Use of seaprose to remove bacterial biofilm

Abstract

A method of disrupting a bacterial biofilm present on a surface, comprising applying a composition that includes a semi-alkaline protease produced by the fermentation of the fungus Aspergillus melleus (Seaprose) to the bacterial biofilm, wherein application of the composition to the bacterial biofilm disrupts the matrix of the bacterial biofilm.

Claims

1. A method of disrupting a bacterial biofilm present on a wound that does not include necrotic tissue, the method consisting of applying a composition consisting of seaprose and a pharmaceutically acceptable carrier to the bacterial biofilm present on the wound, wherein seaprose is a semi-alkaline protease produced by the fermentation of the fungus Aspergillus melleus, wherein application of the composition to the bacterial biofilm disrupts the matrix of the bacterial biofilm, wherein the semi-alkaline protease has a molecular weight of about 31 kDa, wherein the pharmaceutically acceptable carrier is selected from a lotion, cream, emulsion, ointment, gel, paste, solution, aerosol spray, aerosol foam, non-aerosol spray, non-aerosol foam, powder, liquid solution, liquid suspension, film, and sheet, and wherein seaprose is the only enzymatic agent in the composition.

2. The method of claim 1, wherein the wound is a chronic wound.

3. The method of claim 2, 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.

4. The method of claim 1, wherein the composition includes 0.0001 to 1% by weight of seaprose.

5. The method of claim 1, wherein the seaprose is isolated or purified seaprose.

6. The method of claim 1, wherein the pharmaceutically acceptable carrier is a gel, cream, ointment, solution, or paste.

7. The method of claim 1, wherein the composition is topically applied to the wound.

8. The method of claim 1, wherein the bacterial biofilm is a gram-positive bacterial biofilm.

9. The method of claim 8, wherein the bacterial biofilm includes Staphylococcal bacteria.

10. The method of claim 9, wherein the bacteria is Staphylococcus aureus.

11. The method of claim 10, wherein the bacteria is methicillin-resistant Staphylococcus aureus (MRSA).

12. The method of claim 1, wherein applying the composition removes a portion of the bacterial biofilm from the wound.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1. A graph showing the effectiveness of Seaprose in removing a S. aureus bacterial biofilm when compared with collagenase and a control.

(2) FIG. 2. A graph showing the effectiveness of Seaprose in removing a P. aeruginosa bacterial biofilm when compared with collagenase and a control.

(3) FIG. 3. A graph showing the effectiveness of Seaprose in removing a methicillin-resistant S. aureus (MRSA) biofilm in a mouse model.

(4) FIG. 4. Photos of wounds after treatment with Seaprose and comparators (pretreated wounds had dense MRSA biofilm in a mouse model).

(5) FIG. 5. A plot of the results of an in-vitro study comparing the degradation of pig eschar by bromelain, thermolysin, and Seaprose gels at 37° C. within a 24-hour period.

(6) FIG. 6. An image of in vivo pig wounds after 24 hour treatment with a Seaprose hydrogel compared with a control (moist wound care).

(7) FIG. 7. A graph of the results of the in-vivo pig study comparing the debridement of wounds with Seaprose hydrogel compared with a control (moist wound care).

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

(8) Bacterial biofilms are present in several health conditions that afflict the population. Examples of such conditions include urinary tract infections, cystitis, lung infections, skin infections, sinus infections, ear infections, acne, dental caries, periodontitis, nosocomial infections, open wounds, and chronic wounds.

(9) One of the unique aspects of the present invention is the inventors' discovery that Seaprose can be used to disrupt or remove bacterial biofilms from a surface that has a bacterial biofilm. An example of such a surface is a skin wound. These and other non-limiting aspects of the present invention are described in further detail in the following subsections.

(10) A. Compositions

(11) The compositions of the present invention can be used to combat the presence of bacterial biofilms. Such compositions include an effective amount of Seaprose to achieve this result. The compositions can also include a pharmaceutically acceptable carrier (e.g., topical carrier or injectable carrier). The compositions of the invention may further comprise pharmaceutically active ingredients, cosmetically active ingredients, and vulnerary agents (e.g., growth factors) suitable for topical or injectable administration to wounds.

