011514, the content of which is incorporated by reference as if fully set forth herein. The proteolytic enzyme mixture obtained from bromelain comprises at least two of the cysteine proteases present in bromelain: stem bromelain (EC 3.4.22.32) and ananain (EC 3.4.22.31). The proteolytic mixture can further comprise one or more of the 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 3.4.22.31) precursor. The proteolytic mixture can further comprise cysteine protease fragments (see, for example, WO 2006/054309), a jacalin-like lectin, and/or bromelain inhibitors. According to a certain embodiment, the proteolytic mixture obtained from bromelain comprises stem bromelain (EC 3.4.22.32), ananain (EC 3.4.22.31), a cysteine protease precursor of bromelain, and a jacalin-like lectin.

[0114] The proteolytic enzyme mixture can be obtained by the procedure disclosed in WO 2013/011514. As the last step of the preparation, the proteolytic mixture is lyophilized and stored as a lyophilized powder until use.

[0115] The proteolytic enzyme mixture is highly stable and can be stored at 2-8 C. for long periods of time, e.g., up to three years. After this period of time, the proteolytic enzyme mixture maintains at least 90% of the original debriding activity which is determined immediately after the preparation process.

[0116] The proteolytic enzyme mixture is denoted throughout the specification and claims as the active principal ingredient (API). According to the invention, the amount of proteins, or alternatively the amount of API, in the debriding formulation ranges from about 0.5% (w/w) to about 7% (w/w) of the total weight of the debriding formulation. According to additional embodiments, the amount of proteins or API ranges from about 1% (w/w) to about 5% (w/w) , such as of about 1% (w/w), 2%, 3%, 4%, 5%, 6%, 7% of the total weight of the debriding formulation, or alternatively of about 2% (w/w) of the total weight of the debriding formulation.

[0117] The terms dry, dried, lyophilized or powdered composition as used interchangeably throughout the specification and claims refer to the composition which contains water in an amount of no more than about 5% (w/w) of the total weight of the composition, alternatively water is present in an amount of no more than about 3%, 2%, 1%, 0.5%, or further alternatively no more than about 0.1% (w/w) of the total weight of the composition. According to a certain embodiment, the composition is devoid of water.

[0118] The term hydrogel as used herein refers to an aqueous composition capable of maintaining a gel-like form.

[0119] The term homogenous hydrogel means a hydrogel having uniform viscosity (e.g., well mixed throughout).

[0120] The excipients of the debriding formulation are all pharmaceutically acceptable. The term pharmaceutically acceptable means approved by a regulatory agency of the Federal or a state government or listed in the U. S. Pharmacopeia or other generally recognized pharmacopeia for use in humans.

[0121] The term about refers to a value which is 10% above or below the indicated value.

[0122] According to some embodiments, the excipients of the debriding formulation are water-soluble. The term water soluble refers to an agent which typically has solubility in water in the range of 1 gr/ml to 1 gr/30 ml at room temperature.

[0123] The water-soluble gelling agent can be a naturally occurring gelling agent, a semi-synthetic gelling agent, and a synthetic gelling agent. The gelling agents according to the present invention do not include cross-linked polymers of acrylic acid.

[0124] The water-soluble naturally occurring gelling agent include, but are not limited to, water-soluble naturally occurring polysaccharides such as, for example, galactomannans, glucomannans, starches, agar, pectins, alginates, carrageenans, or a combination thereof. Each possibility represents a separate embodiment. Non-limiting examples of galactomannans and glucomannans are guar gum, locust bean gum, xanthan gum, gum acacia, gum tragacanth, gellan gums, and mixtures thereof. Each possibility represents a separate embodiment. According to a certain embodiment, the water-soluble naturally occurring gelling agent is guar gum.

[0125] Other biopolymers include, for example chitin, chitosan, collagens, gelatin, glycosaminoglycans such as, for example, heparin, chondroitin sulfate, dermatan sulfate, and heparan sulfate, proteoglycans, fibronectins, and laminins

[0126] Semi-synthetic gelling agents include, but are not limited to, cellulose ethers (e.g. hydroxyethyl cellulose, methyl cellulose, carboxymethyl cellulose, hydroxy propylmethyl cellulose), polyvinylpyrrolidone, polyvinylalcohol, hydroxypropyl guar gum, and the like.

