STABLE THERMOLYSIN HYDROGEL

Abstract

Disclosed is a hydrogel comprising a hydrophilic gelling agent that includes a nonionic cellulose ether, and active thermolysin, wherein the proteolytic activity of the thermolysin does not decrease by more than 20% when stored at room temperature for 6 months.

Claims

1-25. (canceled)

26. A hydrogel comprising a hydrophilic gelling agent that includes a nonionic cellulose ether, water, and active thermolysin; wherein the proteolytic activity of the thermolysin does not decrease by more than 20% when stored at room temperature for 6 months, and wherein the nonionic cellulose ether is hydroxyethylcellulose, hydroxypropylcellulose, or the combination thereof.

27. The hydrogel of claim 26, wherein the hydrogel further comprises a diol.

28. The hydrogel of claim 27, wherein the diol is propylene glycol.

29. The hydrogel of claim 26, wherein the hydrogel further comprises a preservative.

30. The hydrogel of claim 29, wherein the preservative is methylparaben, propylparaben, or phenoxyethanol, or mixtures thereof.

31. The hydrogel of claim 26, wherein the hydrogel further comprises a metal salt.

32. The hydrogel of claim 31, wherein the metal salt is sodium chloride or calcium chloride or mixtures thereof.

33. The hydrogel of claim 26, further comprising a buffer having a pH of 7.0 to 8.0.

34. The hydrogel of claim 33, wherein the hydrogel comprises a buffer having a pH of about 7.5

35. The hydrogel of claim 26, wherein the proteolytic activity of the thermolysin does not decrease by more than 10% when stored at room temperature for 6 months.

36. The hydrogel of claim 26, wherein the proteolytic activity of the thermolysin does not decrease by more than 10% when stored at room temperature for 24 months.

37. The hydrogel of claim 26, comprising 0.1 to 5% w/w of the thermolysin.

38. The hydrogel of claim 26, wherein the thermolysin is solubilized within the hydrogel.

39. The hydrogel of claim 26, wherein the thermolysin is suspended within the hydrogel.

40. The hydrogel of claim 26, wherein the thermolysin is partially solubilized and partially suspended within the hydrogel.

41. The hydrogel of claim 26, wherein the hydrogel is sterile.

42. A method of debriding a wound comprising topically applying a composition according to claim 26 to a wound in need of debridement.

43. The method of claim 42, wherein the wound is a chronic wound.

44. The method of claim 43, wherein the chronic wound is a diabetic foot ulcer, a venous leg ulcer, an arterial leg ulcer, a decubitus ulcer, a stasis ulcer, a dermal ulcer, a burn, or a pressure ulcer.

45. The method of claim 42, wherein the wound includes necrotic tissue.

46. The method of claim 45, wherein the necrotic tissue is an eschar.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0019] FIG. 1. A graph showing 1 month thermolysin activity expressed as % Target in a hydrogel of the present invention (HEC) and in hydrogels that do not include a nonionic cellulose ether.

[0020] FIG. 2. A graph showing thermolysin activity expressed as % Target over 24 months when thermolysin is provided in an aqueous hydrogel of the present invention and stored at 4° C., room temperature (20-25° C.), and 40° C. The formulation used in FIG. 2 is provided in Table 1.

[0021] FIG. 3. A graph showing the percentage debridement in pig eschar wounds over time following treatment with SANTYL® collagenase ointment, a papain/urea ointment (with 2.5% papain and 10% urea), a thin (˜50,000 cps) thermolysin aqueous hydrogel of the present invention, and a thick (˜70,000 cps) thermolysin aqueous hydrogel of the present invention. The formulations for the thin and thick hydrogels are provided in Tables 2 and 3, respectively.

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

[0022] Effective wound cleansing and debridement help facilitate the healing process. Devitalized tissue present in a wound bed is undesirable—it serves as a reservoir for bacterial growth, contains elevated levels of inflammatory mediators that promote chronic inflammation at the wound site, and impairs cellular migration. The four most common methods used to debride a wound include surgical, autolytic, enzymatic, and mechanical debridement.

[0023] With respect to enzymatic debridement, currently SANTYL® Collagenase Ointment is the only approved enzymatic debriding drug product in the U.S. marketplace. This product is a hydrophobic ointment that utilizes collagenase as the debriding agent. While others have attempted to use different debriding agents and various formulations, one of the problems faced is that of stability. In particular, the instability of debriding agents limits their use to effectively debride wounds.

[0024] The inventors have discovered a solution to the instability issues surrounding the debriding agent, thermolysin. In particular, the inventors discovered a way to stabilize thermolysin in an aqueous environment by using a nonionic cellulose ether to create a hydrogel that contains and stabilizes active thermolysin. These and other non-limiting aspects of the present invention are described in the following subsections.

