COMPOSITION AND METHODS OF MANUFACTURE

Abstract

The present invention relates to compositions comprising Carica papaya derived serine protease, methods of extracting the protease from fruit sources as well as cosmetic and therapeutic uses thereof, and associated kits.

Claims

1. A composition comprising one or more proteolytically active serine proteases extracted from ripe fruit of Carica papaya.

2. A composition comprising one or more isolated proteolytically active serine proteases extracted from ripe fruit of Carica papaya.

3. A composition comprising a mixture of one or more proteolytically active serine proteases and one or more active cysteine proteases extracted from ripe fruit of Carica papaya.

4. A composition comprising a mixture of one or more isolated proteolytically active serine proteases and one or more active cysteine proteases extracted from ripe fruit of Carica papaya.

5. A process for preparing a composition comprising a proteolytically active serine protease, wherein the process comprises treating pulped ripe fruit of Carica papaya with an alkali, without subjecting the pulped ripe fruit to a heating step, and separating soluble protease from insoluble plant material after the alkali treatment.

6. The process according to claim 5, wherein the alkali is a weak alkali.

7. The process according to claim 6, wherein the weak alkali has a pKa of less than 11.

8. The process according to claim 7, wherein the weak alkali is sodium bicarbonate.

9. The process according to claim 5, wherein the composition further comprises a proteolytically active cysteine protease.

10. The process according to claim 5, wherein the composition is further treated to increase the concentration of the protease or proteases.

11. A composition comprising a proteolytically active serine protease obtainable by the process of claim 5.

12. A composition comprising a proteolytically active serine protease obtained by the process of claim 5.

13. A pharmaceutical composition comprising a proteolytically active Carica papaya serine protease together with a pharmaceutically acceptable carrier or diluent, wherein the serine protease is extracted from ripe fruit of Carica papaya.

14. A pharmaceutical composition comprising an isolated proteolytically active Carica papaya serine protease together with a pharmaceutically acceptable carrier or diluent, wherein the serine protease is extracted from ripe fruit of Carica papaya.

15. A pharmaceutical composition comprising a composition according to claim 1, together with a pharmaceutically acceptable carrier or diluent.

16. A cosmetic composition comprising a proteolytically active Carica papaya serine protease together with a cosmetically acceptable carrier or diluent, wherein the serine protease is extracted from ripe fruit of Carica papaya.

17. A cosmetic composition comprising an isolated proteolytically active Carica papaya serine protease together with a cosmetically acceptable carrier or diluent, wherein the serine protease is extracted from ripe fruit of Carica papaya.

18. A cosmetic composition comprising a composition according to claim 1, together with a cosmetically acceptable carrier or diluent.

19. A pharmaceutical composition according to claim 13 for use in wound debridement.

20. Use of the pharmaceutical composition according to claim 13 in the manufacture of a medicament for wound debridement.

21. A cosmetic composition according to claim 16 for use in skin exfoliation.

22. Use of the cosmetic composition of claim 16 in skin exfoliation.

23. A method of preventing, treating, reducing or ameliorating a skin condition, wherein the method comprises administering to a subject the composition of claim 1.

24. A kit for use in, or when used for, preventing, treating, reducing or ameliorating a skin condition, wherein the kit comprises the composition of claim 1, and instructions for use.

Description

DESCRIPTION OF THE FIGURES

[0120] FIG. 1: Overlay-zymography schematic representation. Overlay-zymography process and subsequent excision of bands from enzyme activity spot to process on SDS PAGE gels.

[0121] FIG. 2: Representation of debriding assay using Franz cell diffusion system (Shi, Ermis et al. 2009).

[0122] FIG. 3: Bar chart plot of the L-BApNA proteolytic activity of OPAL A and OPAL B filtrates. OPAL A and OPAL B filtrates on L-BApNA, as measured by the slopes of the time-course. OPAL B filtrate has almost 8-fold more catalytic activity as compared to OPAL A filtrate. Results are represented as the mean±S.D for n=8 experiments. Structural analysis was carried out using student T-test (*** p<0.001).

[0123] FIG. 4: Spectrophotometric plot of enzymatic hydrolysis of L-BApNA by OPAL B filtrate and with addition of E-64. Representative plot showing enzymatic hydrolysis of the substrate L-BAPNA by OPAL B filtrate, and partial activity inhibition by cysteine protease inhibitor E-64, suggesting the presence of other proteases in addition to cysteine proteases.

[0124] FIG. 5: Spectrophotometric plot of the effect of protease inhibitor cocktail (PIC) on the proteolytic activity of OPAL B filtrate. Representative trace showing enzymatic hydrolysis of the substrate L-BAPNA by OPAL B filtrate, and its complete inhibition by the PIC.

[0125] FIG. 6: Bar chart plot of effect of different protease inhibitor classes on the proteolytic activity of OPAL B filtrate.

[0126] FIG. 7: Spectrophotometric plot of the effect of E-64+AEBSF on the proteolytic activity of OPAL B filtrate. Representative trace showing the complete inhibition of L-BAPNA activity in OPAL B filtrate by the combination of cysteine protease inhibitor E-64 and serine protease inhibitor AEBSF.

[0127] FIG. 8: Overlay zymography analysis of OPAL B filtrate.

[0128] FIG. 9: Overlay zymography analysis of OPAL B filtrate and effect on using inhibitors. Native PAGE gel bands in OPAL B samples were obtained and the protein bands were subsequently transferred from gel to nitrocellulose membrane. Zymography analysis shows the two enzymatic activities in control and complete inhibition of upper band ( - - - ) with cysteine protease inhibitor E-64 and inhibition of lower band ( - - - ) with serine protease inhibitor AEBSF.