[0127] The synthetic gelling agents include, but are not limited to, carboxyvinyl polymers, polyvinylpyrrolidone, polyvinyl acetate polymers, polyvinyl chloride polymers, polyvinylidene chloride polymers and the like.

[0128] The debriding composition can further comprise at least one excipient selected from the group consisting of an anti-aggregation agent and a pH adjusting agent.

[0129] The anti-aggregation or anti-agglomeration agent suitable for practicing the present invention is any known anti-aggregation agent, such as a water-soluble oligosaccharide, for example, lactose, sucrose, mannitol, sorbitol, and glucose. Each possibility represents a separate embodiment. According to a certain embodiment, the anti-aggregation agent is lactose.

[0130] The pH adjusting agent preferably has a pKa of above 6.0. In some embodiments, the pH adjusting agent can be any known pH adjusting agent such as, for example, potassium phosphate, potassium carbonate, sodium carbonate, and sodium phosphate. According to some embodiments, the pH adjusting agent is a combination of potassium phosphate monobasic and potassium phosphate dibasic present in an amount ranging from about 2% (w/w) to about 10% (w/w) of the total weight of the debriding formulation. It is now disclosed that higher amounts of potassium phosphate monobasic and potassium phosphate dibasic in the debriding formulation cause bleeding at the application site. It is therefore disclosed that if the pH adjusting agent is a combination of potassium phosphate monobasic and potassium phosphate dibasic, their total amount are preferably not higher than about 10% of the total weight of the debriding formulation in order to achieve efficient debridement without undesirable bleeding.

[0131] The composition can further comprise an anti-foaming agent. Anti-foaming agents are known in the art and include, but not limited to, polyethylene glycols, e.g., PEG-1450, PEG-3350, and the like. The composition can further comprise a preservative such as, for example, benzyl alcohol, parabens, methyl- or propylhydroxybenzoates; and/or an anti-oxidant such as, for example, ascorbic acid, dihydroquinone, butylated hydroxytoluene and dithiothreitol.

[0132] The composition can further comprise an anesthetic agent, an antibacterial agent, an antifungal agent, an anti-inflammatory agent, an analgesic agent, a growth factor and/or an agent promoting healing.

[0133] The anesthetic agents include, but are not limited to, amethocaine (tetracaine), lignocaine (lidocaine), xylocaine, bupivacaine, prilocaine, ropivacaine, benzocaine, mepivocaine, cocaine. Each possibility represents a separate embodiment.

[0134] The antibacterial agents include, but are not limited to, amanfadine hydrochloride, amanfadine sulfate, amikacin, amikacin sulfate, amoglycosides, amoxicillin, ampicillin, amsamycins, bacitracin, beta-lactams, candicidin, capreomycin, carbenicillin, cephalexin, cephaloridine, cephalothin, cefazolin, cephapirin, cephradine, cephaloglycin, chilomphenicols, chlorhexidine, chloshexidine gluconate, chlorhexidine hydrochloride, chloroxine, chlorquiraldol, chlortetracycline, chlortetracycline hydrochloride, ciprofloxacin, circulin, clindamycin, clindamycin hydrochloride, clotrimazole, cloxacillin, demeclocycline, diclosxacillin, diiodohydroxyquin, doxycycline, ethambutol, ethambutol hydrochloride, erythromycin, erythromycin estolate, erhmycin stearate, farnesol, floxacillin, gentamicin, gentamicin sulfate, gramicidin, giseofulvin, haloprogin, haloquinol, hexachlorophene, iminocylcline, iodochlorhydroxyquin, kanamycin, kanamycin sulfate, lincomycin, lineomycin, lineomycin hydrochloride, macrolides, meclocycline, methacycline, methacycline hydrochloride, methenine, methenamine hippurate, methenamine mandelate, methicillin, metonidazole, miconazole, miconazole hydrochloride, minocycline, minocycline hydrochloride, mupirocin, nafcillin, neomycin, neomycin sulfate, netimicin, netilmicin sulfate, nitrofurazone, norfloxacin, nystatin, octopirox, oleandomycin, orcephalosporins, oxacillin, oxyteacline, oxytetracycline hydrochloride, parachlorometa xylenol, paromomycin, paromomycin sulfate, penicillins, penicillin G, penicillin V, pentamidine, pentamidine hydrochloride, phenethicillin, polymyxins, quinolones, streptomycin sulfate, tetracycline, tobramycin, tolnaftate, triclosan, trifampin, rifamycin, rolitetracycline, silver salts, spectinomycin, spiramycin, struptomycin, sulfonamide, tetracyclines, tetracycline, tobramycin, tobramycin sulfate, triclocarbon, triclosan, trimethoprim-sulfamethoxazole, tylosin, vancomycin, and yrothricin. Each possibility represents a separate embodiment.