A. Hydrogels

[0025] The compositions of the present invention are formulated as hydrogels in that that the continuous medium of the formulation is an aqueous environment that has been gelled with a nonionic cellulose ether. The thermolysin can be suspended or solubilized within the gel. It can also be partially solubilized and partially suspended within the gel. Further, other additives can also be added to the hydrogels of the present invention.

[0026] 1. Thermolysin

[0027] Thermolysin is a thermostable metalloproteinase that cleaves at the N-terminus of the hydrophobic residues leucine, phenylalanine, valine, isoleucine, alanine and methionine. The optimal digestion temperature range is 40-80° C. Thermolysin activity is optimal at pH 5.0-8.5. The term “active thermolysin” means thermolysin which exhibits proteolytic activity. Amano Japan is a manufacturer and supplier of thermolysin. The Amano thermolysin was obtained from a bacterial species called Bacillus thermoproteolyticus rokko. The CAS No. for thermolysin is 9073-78-3. The thermolysin used in the Examples of this specification is from Amano.

[0028] 2. Nonionic Cellulose Ethers

[0029] Nonionic cellulose ethers are high-molecular-weight compounds that can be made by replacing the hydrogen atoms of hydroxyl groups in the glucose units of cellulose with alkyl or hydroxylalkyl groups. Non-limiting examples of non-ionic alkyl cellulose ethers include methyl cellulose (MC), ethyl cellulose (EC), and ethyl methyl cellulose (EMC). Non-limiting examples of non-ionic hydroxyalkyl cellulose ethers include hydroxyethyl cellulose (HEC), hydroxylpropyl cellulose (HPC), hydroxymethyl cellulose (HMC), hydroxypropylmethyl cellulose (HPMC), ethylhydroxyethyl cellulose (EHEC), hydroxyethylmethyl cellulose (HEMC), methylhydroxyethyl cellulose (MHEC), methylhydroxypropylcellulose (MHPC), and hydroxyethylcarboxymethyl cellulose (HECMC). There are a wide range of commercial sources for each of these cellulose ethers (e.g., Dow Chemical Company (USA), Ashland (USA), Samsung Fine Chemicals (USA)). Additional commercial sources of these nonionic cellulose ethers can be found in the International Cosmetic Ingredient Dictionary and Handbook, 12.sup.th Edition (2008), volumes 1-3.

[0030] 3. Additives

[0031] In addition to thermolysin, nonionic cellulose ether, and water, the hydrogels can include additional ingredients. For instance, water-soluble solvents such as propylene glycol or other diols can be used. Triols and polyols can also be used in the hydrogels. A non-limiting example of a triol is glycerin. The amount of such solvents can range from 1 to 20% w/w, although more or less can be added to achieve a desired result for the hydrogel. Metal salts such as NaCb or CaCb can be used as stabilization agents. The amounts of salt can range from 0.01 to 1% w/w, although more or less can be added to achieve a desired result for the hydrogel. Preservatives such as methylparaben, propylparaben, and phenoxyethanol can be used to preserve the hydrogel. The amounts of such preservatives can range from 0.01 to 1% w/w, although more or less can be added to achieve a desired result for the hydrogel. Buffers having an appropriate pH range (e.g., 6.5 to 9 or 7 to 8 or about 7.5) for thermolysin can be used. An example of such a buffer is Tris buffer (lO mM, pH=7.5). The water source for the hydrogel can come from the buffer solution. Therefore, the amount of buffer that can be added is an amount that fills out the formulation (e.g., q.s. to 100%). Further additives can be added to achieve a given tactile property or to add an additional functional aspect to the hydrogel (e.g., agents that can further aid in the wound healing process such as vulnerary agents, antimicrobial agents, anti-inflammatory agents, pain-relieving agents, etc.).

[0032] 4. Preparation of the Hydrogel

[0033] The hydrogels of the present invention can be prepared by mixing propylene glycol and preservatives (e.g., methylparaben and propylparaben) in de-ionized (DI) water or Tris buffer (pH=7.5) at 70° C. Upon solubilization (i.e., clear solution is obtained), the solution can be cooled to room temperature (RT) and a gelling agent (e.g., Hydroxyethylcellulose-HEC) can be added. The mixture can then be stirred until homogeneous (i.e., no visible particles of gelling agent, HEC), thereby forming a hydrogel. The active phase can be made by mixing thermolysin, NaCl and CaCh in DI water or Tris buffer (pH=7.5) at appropriate concentrations. Upon homogenization (a white slurry will be obtained), the active phase can then be added to a hydrogel (at appropriate concentration). The final mixture can then be stirred at RT. A milky hydrogel is then obtained.