(12) 1. Seaprose

(13) Seaprose is a semi-alkaline protease produced by the fermentation of the fungus Aspergillus melleus and is commercially available in a powder form from Amano Enzyme, Inc., Japan under the trade name SEAPROSE S®. Seaprose may be prepared by either a liquid or solid fermentation process using techniques known by one of skill in the art. Seaprose has also been referred to as onoprose, promelase, promelasum, Jeoase, FLAMINASE® (Prodotti Formenti S.r.l., Milan Italy), and Aspergillus melleus semi-alkaline proteinase.

(14) The major protease in Seaprose is a semi-alkaline protease with a molecular weight around 31 kDa. It can also contain other enzymes such as amylase, which is a hydrolytic enzyme which breaks down carbohydrates. Alternatively, Seaprose can be purified or isolated by standard techniques known to those of skill in the art. Seaprose shows great stability at an optimal pH range of 5 to 9, and an optimal temperature below 50° C. These conditions are suitably for application of the enzyme in wounds and favorable for drug formulation and manufacture.

(15) Seaprose has previously been used for a variety of medical indications and treatment; however, it has never previously been used in a topical or injectable form for use as an anti-biofilm agent. For example, Seaprose has been shown to possess in-vitro mucolytic activity (Braga 1990) and to effectively treat patients with bronchitis by oral administration of Seaprose capsules (Braga 1993), (Moretti 1993). Seaprose has shown anti-inflammatory activity against many different inflammatory conditions in animal models (Fossati 1991). Seaprose was shown to be effective in treating patients with inflammatory venous disease by oral administration of Seaprose tablets (Bracale 1996). Seaprose has been used to treat abdominal pain due to pancreatitis (U.S. Pat. No. 7,459,155). Seaprose has been used to treat complications of puerperal surgical wounds by oral administration of Seaprose 30 mg tablets (Dindelli 1990).

(16) According to the present invention, Seaprose may be in a dissolved state and/or a dispersed state in the pharmaceutically acceptable carrier. The Seaprose may also be encapsulated. It may also be used neat without a carrier. Seaprose can also be used in a purified or isolated form.

(17) The amount of Seaprose in a composition with a pharmaceutically acceptable carrier is an amount effective for wound debridement and can generally range from about 0.001% w/w to about 10% w/w; or from about 0.01% to about 9%; or from about 0.1% to about 8%; or from about 0.1% to about 0.9%; or from about 0.2% to about 0.8%; or from about 0.3% to about 0.7%; or from about 0.4% to about 0.6%; or about 0.5%; or from about 0.5% to about 7%; or about 1% to about 6%; or from about 1.5% to about 5%; or from about 0.5% to about 1.5%; or from about 0.6% to about 1.4%; or from about 0.7% to about 1.3%; or from about 0.8% to about 1.2%; or from about 0.9% to about 1.1%; or about 1%. Such amount will be that amount which effectively debrides necrotic tissue in wounds. In particular embodiments, a range of 0.0001 to 1% or 0.001 to 1% can be used.

(18) 2. Pharmaceutically Acceptable Carriers

(19) The compositions of the present invention may comprise various pharmaceutically acceptable carriers suitable for topical delivery and compatible with Seaprose. Non-limiting examples include lotions, creams, emulsions, ointments, gels, pastes, solutions, aerosol sprays, aerosol foams, non-aerosol sprays, non-aerosol foams, powders, liquid solutions, liquid suspensions, films, and sheets. The compositions may be impregnated in gauzes, bandages, or other wound dressing materials for topical delivery.

(20) The compositions of the invention may further comprise functional ingredients suitable for use in topical compositions and compatible with Seaprose. Non-limiting examples include absorbents, antimicrobial agents, antioxidants, binders, buffering agents (including Tris buffer solutions), bulking agents, chelating agents, colorants, biocides, deodorant agents, emulsion stabilizers, film formers, fragrance ingredients, humectants, lytic agents, enzymatic agents, opacifying agents, oxidizing agents, pH adjusters, plasticizers, preservatives, reducing agents, emollient skin conditioning agents, humectant skin conditioning agents, moisturizers, surfactants, emulsifying agents, cleansing agents, foaming agents, hydrotopes, solvents, suspending agents, viscosity control agents (rheology modifiers), viscosity increasing agents (thickeners), and propellants. Listings and monographs of the functional ingredients described herein are disclosed in The International Cosmetic Ingredient Dictionary and Handbook (INCI), 12.sup.th Edition, 2008, hereby incorporated by reference.