[0135] The antifungal agents include, but are not limited to, nystatin, clotrimazole, miconazole, ketoconazole, fluconazole, thiabendazole, econazole, clomidazole, isoconazole, tiabendazole, tioconazole, sulconazole, bifonazole, oxiconazole, fenticonazole, omoconazole, sertaconazole, and flutrimazole. Each possibility represents a separate embodiment.

[0136] The anti-inflammatory agent can be non-steroidal, steroidal, or a combination thereof. Non limiting examples of non-steroidal anti-inflammatory agents include oxicams, such as piroxicam, isoxicam, tenoxicam, sudoxicam; salicylates, such as aspirin, disalcid, benorylate, trilisate, safapryn, solprin, diflunisal, and fendosal; acetic acid derivatives, such as diclofenac, fenclofenac, indomethacin, sulindac, tolmetin, isoxepac, furofenac, tiopinac, zidometacin, acematacin, fentiazac, zomepirac, clindanac, oxepinac, felbinac, and ketorolac; fenamates, such as mefenamic, meclofenamic, flufenamic, niflumic, and tolfenamic acids; propionic acid derivatives, such as ibuprofen, naproxen, benoxaprofen, flurbiprofen, ketoprofen, fenoprofen, fenbufen, indopropfen, pirprofen, carprofen, oxaprozin, pranoprofen, miroprofen, tioxaprofen, suprofen, alminoprofen, and tiaprofenic; pyrazoles, such as phenylbutazone, oxyphenbutazone, feprazone, azapropazone, and trimethazone. Extracts of these non-steroidal anti-inflammatory agents may also be employed. Each possibility represents a separate embodiment.

[0137] Non-limiting examples of steroidal anti-inflammatory drugs include corticosteroids such as hydrocortisone, hydroxyl-triamcinolone, alpha-methyl dexamethasone, dexamethasone-phosphate, beclomethasone dipropionates, clobetasol valerate, desonide, desoxymethasone, desoxycorticosterone acetate, dexamethasone, dichlorisone, diflorasone diacetate, diflucortolone valerate, fluadrenolone, fluclorolone acetonide, fludrocortisone, flumethasone pivalate, fluosinolone acetonide, fluocinonide, flucortine butylesters, fluocortolone, fluprednidene (fluprednylidene) acetate, flurandrenolone, halcinonide, hydrocortisone acetate, hydrocortisone butyrate, methylprednisolone, triamcinolone acetonide, cortisone, cortodoxone, flucetonide, fludrocorisone, difluorosone diacetate, fluradrenolone, fludrocortisone, diflurosone diacetate, fluradrenolone acetonide, medrysone, amcinafel, amcinafide, betamethasone and the balance of its esters, chloroprednisone, chlorprednisone acetate, clocortelone, clescinolone, dichlorisone, diflurprednate, flucloronide, flunisolide, fluoromethalone, fluperolone, fluprednisolone, hydrocortisone valerate, hydrocortisone cyclopentylpropionate, hydrocortamate, meprednisone, paramethasone, prednisolone, prednisone, beclomethasone dipropionate, and triamcinolone. Each possibility represents a separate embodiment.

