Composition and method of manufacture

Abstract

This invention provides a method and composition for preparing a composition derived from Carica papaya. [Solution] A method is provided for preparing a composition derived from the fruit of the Carica papaya plant, wherein the composition has a pH of about 7.5 to 9.5, and the method comprises the steps of treating ripe fruit pulp of the Carica papaya plant with alkali without subjecting it to a heating step, and separating soluble proteases from insoluble plant material after the alkali treatment. A composition is also provided comprising one or more serine proteases of proteolytic activity obtainable by the method.

Description

[Technical Field]

[0001] The present invention relates to compositions comprising at least one protease identified in the Carica papaya plant, methods for producing compositions from the Carica papaya plant, compositions comprising at least one protease obtained or obtainable by the aforementioned methods, the use of such compositions in the manufacture of pharmaceuticals and cosmetics, the use of such compositions in the treatment of diseases and disorders, including wounds, the use of such compositions in cosmetic applications, and related kits for carrying out the methods or uses of the present invention. [Background technology]

[0002] The effective management of many types of chronic wounds, including but not limited to diabetic foot ulcers, pressure ulcers, venous ulcers, and arterial ulcers, remains a challenge. Such wounds may take a considerable amount of time to heal or may never truly heal, requiring significant assistance from healthcare professionals. Numerous products have been developed to support wound healing, such as bandages containing ascorbic acid, type I collagen, and D-glucose polysaccharide obtained by hydrolysis of starch (U.S. Patent No. 6,187,743).

[0003] There has also been interest in the use of proteases such as papain and bromelain for wound debridement. [Overview of the Initiative]

[0004] The inventors identified a protease previously unidentified as an active protease in the fruit in a filtrate obtained by treating the pulp of ripe papaya (Carica papaya) with alkali. The inventors confirmed that this protease is a serine protease and that the filtrate contains both serine protease and cysteine ​​protease activity. A serine protease identified in the secretions of medicinal maggots (van der Plas et al., 2014, PLoS ONE 9(3):e92096) has been shown to enhance plasminogen-activator-induced fibrinolysis by degrading plasminogen, suggesting that this serine protease may contribute to the medicinal maggots' ability to debride necrotic tissue wounds.

[0005] Accordingly, in a first embodiment, the present invention provides a composition comprising an active serine protease extracted from the ripe fruit of the Carica papaya plant, such as an active serine protease extracted from alkali-treated, pulped ripe Carica papaya fruit.

[0006] In one embodiment, the active serine protease is substantially free of insoluble Carica papaya-derived material.

[0007] In a second embodiment, the serine protease comprises a continuous amino acid sequence having at least 95, 96, 97, 98, or 99% homology (sequence identity, etc.) with amino acids 113-771 of SEQ ID NO: 1. In a particular embodiment, the serine protease has a molecular weight of 65-75 kDa, for example, about 70 kDa.

[0008] In a third embodiment, the serine protease has a molecular weight of 45-55 kDa and comprises a continuous amino acid sequence having at least 95% homology to amino acids 142-618 of SEQ ID NO: 1. Therefore, the present invention provides a composition comprising an isolated active serine protease having a molecular weight of 45-55 kDa and comprising a continuous amino acid sequence having at least 95% homology to amino acids 142-618 of SEQ ID NO: 1.

[0009] The present invention also provides a composition comprising an isolated active serine protease substantially free of insoluble Carica papaya-derived material, wherein the serine protease has a molecular weight of 45-55 kDa and comprises a continuous amino acid sequence having at least 95% homology to amino acids 142-618 of SEQ ID NO: 1.

[0010] In a second embodiment, the present invention provides a composition comprising a mixture of one or more active serine proteases derived from or derived from Carica papaya and one or more active cysteine ​​proteases derived from or derived from Carica papaya. In one embodiment, one or more active proteases are extracted from ripe Carica papaya. In another embodiment, one or more active proteases are produced using a recombinant expression system.

[0011] The present invention also relates to a method for preparing a composition comprising at least one active serine protease by extracting proteases from ripe Carica papaya. Accordingly, in a third embodiment, the present invention provides a method for preparing a composition comprising an active serine protease, the method comprising the step of treating ripe Carica papaya fruit pulp with an alkali without subjecting the pulp to a heating step. In one embodiment, the alkali is a weak alkali, preferably sodium bicarbonate.

[0012] The composition produced by this method may further contain one or more cysteine ​​proteases derived from or deriveable from Carica papaya.

[0013] In one embodiment, at least one soluble protease is separated from the insoluble plant material after alkaline treatment.

[0014] The composition may be further processed to increase the concentration of one or more proteases, for example, by lyophilization, dialysis, size exclusion chromatography, or a combination thereof.

[0015] In a fourth embodiment, the present invention further provides compositions comprising serine proteases obtained or obtainable by the method of the present invention.

[0016] The compositions may be formulated to be more suitable for pharmaceutical or cosmetic applications. Accordingly, in a fifth embodiment, the present invention provides pharmaceutical compositions comprising active Caricacilla papayaserine protease with a pharmaceutically acceptable carrier or diluent, and cosmetic compositions comprising active Caricacilla papayaserine protease with a cosmetically acceptable carrier or diluent. In related embodiments, the present invention provides compositions of any of the first, second, or fourth embodiments of the present invention with a pharmaceutically acceptable carrier or diluent, and cosmetic compositions comprising compositions of any of the first, second, or fourth embodiments of the present invention with a cosmetically acceptable carrier or diluent.

