ENZYMATIC WOUND DEBRIDING COMPOSITIONS WITH ENHANCED ENZYMATIC ACTIVITY

Abstract

The present invention is directed to topical enzymatic wound debriding compositions with enhanced enzymatic activity. These compositions comprise a dispersed phase comprising at least one proteolytic enzyme and at least one hydrophilic polyol; and a continuous phase comprising a hydrophobic base.

Claims

1-36. (canceled)

37. A method of treating a wound in need of debridement, the method comprising: applying to the wound a wound debriding composition comprising: (a) a hydrophilic dispersed phase comprising a liquid hydrophilic polyol, and an effective debriding concentration of at least one proteolytic enzyme; and (b) a hydrophobic continuous phase comprising a hydrophobic base, wherein the composition is a dispersion of the hydrophilic dispersed phase in the hydrophobic continuous phase, wherein the composition is anhydrous, and wherein the amount of liquid hydrophilic polyol in the wound debriding composition is within 10% w/w of X, wherein X is determined by: (i) obtaining a series of compositions comprising the hydrophilic dispersed phase and the hydrophobic continuous phase, wherein each composition in the series of compositions includes an identical amount of the proteolytic enzyme and a different amount of the liquid hydrophilic polyol, and wherein each composition in the series of compositions is q.s.-ed to 100% with the hydrophobic continuous phase; (ii) determining the enzymatic activity of each composition in the series of compositions; and (iii) determining the highest point on a graph that plots the enzymatic activity versus the amount of liquid hydrophilic polyol included in each composition of the series of compositions, wherein the highest point on the graph correlates to X.

38. The method of claim 37, wherein the amount of liquid hydrophilic polyol in the wound debriding composition is within 7% w/w of X.

39. The method of claim 37, wherein the amount of liquid hydrophilic polyol in the wound debriding composition is within 5% w/w of X.

40. The method of claim 37, wherein the liquid hydrophilic polyol comprises a liquid polyethylene glycol, a liquid poloxamer, or mixtures thereof.

41. The method of claim 37, wherein the hydrophobic base comprises petrolatum.

42. The method of claim 37, wherein the composition is a liquid.

43. The method of claim 37, wherein the composition is a semi-solid.

44. The method of claim 37, wherein the proteolytic enzyme is a metalloprotease, a cysteine protease, a serine protease, or an aspartic peptidase.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0031] FIG. 1. Plot of the in-vitro collagenolysis activity (mg/ml) of a series of compositions comprising a dispersed phase comprising collagenase and PEG-400, dispersed in a hydrophobic phase comprising white petrolatum (y-axis) versus the percentage of the PEG-400 comprised in the series of compositions (x-axis).

[0032] FIG. 2. Plot of the in-vitro collagenolysis activity (mg/ml) of a series of compositions comprising a dispersed phase comprising collagenase and PEG-600, dispersed in a hydrophobic phase comprising white petrolatum (y-axis) versus the percentage of the PEG-600 comprised in the series of compositions (x-axis).

[0033] FIG. 3. Plot of the in-vitro collagenolysis activity (mg/ml) of a series of compositions comprising a dispersed phase comprising collagenase and poloxamer-124, dispersed in a hydrophobic phase comprising white petrolatum (y-axis) versus the percentage of the poloxamer-124 comprised in the series of compositions (x-axis).

[0034] FIG. 4. Plot of the in-vitro collagenolysis activity (mg/ml) of a series of compositions comprising a dispersed phase comprising trypsin and PEG-400, dispersed in a hydrophobic phase comprising white petrolatum (y-axis) versus the percentage of the PEG-400 comprised in the series of compositions (x-axis).

[0035] FIG. 5. Plot of the in-vitro collagenolysis activity (mg/ml) of a series of compositions comprising a dispersed phase comprising papain and PEG-400, dispersed in a hydrophobic phase comprising white petrolatum (y-axis) versus the percentage of the PEG-400 comprised in the series of compositions (x-axis).

[0036] FIG. 6. Plot of the in-vitro collagenolysis activity (mg/ml) of a series of compositions comprising a dispersed phase comprising thermolysin and PEG-400, dispersed in a hydrophobic phase comprising white petrolatum (y-axis) versus the percentage of the PEG-400 comprised in the series of compositions (x-axis).

[0037] FIG. 7. Plot of the in-vitro collagenolysis activity (mg/ml) of a series of compositions comprising a dispersed phase comprising pepsin and PEG-400, dispersed in a hydrophobic phase comprising white petrolatum (y-axis) versus the percentage of the PEG-400 comprised in the series of compositions (x-axis).

[0038] FIG. 8. Plot of the physical release of collagenase (mg) from of a series of compositions comprising a dispersed phase comprising collagenase and PEG-400, dispersed in a hydrophobic phase comprising white petrolatum (y-axis) versus the percentage of the PEG-400 comprised in the series of compositions (x-axis).

[0039] FIG. 9. Enzyme stability in PEG-in-white petrolatum dispersion compared with oil-in-water emulsion cream.

[0040] FIG. 10. Debridement efficacy in Eschar removal in pig burn wound.

DETAILED DESCRIPTION

[0041] One aspect of the present invention provides for topical enzymatic wound debriding compositions with enhanced enzymatic activity. These compositions comprise a dispersed phase comprising at least one proteolytic enzyme and a hydrophilic polyol; and a continuous phase comprising a hydrophobic base. In one aspect of the invention, the hydrophilic polyol is a liquid hydrophilic polyol.

[0042] It was found that the enzymatic activity (e.g., in vitro collagenolysis) of the compositions of the present invention, which are dispersions of a hydrophilic polyol and a proteolytic enzyme in a hydrophobic base, not only was higher than the enzymatic activity of enzyme compositions based solely on a proteolytic enzyme and hydrophobic base combination (i.e., no hydrophilic phase such as a hydrophilic polyol), but also surprisingly higher than those enzyme compositions based solely on a proteolytic enzyme and hydrophilic base combination (i.e., no hydrophobic phase such as petrolatum). Since enzymes are activated in the presence of moisture, it would have been expected to see the highest enzymatic activity in compositions based solely on a proteolytic enzyme and hydrophilic base combination, where the base would be completely miscible in moisture and conditions would be the most favorable for release and activation of the enzyme. However, the dispersion composition of hydrophilic and hydrophobic phases of the present invention had the highest enzymatic activity correlating to an optimum amount of the hydrophilic polyol which was more than 0% and less than 100% of the hydrophilic polyol in the composition.