[0138] Analgesic agents include, but are not limited to, codeine, hydrocodone, oxycodone, fentanyl, and propoxyphene. Each possibility represents a separate embodiment.

[0139] The growth factors include, but are not limited to, epidermal growth factors, fibroblast growth factors, insulin-like growth factors, and the like.

[0140] Agents promoting healing include, but are not limited to, hyaluronic acid and the like.

[0141] The viscosity of the gel formulations of the present invention can be measured by any known means. According to some embodiments, an absolute viscometer with plate plate geometry can be used to calculate the viscosity of the gel formulations described herein. The viscosity ranges referred to herein are all measured at room temperature.

[0142] According to the principles of the present invention, the composition (a) which is present in a dry or powdered form and the water (b) can be placed in a first compartment and a second compartment, respectively, of a single container or can be placed in two separate containers. Before use, the composition (a) and the water (b) are admixed to form the debriding formulation.

[0143] The debriding formulations of the present invention are of low bacterial bioburden, and therefore the formulations of the present invention reduce the risk of further contaminating the wound site. According to some embodiments, the debriding formulations are sterile.

[0144] According to some embodiments, the debriding formulation comprises:

[0145] (a) a composition in a dried or powdered form comprising: [0146] (i) a proteolytic enzyme mixture obtained from bromelain comprising stem bromelain (EC 3.4.22.32) and ananain (EC 3.4.22.31); [0147] (ii) a water-soluble gelling agent, wherein the water-soluble gelling agent is other than a cross-linked polymer of acrylic acid; [0148] (iii) an anti-aggregation agent; [0149] (iv) a pH adjusting agent; and

[0150] (b) water,

[0151] wherein the composition (a) being admixed with the water (b) to form a debriding formulation characterized by being a homogenous hydrogel having a viscosity in the range of about 2,000,000 cP to about 8,500,000 cP and a pH ranging from about 6.0 to about 8.0, and wherein the amount of proteins in the debriding formulation ranges from about 0.5% (w/w) to about 7% (w/w) of the total weight of the debriding formulation.

[0152] According to some embodiments, the debriding formulation comprising: [0153] (a) a composition in a dried or powdered form, present in a first compartment of a container or in a first container, the composition comprising: [0154] (i) the proteolytic enzyme mixture obtained from bromelain comprising stem bromelain (EC 3.4.22.32) and ananain (EC 3.4.22.31); [0155] (ii) guar gum in an amount ranging from about 0.25% (w/w) to about 5% (w/w) of the total weight of the debriding formulation; [0156] (iii) lactose in an amount ranging from about 10% (w/w) to about 25% (w/w) of the total weight of the debriding formulation; [0157] (iv) a pH adjusting agent; and [0158] (b) water in an amount ranging from about 55% (w/w) to about 90% (w/w), present in

[0159] the second compartment of the container or in the second container.

[0160] It is to be understood that the debriding formulations of the present invention are formulated as gels, i.e., hydrogels, and as such are applied on to the wound site. Preferably, the debriding formulations are not patch formulations. According to some embodiments, the formulations are devoid of adhesive agents, and thus the formulations are non adhesive.

Uses of the Debriding Formulation

[0161] The present invention provides a method for debridement of a skin wound and/or treating a skin wound comprising a step of topically applying to a wound site of a subject in need of such treatment a therapeutically effective amount of a debriding formulation in a regimen of up to ten applications during a time period of up to four weeks, wherein the debriding formulation present in the form of a hydrogel comprising: (i) a proteolytic enzyme mixture obtained from bromelain 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 other than a cross-linked polymer of acrylic acid, and wherein said debriding formulation is maintained in contact with the wound site for at least four hours per application.

[0162] According to some embodiments, the wound is a chronic or hard to heal wound.

[0163] The terms chronic wound, chronic skin wound or a hard to heal wound as used interchangeably throughout the specification and claims refer to a wound that has failed to proceed through an orderly and timely series of events to produce a durable structural, functional, and/or cosmetic closure as wounds do. Wounds that do not heal within one month are considered chronic wounds.