(21) Non-limiting examples of antimicrobial agents include anti-fungal agents such as Miconazole Nitrate, Econazole Nitrate, and others, and antibiotics such as Neomycin, Bacitracin, Polymixin, etc. Additional non-limiting antimicrobial agents that can be used include Benzalkonium Chloride, Benzethonium Chloride, Benzoic Acid or salt form thereof, Benzoyl Peroxide, Benzyl Alcohol, Bispyrithione Salt, Borage Oil, Boric Acid, Cadexomer-Iodine, Camphorated Metacresol, Camphorated Phenol, Chlorhexidine Gluconate, Chlorobutanol, Cloflucarban, Dapsone, Dehydroacetic Acid or salt form thereof, Ethyl Alcohol, Eucalyptol, Extracts of Lavender Oil, Free fatty acids having from six to eighteen carbons, Glyceryl Laurate, Hexachlorophene, Hexitidine, Hexylresorcinol, Hydrogen Peroxide, Hydroxybenzoic Acids or salt forms thereof, Iodine Complexed with Phosphate Ester of Alkylaryloxy Polyethylene, Iodine Tincture, Iodine Topical Solution, lodoquinol, Isopropyl Alcohol, Lipacide CG, Mafenide Acetate, Magnesium Pyrithione, Menthol, Merbromin, Mercufenol Chloride, Methyl Salicylate, Methylbenzethonium Chloride, Methylparaben, Metronidazole, Metronidazole derivatives, Nitrofurazone, Nonyl Phenoxypoly Ethanol-Iodine, n-Propanol, Organic Peroxides, p-chloro-m-xylenol, Phenol, Phenoxyethanol, Phenyl Alcohol, Poloxamer-iodine complex, Povidone Iodine, PVP-Iodine, Rose Hips Oil, Salicylic Acid, Secondary Amyltricresols, Selenium sulfide, Silver or salt form thereof, Silver Sulfadiazine, Sodium Oxychlorosene, Sodium Sulfacetmide, Sorbic Acid or salt form thereof, Sulfur, Tetrachlorosalicylanilide, Thymol, Tribromsalan, Triclocarbon, Triclosan, Undecoylium Chloride-iodine Complex, Zinc Pyrithione. In addition, antimicrobial peptides and proteins can be used.

(22) Suitable pharmaceutically acceptable topical carriers include an anhydrous hydrophilic wound debrider composition as disclosed in: U.S. Pat. No. 6,548,556 herein incorporated by reference; a spray-on topical wound debrider composition as disclosed in U.S. Pat. No. 7,785,584 herein incorporated by reference; an enzymatic wound debriding composition as disclosed in international PCT application PCT/US10/59409 herein incorporated by reference; a hydrogenated castor oil ointment as disclosed in U.S. Pat. No. 6,479,060 herein incorporated by reference; an anhydrous hydrophilic absorbent wound dressing as disclosed in U.S. Pat. No. 6,399,092 herein incorporated by reference; and a hydrogel wound dressing as disclosed in U.S. Pat. No. 5,902,600 herein incorporated by reference.

(23) The compositions of the present invention may also comprise various pharmaceutically acceptable carriers suitable for injectable delivery compatible with Seaprose.

(24) The compositions of the present invention may be packaged in any package configuration suitable for topical or injectable products. Non-limiting examples for topical products include bottles, lotion pumps, toddles, tubes, jars, non-aerosol pump sprayers, aerosol containers, syringes, pouches, and packets. The packages may be configured for single-use (one dose) or multiple-use administration. Non-limiting examples for injectable products include vials, syringes, micro-needle syringes, or bags.

(25) The compositions of the present invention may also be sterile. They may be sterilized via an aseptic manufacturing process or sterilized after packaging by methods known in the art.

(26) 3. Manufacture

(27) The compositions of the present invention may be manufactured by suitable processing methods known by one of skill in the art for topical and/or injectable products. For example, Seaprose can be admixed with the pharmaceutically acceptable carrier. Further, the compositions can be sprayed onto a surface. Alternatively, Seaprose can be applied to a bacterial biofilm in a neat form (e.g., without carrier).