[0043] It was found, expectedly, that the physical enzyme release in compositions based solely on a hydrophilic vehicle was greater than the release of the enzyme in compositions based solely on a hydrophobic vehicle, and also more than compositions of the present invention. As seen in FIG. 8, the enzyme release profile generally increased with the increasing percentage of hydrophilic polyol (PEG-400), with the highest release at 100% and the lowest release at 0%. However, surprisingly, the enzymatic activity was greater with the dispersion compositions of the present invention (see FIGS. 1-7). Thus the enzymatic activity profile of these dispersion compositions does not correlate with the physical enzyme release profile as would be expected.

[0044] The compositions of the present invention are suitable for treatment of a wound in need of debridement by applying to the wound a composition comprising a dispersed phase comprising a hydrophilic polyol, and an effective debriding concentration of at least one proteolytic enzyme; and a continuous phase comprising a hydrophobic base; wherein the amount of hydrophilic polyol is within 10% w/w of the optimum amount, or 9%, 8%, 7%, or 6% w/w of the optimum amount, or 5%, 4%, 3%, 2%, 1%, 0.5%, or 0.1% w/w of the optimum amount of hydrophilic polyol.

[0045] These and other non-limiting aspects of the present invention are discussed in further detail in the following sections.

A. Method for Determining the Optimum Amount of Liquid Hydrophilic Polyol

[0046] The following protocol can be used to prepare a series of compositions (referred to as Series of Compositions) and to subsequently determine the optimum amount of liquid hydrophilic polyol that can be used in a dispersion of the present invention:

[0047] Eleven (11) compositions can be used to create the Series of Compositions. Note that the amount (% w/w) of proteolytic enzyme in the series of compositions is held constant. The following steps can be used to prepare the eleven (11) compositions:

[0048] (i) Determine the ingredients (i.e., liquid hydrophilic polyol, proteolytic enzyme, and hydrophobic base) to be used in the Series of Compositions and select the amount of proteolytic enzyme to be used. By way of example, liquid hydrophilic polyol (e.g., PEG 400), proteolytic enzyme (e.g., collagenase at 1% w/w), and hydrophobic base (e.g., white petrolatum).

[0049] (ii) For composition one in the Series of Compositions, use 0% of the liquid hydrophilic polyol, use the selected amount of proteolytic enzyme, and q.s the batch with the hydrophobic base to 100%. For example, and referring to step (i) above, composition one of the Series of Compositions would have: 0% w/w PEG 400, 99% w/w of white petrolatum, and 1% w/w of collagenase.

[0050] (iii) For composition two in the Series of Compositions, use 10% w/w of the liquid hydrophilic polyol, the same amount of the proteolytic enzyme, and q.s. the batch with the hydrophobic base to 100%. (Note that it is permissible to use some solid hydrophilic polyol in the makeup of the liquid hydrophilic polyol as necessary to produce a physically stable dispersion for compositions in the Series of Compositions).

[0051] (iv) For composition three in the Series of Compositions, use 20% w/w of the liquid hydrophilic polyol, the same amount of the proteolytic enzyme, and q.s. the batch with the hydrophobic base to 100%.

[0052] (v) For composition four in the Series of Compositions, use 30% w/w of the liquid hydrophilic polyol, the same amount of the proteolytic enzyme, and q.s. the batch with the hydrophobic base to 100%.

[0053] (vi) For composition five in the Series of Compositions, use 40% w/w of the liquid hydrophilic polyol, the same amount of the proteolytic enzyme, and q.s. the batch with the hydrophobic base to 100%.

[0054] (vii) For composition six in the Series of Compositions, use 50% w/w of the liquid hydrophilic polyol, the same amount of the proteolytic enzyme, and q.s. the batch with the hydrophobic base to 100%.

[0055] (viii) For composition seven in the Series of Compositions, use 60% w/w of the liquid hydrophilic polyol, the same amount of the proteolytic enzyme, and q.s. the batch with the hydrophobic base to 100%.

[0056] (ix) For composition eight in the Series of Compositions, use 70% w/w of the liquid hydrophilic polyol, the same amount of the proteolytic enzyme, and q.s. the batch with the hydrophobic base to 100%.

[0057] (x) For composition nine in the Series of Compositions, use 80% w/w of the liquid hydrophilic polyol, the same amount of the proteolytic enzyme, and q.s. the batch with the hydrophobic base to 100%.

[0058] (xi) For composition ten in the Series of Compositions, use 90% w/w of the liquid hydrophilic polyol, the same amount of the proteolytic enzyme, and q.s. the batch with the hydrophobic base to 100%.

[0059] (xii) For composition eleven in the Series of Compositions, use 0% of the hydrophobic base, the same amount of the proteolytic enzyme, and q.s. the batch with the hydrophilic polyol.

[0060] (xiii) determine the enzymatic activity of each of the eleven compositions in the Series of Compositions by using the in vitro artificial eschar testing model for the following sample collection times: 6, 12, 18 and 24 hours, as described in Section H of the Detailed Description section of this specification.

[0061] (ivx) plot a curve of the enzymatic activity of each composition versus the correlating amount of liquid hydrophilic polyol(s) present in each composition of the Series of

[0062] Compositions cumulatively for each data collection time. The highest point on the curve for the cumulative 24-hour data collection time correlates to the optimum amount of liquid hydrophilic polyol that can be used in a dispersion.

[0063] Further, given that multiple ingredients can be included in the Series of Compositions (e.g., polyol(s) proteolytic enzyme(s), hydrophobic base, and additional ingredients within the dispersed phase, and/or additional ingredients within the continuous hydrophobic phase), the Series of Compositions can be created by (1) varying the amount of hydrophilic polyol as discussed above for each composition in the series, (2) using the determined amount of proteolytic enzyme, and (3) q.s.-ing the batch to 100% with the amount of the additional ingredients including the hydrophobic base; except for composition eleven, where the batch would be q.s.-ed to 100% with the amount of the additional ingredients including the hydrophilic polyol.