B. Methods of Using the Hydrogels

[0034] The hydrogels of the present invention may be used in a variety of methods. For instance, they can be used to debride wounds such as chronic wound. Such methods include applying to the wound a composition comprising thermolysin (e.g., via topical application). After application, the wound may be covered with a wound dressing such as a gauze pad. The hydrogel may be applied to a dressing such as a gauze pad first and then applied to the wound surface. The application amount depends on the severity and type of the wound and nature of the subject.

[0035] In certain aspects, the hydrogel can be applied to the wound periodically, for example, daily, twice daily, once every other day, once a week, as needed. A therapeutic regimen can be used that includes periodic dressing changes with wound cleansing and application of fresh composition between changes until the debridement of the necrotic tissue is complete.

[0036] A wide variety of wounds can be treated with the hydrogels of the present invention. Examples include burns, acute wounds, or chronic wounds. Non-limiting examples of chronic wounds include diabetic foot ulcers, venous leg ulcers, arterial leg ulcers, decubitus ulcers, stasis ulcers, dermal ulcers, burns, and pressure ulcers.

C. Kits

[0037] Kits are also contemplated as being used in certain aspects of the present invention. For instance, a hydrogel of the present invention can be included in a kit. A kit can include a container. Containers can include a bottle, a metal tube, a laminate tube, a plastic tube, a dispenser, a pressurized container, a barrier container, a package, a compartment, or other types of containers such as injection or blow-molded plastic containers into which the hydrogels are retained. The kit can include indicia on its surface. The indicia, for example, can be a word, a phrase, an abbreviation, a picture, or a symbol.

[0038] The containers can dispense a pre-determined amount of a composition. In other embodiments, the container can be squeezed (e.g., metal, laminate, or plastic tube) to dispense a desired amount of the composition. The composition can be dispensed as a spray, foam, an aerosol, a liquid, a fluid, or a semi-solid. The containers can have spray, pump, or squeeze mechanisms. The containers can be designed for single-dose or multiple-dose dispensing. A kit can also include instructions for using the kit and/or compositions.

[0039] Further, the hydrogels of the present invention may also be sterile, and the kits containing such hydrogels can be used to preserve the sterile hydrogel. The hydrogels may be sterilized via an aseptic manufacturing process or sterilized after packaging by methods known in the art.

D. Examples

[0040] The following examples are included to demonstrate certain non-limiting aspects of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples that follow represent techniques discovered by the applicants to function well in the practice of the invention. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments that are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.

[0041] 1. Exemplary Formulations

[0042] The following Tables provide non-limiting examples of various hydrogel formulations that include thermolysin and a cellulosic ether.

TABLE-US-00001 TABLE 1 Thermolysin/HEC Hydrogel (in FIG. 2) % Concentration Ingredient (by weight) Hydroxyethylcellulose (HEC) 2.68 NaCl 0.28 CaCb 0.11 Propylene Glycol 14.2 Methyl Paraben 0.24 Propyl Paraben 0.05 Thermolysin 0.3917 Tris buffer (10 mM, pH 7.5) q.s. Total 100

[0043] Process: HEC used was Natrosol® 250 Pharm from Ashland (USA). The buffer used was Tris buffer (10 mM, pH 7.5). The hydrogel was prepared by mixing propylene glycol and preservatives (e.g., methyl and propyl paraben) in Tris Buffer at 70° C. Upon solubilization (i.e., clear solution is obtained), the solution is cooled to room temperature (RT) and gelling agent (HEC) was added. The mixture is stirred until homogeneous (i.e., no visible particles of gelling agent, HEC), a hydrogel is formed. The active phase is made by mixing thermolysin, NaCl and CaCb in DI water or Tris buffer (pH=7.5) at appropriate concentration. Upon homogenization (a white slurry will be obtained), the active phase is added to a hydrogel (at appropriate concentration). The final mixture is stirred for at least 2 h at RT. Milky hydrogel is obtained having an average viscosity of 54,876 cps (standard deviation of 251.66, and based on three separate measurements from three samples of same hydrogel), as measured on a Brookfield Viscometer at lO rpm, spindle #14, RT, 30 sec. Thermolysin was stable in this formulation for 6 months, 12 months, 18 months and 24 months at room temperature and 4° C. as measured by Casein digestion method.