(28) B. Methods of Use

(29) The composition of the present invention may be used in methods for treating, disrupting, or removing bacterial biofilms from a surface or with preventing or limiting the formation of a bacterial biofilm on a surface that is susceptible of developing a bacterial biofilm (such as, for example, a wound, a surgical incision or wound, an implanted device, etc.). Such methods can include applying (e.g., topical, injectable, sprayable etc.) to a bacterial biofilm or target surface a composition comprising Seaprose. After application, the bacterial biofilm can be covered with a dressing such as a gauze pad. Alternatively, or additionally, the surface can then be treated with a traditional anti-microbial agent to attack the bacteria remaining within the bacterial biofilm. The composition can also or alternatively be applied to a dressing such as a gauze pad first and then applied to a bacterial biofilm. The application amount can depend on the type and severity of the bacterial biofilm. Further, application of the composition can be in the form of a regimen with period application (e.g., hourly, daily, weekly, etc.). As explained above, a wide range of surfaces that have bacterial biofilms can be treated with the compositions of the present invention. For instance, wound surfaces present on a person's skin can be treated. Such wound surfaces can be, by way of example, burns, acute wounds, or chronic wounds that include a bacterial biofilm or are susceptible to formation of a bacterial biofilm. Other types of surfaces that could have bacterial biofilms (e.g., living tissue, bodily surfaces, inanimate objects) or are susceptible in developing bacterial biofilms (e.g., wounds, surgical incision or wounds, medical implant devices, etc.) can be treated with the compositions of the present invention.

EXAMPLES

(30) 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

Exemplary Formulations

(31) The following Tables provide non-limiting examples of formulations containing Seaprose of the present invention:

(32) TABLE-US-00001 TABLE 1 Gel* % Concentration Ingredient (by weight) SEAPROSE S 1.0 Tris Buffer Solution (TBS) 10 mM (pH 7.5) 96.4 Hydroxyethylcellulose (HEC) 2.6 TOTAL 100 *Procedure: A gel was made with the HEC and Tris buffer. SEAPROSE S was admixed with the HEC gel. The viscosity of the gel gradually reduced over time possibly due to the amylase present in the Seaprose material degrading the HEC.

(33) TABLE-US-00002 TABLE 2 Gel* % Concentration Ingredient (by weight) SEAPROSE S 1.0 CURASOL ® Gel Wound Dressing 99.0 TOTAL 100 *Procedure: SEAPROSE S was admixed with the CURASOL Gel Wound Dressing to form a clear gel. The viscosity was maintained over time.

(34) TABLE-US-00003 TABLE 3 Cream* % Concentration Ingredient (by weight) SEAPROSE S 0.5 Tris Buffer Solution (TBS) 10 mM (pH 7.5) 71.52 Glycerin 7.0 Methylparaben 0.2 Propylparaben 0.08 Emulsifying Wax 15.2 Isopropyl Palmitate NF 5.5 TOTAL 100 *Procedure: Methylparaben, propylparaben and glycerin were dissolved in the Tris buffer solution at 70° C. Emulsifying wax and isopropyl palmitate were added to the above solution at 70° C. and mixed to form an emulsion, The emulsion was cooled to 35° C. at which time SEAPROSE S was admixed with the emulsion. A white cream was obtained.

(35) TABLE-US-00004 TABLE 4 Ointment* % Concentration Ingredient (by weight) SEAPROSE S 0.5 White Petrolatum 78.5 PEG-600 20.0 Poloxamer-407 1.0 TOTAL 100 *Procedure: An Active Phase was made by melting a mixture of half of the amount of PEG-600 and half of the amount of poloxamer-407 at 70° C., cooling the mixture to 35° C. at which time SEAPROSE S was admixed with the mixture. A Main Phase was made by melting a mixture of white petrolatum and the remaining half of the amount of PEG-600, and the remaining half of the amount of poloxamer-407 at 70° C., cooling the mixture to 35° C. after the homogenization and melting of poloxamer-407. The Active Phase was then admixed with the Main Phase. The resulting mixture was mixed at RT for 45 minutes.