B. Method for Determining Whether A Composition Has +/10% w/w of the Optimum Amount of Liquid Hydrophilic Polyol

[0064] It can be determined if a composition comprising (a) a dispersed phase including a liquid hydrophilic polyol and at least one proteolytic enzyme; and (b) a continuous phase comprising a hydrophobic base (referred to as Composition of Interest) is within 10% of the Optimum Amount of liquid hydrophilic polyol by using the following protocol:

[0065] Step One: Obtain a Composition of Interest that includes: (i) a dispersed phase including a liquid hydrophilic polyol(s) and a proteolytic enzyme and (ii) a continuous phase including a hydrophobic base.

[0066] Step Two: Prepare a series of compositions (referred to as Series of Compositions) based on the Composition of Interest. Note that the amount (% w/w) of proteolytic enzyme in the Series of Compositions is held constant and is the same as the amount (% w/w) present in the Composition of Interest. The following steps can be used to prepare the Series of Compositions:

[0067] (i) Determine the amount of all ingredients in the Composition of Interest (% w/w).

[0068] (ii) Determine the total amount of the continuous phase in the Composition of Interest (% w/w). By way of example, if the Composition of Interest includes 15% w/w liquid hydrophilic polyol (e.g., PEG 400), 1% w/w proteolytic enzyme (e.g., collagenase), and 84% w/w hydrophobic base (e.g., white petrolatum), then the Composition of Interest would be 84% w/w continuous phase and 16%w/w dispersed phase.

[0069] Step Three: Prepare the Series of Compositions in a manner described above in Section A of this specification (e.g., this would include preparing 11 compositions in a manner described in Section A of this specification).

[0070] Step Four: Determine the enzymatic activity of each of the eleven compositions in the Series of Compositions by using the in vitro artificial eschar testing model for each of the following sample collection times: 6, 12, 18 and 24 hours as described in Section H of the Detailed Description section of this specification.

[0071] Step Five: Plot a curve of the enzymatic activity of each composition versus the correlating amount of liquid hydrophilic polyol(s) present in each composition of the Series of Compositions cumulatively for each data collection time. The highest point on the curve for the cumulative 24-hour data collection time correlates to the optimum amount of liquid hydrophilic polyol for the Composition of Interest.

[0072] Step Six: Compare the amount of liquid hydrophilic polyol present within the Composition of Interest to determine whether it is within 10% w/w of the optimum amount of liquid hydrophilic polyol for the Composition of Interest.

C. Proteolytic Enzymes

[0073] Any proteolytic enzyme useful for wound debridement is suitable for the present invention. Proteolytic enzymes (proteases) break down protein by hydrolysis of the peptide bonds that link amino acids together in the polypeptide chain of a protein. They are divided into four major groups on the basis of catalytic mechanism: serine proteases, cysteine proteases. metalloproteases, and aspartic proteases. Some proteases have been identified with other catalytic amino acids in the active site, such as threonine and glutamic acid; however, they do not form major groups.

[0074] 1. Serine proteases

[0075] Serine proteases depend upon the hydroxyl group of a serine residue acting as the nucleophile that attacks the peptide bond. The major clans found in humans include the chymotrypsin-like, the subtilisin-like, the alpha/beta hydrolase, and signal peptidase clans. In evolutionary history, serine proteases were originally digestive enzymes. In mammals, they evolved by gene duplication to serve functions in blood clotting, the immune system, and inflammation. These proteases have a broad substrate specificity and work in a wide pH range. Non-limiting examples of serine proteases include trypsin, chymotrypsin, subtilisin, sutilains, plasmin, and elastases.

2. Cysteine Proteases

[0076] Peptidases in which the nucleophile that attach the scissile peptide bond in the sulfhydryl group of a cysteine residue are known as cysteine proteases. Cysteine proteases are commonly encountered in fruits including papaya, pineapple, and kiwifruit. Cysteine proteases have a broad specificity and are widely used under physiological conditions. In this family, papain has been used extensively for wound debridement for a long time. Other cysteine proteases, such as bromelain and analain, have also been investigated for the applications in wound debridement. Other non-limiting examples of cysteine proteases include calpain, caspases, chymopapain, and clostripain.

3. Metalloproteases

[0077] Metalloproteases are among the proteases in which the nucleophilic attach on a peptide bond is mediated by a water molecule, while a divalent metal cation, usually zinc but sometimes cobalt, manganese, nickel or copper, activates the water molecule. The metal ions are extremely important for the activity. Any compounds that have potential to interact with the metal ion, chelating or oxidation, will affect the enzymatic activity. Non-limiting examples of metalloproteases in this family include thermolysin, collagenases, matrix metallo proteinases (MMPs), bacillolysin, dispase, vibriolysin, pseudolysin, stromelysin, and various bacterial derived neutral metalloproteases.

4. Aspartic Peptidases

[0078] Aspartic peptidases are so named because aspartic acid residues are the ligands of the activated water molecule. In most enzymes in this family, a pair of aspartic residues act together to bind and activate the catalytic water molecule. All or most aspartic peptidases are endopeptidases. Most aspartic peptidases have a broad specificity. However, the optimum pH of most aspartic peptidases is in the acidic range. Non-limiting examples of aspartic peptidases are pepsin, chymosin, beta-secretase, plasmepsin, plant acid proteases and retroviral proteases.

5. Collagenase

[0079] A suitable proteolytic enzyme for wound debridement is the metalloprotease collagenase. The collagenase can be substantially pure or it may contain detectable levels of other proteases.

[0080] The potency assay of collagenase, and meaning of collagenase units as used herein, is based on the digestion of undenatured collagen from (bovine Achilles tendon) at pH 7.2 and 37 C. for 24 hours. The number of peptide bonds cleaved is measured by reaction with ninhydrin. Amino groups released by a trypsin digestion control are subtracted. One net collagenase unit will solubilize ninhydrin reactive material equivalent to 1 nanomole of leucine equivalents per minute.

[0081] The amount (potency or concentration) of collagenase in the compositions of the present invention is at an effective level to debride the wound. Generally, the potency of collagenase in the compositions can vary from about 1 to about 10,000 collagenase units per gram of product, based on the activity of the collagenase used in the product. In various embodiments, the potency, expressed as collagenase units per gram of product, is from about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500 to about 10000, or any range or numerical amount derivable therein.