[0129] FIG. 10: Silver staining of SDS PAGE gel run with elutions 2-10. The high intensity band present in the 6.sup.th fraction at 80 kDa as well as bands at 50 k Da and 30 kDa, were analyzed using LC/MS analysis and revealed as a subtilase.

[0130] FIG. 11: SDS PAGE western blotting analysis of OPAL B filtrate treated with serine protease probe using various treatments. SDS PAGE western blotting analysis of OPAL B filtrate treated with 1 μl probe (per 100 μl) with no tris-HCl (lane 2) and with 5 mg/ml tris-HCl (lane 4), 10 mg/ml tris-HCL (lane 6) and respectively with AEBSF (lanes 3, 5 and 7). OPAL B filtrates with 1 μL probe and 10-mg/mL tris-HCl with E-64 (lane 6) and with E-64 and AEBSF (lane 7) showing no background disturbance.

[0131] FIG. 12: Differential digestions of AWE substrate (percentage activity) by OPAL A filtrate as compared to latex-papain, showing OPAL A is better at digesting collagen and fibrin whereas control latex papain is better at digesting elastin. Results are represented as the mean±SD for n=3 experiments. Structural analysis was carried out using student T-test (** p<0.01, *** p<0.001).

[0132] FIG. 13: Differential digestion of AWE substrate (percentage activity) by dialyzed OPAL A and OPAL A+10% urea as compared to native OPAL A filtrate. Dialyzed OPAL A had enhanced debriding activity on both fibrin and elastin but not collagen which correlates with the lower activity of OPAL A towards collagen. OPAL A addition of urea inhibited the activity of enzymes towards Fibrin and Elastin in the AWE assay. Results are represented as the mean±S.D for n=3 experiments. Structural analysis was carried out using student T-test (** p<0.01, *** p<0.001).

[0133] FIG. 14: In vivo case study regarding management of severe pressure ulcer with OPAL B, pre-commencement of treatment with Opal B. Central area of necrosis. Failed attempt to reduce the size of the ulcer with a romboid flap. Distal part of the flap dusky. Warning—graphic images.

[0134] FIG. 15: In vivo case study regarding management of severe pressure ulcer with OPAL B, treatment day 2. Delineation of area that will become necrotic from that which will be viable. Wound edge pink and better blood flow to the skin flap. Warning—graphic images.

[0135] FIG. 16: In vivo case study regarding management of severe pressure ulcer with OPAL B, treatment day 4. Clear demarcation between viable and non-viable tissue. Viable tissue looks clean and pink. Skin flap looks fully viable. Warning—graphic images.

[0136] FIG. 17: In vivo case study regarding management of severe pressure ulcer with OPAL B, treatment day 16. Wound debrided surgically, sutures removed and flap excised. Early granulation of the clean wound surface is evident. Wound edge is pink and suggestion of new skin growth (exposure adjusted to match skin colour of previous images). Warning—graphic images.

[0137] FIG. 18: In vivo case study regarding management of severe pressure ulcer with OPAL B, treatment day 19. Skin and wound colour becoming dusky, particularly wound edges. Wound remains clean (exposure adjusted to match skin colour of earlier images). Warning—graphic images.

EXAMPLES

[0138] Preparation of OPAL A and OPAL B

[0139] The manufacturing process of OPAL A filtrate was performed in sterile conditions. The ends of the ripe fruit of the Carica papaya plant were trimmed and the skin was peeled. The flesh of the fruit was quartered and seeds were then excised. The papaya pieces were mixed in a blender until a smooth pulp was obtained. The pulp was placed in a beaker, which was inside a water bath set at 80° C. and stirred continuously until the temperature of the pulp was 55° C. (measured using a glass thermometer). 10% by weight dry sodium bicarbonate was added to the beaker. The beaker was then placed in the water bath, set at 55° C. and stirred slowly for 5 minutes. After the pulp containing sodium bicarbonate was centrifuged at 12000×g for 30 minutes, the supernatant was collected and transferred into sterile universal tubes by filtering using 0.22 μm filters (FDR-050-071N-Fisher Scientific). OPAL B filtrates were prepared with a one step change in the manufacturing process in that the heating step was omitted.

[0140] Gel Filtration Chromatography Using Sephadex G-100 Superfine

[0141] Gel filtration chromatography was used to separate the proteins specifically on the basis of molecular weight. 14 mL length chromatography columns (C3919-1EA-sigma Aldrich) with Sephadex G-100 (G10050-Sigma) (separation between 4 kDa-150 kDa) were packed and washed with 100 mM Tris-HCl with 0.15M NaCl at pH 8 overnight. 1 mL of the required samples was passed through the column separately in order to separate the proteins on the basis of molecular weight. The required number of 0.5 mL fractions were collected and A280 was measured. 50 μL of each fraction was then plated in 96 well plates and 50 μL of 1.5 mM L-BApNA buffer (made with 10 mM Tris-HCl pH 8.5 buffer) was added. L-BApNA activity at A410 was then measured both immediately and then after 2 hours using a Synergy HT microplate reader (cat. 12926527, Bio-Tek Instruments, Thermo Fisher Scientific, UK). The difference in values was plotted and the fractions with higher L-BApNA activity were run through SDS PAGE gels, which were then stained using Coomassie or silver staining.