(36) TABLE-US-00005 TABLE 5 Capmul Oil Based Formulation* % Concentration Ingredient (by weight) SEAPROSE S 0.5 Capmul MCM, NF 20 Tris Buffer Solution (TBS) 10 mM (pH 7.5) q.s. Poloxamer-407 12.75 TOTAL 100 *Procedure: Poloxamer-407 was solubilized at 4° C. in TBS buffer (10 mM TBS), upon solubilization, the oil, Capmul MCM NF, from Abitec, was added and the mixture was mixed at RT under high shear until homogeneous. Seaprose S was solubilized at calculated concentration in TBS, upon solubilization, the solution was added to the cream and mixed for 30 min at RT. Off-white cream was obtained.

Example 2

In Vitro Biofilm Removal Data

(37) An in vitro assay was performed to demonstrate the bacterial biofilm disruption and removal capabilities of Seaprose. In this assay, S. aureus ATCC 6538 was suspended in tryptic soy broth supplemented with 0.25% glucose for optimal bacterial biofilm formation. The suspension was transferred to the wells of sterile 96 well plates and incubated for 22 hours at 37° C. with one change of media. After bacterial biofilm formation the media was replaced with enzyme treatments prepared in growth media. After 16 hours of treatment (treatment composition included Seaprose S+above mentioned growth medium) the remaining attached bacteria were quantified by aspirating media and washing the plate thoroughly followed by crystal violet staining and recording the absorbance at 570 nm. The crystal violet stains the remaining attached bacteria and a decreased absorbance compared to control indicates removal of attached bacteria. FIG. 1 provides a summary of these data. As illustrated in FIG. 1, Seaprose was far more effective at disrupting and removing the bacterial biofilm than collagenase at levels ranging from 1% w/w to 0.0000001% w/w, with a surprising level of efficacy at levels of 1% w/w to 0.0001% w/w, and even more surprising levels of 1% w/w to 0.001% w/w. The surprising nature of this discovery is based on the general knowledge that proteases are not thought to be overly effective on their own in disrupting or removing bacterial biofilms.

(38) An in vitro assay was also performed to demonstrate the bacterial biofilm disruption and removal capabilities of Seaprose on P. aeruginosa. In this assay, P. aeruginosa ATCC 15442 was suspended in phosphate buffered saline with 10% Tryptic soy broth and 0.45% glucose. The suspension was transferred (200 microliters) to the wells of sterile 96 well plates and incubated for 26 hours at 37° C. with one change of media. After biofilm formation, the media was replaced with enzyme treatments previously prepared in growth media. After 18 hours of treatment at 37° C. (treatment composition included Seaprose S+above mentioned growth medium) the remaining attached bacteria were quantified by aspirating the media and washing the plate thoroughly followed by crystal violet staining and recording the absorbance at 570 nm. The crystal violet stains the remaining attached bacteria and a decreased absorbance compared to the growth control indicates removal of attached bacteria. FIG. 2 provides a summary of these data.

Example 3

In Vivo Biofilm Removal Data

(39) An in vivo assay was performed to demonstrate the bacterial biofilm disruption and removal capabilities of Seaprose on methicillin-resistant S. aureus (MRSA) containing biofilms. The assay was similar to that described in E. D. Roche, P. J. Renick, S. P. Tetens, and D. L. Carson, 2012, A Model for Evaluating Topical Antimicrobial Efficacy against Methicillin-Resistant Staphylococcus aureus Biofilms in Superficial Murine Wounds, Antimicrobial Agents and Chemotherapy, 56, 4508-10.