[0164] According to some embodiments, the chronic wound is selected from the group consisting of a diabetic ulcer, a venous stasis ulcer, an arterial insufficiency ulcer, a pressure ulcer, a post-operative and a post trauma wound. Each possibility represents a separate embodiment. According to further embodiments, the chronic wound is a diabetic lower extremity ulcer or a venous leg ulcer.

[0165] The term debridement of a wound as used herein refers to the removal of nonviable tissue: necrotic eschar, slough or fibrin, foreign material, and bacteria/biofilm from a wound. Necrotic eschar is a thin or thick, leathery, devitalized, black, brown or tan tissue, whereas slough and biofilm are exudative, white or yellow-greenish mottled, tenuous tissue on the wound bed. Necrotic tissue, foreign material and bacteria impede the body's attempt to heal by producing or stimulating the production of metalloproteases that interfere with the local wound-healing process. This hostile environment allows bacteria to proliferate, further colonize the wound within the exudates, debris, and purulent discharges (slough) that cover the wound bed. In addition, the bacteria secrete structural products that together with the slough form the biofilm, thus protect their colonies from potential destruction. The bacteria produce their own wound-inhibiting enzymes and, more significantly, consume much of the scarce, available local resources that are necessary for wound healing.

[0166] According to some embodiments, debridement of a wound refers to removal of at least 50%, alternatively of at least 75% of the non-viable tissue which is present prior to treatment. Each possibility represents a separate embodiment of the invention. According to certain embodiments, debridement of a wound refers to removal of at least 90%, or of at least 95%, and preferably of 100% of the non-viable tissue which is present prior to treatment; such debridement, namely of 90% or more of the non-viable tissue present prior to treatment is referred throughout the specification and claims as complete debridement of a wound.

[0167] In chronic or hard to heal wounds several different factors may play an important role. Exposed surfaces such as bone, tendons, fascia or even fat do not support cellular proliferation and they dry and become foreign bodies such as synthetic implants. Any interference with local blood supply (arterial, venous, lymphatic, pressure etc.) may cause a wound to become hard to heal and chronic. Granulation tissue may become recalcitrant, atrophic, lose its rich vascular matrix, become darker and opaque in color and will not take any part in the wound healing and closure processes.

[0168] The term wound bed preparation as used herein refers to a wound bed which results from a proper debridement in order to accelerate endogenous healing or to facilitate the effectiveness of other therapeutic measures. It is a process of debriding, removing various burdens within both the wound and the patient that impede healing. Burdens within the wound include exudate, bacteria, biofilm and necrotic/cellular debris. The overall health status of the patient is important to the healing process. In chronic or hard to heal wounds complete removal of the offending eschar, slough or biofilm may result in a clean wound bed, yet such a wound bed may still be inadequate for future healing if the patient's systemic or the extremity's condition cannot support it.

[0169] A wound bed prepared for healing is one without eschar, slough, fibrin or biofilm that also has a viable bed of healthy tissues and/or healthy granulation tissue (level >7 in the granulometer scale) that will allow the wound to close spontaneously by scarring and contracture-epithelialization (optionally using modalities such as biological dressings, wound-healing enhancing dressings, synthetic wound dressings, vacuum or ozone wound healing systems) over the viable, clean bed or will support autologous STSG (Split Thickness Skin Graft) or skin allografting.

[0170] The term wound closure refers to the process of regenerating the covering cell layers of a tissue. Thus, promoting wound closure means creating a positive effect in the regeneration of the covering cell layers. The positive effect can be an acceleration of the regeneration process or a decrease of the damaged area of the wound. Wound closure is also defined as full epithelialization without drainage, and without need for additional dressing, confirmed at two consecutive study visits 2 weeks apart

[0171] The term therapeutically effective amount is that amount of the proteolytic enzyme mixture which is sufficient to provide a beneficial effect to the subject to which the composition is administered.

[0172] According to some embodiments, the debriding formulation can be applied to a wound site up to 10 applications, wherein the debriding formulation is maintained in contact with the wound site for at least four hours per application per day.