[0142] L-BApNA (N-benzoyl-L-arginine 4-nitroanilide hydrochloride) Assays

[0143] A 1.5 mM stock solution of L-BApNA (B-3133 sigma) was prepared by dissolving 63 mg of L-BApNA in 1.5 ml dimethyl sulfoxide (DMSO), and then made up to 100 ml with water. Hydrolysis of the L-BApNA at the bond between the arginine and the p-nitroaniline moieties releases the chromophore p-nitroaniline, which can be detected by spectroscopy at an absorbance of 410 nm in International Units (IU). IU is defined as the amount of enzyme that causes an increase in absorbance of 0.01 units/min under excess substrate conditions (zero order kinetics). Solids specific activity is expressed in IU/mg, while in the case of liquid formulations, the activity is expressed in IU/mL.

[0144] To determine the optimal substrate concentration to maintain zero order kinetics throughout experiments, a series of titration runs were carried out with a range of substrate concentrations. The minimum concentration thus found is 0.2 mM or 1:6 dilution of the stock 1.5 mM solution. Table 1 shows reagent doses used for all kinetic experiments. Phosphate buffer at pH 6 was used in the L-BApNA assays because it gave the highest enzymatic activity reading compared to other buffers.

TABLE-US-00003 TABLE 1 L-BApNA assay final reagent concentrations Contents Reference Sample pH 6.0 Buffer 333 333 Water 501 333 Substrate (L-BApNA) 166 166 Zero at 410 nm Enzyme (OPAL A/B) 0 166 Record A.sub.410

[0145] To verify substrate purity, a controlled total hydrolysis catalyzed by alkali was carried out under the following conditions.

TABLE-US-00004 TABLE 2 L-BApNA substrate purity assay compositions Reference cell (μl) Sample Cell (μl) 5M NaOH 0 966 Water 966 0 Zero at 410 nm Substrate 33 33 Record A.sub.410 [00001]$T\mathrm{hus}\left[\mathrm{subtrate}\right]=\frac{\left(A_{410}\times 30\left(\mathrm{dilution}\mathrm{factor}\right)\right)}{8800\left(\begin{array}{c}\mathrm{molar}\mathrm{coefficient}\mathrm{of}\mathrm{extinction}\mathrm{of}p-\mathrm{nitroanaline}\\ \mathrm{at}410\mathrm{nm}\end{array}\right)}$

[0146] Results confirmed 99% purity of the substrate as stated by manufacturer.

[0147] Inhibitors

[0148] E-64 (IU PAC name: (1S,2S)-2-(((S)-1-((4-Guanidinobutyl)amino)-4-methyl-1-oxopentan-2-yl) carbamoyl) cyclopropane carboxylic acid) is a strong and irreversible specific inhibitor of cysteine proteases. The trans-epoxysuccinyl group (active moiety) of E-64 irreversibly binds to the active thiol group of cysteine proteases, thereby inhibiting them. E-64 does not react with non-protease enzymes and does not inhibit serine proteases.

[0149] 2,2′-dipyridyldisulfide (2DPS) is used as a thiol-specific reversible inhibitor for cysteine proteases, including papain, bromelain, and ficin.

[0150] Protease inhibitor cocktails (PICs) were formulated as shown in Table 3:

TABLE-US-00005 TABLE 3 The components of protease inhibitors in protease inhibitor cocktails Protease inhibitor Enzymes targeted Reagent (stock concentration) by inhibitor Protease Inhibitor Cocktail E-64 (1.4 mM) Cysteine Proteases (P2714, Sigma) Leupeptin (2 mM) AEBSF (104 mM) Serine Proteases Aprotinin (80 μM) Leupeptin (2 mM) Pepstatin A (1.5 mM) Acid Proteases Bestatin (4 mM) Amino peptidases

[0151] Overlay Zymography

[0152] Overlay-zymography was carried out according to the procedure in Vinokurov et al. (2005) with some modifications (FIG. 1). Briefly, a nitrocellulose membrane was soaked in a 1.2 mg/mL L-BApNA solution in water. The membrane was subsequently left to air dry for 5 minutes and then laid on top of the native PAGE gel after running the samples through, which were slightly soaked in their respective running buffers. Two additional experiments were carried out in which membranes were soaked in 35M R-alanine, 0.14 M acetic acid, pH 4.3, with either 10 mM DTT or 25 mM cysteine. All zymographies were then incubated in a closed chamber at 37° C. for 30-60 min. The membrane was removed and again left to dry for 5 minutes and the p-nitroaniline was visualized by diazotization.

[0153] Diazotization was performed by following the protocol detailed in Hosseininaveh et al. (2009). Briefly, the membrane was soaked sequentially for 5 minutes in a sodium nitrite solution (1 mg/mL in 1 M HCl), then an ammonium sulfamate solution (5 mg/mL in 1M HCl) and finally a NNED solution (N-(1-naphthyl)-ethylenediamine dihydrochloride) (0.5 mg/mL in 48% v/v ethanol/water), for about 30 seconds to 1 minute until any diazotized p-nitroaniline became clearly visible as a purple smear/bands.

[0154] Mass Spectrometry

[0155] Protein bands identified with colloidal-coomassie staining were analyzed by MALDI-Mass fingerprinting at the PNAC Facility, Department of Biochemistry at the University of Cambridge. The gel bands were excised and subjected to the following treatment—30 min per step, 20° C., in 200 μL 100 mM ammonium bicarbonate/50% acetonitrile: 1) Reduction with 5 mM Tris (2-carboxyethyl) phosphine; 2) Alkylation by addition of iodoacetamide (25 mM final concentration); and 3) Removal of liquid and then wash.