TABLE-US-00002 TABLE 2 Thermolysin/HEC Hydrogel (“Thin Gel” in FIG. 3) % Concentration Ingredient (by weight) Hydroxyethylcellulose (HEC) 3 NaCl 0.28 CaCb 0.11 Propylene Glycol 14.9 Methyl Paraben 0.25 Propyl Paraben 0.05 Thermolysin 0.795 Tris buffer (10 mM, pH 7.5) q.s. Total 100

[0044] Process: HEC used was Natrosol® 250 Pharm from Ashland (USA). The buffer used was Tris buffer (10 mM, pH 7.5). The hydrogel was prepared by mixing propylene glycol and preservatives (e.g., methyl and propyl paraben) in Tris buffer at 70° C. Upon solubilization (i.e., clear solution is obtained), the solution is cooled to room temperature (RT) and gelling agent (HEC) was added. The mixture is stirred until homogeneous (i.e., no visible particles of gelling agent, HEC), a hydrogel is formed. The active phase is made by mixing thermolysin, NaCl and CaCb in DI water or Tris buffer (pH=7.5) at appropriate concentration. Upon homogenization (a white slurry will be obtained), the active phase is added to a hydrogel (at appropriate concentration). The final mixture is stirred for at least 2 h at RT. Milky hydrogel is obtained having an average viscosity of 64,900 cps (standard deviation of 173.21 based on three separate measurements from three samples of same hydrogel), as measured on a Brookfield Viscometer at lO rpm, spindle #14, RT, 30 sec.

TABLE-US-00003 TABLE 3 Thermolysin/HEC Hydrogel (“Thick Gel” in FIG. 3) % Concentration Ingredient (by weight) Hydroxyethylcellulose (HEC) 4 NaCl 0.28 CaCb 0.11 Propylene Glycol 14.77 Methyl Paraben 0.25 Propyl Paraben 0.05 Thermolysin 0.797 Tris buffer (10 mM, pH 7.5) q.s. Total 100

[0045] Process: HEC used was Natrosol® 250 Pharm from Ashland (USA). The buffer used was Tris buffer (10 mM, pH 7.5). The hydrogel was prepared by mixing propylene glycol and preservatives (e.g., methyl and propyl paraben) in Tris buffer at 70° C. Upon solubilization (i.e., clear solution is obtained), the solution is cooled to room temperature (RT) and gelling agent (HEC) was added. The mixture is stirred until homogeneous (i.e., no visible particles of gelling agent, HEC), a hydrogel is formed. The active phase is made by mixing thermolysin, NaCl and CaCb in DI Water or Tris buffer (pH=7.5) at appropriate concentration. Upon homogenization (a white slurry will be obtained), the active phase is added to a hydrogel (at appropriate concentration). The final mixture is stirred for at least 2 h at RT. Milky hydrogel is obtained having an average viscosity of 170,067 cps (standard deviation of 25,516, and based on three separate measurements from three samples of same hydrogel), as measured on a Brookfield Viscometer at 5 rpm, spindle #14, RT, 60 sec.

TABLE-US-00004 TABLE 4 Thermolysin/HPMC Hydrogel % Concentration Ingredient (by weight) Hydroxymethylpropylcellulose (HPMC) 1.05 Propylene Glycol 20.17 Methyl Paraben 0.27 Propyl Paraben 0.05 Thermolysin 0.202 DI Water q.s. Total 100

[0046] Process: HPMC used was Methocel K-15M from DOW (USA). The suspending media was DI water. The hydrogel was prepared by mixing propylene glycol and preservatives (e.g., methyl and propyl paraben) in Iris buffer at 70 C. Upon solubilization (i.e., clear solution is obtained), the solution is cooled to room temperature (RI) and gelling agent (HPMC) was added. The mixture is stirred until homogeneous (i.e., no visible particles of gelling agent, HPMC), a hydrogel is formed. The active phase is made by mixing thermolysin, in DI water at appropriate concentration. Upon homogenization (a white slurry will be obtained), the active phase is added to a hydrogel (at appropriate concentration). The final mixture is stirred for at least 2 h at RI. Milky hydrogel is obtained having an average viscosity of 9,400 cps (standard deviation of 100, and based on three separate measurements from three samples of same hydrogel), as measured on a Brookfield Viscometer at lO rpm, spindle #14, RT, 30 sec. Thermolysin was stable in this formulation for 10 months at room temperature and 4° C. as measured by Casein digestion method.

TABLE-US-00005 TABLE 5 Thermolysin/HPC Hydrogel Ingredient % Concentration (by weight) Hydroxypropylcellulose (HPC) 1.55 Glycerin 3.41 Phenoxyethanol 0.73 Thermolysin 0.205 NaCl 0.82 DI Water (10% PBS pH = 7.5) q.s. Total 100

[0047] Process: HPC (Klucel) used was from Ashland Inc. (USA). The suspending media was DI water. The hydrogel was prepared by mixing glycerin, phenoxy ethanol and gelling agent (HPC) in DI water at RT. Upon solubilization a hydrogel was formed. The active phase is made by mixing thermolysin and NaCl, in DI eater at appropriate concentration. Upon homogenization (a white slurry will be obtained), the active phase is added to a hydrogel (at appropriate concentration). The final mixture is stirred for at least 2 h at RT. Milky hydrogel is obtained having an average viscosity of 42,633 cps (standard deviation of 1,205.5, and based on three separate measurements from three samples of same hydrogel), as measured on a Brookfield Viscometer at lO rpm, spindle #14, RT, 30 sec. Thermolysin was stable in this formulation for 10 months at room temperature and 4° C. as measured by Casein digestion method.