[0173] According to additional embodiments, the debriding formulation can be applied in a regimen of up to 10 times to a wound site, wherein the debriding formulation is maintained in contact with the wound site for about 24 hours per application. Thus, the debriding formulation can be applied daily for up to 10 consecutive days so as to be maintained in contact with the wound site for about 24 hours per application or can be applied continuously 1, 2, 3, 4, 5, 6, 7, 8, or 9 applications for about 24 hours per application with a halt of application in between of one day or more as required.

[0174] According to additional embodiments, the debriding formulation can be applied in a regimen of up to 10 times to a wound site, wherein the debriding formulation is maintained in contact with the wound site for about 48 hours per application. Thus, the debriding formulation can be applied every other day for up to 20 consecutive days or can be applied 1, 2, 3, 4, 5, 6, 7, 8, or 9 times every other day with a halt of application in between of one day or more as required.

[0175] According to further embodiments, the debriding formulation can be applied in a regimen of up to 10 times to a wound site, three times a week, wherein the debriding formulation is maintained in contact with the wound site for a duration selected from the group consisting of 48 hours per application and 72 hours per application.

[0176] According to additional embodiments, the debriding formulation can be applied in a regimen of up to 10 times to a wound site, wherein the debriding formulation is maintained in contact with the wound site for about 72 hours per application.

[0177] According to certain embodiments, the debriding formulation is maintained in contact with the wound site up to about 72 hours per application.

[0178] After the contact of the debriding formulation with the wound site for the indicated application time, such as after at least 4 hours treatment, or after the 24 hours treatment, or after the 48 hours treatment, or after the 72 hours treatment, the wound site can be washed. Thus, the methods of the present invention can further comprise a step of washing the wound site after said contact, prior to a subsequent application of the debriding formulation. If a halt of application is performed, the wound site can be covered with a moist dressing such as moist saline gauze.

[0179] According to some embodiments, the methods of the present invention can further comprise a step of covering the debriding formulationwith an occlusive layer or dressing to maintain or hold the composition at the wound site.

[0180] According to additional embodiments, the method of the present invention can further comprise a step of protecting the wound edges and the peri-wound skin during debridement.

[0181] The ranges of numerical values indicated throughout the specification and claims include any integer in between.

[0182] It is to be understood that the regimens defined in any of the above can be repeated one, two, three or more times until the eschar/necrotic tissue is completely debrided, optionally with a halt of application. The halt of application can be of days, weeks or months. The regimen of application of the debriding formulation can be repeated as necessary to debride eschar. If eschar reoccurs, the regimen of application of the debriding formulation can be repeated as necessary to debride eschar.

[0183] The present invention encompasses combination therapy wherein the methods of the present invention can be combined with known debridement methods, such as, surgical or sharp debridement. According to some embodiments, the methods of the present invention can be performed prior to surgical or sharp debridement. Alternatively, the methods of the present invention can be performed after surgical or sharp debridement.

[0184] According to some embodiments, the amount of API applied ranged between about 0.1 gr to about 2 gr of the sterile lyophilized proteolytic enzyme mixture per 100 cm.sup.2 of wound surface. According to additional embodiments, the amount of hydrogel applied to a wound site is of about 20 gr per 100 cm.sup.2 of wound surface.

EXAMPLE 1

Gel Formulation

[0185] The following debriding formulations were developed:

TABLE-US-00003 % (w/w) in Ingredient formulation Function Proteolytic enzyme mixture 5* Active ingredients obtained from bromelain (API) (API) Guar gum 3.5 Gelling agent Lactose 15** Anti-agglomeration agent Potassium phosphate dibasic 2.5 pH adjusting agent Potassium phosphate 0.8 pH adjusting agent monobasic PEG-3350 2 Anti-foaming agent Water for injection Complete to 100% *Other amounts of API (w/w) which were evaluated: 0.1%; 0.5%; 1%; and 2%. **The amount of lactose was adjusted according to the amount of API.