[0156] Once washed, the gel pieces were dried in vacuo for 10 min and then 25 μl 100 mM ammonium bicarbonate containing 5 μg/mL modified trypsin (Promega) was added. Digestion was performed for 17 h at 32° C. Peptides were recovered and desalted using μC18 ZipTip (Millipore) and eluted to a maldi target plate using 1-2 μL alpha-cyano-4-hydroxycinnamic acid matrix (Sigma) in 50% acetonitrile/0.1% trifluoroacetic acid. Peptide masses were determined using a Bruker ultrafleXtreme Maldi mass spectrometer in reflectron mode and ms/ms fragmentation peroformed in LIFT mode. Data analysis was undertaken with FlexAnalysis, BioTools and ProteinScape software (Bruker). Database searches of the combined mass fingerprint data were performed using Mascot (Matrix Science). Where required, additional manipulation was performed through Protein Prospector.

[0157] Debriding Assay

[0158] The AWE debriding assay is an in vitro surrogate of wound necrotic tissue proteolysis activity developed by Health Point (Shi, Ermis et al. 2009). It has been shown to compare well to in vivo animal data. The AWE substrate consists of a pellet of three wound related extra cellular matrix proteins (collagen, elastin and fibrin), each tagged with a different fluorophore. Gradual degradation of this matrix can be measured by progressive increase in fluorescence intensity in a Franz diffusion cell setup (FIG. 2). The final readings for the experiments are taken at 24 hours.

[0159] Materials for the debriding assay were: Collagen-Fluorescein Isothiocyanate (FITC) (CF308), Elastin-Rhodamine (R144) from Elastin products company, USA. Thrombin and fibrinogen (605157, 341573) from Merck chemicals limited. Tris Buffer containing 50 mM Tris with 100 mM NaCl and 10 mM CaCl.sub.2, magnetic stirrer bar (Z328936), L-cysteine (W326305), Elastin-bovine (E1625), 7-Amino-4-Methyl Coumarin (A9891) and papain from papaya latex (P3375) all from Sigma.

[0160] Preparation of Fibrin-Coumarin: Fibrinogen was labelled with 7-amino-4-methyl coumarin by mixing fibrinogen in a coumarin solution (0.02 mg/mL in Tris buffer) with a final fibrinogen concentration of 10 mg/mL (in Tris buffer). The mixture was incubated at room temperature with rotary shaking for 1 hour. The coumarin-labelled fibrin was derived by adding a thrombin solution (2.5 units/mL) to the fibrinogen-coumarin solution and allowed to clot for 2 hours.

[0161] The formed clots were washed 3 times with water and allowed to stand overnight in distilled water and methanol (1:1) to remove excess dye. The clots were then transferred to a glass container with transparent non-sticky filter paper and allowed to dry for 3 days. The dried fibrin-coumarin was ground into fine powder using a mortar and pestle.

[0162] Preparation of Artificial Wound Eschar (AWE) Substrate

[0163] Collagen-FITC, elastin-rhodamine and fibrin-coumarin were mixed according to the composition referred to in Table 4. Except fibrinogen, all the materials were weighed into a 50 mL conical centrifuge tube. Using a tissue tearer the materials were homogenised in 10 mL Tris buffer for 3-5 minutes. A 10 mL, 15-mg/mL fibrinogen solution was prepared in Tris buffer pH 6.8 in a separate tube. The two solutions were combined and thoroughly mixed by using the tissue tearer for about 2 minutes. Thrombin solution (50 U/mL) was added and quickly mixed, and the solution was then poured into a petri dish containing a 90 mm non-reactive membrane and allowed to clot for 1 hour. The clotted substrate was then rinsed with water 3 times (5 minutes each) to remove thrombin. After washing, excess water was removed by using tissue paper and stored at 4° C. until further use.

[0164] Franz Diffusion Cell System

[0165] With the AWE substrate still attached to the membrane, a 9 mm diameter piece was punched out using a biopsy punch. The 9 mm AWE substrate was placed on the top of a non-reactive nitrocellulose membrane and placed in the Franz diffusion cell between the two chambers, of which the lower (receptor cell) was filled with Tris buffer containing 1% (v/v) penicillin-streptomycin (FIG. 2). The sample holder was placed on the top and the whole assembly was fastened with a screw clamp to the receptor cell and surrounded with a 35° C. water bath. The sample was then loaded in the upper chamber of the sample holder and covered with parafilm in which an air hole was made. The solution was mixed using a magnetic stirrer at 500 rpm throughout the experiment. At time zero and regular intervals up to 24 hours, 100 μL of solution was sampled from the receptor cell. The collected samples were loaded into a 96 well micro plate for immediate fluorescent measurement using a fluorescent plate reader (Synergy HT KC4 v3.4). After measurement, 100 μL of sample was replaced in the receptor cell.

[0166] The following equation was applied to determine the total cumulative digestion of all three-protein components in the substrate:

CD.sub.n=In×V.sub.cell

[0167] Where n=time in hours, CD.sub.n and In are the cumulative digestion parameter and fluorescent intensity at hour n, and V.sub.cell is the volume of the cell (5.1 mL).