(40) In particular, twenty-eight female, SKH1 mice were administered Cytoxan injections four days prior to the wounding. Overnight cultures were prepared of the MRSA 33592, which were streaked to confirm purity. Prior to wounding, the inoculum was prepared and adjusted to 2.0×10.sup.9 cfu/mL (colony forming unit/milliliter). The inoculum was spot plated to confirm the challenge cfu and placed on ice for the duration of the wound creation and inoculation procedures. All wounding, treatment applications, and dressing changes were conducted with the animals under anesthesia, administered via isoflurane inhalant. The surgical field was sterilized with povidone-iodine followed by an alcohol swab. The skin was blotted dry with sterile gauze before wounding. Using a pre-cut template, the wounds were created using a rotary tool, on a low speed, by repeatedly touching the skin for 5 seconds at a time. The wounds were wiped with saline moistened gauze to clear the wound of any debris created by the rotary tool prior to inoculation. Each wound was inoculated with 10 μL of inoculum and dressed with a pre-moistened spot band-aid. Secondary dressings of a layer of Surgilast® size 1 dressing secured at the distal end with a strip of Elastikon® were applied after dressing the inoculated wounds. Each mouse was placed in a heated recovery tub until conscious and moving before being returned to the animal room.

(41) The mice were organized in seven groups, with four mice in each group. Each treatment was present in two treatment groups, opposite a different treatment in each case. Initial treatments, at timepoint 0, were applied 24 hours after inoculation. A second treatment was applied at 24 hours. The wounds were photographed at wounding, each treatment application, and study end. Three mice from each group were sampled for microbiology 48 hours after the initial treatment was applied. The microbiology samples were obtained with 4 mm biopsy punches and placed in pre-labeled, pre-weighed tubes containing PBS (phosphate buffered saline) solution. The samples were placed on ice until further processing could take place. Once all the samples were obtained, the samples were allowed to warm to room temperature for re-weighing. Once re-weighed, the samples were homogenized at 30,000 rpm for 10 second intervals until the sample was completely disrupted. Once fully homogenized, the sample was placed back on ice until all samples were processed. The samples were used to create dilution plates, with duplicates of all samples, for spot plating on TSA and Charcoal agars. The spot plates were grown overnight in a 37° incubator before the colonies were counted. The colony counts were converted into log cfu/g and graphed. Data is presented in FIGS. 3 and 4, which confirms Seaprose had an effect in reducing MRSA bioburden in a mouse MRSA wound biofilm model beyond the oil base in which it was present. The oil base+Seaprose (referenced as “Seaprose (in Base)” in FIGS. 3 and 4) also trended toward a greater effect than a silver gel (SilvaSorb® Gel obtained from Medline Industries, Inc., Mundelein, Ill. (USA).

(42) The oil base and oil base-Seaprose formulas referenced above and noted in FIGS. 3 and 4 are provided in Tables 6 and 7, respectively.

(43) TABLE-US-00006 TABLE 6 Oil Base* % Concentration Ingredient (by weight) Poloxamer-407 13.513 Capmul MCM NF 20.033 Tris Buffer Solution (TBS) 10 mM (pH 7.5) 66.454 TOTAL 100 *Procedure: Poloxamer-407 was solubilized at 4° C. in TBS buffer (10 mM TBS), upon solubilization, the oil, Capmul MCM NF, from Abitec, was added and the mixture was mixed at RT under high shear until homogeneous. Off-white cream was obtained.

(44) TABLE-US-00007 TABLE 7 Oil Base + Seaprose S % Concentration Ingredient (by weight) Poloxamer-407 12.761 Capmul MCM NF 19.974 SEAPROSE S 0.501 Tris Buffer Solution (TBS) 10 mM (pH 7.5) 66.764 TOTAL 100 *Procedure: Poloxamer-407 was solubilized at 4° C. in TBS buffer (10 mM TBS), upon solubilization, the oil, Capmul MCM NF, from Abitec, was added and the mixture was mixed at RT under high shear until homogeneous. Seaprose S was solubilized at calculated concentration in TBS, upon solubilization, the solution was added to the cream and mixed for 30 min at RT. Off-white cream was obtained.

Example 4

In Vitro Digestion of Pig Burn Eschar

(45) The gel formula in Table 1 (1% Seaprose Gel) and each of the following two gel formulas (1% Thermolysin Gel (Table 8) and 10% Bromelain Gel (Table 9)) were used in an in-vitro study to compare the degradation of pig eschar by each gel formula.