[0082] The concentration of collagenase in the compositions generally can vary from about 0.001% w/w to about 8% w/w. In various embodiments, the concentration, expressed as percentage weight by weight, is from about 0.001, 0.002, 0.003, 0.004, 0.005, 0.006, 0.007, 0.008, 0.009, 0.010 0.015, 0.020, 0.025, 0.030, 0.035, 0.040, 0.050, 0.055, 0.060, 0.065, 0.070, 0.075, 0.080, 0.085, 0.090, 0.095, 0.100, 0.125, 0.150, 0.175, 0.20, 0.25, 0.30 ,0.35, 0.40, 0.45, 0.50, 0.55, 0.60, 0.65, 0.70, 0.75, 0.80, 0.85, 0.90, 0.95, 1, 2, 3, 4, 5, 6, 7 to about 8 or any range or numerical amount derivable therein.

[0083] In one embodiment, the collagenase is derived from Clostridium histolyticum; however, in other embodiments the collagenase can be derived from other sources. Methods for producing a suitable collagenase are disclosed in U.S. Pat. Nos. 3,705,083; 3,821,364; 5,422,261; 5,332,503; 5,422,103; 5,514,370; 5,851,522; 5,718,897; and 6,146,626 all of which are herein incorporated by reference.

6. Trypsin

[0084] Another suitable proteolytic enzyme for wound debridement is the serine protease trypsin. Typically, trypsin is derived from the pancreas of healthy bovine or porcine animals, or both. Trypsin can also be derived from recombinant sources. The pharmaceutical grade (USP/NF) of trypsin is known as Crystallized Trypsin. It contains not less than 2500 USP Trypsin Units per mg, calculated on the dried basis, and not less than 90.0% and not more than 110.0% of the labeled potency. The potency assay of trypsin as well as the definition of a USP Trypsin Unit are found in the Crystallized Trypsin monograph of the USP 31 (Official Aug. 1, 2008) herein incorporated by reference.

[0085] The amount (potency or concentration) of trypsin in the compositions of the present invention is at an effective level to debride the wound. Generally, the potency of trypsin in the compositions can vary from about 90 to about 60,000 USP Trypsin Units per gram of product. In various embodiments the potency of trypsin, expressed as USP Trypsin Units per gram of product, is from about 90, 100, 150, 200, 250, 300, 320, 350, 375, 400, 500, 600, 675, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 10000, 20000, 30000, 40000, 50000 to about 60000, or any range or numerical amount derivable therein.

[0086] The concentration of trypsin in the compositions generally can vary from about 0.0025% w/w to about 1% w/w. In various embodiments, the concentration of trypsin, expressed as percent weight by weight, is from about 0.0025, 0.0050, 0.010, 0.015, 0.020, 0.025, 0.030, 0.035, 0.040, 0.045, 0.050, 0.055, 0.060, 0.065, 0.070, 0.075, 0.080, 0.085, 0.090, 0.095, 0.10, 0.15, 0.20 0.25, 0.30, 0.35, 0.40, 0.45, 0.50, 0.55, 0.60, 0.65, 0.70, 0.75, 0.80, 0.85, 0.90, 0.95 to about 1, or any range or numerical amount derivable therein.

D. Hydrophilic Polyols

[0087] Hydrophilic polyols of the present invention are water-soluble, polar aliphatic alcohols with at least two hydroxyl groups, and include polymeric polyols, e.g., polyethylene glycols and poloxamers. In one aspect of the invention, the hydrophilic polyol in the dispersed phase is a liquid hydrophilic polyol. In some embodiments, the liquid hydrophilic polyol is a liquid polyethylene glycol or a liquid poloxamer, or mixtures thereof. Solid hydrophilic polyols such as solid polyethylene glycols or solid poloxamers can also be added to the dispersed phase of the invention to help physically stabilize the dispersion. Other examples of liquid hydrophilic polyols include but are not limited to propylene glycol, butylene glycol, pentylene glycol, hexylene glycol, glycerin, hexylene glycol, methoxy polyethylene glycol, propylene carbonate, and ethoxydiglycol, and these may also be added to the dispersed phase.

1. Polyethylene Glycols

[0088] Polyethylene glycols are homo-polymers of ethylene glycol and water represented by the formula:

H(OCH.sub.2CH.sub.2).sub.nOH

in which n represents the average number of oxyethylene groups. Polyethylene glycols can be either liquid or solid at 25 C. depending on their molecular weights.

[0089] The following suitable non-limiting examples of liquid polyethylene glycols are described using USP nomenclature: polyethylene glycol 200, polyethylene glycol 300, polyethylene glycol 400, polyethylene glycol 500, and polyethylene glycol 600.

[0090] The following suitable non-limiting examples of solid polyethylene glycols are described using USP nomenclature: polyethylene glycol 700, polyethylene glycol 800, polyethylene glycol 900, polyethylene glycol 1000, polyethylene glycol 1100, polyethylene glycol 1200, polyethylene glycol 1300, polyethylene glycol 1400, polyethylene glycol 1450, polyethylene glycol 1500, polyethylene glycol 1600, polyethylene glycol 1700, polyethylene glycol 1800, polyethylene glycol 1900, polyethylene glycol 2000, polyethylene glycol 2100, polyethylene glycol 2200, polyethylene glycol 2300, polyethylene glycol 2400, polyethylene glycol 2500, polyethylene glycol 2600, polyethylene glycol 2700, polyethylene glycol 2800, polyethylene glycol 2900, polyethylene glycol 3000, polyethylene glycol 3250, polyethylene glycol 3350, polyethylene glycol 3750, polyethylene glycol 4000, polyethylene glycol 4250, polyethylene glycol 4500, polyethylene glycol 4750, polyethylene glycol 5000, polyethylene glycol 5500, polyethylene glycol 6000, polyethylene glycol 6500, polyethylene glycol 7000, polyethylene glycol 7500, and polyethylene glycol 8000.

[0091] The liquid and solid polyethylene glycols are available commercially from the DOW Chemical Company under the CARBOWAX tradename and from the BASF Corporation under LUTROL E and PLURACARE E tradenames. Both pharmaceutical grade (USP/NF) and cosmetic grade polyethylene glycols are suitable for the present invention.