TABLE-US-00006 TABLE 6 Thermolysin/Carbopol 940 Hydrogel Ingredient % Concentration (by weight) Carbopol 940 0.56 Thermolysin 0.1 lMNaOH 10 Tris Buffer (lOmM, pH = 7.5) q.s. Total 100

[0048] Process: Carbopol 940 used was obtained from Lubrizol (USA). The suspending media was Tris buffer (lO mM, pH=7.5). The hydrogel was prepared by mixing gelling agent (Carbopol 940) in Tris buffer at RT. Upon solubilization a blurry solution was formed. The hydrogel was formed upon the neutralization of the solution with IM NaOH. The active phase is made by mixing thermolysin in Tris buffer at appropriate concentration. The active phase was added to a hydrogel. The final mixture is stirred for at least 2 h at RT. Milky hydrogel is obtained. Thermolysin activity was inhibited in this formulation as measured by Casein digestion method.

TABLE-US-00007 TABLE 7 Thermolysin/Polyquaternium-10 Hydrogel Ingredient % Concentration (by weight) Polyquatemium-10 1.49 Thermolysin 0.104 Tris Buffer (l0 mM, pH = 7.5) q.s. Total 100

[0049] Process: Polyquaternium-10 (U-Care Polymer JR-30M) used was from DOW (USA). The suspending media was Tris buffer (lO mM, pH=7.5). The hydrogel was prepared by mixing gelling agent (Polyquaternium-10) in Tris buffer at RT. Upon solubilization a hydrogel was formed. The active phase is made by mixing Thermolysin in Tris buffer at appropriate concentration. The active phase was added to a hydrogel. The final mixture is stirred for at least 2 h at RT. Milky hydrogel is obtained. Thermolysin was unstable in this formulation. About 60% loss in the activity was measured after 1 month storage at room temperature by Casein digestion method.

TABLE-US-00008 TABLE 8 Thermolysin/Hispagel Hydrogel Ingredient % Concentration (by weight) Hispagel-200 43.8 Thermolysin 0.106 lMNaOH 6.56 Tris Buffer (lOmM, pH = 7.5) q.s. Tris Buffer Solid 1.01 Total 100

[0050] Process: Hispagel-200 used was obtained from Cognis (USA). The suspending media was Tris buffer (lO mM, pH=7.5). The hydrogel was prepared by mixing gelling agent (Hispagel-200) in Tris buffer at RT. Upon solubilization a hydrogel was formed. The active phase is made by mixing thermolysin in Tris buffer at appropriate concentration. The active phase was added to a hydrogel. The final mixture is stirred for at least 2 h at RT. The final pH was adjusted by IM NaOH and solid Tris buffer. Milky hydrogel is obtained. Thermolysin activity was inhibited in this formulation as measured by Casein digestion method.

TABLE-US-00009 TABLE 9 Thermolysin/Aristoflex Hydrogel Ingredient % Concentration (by weight) Aristoflex AVC 0.96 Thermolysin 0.1 Tris Buffer (l0 mM, pH = 7.5) q.s. Total 100

[0051] Process: Aristoflex AVC used was from Clariant (USA). The suspending media was Tris buffer (lO mM, pH=7.5). The hydrogel was prepared by mixing gelling agent (Aristoflex AVC) in Tris buffer at RT. Upon solubilization a hydrogel was formed. The active phase is made by mixing thermolysin in Tris buffer at appropriate concentration. The active phase was added to a hydrogel. The final mixture is stirred for at least 2 h at RT. Milky hydrogel is obtained. Thermolysin activity was inhibited in this formulation as measured by Casein digestion method.

TABLE-US-00010 TABLE 10 Thermolysin/Ultragel-300 Hydrogel Ingredient % Concentration (by weight) Ultragel-300 0.55 Thermolysin 0.1 Tris Buffer (l0 mM, pH = 7.5) q.s. Total 100

[0052] Process: Ultragel-300 (polyquaternium-37) used was obtained from Cognis (USA). The suspending media was Tris buffer (lO mM, pH=7.5). The hydrogel was prepared by mixing gelling agent (Ultragel-300) in Tris buffer at RT. Upon solubilization a hydrogel was formed. The active phase is made by mixing thermolysin in Tris buffer at appropriate concentration. The active phase was added to a hydrogel. The final mixture is stirred for at least 2 h at RT. Milky hydrogel is obtained. Thermolysin was unstable in this formulation. About 80% loss in the activity was measured after 1 month storage at room temperature by Casein digestion method.