TABLE-US-00006 TABLE 4 Composition of artificial wound Eschar (AWE) substrate Wavelengths Component excitation/emission Percentage (%) Collagen-FITC 485 nm/520 nm 65 Fibrin-Coumarin 365 nm/440 nm 10 Elastin-Rhodamine 550 nm/570 nm 10 Fibrinogen 15

Example 1—OPAL B Filtrate—a Variant of OPAL a Filtrate and its Proteolytic Activity

[0168] Carica papaya belongs to the small family of Caricaceae and is one of the major fruits cultivated in tropical and sub tropical zones for its edible fruit and latex. The fruits used in these examples are ripe papaya fruits, mainly obtained from Brazil and Jamaica, purchased in local UK-based supermarkets. Most of the well-characterized and intensively studied cysteine proteases are members of the papain family. Papain, chymopapain A and B, chymopapain M, and caricain have all been extracted from the latex of Carica papaya but have never been characterized from the ripe fruit.

[0169] We have previously carried out mass spectrometry work (Richard J. Lipscombe—Proteomics International laboratory, University of Western Australia) and showed that OPAL A filtrate from Carica papaya ripe fruit extract contains cysteine (thiol dependent) proteases. These cysteine proteases are traditionally extracted only from papaya latex or from the skin of papaya (Proteomics International, personal communication). To functionally identify cysteine proteases in OPAL A filtrates, spectrophotometric assays were performed. These experiments involved monitoring catalytic hydrolysis of the chromogenic substrate L-BApNA (Nα-benzoyl-L-arginine 4-nitroanilide hydrochloride) by the OPAL A filtrate.

[0170] Hydrolysis of L-BApNA in the presence of protease, at the bond between the arginine and p-nitroaniline moieties, releases the chromophore p-nitroaniline, which can then be detected by spectroscopy at a wavelength of 410 nm. The procedure for the L-BApNA activity determination is described in the methods above. Under the optimized conditions employed, the excess substrate yields zero order kinetics, which are apparent as a straight line, the slope of which is proportional to enzymatic activity.

[0171] We have previously established (unpublished data) that OPAL A has cysteine protease activity that is essentially abolished by the addition of the inhibitor E-64, that specifically inhibits cysteine proteases. We therefore previously concluded that the protease activity in OPAL A is essentially due only to the presence of active cysteine proteases. To determine the effect of omitting the heat treatment step, the protease activity in both OPAL A and OPAL B filtrates was tested using an L-BApNA assay. After 10 minutes, E-64 inhibitor was added.

[0172] OPAL B appeared to exhibit an almost 8-fold increased catalytic activity (linear increase in A.sub.410 substrate release over time), as compared to OPAL A (FIG. 3). Interestingly, addition of E-64 to OPAL B filtrate in excess did not result in complete inhibition of activity, as in OPAL A. Therefore, and without wishing to be bound by theory, the increase of A.sub.410 values with substrate could be due to non-thiol-dependent (non-cysteine proteases) catalysis. The fact that treatment with the cysteine protease inhibitor E-64 did not completely abolish protease activity suggests that both cysteine and non-cysteine proteases were present in the OPAL B filtrate, as shown in FIG. 4.

Example 2—Identification of Cysteine and Serine Proteases in OPAL B Filtrate Via Enzyme Kinetics

[0173] To better understand the non-thiol dependent or non-cysteine proteases in the OPAL B filtrate, enzymatic assays were carried out initially using a Protease Inhibitor Cocktail (PIC) (sigma P2714), containing all the essential protease inhibitors (see Table 3). Adding 20 μL of PIC to the OPAL B filtrate achieved complete inhibition of the protease activity, as shown in FIG. 5.

[0174] Based on these data, we next investigated the effect of each active inhibitor by individually testing each component of PIC that may be responsible for the enhanced activity of OPAL B. The components of the protease inhibitors present in the cocktail are shown in Table 3.

[0175] After testing each inhibitor present in the PIC with OPAL B filtrates, only two inhibitors were able to halt proteolytic activity. These were E-64 and AEBSF, as shown in FIG. 6, which are cysteine protease and serine protease inhibitors, respectively.

[0176] The combination of both inhibitors completely abrogates enzyme activity, as shown in FIG. 7. This suggests that the enzyme activity in OPAL B filtrate consists solely of cysteine and serine proteases.

Example 3—Zymography Assays to Functionally Identify Proteases Determined by Enzyme Kinetics

[0177] OPAL B filtrate appears to be a complex mixture of many proteins. Therefore, to individually visualize the protease activities that were detected by spectrophotometry assays, we used a technique called overlay zymography. In this technique, proteins are first separated on a standard native PAGE gel, which preserves enzymatic activity. A membrane containing substrate solution (L-BApNA at 1.2 mg/mL) is then overlaid onto the gel, and developed using a colour-enhancing agent. This enables active proteases to be directly visualized on the membrane.

[0178] As shown in FIG. 8, Colloidal Blue stained native PAGE gel revealed bands in the OPAL B filtrate in unidirectional phase (anodic side). Transfer of protein from gel to nitrocellulose membrane and subsequent zymography analysis showed a stark increase in enzymatic activity (which shows two enzymatic activities).

[0179] We further characterized the two-zymography bands obtained in FIG. 8 to verify that the observed two signals or bands are cysteine and serine proteases, respectively. The method was modified by including specific inhibitors (E-64 for cysteine proteases, and AEBSF for serine proteases), onto the nitrocellulose membrane before placing the membrane on top of the native PAGE gel. The data obtained clearly revealed that the bands are indeed cysteine protease(s) and serine protease(s) in the OPAL B concentrated filtrate. The cysteine protease(s) correspond to the upper band and the serine protease(s) correspond to the lower band, as shown in FIG. 9.