2. Poloxamers

[0092] Poloxamers are synthetic block copolymers of ethylene oxide and propylene oxide represented by the formula:

HO(C.sub.2H.sub.4O).sub.a(C.sub.3H.sub.6O).sub.b(C.sub.2H.sub.4O).sub.aH

in which formula a and b represent the number of repeat units. Generally a is from 2 to 150 and b is from 15 to 70 depending on the particular poloxamer. Poloxamers can be either liquid or solid at 25 C. depending on their molecular weights.

[0093] The following suitable non-limiting examples of liquid poloxamers are described using CTFA/INCI nomenclature: poloxamer 101, poloxamer 105, poloxamer 122, poloxamer 123, poloxamer 124, poloxamer 181, poloxamer 182, poloxamer 183, poloxamer 184, poloxamer 212, poloxamer 231, poloxamer 282, poloxamer 331, poloxamer 401, and poloxamer 402.

[0094] The following suitable non-limiting examples of solid poloxamers are described using CTFA/INCI nomenclature: poloxamer 108, poloxamer 188, poloxamer 217, poloxamer 237, poloxamer 238, poloxamer 288, poloxamer 338, poloxamer 407, poloxamer 185, poloxamer 215, poloxamer 234, poloxamer 235, poloxamer 284, poloxamer 333, poloxamer 334, poloxamer 335, and poloxamer 403.

[0095] The liquid and solid poloxamers are available commercially from the BASF Corporation under the PLURONIC and LUTROL tradenames and from the UNIQEMA Corporation under the SYNPERONIC trademark. Pharmaceutical grade (USP/NF) poloxamers are poloxamer 124, poloxamer 188, poloxamer 237, poloxamer 338, and poloxamer 407. Both pharmaceutical grade and cosmetic grade poloxamers are suitable for the present invention.

E. Hydrophobic Bases

[0096] The hydrophobic bases of the present invention can comprise, but are not limited to, plant, animal, paraffinic, and synthetic derived fats, butters, greases, waxes, solvents, and oils; mineral oils, vegetable oils, petrolatum, water insoluble organic esters and triglycerides, silicones, or fluorinated compounds; or mixtures thereof. In one embodiment of the present invention the hydrophobic phase comprises petrolatum.

[0097] Plant derived materials include, but are not limited to, arachis (peanut) oil, balsam Peru oil, carnauba wax, candellila wax, castor oil, hydrogenated castor oil, cocoa butter, coconut oil, corn oil, cotton seed oil, jojoba oil, macadamia seed oil, olive oil, orange oil, orange wax, palm kernel oil, rapeseed oil, safflower oil, sesame seed oil, shea butter, soybean oil, sunflower seed oil, tea tree oil, vegetable oil, and hydrogenated vegetable oil.

[0098] Non-limiting examples of animal derived materials include beeswax, cod liver oil, emu oil, lard, mink oil, shark liver oil, squalane, squalene, and tallow.

[0099] Non-limiting examples of paraffinic materials include isoparaffin, microcrystalline wax, heavy mineral oil, light mineral oil, ozokerite, petrolatum, and paraffin.

[0100] Suitable non-limiting examples of organic esters and triglycerides include C12-15 alkyl benzoate, isopropyl myristate, isopropyl palmitate, medium chain triglycerides, trilaurin, and trihydroxystearin.

[0101] Non-limiting examples of silicones are dimethicone and cyclomethicone. A non-limiting example of a fluorinated compound is polytetrafluoroethylene (PTFE).

1. Petrolatum

[0102] Petrolatum is a purified mixture of semisolid hydrocarbons obtained from petroleum and varies from dark amber to light yellow in color. White petrolatum is wholly or nearly decolorized petrolatum and varies from cream to snow white in color. Petrolatum and White Petrolatum can also vary in melting point, viscosity, and consistency.

[0103] Various grades are available commercially from the PENRECO Corporation under the tradenames: PENRECO ULTIMA, PENRECO SUPER, PENRECO SNOW, PENRECO REGENT, PENRECO LILY, PENRECO CREAM, PENRECO ROYAL, PENRECO BLOND, and PENRECO AMBER. Various grades are also available commercially from the SONNEBORN Corporation under the tradenames: ALBA, SUPER WHITE PROTOPET, SUPER WHITE FONOLINE, WHITE PROTOPET 1S, WHITE PROTOPET 2L, WHITE PROTOPET 3C, WHITE FONOLINE, PERFECTA, YELLOW PROTOPET 2A, YELLOW FONOLINE, PROTOLINE, SONOJELL #4, SONOJELL #9, MINERAL JELLY #10, MINERAL JELLY #14, MINERAL JELLY #17, AND CARNATION TROUGH GREASE.

[0104] Petrolatum and White Petrolatum are available in cosmetic grade and pharmaceutical (USP/NF) grade and both are suitable for the present invention.

F. Topical Compositions

[0105] The topical compositions of the present invention are dispersions comprising a hydrophilic dispersed phase in a hydrophobic continuous phase. The dispersed phase comprises a proteolytic enzyme and a hydrophilic polyol. In an aspect of the invention, the hydrophilic polyol is a liquid hydrophilic polyol. In some embodiments, the liquid hydrophilic polyol is a liquid polyethylene glycol or a liquid poloxamer, or mixtures thereof. The continuous phase comprises a hydrophobic base. The hydrophobic base can be petrolatum. The compositions are useful for treatment of wounds for wound debridement.

[0106] The compositions can be anhydrous as defined herein. The compositions can be semisolid or liquid. The composition can be impregnated on a pad, gauze, or sponge. The compositions can also be sterile.

[0107] The compositions can include additional materials known in the art that are suitable for topical compositions of this nature, e.g., absorbents, deodorizers, surfactants, solvents, rheology modifiers, film formers, stabilizers, emollients, moisturizers, preservatives, antimicrobials, antioxidants, chelating agents, fragrances, and colorants.

[0108] The compositions can also include additional pharmaceutical active ingredients known in the art that are suitable for topical compositions of this nature, e.g., antimicrobial agents, wound healing agents, anesthetic agents, vulnerary agents, and haemostatic agents. A non-limiting example of a vulnerary agent is balsam Peru.

[0109] The compositions can be packaged in any package suitable for dispensing a wound debrider. The compositions can be packaged in multi-use, single-dose, or metered dose packages. Non-limiting examples include a tube, bottle, jar, pump container, pressurized container, bladder container, aerosol container, aerosol spray container, non-aerosol spray container, syringe, pouch, or sachet.