[0186] The debriding formulations were prepared by admixing the dried or powdered composition which contained API, guar gum, lactose, potassium phosphate dibasic and monobasic, and PEG-3350, with water to form the hydrogel having a homogenous appearance and which has a viscosity ranging from 2,40,000 cP to 6,200,000 cP.

EXAMPLE 2

Debridement of Eschar by the Gel Formulation

[0187] The aim of this study was to determine the dose of the active ingredients in the gel formulation which provides maximal efficacy of eschar debridement of chronic wounds.

[0188] A chronic wound model was established in crossbred domestic pigs.

[0189] Prior to application of the gel formulation, wound edges were protected with thick layer of Vaseline. Each wound site received 2 g of the gel formulation to cover the wound for 24 hours, and bandaged with non absorbing dressing. Each wound was photographed before and after each application. The following doses were examined: placebo (0%), 0.1%, 0.5%, 1%, 2%, 5%.

[0190] This procedure was followed for up to 11 consecutive daily treatments or until clean wound bed was achieved. This period was denoted the Treatment period. The treatment period was followed by two weeks recovery period with no treatments. In the recovery period the wounds were photographed 3 times a week.

[0191] The wound area, clean area and eschar volume were evaluated visually, measured by ImageJ software (NIH, MD, USA) and analyzed by JMP statistical software (SAS Inc., NC, USA).

[0192] On the first treatment day, all wounds were covered by a full eschar. The eschar composed of 2 distinct areas: [0193] a. the center of the wound, which was covered with thin eschar layer; [0194] b. the wound edges, which were characterized by fully necrotic tissue.
Representative photographs of the wound before treatment and after treatment 7 or 10 days with the gel formulation or with the gel vehicle are shown in FIGS. 1A-1F.

[0195] At the beginning of the treatment period, the chronic wounds already developed eschar. FIGS. 1A and 1D are representative photographs showing the chronic wound before treatment. The eschar was composed of two distinct areas: the center of the wound where the skin was fully excised, and the wound edges. In the center, a thin eschar layer was developed, while in the edges, the eschar was composed of necrotic skin. In chronic wounds treated with the gel formulation containing API, the eschar softened and gradually dissolved from treatment to treatment until the dissolution at the circumference caused the eschar to wholly disconnect from the viable tissue (FIGS. 1B and 1C). In contrast, chronic wounds treated with the gel vehicle barely changed their appearance and consistency during the treatment period (FIGS. 1E and 1F).

[0196] The clean area was calculated as percent from total wound size. The volume of the eschar was calculated as percentage from the eschar volume just before the first treatment, taking into account both the area and the thickness of eschar.

[0197] The efficacy of the treatments was evaluated by measuring the area under the curve (AUC) where the x axis is the day of treatment and y axis is the percent clean area or percent eschar volume. The more effective the treatment, the larger is the area under the curve for percent clean area and the smaller the area under the curve for eschar volume.

[0198] To assess the irritation caused by the formulation, five blinded assessors scored each wound according to the photographs of the entire experiment.

TABLE-US-00004 TABLE 1 Summary of the debridement results Parameter (Y) Gel formulation Placebo % clean area 82 52 % eschar from day 0 7 31 AUC clean 454 274 AUC eschar 531 850

Percent Clean Area Out of the Wound Area at the End of the Treatment Period:

[0199] The percent of clean area at the end of treatments out of the initial wound area was found to be significantly dependent on the amount of API. The dependency on the amount of API was linear (FIG. 2).

[0200] At a dose of 5% of API in the gel formulation, an average of 82% of the wound was clean.

Percent of Eschar Out of the Initial Amount of the Wound at the End of the Treatment Period:

[0201] The percent of eschar at the end of treatments out of the initial amount was found to be significantly dependent on the amount of API. The dependency on the amount of API was linear.

[0202] At a dose of 5% of API in the gel formulation, an average of 93% of the eschar was removed.

The Area Under the Curve (AUC) where the x Axis is Day of Treatment and the y Axis is the Percent Clean Area:

[0203] This parameter shows the cleaning efficacy of the treatments: the more effective the treatments are, the larger the AUC. The AUC of percent clean area during treatments was significantly dependent on the amount of API. As shown in FIG. 3, the dependency of AUC percent clean area on API amount was linear.