TABLE-US-00011 TABLE 11 Thermolysin/Gellan Gum Hydrogel Ingredient % Concentration (by weight) Gellan Gum 0.2454 Thermolysin 0.101 Tris Buffer (lOmM, pH = 7.5) q.s. Total 100

[0053] Process: Gelan gum used was obtained from C.P.Kelco (USA). The suspending media was Tris buffer (lO mM, pH=7.5). The hydrogel was prepared by mixing gelling agent (Gelan gum) in Tris buffer at RT. Upon solubilization a hydrogel was formed. The active phase is made by mixing thermolysin in Tris buffer at appropriate concentration. The active phase was added to a hydrogel. The final mixture is stirred for at least 2 h at RT. Milky hydrogel is obtained. Thermolysin was unstable in this formulation. About 50% loss in the activity was measured after 1 month storage at room temperature by Casein digestion method.

TABLE-US-00012 TABLE 12 Thermolysin/Cosmedia Guar Hydrogel Ingredient % Concentration (by weight) Cosmedia Guar 1.217 Thermolysin 0.102 Tris Buffer (lOmM, pH = 7.5) q.s. Total 100

[0054] Process: Cosmedia guar (guar hydroxypropyltrimonium chloride) used was obtained from Cognis (USA). The suspending media was Tris buffer (lO mM, pH=7.5). The hydrogel was prepared by mixing gelling agent (Cosmedia Guar) in Tris buffer at RT. Upon solubilization a hydrogel was formed. The active phase is made by mixing thermolysin in Tris buffer at appropriate concentration. The active phase was added to a hydrogel. The final mixture is stirred for at least 2 h at RT. Milky hydrogel is obtained. Thermolysin was unstable in this formulation. About 60% loss in the activity was measured after 1 month storage at room temperature by Casein digestion method.

TABLE-US-00013 TABLE 13 Thermolysin/Xanthan Gum Hydrogel Ingredient % Concentration (by weight) Xanthan Gum 1.5 Thermolysin 0.102 Tris Buffer (l0 mM, pH = 7.5) q.s. Total 100

[0055] Process: Xanthan gum used was obtained from TIC Gums (USA). The suspending media was Tris buffer (lO mM, pH=7.5). The hydrogel was prepared by mixing gelling agent (xanthan gum) in Tris buffer at RT. Upon solubilization a hydrogel was formed. The active phase is made by mixing thermolysin in Tris buffer at appropriate concentration. The active phase was added to a hydrogel. The final mixture is stirred for at least 2 h at RT. Milky hydrogel is obtained. Thermolysin was unstable in this formulation. About 70% loss in the activity was measured after 1 month storage at room temperature by Casein digestion method.

TABLE-US-00014 TABLE 14 Thermolysin/CMC Ticalose Hydrogel Ingredient % Concentration (by weight) CMC Ticalose (Sodium 2.03 Thermolysin 0.102 Tris Buffer (lOmM, pH = 7.5) q.s. Total 100

[0056] Process: CMC Ticalose (sodium carboxymethylcellulose-anionic) used was obtained from TIC Gums (USA). The suspending media was Tris buffer (lO mM, pH=7.5). The hydrogel was prepared by mixing gelling agent (CMC Ticalose) in Tris buffer at RT. Upon solubilization a hydrogel was formed. The active phase is made by mixing thermolysin in Tris buffer at appropriate concentration. The active phase was added to a hydrogel. The final mixture is stirred for at least 2 h at RT. Milky hydrogel is obtained. Thermolysin activity was inhibited in this formulation.

TABLE-US-00015 TABLE 15 Thermolysin/PolyOx WSR Hydrogel Ingredient % Concentration (by weight) PolyOx WSR 2 Thermolysin 0.1 Tris Buffer (l0 mM, pH = 7.5) q.s. Total 100

[0057] Process: PolyOx (PEG) WSR used was obtained from TIC Gums (USA). The suspending media was Tris buffer (lO mM, pH=7.5). The hydrogel was prepared by mixing gelling agent (PolyOx WSR) in Tris buffer at RT. Upon solubilization a hydrogel/thick liquid was formed. The active phase is made by mixing Thermolysin in Tris buffer at appropriate concentration. The active phase was added to a hydrogel. The final mixture is stirred for at least 2 h at RT. Milky hydrogel/thick liquid is obtained. Thermolysin was unstable in this formulation. About 80% loss in the activity was measured after 1 month storage at room temperature by Casein digestion method.