G. Manufacturing Process

[0110] The compositions of the present invention can be prepared by techniques and methods known by one of ordinary skill in the art by dissolving or suspending the proteolytic enzyme in part or all of the available hydrophilic polyol. The resulting solution or suspension can be mixed with a hydrophobic base to form a dispersion, wherein the hydrophobic base becomes the continuous phase and the hydrophilic polyol/enzyme phase becomes the dispersed phase. These compositions can be prepared using processing equipment known by one of ordinary skill in the art, e.g., blenders, mixers, mills, homogenizers, dispersers, dissolvers, etc.

H. In vitro Artificial Eschar Testing Model

[0111] Enhancement of the enzymatic activity of the compositions was established by testing the compositions using an in vitro artificial eschar model as described below and in the publication Study on the debridement efficacy of formulated enzymatic wound debriding agents by in vitro assessment using artificial wound eschar and by an in vivo pig model, Shi et. al., Wound Repair Regen, 2009, 17(6):853, herein incorporated by reference. Bovine collagen (Type I), bovine fibrinogen, and elastin were used to make an Artificial Wound Eschar (AWE) substrate. Collagen-FITC labeled, elastin-rhodamine, and fibrin-coumarin were the raw materials used for producing the AWE substrate. To prepare 1 gram of AWE substrate, 650 mg Collagen-FITC and 100 mg each of elastin-rhodamine and fibrin-coumarin were weighed into a 50 mL tube and homogenized in 10 mL of Tris buffer saline. In a separate tube, 10 mL of fibrinogen solution was prepared at 15 mg/mL with Tris buffer saline. The two solutions were combined and thoroughly mixed. A thrombin solution (0.25 mL at 50 U/mL) was added, quickly mixed, and the solution was poured into a Petri dish containing a 90 mm nonreactive membrane filter. As a result of the thrombin-induced fibrinogen polymerization, the material began to form a soft sheet on top of the membrane filter by clotting the dyed proteins into a solid matrix. The clotted AWE substrate was allowed to solidify for 30 minutes and then rinsed with water for 15 minutes to remove the thrombin. The AWE substrate was further dehydrated to 75% moisture content in preparation for use.

[0112] With the AWE substrate still attached to the membrane, a 35 mm diameter piece was punched out using a hole punch. The AWE substrate punch was placed on the top flat face of a Franz Diffusion Cell System (Hanson Research, Chatsworth, Calif.), and a TEFLON sample holder placed on top. The debriding ointment samples were loaded in the center of the sample holder, and any excess sample was removed by scraping. The solution in the receptor cells was Tris buffer at a pH of 7.4 for samples containing collagenase, papain, thermolysin, or trypsin; and was sodium acetate buffer at a pH of 2 for samples containing pepsin. The solution in receptor cells was sampled in 1 mL increments at the following sample collection times: 0, 1, 2, 3, 6, 12, 18 and 24 hours. Once finished, the samples were analyzed by fluorescence measurement of FITC dye at 485 nm (excitation wavelength) and 520 nm (emission wavelength) to determine the digestion of collagen (collagenolysis) reported in mg/ml.

I. In-vitro Physical Enzyme Release Test

[0113] The release of enzyme from the compositions was determined by a Franz cell diffusion study using PVDF (0.45 micron) filters. This study was performed at 35 C. and lasted for 6 hours. The solution samples in the receptor cells were subjected to a total protein analysis.

[0114] The protein concentration was determined by a BCA assay (Peirce) while the same collagenase was used as the reference standard. The details are described as follows.

[0115] The BCA Protein Assay combines the well-known reduction of Cu.sup.2+ to Cu.sup.1+ by protein in an alkaline medium with the highly sensitive and selective colorimetric detection of the cuprous cation (Cu.sup.1+) by bicinchoninic acid. The first step is the chelation of copper with protein in an alkaline environment to form a blue-colored complex. In this reaction, known as the biuret reaction, peptides containing three or more amino acid residues form a colored chelate complex with cupric ions in an alkaline environment containing sodium potassium tartrate. This became known as the biuret reaction because a similar complex forms with the organic compound biuret (NH2CONHCONH2) and the cupric ion. Biuret, a product of excess urea and heat, reacts with copper to form a light blue tetradentate complex. In the second step of the color development reaction, BCA, a highly sensitive and selective colorimetric detection reagent reacts with the cuprous cation (Cu.sup.1+) that was formed in step 1. The purple-colored reaction product is formed by the chelation of two molecules of BCA with one cuprous ion. The BCA/copper complex is water-soluble and exhibits a strong linear absorbance at 562 nm with increasing protein concentrations. The purple color may be measured at any wavelength between 550 nm and 570 nm with minimal (less than 10%) loss of signal. See the following reference herein incorporated by reference: Smith, P. K., Krohn, R. I., Hermanson, G. T., Mallia, A. K., Gartner, F. H., Provenzano, M. D., Fujimoto, E. K., Goeke, N. M., Olson, B. J. and Klenk, D. C. (1985). Measurement of protein using bicinchoninic acid. Anal. Biochem. 150, 76-85.

EXAMPLES

[0116] 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 which 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 which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.

Example 1: Dispersions of Collagenase/PEG 400 in Petrolatum

[0117] The dispersions in TABLE 1 were prepared with varying concentrations of Polyethylene Glycol 400 (PEG-400) dispersed in Petrolatum.

TABLE-US-00001 TABLE 1 Disper- PEG 400 PEG-1450 Wht Petro- Poloxamer Collagenase sion % w/w % w/w latum % w/w 407 % w/w % w/w A 0 0 99.8 0 0.2 B 10 0 89.8 0 0.2 C 15 0 84.8 0 0.2 D 20 0 79.9 0.4 0.2 E 30 0 69 0.9 0.2 F 50 0 49.3 1.2 0.2 G 68 0 29.8 2.0 0.2 H* 83 12.5 4.5 0 0.2 I* 70 29.8 0 0 0.2 *PEG-1450 was added to PEG-400 to form a semi-solid resulting in approximate total PEG of 96% and 100% respectively.

[0118] The enzymatic debridement activity of each dispersion was determined by the in-vitro artificial eschar model described above and the results plotted in FIG. 1. As can be seen by the results in FIG. 1, the optimum amount of PEG-400 based on the 24 hour curve is about 20% w/w PEG-400.