Example 4—Size Exclusion Chromatography to Identify Serine Protease

[0180] To purify serine protease(s), OPAL B was concentrated and purified through size exclusion chromatography using a sephadex G-100 column that separates proteins in the range of 4 kDa to 150 kDa. Dialysed OPAL B pre-treated with E-64 and with 100 mM L-Arginine (incubated for 2 hours) was run through a 14 mL sephadex G-100 column. Twenty-four elution fractions were collected and the 96 well plate L-BApNA assay was performed (data not shown). The fractions comprising the highest L-BApNA activity were selected for SDS PAGE gels, which were then silver stained, as shown in FIG. 10. The bands in the fractions, the abundance of which correlated with L-BApNA activity, were selected and analysed by LC/MS-MS analysis (namely the intensity bands present in the 6.sup.th fraction at the 80 kDa, 50 kDa and 30 kDa bands). These were matched to a Carica papaya sequence shown in SEQ ID NO: 1 that had been putatively identified as a subtilase.

[0181] This result is consistent with an analysis of complete OPAL B filtrate which identified 4 fragments within SEQ ID NO: 1 as follows.

TABLE-US-00007 MAVSNPTLYL LSFLLFSISL TPVIASKSSY VVYLGAHSHG LELSSADLDR  50 VKESHYDFLG SFLGSPEEAQ ESIFYSYTKH INGFAAELND EVAAKLAKHP 100 KVVSVFLNKG RK              LHTTRSWD FLGLEQNGVV PSSSIWKKAR FGEDTIIGNL 150 DTGVWPESKS FSDEGLGPIP SKWRGICDHG KDSSFHCNRK LIGARFFNRG 200 YASAVGSLNS SFESPRDNEG HGTHTLSTAG GNMVANASVF GLGKGTAKGG 250 SPRARVAAYK VCWPPVLGNE CFDADILAAF DAAIHDRVDV LSVSLGGTAG 300 GFFNDSVAIG SFHAVKHGIV VVCSAGNSGP DDGSVSNVAP WQITVGASTM 350 DREFPSYVLL GNNMSFKGES LSDAVLPGTN FFPLISALNA KATNASNEEA 400 ILCEAGALDP KKVKGKILVC LRGLNARVDK GQQAALAGAV GMILANSELN 450 GNEIIADAHV LPASHISFTD GLSVFEYINL TNSPVAYMTR PKTKLPTKPA 500 PVMAAFSSKG PNIVTPEILK PDITAPGVNV IAAYTRAQGP TNQNFDRRRV 550 QFNSVSGTSM SCPHVSGIVG LLKTLYPSWS PAAIRSAIMT SATTMDNINE  600 SILNASNVKA TPFSYGAGHV QPNQAMNPGL VYDLNTKDYL KFLCALGYSK  650 TLISIFSNDK FNCPRTNISL ADFNYPSITV PELKGLITLS RKVKNVGSPT 750 TYRVTVQKPK GISVTVKPKI LKFKKAGEEK SFTVTLKMKA KNPTKEYVFG 750 SEQ ID NO: 1 ELVWSDEDEH YVRSPIVVKA A 771 SEQ ID NO: 2 SFSDEGLGPI PSK  SEQ ID NO: 3 GESLSDAVLP GTNFFPLISA LNAK   SEQ ID NO: 4 GPNIVTPEIL KPDITAPGVN VIAAYTR SEQ ID NO: 5 TLYPSWSPAA IR 

Example 5—Reactivity Probe to Identify Active Form of Serine Protease

[0182] To establish which of the different bands corresponded to a proteolytically active form of subtilase in the OPAL B filtrate, we used a reactivity probe, which specifically labels active serine proteases via a fluorosulfonate group linked to biotin. This reactivity probe can then be visualized on a regular SDS PAGE gel via western blotting technique, using streptavidin-conjugated enzymes such as alkaline phosphatase. Using this technique allowed the visualization of the active serine protease band inhibited by AEBSF treatment at around 50 kDa as shown in FIG. 11. While this band corresponds to one of the observed bands from the chromatography experiments identified as papaya subtilase via mass spectrometry, its molecular weight does not match the published value of either the full-length protein or pro enzyme (70 and 85 kDa respectively). Without wishing to be bound by theory, the fact that we observed several bands originating from the same protein suggests there may be different isoforms of which only the 50 kDa form is proteolytically active.

[0183] Overall Conclusions

[0184] We have previously described in WO 2004/008887 a process for producing a composition known as OPAL A from the flesh of ripe papaya (Carica papaya). This process involves a heating step followed by treatment with sodium bicarbonate and then a filtration step. Our previous analysis of OPAL A identified cysteine protease activity. OPAL A has shown activity in treating a range of disorders. We have modified the OPAL A process by omitting the heating step to produce OPAL B. We have surprisingly and unexpectedly found that OPAL B contains additional protease activity that is not related to cysteine proteases. We have characterized this as being due to the presence of at least one serine protease which is not present in OPAL A. This protein appears to be present in more than one form, but only a protein having an average molecular weight of 50 kDa as determined by SDS-PAGE showed serine protease activity. LC-MS/MS analysis showed the proteins as having sequences identical to the sequence of a C. papaya sequence putatively identified as a subtilase (Othman and Nuraziyan, 2010), but this was not confirmed by biochemical characterization. Further, this subtilase when expressed recombinantly has a molecular weight of about 70 kDa compared to the 50 kDa molecular weight of the active serine protease that we have presently identified.