TABLE-US-00016 TABLE 16 Thermolysin/Oil-in-Water Emulsion Cream Ingredient % Concentration (by weight) Emulsifying Wax 11 Thermolysin 0.2 Isopropyl Myristate 4 1% KH2PO4 (pH = 7.5) q.s. Glycerin 5 Methyl paraben 0.2 Propyl Paraben 0.08 Total 100

[0058] Process: Emulsifying Wax (Polowax) was obtained from Croda (USA). The suspending media was KH.sub.2PO.sub.4 (1%, w/w, pH=7.5). The creaml was prepared by Solubilizing Glycerin and preservatives in buffer at 70 C. Upon solubilization (clear solution) emulsifying agent (Emulsifying Wax) and Oil (Isopropyl Myristate) were added. Upon mixing (l h at 70 C), the mixture was cooled to RT. Milky white cream was obtained. Thermolysin was found unstable in this formulation after 4 months at room temperature as measured by Casein digestion method.

TABLE-US-00017 TABLE 17 Thermolysin/Carbopol Aqua SF-1 Hydrogel Ingredient % Concentration (by weight) Carbopol Aqua SF-I 8.53 Thermolysin 0.2 lMNaOH 10 DI Water q.s. Methyl Paraben 0.19 Propyl Paraben 0.08 Glycerin 4.8 Total 100

[0059] Process: Carbopol Aqua SF-I used was obtained from Lubrizol (USA). The suspending media was DI Water. The hydrogel was prepared by mixing gelling agent (Carbopol Aqua SF-I) in DI Water along with preservatives and Glycerin at 70 C. Upon solubilization the solution was cooled to RI. The hydrogel was formed upon the neutralization of the solution with IM NaOH. The active phase is made by mixing Thermolysin in DI Water at appropriate concentration. The active phase was added to a hydrogel. The final mixture is stirred for at least 2 h at RI. Translucent hydrogel was obtained. Thermolysin activity was inhibited in this formulation.

TABLE-US-00018 TABLE 18 Thermolysin/Poloxamer-407 based Hydrogel Ingredient % Concentration (by weight) Poloxamer-407 18 Thermolysin 0.2 1% KH2PO4 q.s. Phenoxy Ethanol 0.53 Total 100

[0060] Process: Poloxamer-407 used was obtained from BASF (USA). The suspending media was 1% KH.sub.2PO.sub.4 Buffer. The hydrogel was prepared by mixing gelling agent (Poloxamer-407) in buffer along with preservatives at 4 C. Upon solubilization the solution was equilibrated to RT. The hydrogel was formed upon temperature from 4 C to RT. Clear hydrogel was obtained. Thermolysin activity was inhibited in this formulation.

TABLE-US-00019 TABLE 19 Thermolysin/Poloxamers based hydrophilic dispersion Ingredient % Concentration (by weight) Poloxamer-407 15 Thermolysin 0.2 Poloxamer-124 q.s. Poloxamer-188 6 Total 100

[0061] Process: Poloxamer-407/Poloxamer-124 and Poloxamer-188 used were obtained from BASF (USA). The dispersion was made by heating and mixing the Poloxamers at 70 C until homogeneous. The active phase was prepared by suspending the enzyme in Poloxamer-124 and adding it to a previously cooled (to RT) main phase. White cream was obtained. Thermolysin was found unstable in this formulation after 6 months at room temperature as measured by Casein digestion method.

[0062] 2. Thermolysin Stability Data

[0063] The stability of thermolysin in the Table 1 Thermolysin/HEC hydrogel was tested over a one-month period while being stored at room temperature (20 to 25° C.). For comparison purposes, various thermolysin hydrogels were also prepared with gelling agents other than nonionic cellulose ethers. Such gelling agents included polyquaternium-10, Ultragel 300, gellan gum, cosmedia guar, xanthan gum, anionic cellulose ether, Carbopols, polaxamers, polyox WSR, and others. The additional hydrogels were also stored at room temperature (20 to 25° C.).

[0064] FIG. 1 provides a summary of the stability results. The thermolysin activity in hydrogels (e.g., HEC hydrogel) might be slightly different than the expected activity, due to favorable or unfavorable influence of the formulation excipients on enzyme activity.

[0065] Data presented in FIG. 1 suggest that, surprisingly, the thermolysin activity for the Table 1 Thermolysin/HEC hydrogel remained the same/did not decrease over the one-month storage period. By comparison, the thermolysin activity in hydrogels that did not include a nonionic cellulose ether gelling agent decreased substantially over this period (over 50% decrease in activity). Without wishing to be bound by theory, it is thought that the nonionic structure and weak amphiphilic (i.e., both polar and non-polar parts of the cellulose ethers) character of cellulose ethers as the gelling agent for the hydrogel creates an environment that favors the stability of thermolysin.