Example 2: Dispersions of Collagenase/PEG 600 in Petrolatum

[0119] The dispersions in TABLE 2 were prepared with varying concentrations of Polyethylene Glycol 600 (PEG-600) dispersed in Petrolatum.

TABLE-US-00002 TABLE 2 PEG 600 Wht Petrolatum Poloxamer 407 Collagenase Dispersion % w/w % w/w % w/w % w/w J 0 99.8 0 0.2 K 10 89.525 0.275 0.2 L 20 79.248 0.552 0.2 M 30 68.973 0.827 0.2 N 50 48.42 1.38 0.2 0 80 17.59 2.21 0.2 P 97 0 2.8 0.2

[0120] The enzymatic debridement activity of each dispersion was determined by the in-vitro artificial eschar model described above. The results are plotted in FIG. 2. As can be seen by the results in FIG. 2, the optimum amount of PEG-600 based on the 24 hour curve is about 30% w/w PEG-600.

Example 3 Dispersions of Collagenase/Poloxamer 124 in Petrolatum

[0121] The dispersions in TABLE 3 were prepared with varying concentrations of Poloxamer 124 dispersed in Petrolatum.

TABLE-US-00003 TABLE 3 Poloxamer 124 Wht Petrolatum Poloxamer 407 Collagenase Dispersion % w/w % w/w % w/w % w/w Q 0 99.8 0 0.2 R 10 89.8 0 0.2 S 20 79.8 0 0.2 T 30 69.8 0 0.2 U 50 48.14 1.66 0.2 V 80 17.14 2.66 0.2 W 85* 0 15 0.2 *Poloxamer 407 was added to Poloxamer 124 to form a semi-solid resulting in approximate total of Poloxamer of 100%

[0122] The enzymatic debridement activity of each dispersion was determined by the in-vitro artificial eschar model described above. The results are plotted in FIG. 3. As can be seen by the results in FIG. 3, the optimum amount of Poloxamer 124 based on the 24 hour curve is about 30% w/w Poloxamer 124.

Example 4: Dispersions of Trypsin/PEG 400 in Petrolatum

[0123] The dispersions in TABLE 4 were prepared with varying concentrations of Polyethylene Glycol 400 (PEG-400) dispersed in Petrolatum.

TABLE-US-00004 TABLE 4 Disper- PEG 400 PEG 1450 Wht Petrolatum Poloxamer 407 Trypsin sion % w/w % w/w % w/w % w/w % w/w X 0 0 99.8 0 0.2 Y 14 0 84.9 0.4 0.2 Z 29 0 69.8 0.9 0.2 AA 59 0 39.16 1.64 0.2 BB 80 0 17.06 2.74 0.2 CC 82* 15.2 0 2.6 0.2 *PEG-1450 was added to PEG-400 to form a semi-solid resulting in approximate total PEG of 97%

[0124] The enzymatic debridement activity of each dispersion was determined by the in-vitro artificial eschar model described above. The results are plotted in FIG. 4. As can be seen by the results in FIG. 4, the optimum amount of PEG-400 based on the 24 hour curve is about 14% w/w PEG-400.

Example 5: Dispersions of Papain/PEG 400 in Petrolatum

[0125] The dispersions in TABLE 5 were prepared with varying concentrations of Polyethylene Glycol 400 (PEG-400) dispersed in Petrolatum.

TABLE-US-00005 TABLE 5 Disper- PEG 400 PEG 1450 Wht Petrolatum Poloxamer 407 Papain sion % w/w % w/w % w/w % w/w % w/w DD 0 0 99.85 0 0.15 EE 15 0 85.05 0.4 0.15 FF 29 0 69.82 0.83 0.15 GG 43 0 54.85 1.24 0.15 HH 59 0 39.694 1.636 0.15 II 82* 15.01 0 2.67 0.15 *PEG-1450 was added to PEG-400 to form a semi-solid resulting in approximate total PEG of 97%

[0126] The enzymatic debridement activity of each dispersion was determined by the in-vitro artificial eschar model described above. The results are plotted in FIG. 5. As can be seen by the results in FIG. 5, the optimum amount of PEG-400 based on the 24 hour curve is about 29% w/w PEG-400.

Example 6: Dispersions of Thermolysin/PEG 400 in Petrolatum

[0127] The dispersions in TABLE 6 were prepared with varying concentrations of Polyethylene Glycol 400 (PEG-400) dispersed in Petrolatum.

TABLE-US-00006 TABLE 6 Disper- PEG 400 PEG 1450 Wht Petro- Poloxamer Thermolysin sion % w/w % w/w latum % w/w 407 % w/w % w/w JJ 0 0 99.85 0 0.15 KK 14 0 85.05 0.4 0.15 LL 29 0 69.82 0.83 0.15 MM 59 0 39.694 1.636 0.15 NN 82* 15.01 0 2.67 0.15 *PEG-1450 was added to PEG-400 to form a semi-solid resulting in approximate total PEG of 97%

[0128] The enzymatic debridement activity of each dispersion was determined by the in-vitro artificial eschar model described above. The results are plotted in FIG. 6. As can be seen by the results in FIG. 6, the optimum amount of PEG-400 based on the 24 hour curve is about 29% w/w PEG-400.

Example 7: Dispersions of Pepsin/PEG 400 in Petrolatum

[0129] The dispersions in TABLE 7 were prepared with varying concentrations of Polyethylene Glycol 400 (PEG-400) dispersed in Petrolatum.

TABLE-US-00007 TABLE 7 Disper- PEG 400 PEG 1450 Wht Petrolatum Poloxamer 407 Pepsin sion % w/w % w/w % w/w % w/w % w/w OO 0 0 99 0 PP 15 0 84.2 0.4 1 QQ 29 0 68.97 0.83 1 RR 44 0 54.005 1.24 1 SS 58 0 38.844 1.636 1 TT 81* 15.01 0 2.67 1 *PEG-1450 was added to PEG-400 to form a semi-solid resulting in approximate total PEG of 96%

[0130] The enzymatic debridement activity of each dispersion was determined by the in-vitro artificial eschar model described above. The results are plotted in FIG. 7. As can be seen by the results in FIG. 7, the optimum amount of PEG-400 based on the 24 hour curve is about 58% w/w PEG-400.