(46) TABLE-US-00008 TABLE 8 1% Thermolysin Gel % Concentration Ingredient (by weight) Thermolysin (Sigma-Aldrich) 1.0 Tris Buffer Solution 10 mM (pH 7.5) 95.1 Hydroxyethylcellulose (HEC) 2.9 Sodium Chloride 0.9 Calcium Chloride 0.1 TOTAL 100

(47) TABLE-US-00009 TABLE 9 10% Bromelain Gel % Concentration Ingredient (by weight) Bromelain (Spectrum) 10.0 Water 84.6 Carbomer 980K 1.9 Disodium Phosphate 2.6 4-Chloro-3-Methylphenol 0.1 Sodium Hydroxide 0.8 TOTAL 100

(48) The study was conducted in-vitro using eschar materials obtained from pig burn wounds. The eschar materials were dried completely. The dry weight was used as baseline. Samples of the dried eschar weighing 40-60 mg were moisturized with 50 μl of Tris buffered saline. The moisturized eschar samples were immersed in 3 g of each of the three gel formulas. The gels with eschar were stored at 37° C. for 24 hours. After 24 hours, the samples were centrifuged at 5000 rpm for 5 minutes. The supernatant was discarded and water was added to wash the precipitates. The samples were centrifuged again. Another wash step was performed and then the precipitates were freeze-dried. The dry weights of the precipitates were used to calculate the degradation percentage based on the baseline dry weights. The results are presented in FIG. 5.

(49) The results in FIG. 5 demonstrate that the Seaprose gel was more effective and exhibited superior potency in digesting the eschar material as compared to the 1% thermolysin gel (Table 8) and 10% bromelain gel (Table 9) within the 24 hour period. The quickness at which the Seaprose gel digested the eschar as compared to the 1% thermolysin gel and the 10% bromelain gel was unexpected, because thermolysin and bromelain are both known in the art to be a fast debriding enzymes (see, e.g., U.S. Patent Publication 2003/0198631 and U.S. Pat. No. 8,119,124, respectively). The results of the in-vitro study indicate that Seaprose can efficiently and effectively target and digest eschar proteins and therefore, it is suitable as a superior enzymatic wound debrider which can be used for the treatment of wounds in need of debridement.

Example 5

In Vivo Debridement of Pig Burn

(50) In this in vivo pig study, eschars were formed on the backs of pigs by introducing burn wounds using heated brass rods and allowing the formation of dry eschars over several days. There was a visual effect of Seaprose (SAP) on many wounds in comparison to control after one day of treatment (FIG. 6). Overall, SAP exhibited more rapid complete debridement of the eschars when compared against a control (non-adherent pre-moistened wound dressing with saline) (FIG. 7).

(51) The particulars of this in vivo study are as follows. Pigs were anesthetized, the torso 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, photographed, treated according to the treatment randomization 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.

(52) Treatment regimen for this study included use of a Seaprose containing formulation prepared in the following manner and a control which consisted of a non-adherent pre-moistened wound dressing with saline): (1) Seaprose S powder was prepared (see Table 10 below) and 100 mg of said powder was directly applied to the wound; and (2) a gel was prepared (see Table 11 below) and 400 mg of said gel was applied on top of the Seaprose S powder. Treatments were performed once a day for a fifteen day period. After the initial 24 hours of treatment, visual differences were apparent for many Seaprose-treated wounds, including pitting of the eschar and in some cases limited exposure of healthy wound tissue (FIG. 6). Over the fifteen day treatment period, Seaprose treatment produced a consistent trend of complete debridement of more wounds than the control (Seaprose treatment achieved statistical significance (p<0.05) versus the control on day 13 of treatment) (FIG. 7).

(53) TABLE-US-00010 TABLE 10 Seaprose S Powder* % Concentration Ingredient (by weight) SEAPROSE S 2.0 Sorbitol 98.0 TOTAL 100 *Process: Seaprose S and sorbitol were mixed at room temperature (approximately 20 to 25° C.) to obtain a homogenous powder.

(54) TABLE-US-00011 TABLE 11 Gel* % Concentration Ingredient (by weight) Hispagel-200 31.86 Tris Buffer Solution 10 mM (pH 7.5) 58.37 Imidurea 0.14 Glycerin 9.45 Methylparaben 0.16 Propylparaben 0.02 TOTAL 100 *Process: Preservatives were mixed in Tris Buffer at high temperature (>70° C.) along with glycerin. Upon cooling, Hispagel-200 was added. Clear and transparent gel was obtained.

REFERENCES

Publications

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