[0185] Accordingly, we have shown that OPAL B contains a mixture of both one or more proteolytically active serine proteases and one or more proteolytically active cysteine proteases, whereas until now the only known active proteases that have been recognised in Carica papaya have been cysteine proteases.

Example 6—Quantifying Debriding Potency of OPAL A and OPAL B Filtrate In Vitro

[0186] Debridement is the removal of necrotic (dead) tissue and foreign material from wounds to expose underlying viable tissue. This process promotes and accelerates wound healing. We investigated whether the cysteine proteases identified in the OPAL A filtrates would be active in debridement measured using an Artificial Wound Eschar (AWE) debriding assay.

[0187] The AWE debriding assay is an in vitro surrogate of wound necrotic tissue proteolysis activity developed by Health Point (Shi, Ermis et al. 2009). It has been shown to compare well to in vivo animal data and was therefore used to assess debriding efficacy of OPAL A filtrate formulations. The AWE substrate consists of a pellet of three wound-related extra cellular matrix proteins, collagen, elastin and fibrin, each tagged with a different fluorophore. Gradual degradation of this matrix can be measured by progressive increase in fluorescence intensity in a Franz diffusion cell setup. The final readings for the experiments are taken at 24 hours.

[0188] In the first set of experiments, the proteolytic efficacies of native OPAL A filtrate (0.7 mg/mL protein equivalent) was compared against the control crude papain from papaya latex (at 10 mg/mL, Sigma), using a Franz cell diffusion system. The results shown in FIG. 12 clearly indicate that the OPAL A filtrate sample digestion of collagen and fibrin is consistently better than latex papain (10 mg/mL), but weaker with respect to elastin.

Example 7—Concentrating OPAL a Filtrate Activity Using Dialysis to Improve Debriding Efficacy

[0189] The L-BApNA activity of freshly prepared OPAL A filtrate is on average 0.60 IU/mL. The protein content of fresh OPAL A filtrate measures around 0.7 mg/mL, corresponding to a specific solid activity of around 1 IU/mg. Compared to the activity of pure latex papain of around 10 IU/mg, this approximates to about 9% papain content of the OPAL solids. Remarkably the AWE debriding assay performed in Example 6, as shown in FIG. 12, reveals a significantly higher activity of OPAL A than would be expected from its nominal papain content (0.6 IU/mL versus 30 IU/mL for latex papain).

[0190] The solid residue content of OPAL A was measured via freeze-drying the extract and yields a consistent value across batches of 0.216±0.005 g/mL. It is therefore clear that the vast majority of OPAL A filtrate solids are potentially non-proteolytic in nature. It may therefore be possible to increase specific activity of the extract by removing the solids from the extracts. To investigate this, we subjected fresh OPAL A filtrate to dialysis using membranes with cut-off sizes of 12 kDa and 25 kDa. Dialysis was carried out for 24 hours at 4° C., after which residual proteolytic activity of the solution was measured via the L-BApNA assay. An aliquot of the solution was also freeze-dried to measure solid content. The treatment caused a small (20%) osmotic volume increase, which was taken into account in the calculations.

[0191] Results in Table 5 show almost full retention of activity for either dialysis using cut-off sizes of 12 and 25 kDa and combined with a solid reduction of around 86%.

TABLE-US-00008 TABLE 5 Specific activity and solid content of native OPAL A filtrate and dialyzed OPAL A filtrates with 12 kDa and 25 kDa cut-off molecular sizes. Activity Volume Specific activity Solid content Sample (IU) (mL) (U/mL) mg/mL OPAL A 0.216 0.166 1.301 206 Dialyzed 6.218 0.166 1.314 31 OPAL A 12 kDa Dialyzed 0.205 0.166 1.232 32 OPAL A 25 kDa

[0192] This result is consistent with the commonly accepted sizes of Carica papaya proteolytic enzymes (23-30 kDa) and the fact that OPAL A filtrate contains large amounts of low molecular weight sugars, which are removed upon dialysis. The fact that solid content does not significantly change from 12 kDa to 25 kDa dialysis indicates the absence in the composition of molecules with sizes intermediate between these two values. We therefore used the 12 kDa cut-off dialysis membranes for further experiments.

[0193] Debriding strength of the OPAL A filtrate versus native Opal A was measured as per Example 6. The data in FIG. 13 show that dialyzed OPAL A is better than native OPAL A at digesting fibrin and elastin but inferior on collagen.

[0194] In summary, dialyzed OPAL A filtrate showed full retention of proteolytic activity in comparison with the native OPAL A filtrate, but a solid reduction of 86% less than control. This establishes that the majority of solids in OPAL A filtrate are non-proteolytic in nature, and exist as low molecular weight sugars that can be removed through dialysis. Dialyzed OPAL A filtrate had enhanced debriding activity on both fibrin and elastin as compared to latex papain and native OPAL A, but a marginally minor effect on collagen compared to native OPAL A.

Example 8—In Vivo Case Study Regarding Management of Severe Pressure Ulcer with OPAL B

[0195] This case study discloses rapid treatment of a severe ulcer involving tissue necrosis in an elderly patient through the topical application of OPAL B. OPAL B therefore appears to provide a therapeutic benefit in the healing of severe ulcers through a multifactorial mode of action involving at least tissue regenerative and anti-necrotic/debriding factors.

[0196] A bed-ridden nursing home patient in her 70s with dementia had developed severe pressure ulcers. The ulcers extended laterally across the majority of the patient's lower back and showed significant tissue necrosis. Upon transfer to a hospital, a vacuum dressing was applied in an attempt to clear excessive exudate produced by the ulcerated wound. Additional surgical intervention to reduce the size of the ulcer with a rhomboid flap was also attempted. Despite continued treatment efforts by hospital staff, the severity of the wound persisted (FIG. 14).