Example 8: Dispersions of Collagenase/PEG 400 in Petrolatum for Physical Release of Enzyme

[0131] The dispersions in TABLE 8 were prepared with varying concentrations of Polyethylene Glycol 400 (PEG-400) dispersed in Petrolatum.

TABLE-US-00008 TABLE 8 PEG 400 PEG 1450 Wht Petrolatum Collagenase Dispersion % w/w % w/w % w/w % w/w UU 0 0 99.8 0.2 VV 5 0 94.8 0.2 WW 10 0 89.8 0.2 XX 15 0 84.8 0.2 YY 83* 12.5 4.5 0.2 ZZ 70* 29.8 0 0.2 *PEG-1450 was added to PEG-400 to form a semi-solid resulting in approximate total PEG of 83% and 100% respectively.

[0132] The physical release of enzyme was determined by the In-vitro Physical Enzyme Release Test as described above. The results are plotted in FIG. 8. As can be seen by the results in FIG. 8, the physical release of collagenase generally increased as the concentration of PEG-400 in the dispersion increased with the highest release at 100% and the lowest release at 0% PEG-400.

[0133] As can be seen by the results shown herein, the physical enzyme release profile of the dispersions as a function of increased concentration of hydrophilic polyol does not correlate to the enzymatic activity profile of the enzyme as a function of increased concentration of hydrophilic polyol.

Example 9: Stability and Efficacy Data

[0134] FIG. 9 provides data comparing the stability of collagenase in a dispersion of the present invention (30% PEG in WP dispersion) and an oil-in-water emulsion (Aqueous cream). These data suggest that collagenase was more stable in the 30% PEG in WP dispersion when compared to the Aqueous cream. Tables 9-10 provide descriptions of the 30% PEG in WP dispersion and Aqueous cream formulations.

TABLE-US-00009 TABLE 9 (30% PEG in WP dispersion)* Ingredients wt % PEG-600 30.059774 Poloxamer-407 1.5078044 White Petrolatum 68.309516 Collagenase 0.1228163 TOTAL 100 *PEG in WP dispersion was prepared as follows: (A) Active Phase: (1) 9.71 grams of PEG-600 and 0.2361 grams of collagenase were mixed for 20 minutes at room temperature (20-25 C.) for 45 min. (B) Main Phase: (1) 102.784 grams of white petrolatum, 37.65 grams of PEG-600, and 2.27 grams of poloxamer-407 were mixed at 70 C. until uniform; (2) the mixture was cooled to 40-45 C. Added 7.79 grams of the Active Phase was added to the Main Phase followed by stirring for 30 minutes or until homogenous mixture obtained.

TABLE-US-00010 TABLE 10 (Aqueous cream)* Ingredients wt % Isopropyl Myristate 30.57437 Emulsifying Wax 4.502116 White Petrolatum 20.369574 Incroquat TMS 4.502116 Water 20.009404 Glycerin (96%) 19.839324 Collagenase 0.2030955 TOTAL 100 *Aqueous cream was prepared as follows: (A) Active Phase: (1) 0.2 grams of collagenase was mixed with 20 grams of deionized water. (B) Main Phase: (1) 20.36 grams of white petrolatum was mixed with 4.5 grams of emulsifying wax, 4.5 grams of Incroquate TMS, and 19.83 grams of glycerin (96%) at 70 C. until uniform; (2) the mixture was cooled to 35-40 C. Added Active Phase to Main Phase followed by stirring for 30 minutes or until homogenous mixture obtained.

[0135] FIG. 10 provides data comparing enzyme debridement efficacy in eschar removal in pig burn wounds of a dispersion of the present invention (PEG-in-White PetrolatumTable 11) to the following three formulations: (1) an Aqueous creamTable 12; (2) SANTYL (Commercial product, which is a mixture of collagenase and white petrolatum); and a hydrogel formulationTable 13. The burn wounds were created on pigs and hard eschars formed after several days. Formulation was applied to the hard eschars one a day for two weeks. Only fully debrided wounds were counted as complete debridement. There were a total of 20 wounds per treatment.

TABLE-US-00011 TABLE 11 (PEG-in-White Petrolatum)* Ingredients wt % Poloxamer-407 0.99891551 White Petrolatum 78.7544989 Thermolysin 0.20168104 PEG-600 20.0449046 TOTAL 100 *PEG-in-White Petrolatum was prepared as follows: (A) Active Phase: (1) 32.67 grams of PEG-600 and 1.63 grams of Poloxamer-407 were homogenized at 70 C. until mixture was clear; (2) mixture was cooled to about 35 C.; and (3) thermolysin was and mixed for at least 30 min.. (B) Main Phase: (1) 236.52 grams of white petrolatum, 30.05grams of PEG-600, and 1.5 grams of poloxamer-407 were homogenized at 70 C.; and (2) mixture was cooled to about 35 C. The Active Phase (B) was added to the Main Phase (B) and mixed at room temperature (20-25 C.) for 45 min.

TABLE-US-00012 TABLE 12 (Aqueous cream)* Ingredients wt % Emulsifying Wax 14.993927 1% KH2PO4 in water (pH = 7.5) 74.057507 Isopropyl Palmitate, NF 5.4571649 Glycerin 5.0104708 Thermolysin 0.2001065 Methyl paraben 0.2007937 Propyl paraben 0.0800301 TOTAL 100 *Aqueous cream was prepared as follows: (1) parabens were melted in buffer at high temperature (>70 C.) along with glycerin; (2) emulsifying wax and isopropyl palmitate were added; (3) the mixture was mixed at high temperature for 45 min and then cooled to about 35 C.; (4) thermolysin was added as a slurry in the buffer; (5) the mixture was cooled to room temperature (20-25 C.).

TABLE-US-00013 TABLE 13 (Hydrogel)* Ingredients wt % Hydroxypropylmethylcellulose 2.250621745 1% KH2PO4 in water (pH = 7.5) 77.96851753 Thermolysin 0.202530294 Methyl paraben 0.244719829 Propyl paraben 0.0480663 Propylene glycol 19.28554438 TOTAL 100 *Hydrogel was prepared as follows: (1) parabens and propylene glycol were solubilized in water at 70 C.; (2) HPMC was added at room temperature (20-25 C.); (3) Thermolysin was added and a milky viscous solution formed.