[0066] A further stability study was performed on each of the hydrogels exemplified in Tables 1, 4, and 5 and compared with various thermolysin formulations ranging from a cream (oil-in-water emulsion), anhydrous gel (poloxamer), hydrogels (polyacrylate-Carbopol) and various other hydrogel bases (e.g., Polyquaternium-10). The results of this study are provided in descriptions of Tables 1 and 4-19. Tables 1, 4, and 5, which are non-limiting hydrogels of the present invention, are superior over all of the other tested thermolysin formulations. The initial activity for these other formulas was less than 85% of the targeted amount, and some were even 0% of the targeted amount initially.

[0067] The method used to determine thermolysin activity (stability) for the hydrogels in Tables 1 and 4-19 is the Casein Digestion Method: Thermolysin activity is determined by measuring the absorbance increase of digested Casein fragments upon digestion relative to the reference standard. The thermolysin hydrogel (250 mg), was solubilized in 25 ml of TBS buffer (50 mM Tris, 100 mM NaCl, 13.5 mM CaCb, pH=7.4). Upon solubilization, the solution is diluted 1:10 using TBS. The Standard solutions were prepared using the Thermolysin raw material and TBS, prepared in a series of concentrations. The substrate was a Casein solution (2 g in 100 ml of Phosphate Buffer 50 mM Na.sub.2HPO.sub.4). The digestion procedure was as follows: 2.5 ml of Casein solution, 1.0 ml of analyte or standard were incubated in water bath at 37° C. for 30 min. Upon digestion, the stop solution, 1.5 ml Trichloroacetic acid (TCA, 30 g in 80 ml of DI water) was added. The blank solution was made as follows: 2.5 ml Casein solution, 1.5 ml of TCA and 1.0 ml of analyte or standard (added after TCA). Samples were left at 37° C. for 45 min., followed by filtration through 0.45 um nylon filter and analysis at 280 nm spectrophotometrically.

[0068] The standard curve of thermolysin was calculated by using third order polynomial curve fitting. The amount of thermolysin in each formulation at different storage conditions is calculated using the standard curve.

The % Target was calculated by: % Target=(Tested activity units/g of formulation)*100I(Tested activity units of thermolysin standard in mg*Thermolysin concentration in the formulation, mg/g).

[0069] FIG. 2 provides an additional thermolysin stability assay, in which the hydrogel from Table 1 was tested and subjected to storage conditions ranging from 4° C., room temperature (20 to 25° C.), and 40° C. for 6 months, 9 months, 12 months, 18 months and 24 months. The hydrogel was stable at room temperature as indicated by a decrease of activity less than 20% at 6 months, 9 months, 12 months, 18 months and 24 months (i.e., retained at least 80% activity). Similarly, the hydrogel was stable for 24 months after storage at 4° C. Approximately 55-60% of the thermolysin remained stable when stored at an elevated temperature of 40° C. for 18 months and 40% of the thermolysin remained stable when stored at an elevated temperature of 40° C. for 24 months—this equates to long-term shelf life/room temperature storage conditions of the hydrogel, as elevated temperatures are used to confirm the shelf life stability of a given product over a shorter testing period. This also explains the increasing loss of stability at 24 months when stored at 40° C.

[0070] 3. In Vivo Debridement of Pig Burn Wounds

[0071] The hydrogels in Tables 2 and 3 were evaluated for efficacy in debriding burn wounds in pigs. In this in vivo study, burn wounds were generated on the backs of pigs. Pigs were anesthetized, and the torso was shaved with clippers and a razor and washed with vedadine. Then an isopropyl rinse was performed to sterilize the surgical field. Twenty 2-cm wounds were created on the dorsum of each pig. The wounds were created using solid brass rods, heated to 100° C. in sand baths, held on the skin for 45 seconds. The wounds were left to dry for five days, giving the eschars time to form, with protective foam dressings being replaced every other day during eschar formation. After eschar formation and on a daily basis for treatments, the wounds were cleaned with saline, photographed, treated according to the treatment scheme, and dressed with non-adherent dressings (pre-moistened with saline) secured with Transpore tape and occlusive secondary dressings. Statistical significance for the number of eschars fully debrided was determined using Fisher's Exact test.

[0072] Following eschar formation, all wounds were treated once a day with either: a papain/urea ointment (with 2.5% papain and 10% urea); SANTYL collagenase ointment; a thin thermolysin hydrogel (Table 2); a thick thermolysin hydrogel (Table 3); or a hydrogel placebo (i.e., a hydrogel lacking thermolysin). As shown in FIG. 3, 20% of the wounds were fully debrided after three days of treatment with the Table 2 and 3 thermolysin hydrogels. At the three day time point, no wounds were fully debrided following treatment with papain/urea ointment or SANTYL collagenase ointment. After four or five days, 30-35% of the wounds were fully debrided following treatment with the Table 2 and 3 thermolysin hydrogels, while only 5% of the wounds were fully debrided following treatment with papain/urea ointment. No wounds were fully debrided following five days of treatment with SANTYL collagenase ointment.