[0197] With the consent of the patient's treating surgeon, the vacuum dressing treatment and other surgical interventions were postponed in favour of daily application of fresh, undiluted OPAL B directly to the wound surface, together with daily application of a 30% diluted OPAL B aqueous cream to the skin immediately surrounding the wound.

[0198] By day 2-4 of Opal B treatment, the wound appeared cleaner and less inflamed (FIGS. 15 and 16). Although some necrotic tissue was still observed, it appeared to be localised to a smaller area of the wound. By days 16-19, further improvement involving reduction in wound size was observed (FIGS. 17 and 18). Upon stabilisation, the wound was surgically debrided. The extremely fast arrest in deterioration/necrosis and the progress made in treating what had been hitherto an extremely severe and untreatable ulcer is surprising and remarkable.

[0199] Without wishing to be bound by theory, it is believed that OPAL B may have a number of modes of action: OPAL B appeared to localise tissue that was not viable, and to reverse marginal ischaemia, thereby reducing the amount of necrotic tissue. There is also evidence that OPAL B had begun to debride the slough in the ulcer.

[0200] The various features and embodiments of the present invention, referred to in individual sections above apply, as appropriate, to other sections, nutatis mutandis. Consequently, features specified in one section may be combined with features specified in other sections, as appropriate.

[0201] All publications mentioned in the above specification are herein incorporated by reference. All of the compositions and/or methods disclosed and claimed in this specification can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions and/or methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention.

Example 9—Proteome Mapping of OPAL B

[0202] In Proteome Mapping of OPAL B using Protein Pilot™ searching the UniProt Viridplantae database, the peptide fragments that were detected are those shown in bold below:

[0203] SEQ ID NO: 1 (catalytic triad, Asn 336 and peptides of SEQ ID NOs: 2 to 5 shown in bold)

TABLE-US-00009 MAVSNPTLYL LSFLLFSISL TPVIASKSSY VVYLGAHSHG LELSSADLDR  50 VKESHYDFLG SFLGSPEEAQ ESIFYSYTKH INGFAAELND EVAAKLAKHP 100 KVVSVFLNKG RKLHTTRSWD FLGLEQNGVV PSSSIWKKAR FGEDTIIGNL 150 DTGVWPESKS FSDEGLGPIP SKWRGICDHG KDSSFHCNRK LIGARFFNRG 200 YASAVGSLNS SFESPRDNEG HGTHTLSTAG GNMVANASVF GLGKGTAKGG 250 SPRARVAAYK VCWPPVLGNE CFDADILAAF DAAIHDRVDV LSVSLGGTAG 300 GFFNDSVAIG SFHAVKHGIV VVCSAGNSGP DDGSVSNVAP WQITVGASTM 350 DREFPSYVLL GNNMSFKGES LSDAVLPGTN FFPLISALNA KATNASNEEA 400 ILCEAGALDP KKVKGKILVC LRGLNARVDK GQQAALAGAV GMILANSELN 450 GNEIIADAHV LPASHISFTD GLSVFEYINL TNSPVAYMTR PKTKLPTKPA 500 PVMAAFSSKG PNIVTPEILK PDITAPGVNV IAAYTRAQGP TNQNFDRRRV 550 QFNSVSGTSM SCPHVSGIVG LLKTLYPSWS PAAIRSAIMT SATTMDNINE 600 SILNASNVKA TPFSYGAGHV QPNQAMNPGL VYDLNTKDYL KFLCALGYSK 650 TLISIFSNDK FNCPRTNISL ADFNYPSITV PELKGLITLS RKVKNVGSPT 700 TYRVTVQKPK GISVTVKPKI LKFKKAGEEK SFTVTLKMKA KNPTKEYVFG 750 ELVWSDEDEH YVRSPIVVKA A  771

[0204] Amino acids 1-25 are a putative signal sequence and amino acids 26 to 112 are a putative pro region that is cleaved to form the mature protein.

TABLE-US-00010 SEQ ID NO: 6 SWDFLGLEQN GVVPSSSIWK SEQ ID NO: 7 FGEDTIIGNL DTGVWPESKS FSDEGLGPIP SK SEQ ID NO: 8 GICDHGKDSS FHCNR SEQ ID NO: 9 GARFFNRGYA SAVGSLNSSF ESPR SEQ ID NO: 10 VCWPPVLGNE CFDADILAAF DAAIHDR SEQ ID NO: 11 HGIVVVCSAG NSGPDDGSVS NVAPWQITVG ASTMDR SEQ ID NO: 12 FKGESLSDAV LPGTNFFPLI SALNAKATNA SNEEAILCEA GALDPK SEQ ID NO: 13 ILVCLR SEQ ID NO: 14 TLPTKPAPVM AAFSSKGPNI VTPEILKPDIT APGVNVIAA  YTRAQGPTNQ NFDR SEQ ID NO: 15 VQFNSVSGTS MSCPHVSGIV GLLKTLYPSWS PAAIR SEQ ID NO: 16 ATPFSYGAGH VQPNQAMNPG LVYDLNTK SEQ ID NO: 17 TLISIFSNDK FNCPRTNISL ADFNYPSITV PELK SEQ ID NO: 18 GISVTVKPK  SEQ ID NO: 19 EYVFGELVWS DEDEHYVR

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