The Area Under the Curve (AUC) where the x Axis is Day of Treatment and the y Axis is the Percent of Eschar Out of the Initial Amount of Eschar:

[0204] This parameter shows the eschar removal efficacy of the treatments: the more effective the treatments are, the smaller the AUC. This parameter was found to be significantly dependent on the mount of API. The dependency on API amount was linear (FIG. 4).

[0205] Taken together, these results indicated that the effect of API was does and time dependent.

Irritation

[0206] Five treatment-blinded assessors scored each wound based on the photographs of the wounds through the entire experiment. The irritation caused by the placebo was found to be significantly lower than that of the treatment groups. Irritation was dependent on API amount and disappeared completely after a day or two in the follow up period in all treatments.

EXAMPLE 3

Efficacy and Safety of API in the Gel FormulationClinical Study

[0207] The aim of this study is to assess the safety and the efficacy of two doses: 2% (w/w) and 5% (w/w) of the gel formulation disclosed herein above in Example 1, also designated EX-02, compared to placebo in debridement of chronic venous leg ulcers and of diabetic lower extremity ulcers.

[0208] The study is a multicenter, prospective, randomized, placebo controlled, double-blind, international study.

[0209] Adults with >50% necrotic/slough/fibrin non-viable tissue on a chronic wound (venous leg ulcer, diabetic lower extremity ulcer) between 3 cm.sup.2 and 200 cm.sup.2 (surface area) are enrolled into the study.

[0210] Patients are randomized to EX-02 Low dose (2% w/w), EX-02 high dose (5% w/w), or Placebo treatment group. Treatment is performed three times a week up to 10 applications (up to 10 visits) or until complete debridement is achieved, whichever occurs first. The duration of each application is 242 hours or three times a week, namely 484 hours and 724 hours per application. Following each application the wound is washed, photographed and assessed for wound size and removal of nonviable tissue (by digital planimetry software) and change in granulation tissue, wound status, and safety parameters. The 24 hour treatments are performed successively during week days. During weekends the wound are dressed with moist-to-moist saline gauze.

[0211] Following completion of the debridement treatment period, patients are treated according to standard procedures and evaluated (wound assessments) once a week until complete wound closure for up to 12 weeks from last application (up to 12 visits). For patients who achieved wound closure, additional 3 monthly follow- up visits of wound closure confirmation are conducted; the first monthly visit is performed 2 weeks after reaching wound closure. For patients who didn't achieve wound closure during the 12-weeks follow-up visits, only the 3-months follow-up visit (week 30) is conducted. The placebo is prepared as a powder of the excipients only and water for the preparation of a gel.

[0212] The following endpoints are evaluated and compared between EX-02 and Placebo for all wounds:

Primary Endpoint

[0213] Incidence of complete debridement (non-viable tissue removal) at the end of the debridement period (up to 8 treatment days)

Secondary Endpoints

[0214] 1. Time to achieve complete debridement (within up to 8 treatments); [0215] 2. Number of applications/treatment days to achieve complete debridement; [0216] 3. Assessment of changes in wound debridement status during treatment period : percentage reduction in non viable tissue (daily, during 8 treatments); [0217] 4. Time to achieve complete granulation (up to 12 weeks); [0218] 5. Incidence of complete granulation (on week 12); [0219] 6. Percent of change in granulation tissue over time (weekly, during baseline-12 weeks); [0220] 7. Incidence of complete wound closure (up to 12 weeks). Wound closure is defined as full epithelialization without drainage, and without need for additional dressing, confirmed at two consecutive study visits 2 weeks apart; [0221] 8. Time to complete wound closure (up to 12 weeks); [0222] 9. Wound area reduction: percentage reduction in wound size over time (weekly, from baseline up to 12 weeks). [0223] 10. Incidence of infection.

[0224] It will be appreciated by persons skilled in the art that the present invention is not limited by what has been particularly shown and described herein above. Rather the scope of the invention is defined by the claims that follow.