[0017] The compositions of the present invention can be used within the scope of pharmaceutical and cosmetic applications. Accordingly, in a sixth embodiment, the present invention also provides a method for treating a patient suffering from a skin condition, the method comprising the step of administering the composition of the present invention to the affected area of ​​the patient. In some embodiments, the composition is used for the treatment of diseases and disorders, including wounds. In some embodiments, the composition is used for debridement. In some embodiments, the composition is used to treat burns. In some embodiments, the composition is used to treat ulcers. In some embodiments, the composition is used to treat gangrene. In some embodiments, the composition is applied topically.

[0018] In a seventh embodiment, the present invention further provides a method for achieving desired cosmetic outcomes such as smoothing and regenerating the epidermis of an individual, the method comprising the step of applying the composition of the present invention to a lesion area of ​​a patient. The present invention also provides cosmetic methods using the composition of the present invention. In some embodiments, the composition is used for exfoliation, to lighten the skin, or to apply to wrinkles, blemishes, freckles, pimples, acne, rosacea, sunspots, scars, or varicose veins, or to apply to dry, aged, or damaged skin.

[0019] In an eighth embodiment, the present invention provides the use of compositions of the first, second, fourth, or fifth embodiment in the manufacture of a pharmaceutical. In some embodiments, the pharmaceutical is for the treatment of diseases and disorders, including wounds. In some embodiments, the pharmaceutical is for debridement. In some embodiments, the pharmaceutical is for the treatment of burns. In some embodiments, the pharmaceutical is for the treatment of ulcers. In some embodiments, the pharmaceutical is for the treatment of gangrene.

[0020] In a ninth aspect, the present invention provides for the use of a composition of the first, second, fourth, or fifth aspect in the manufacture of a cosmetic. In some embodiments, the cosmetic is for exfoliation.

[0021] In a tenth aspect, the present invention provides a composition of the first, second, fourth, or fifth aspect for use in the treatment of diseases and disorders including wounds. In some embodiments, the treatment is debridement. In some embodiments, the treatment is for ulcers. In some embodiments, the composition is applied topically.

[0022] In an eleventh aspect, the present invention provides a composition of the first, second, fourth, or fifth aspect for use in exfoliation.

[0023] In a twelfth aspect, the present invention provides a kit comprising a composition of the first, second, fourth, or fifth aspect. In one embodiment, the kit is used to carry out the method of the present invention.

[0024] The composition of the present invention can be formulated, for example, as capsules, tablets, creams, ointments, solutions, pastes, droplets, sprays, aerosols, vapors, wipes, patches, gauzes, gels, or liquids. Thus, in one embodiment, the composition of the present invention is provided or packaged as capsules, tablets, creams, ointments, solutions, pastes, droplets, sprays, aerosols, vapors, wipes, patches, gauzes, gels, or liquids and need not be reconstituted before use.

Brief Description of the Drawings

[0025] [Figure 1] Schematic representation of overlay zymography. The overlay zymography process, and subsequent excision of bands from the enzyme activity spots and process onto an SDS PAGE gel. [Figure 2] Representation of a debride assay using a Franz-type cell diffusion system (Shi, Ermis et al., 2009). [Figure 3] Bar graph plot of the proteolytic activity of OPAL A and OPAL B filtrates against L-BApNA. OPAL A and OPAL B filtrates against L-BApNA measured by the slope over time. The OPAL B filtrate has approximately 8-fold greater catalytic activity compared to the OPAL A filtrate. Results are presented as mean ± S.D for an experiment with n = 8. Structural analysis was performed using the Student T test (***p < 0.001). [Figure 4] Spectrophotometric plot of the enzymatic hydrolysis of L-BApNA with OPAL B filtrate and addition of E-64. A representative plot shows the enzymatic hydrolysis of the substrate L-BAPNA by the OPAL B filtrate and partial activity inhibition by the cysteine protease inhibitor E-64, suggesting the presence of other proteases in addition to cysteine proteases. [Figure 5] Spectrophotometric plot of the effect of protease inhibitor cocktail (PIC) on the proteolytic activity of OPAL B filtrate. A representative trace shows the enzymatic hydrolysis of the substrate L-BAPNA by the OPAL B filtrate and complete inhibition of enzymatic hydrolysis by PIC. [Figure 6] Bar graph plot of the effect of various protease inhibitor classes on the proteolytic activity of OPAL B filtrate. [Figure 7] Spectrophotometric plot of the effect of E-64 + AEBSF on the proteolytic activity of OPAL B filtrate. A representative trace shows complete inhibition of L-BAPNA activity in the OPAL B filtrate by the combination of the cysteine protease inhibitor E-64 and the serine protease inhibitor AEBSF. [Figure 8] Figure of the overlay zymography analysis of OPAL B filtrate. [Figure 9]This figure shows the effects of superimposed zymography analysis of OPAL B filtrate and the use of inhibitors. Native PAGE gel bands were obtained from OPAL B samples, and then the protein bands were transferred from the gel to a nitrocellulose membrane. Zymography analysis shows the activity of two enzymes in the control, as well as complete inhibition of the upper band with the cysteine ​​protease inhibitor E-64 (---) and inhibition of the lower band with the serine protease inhibitor AEBSF (---). [Figure 10] This image shows the silver staining of an SDS-PAGE gel run using elutions 2-10. The high-intensity band present in the sixth fraction at 80 kDa, as well as the bands at 50 kDa and 30 kDa, were analyzed using LC / MS and identified as subtilases. [Figure 11] This figure shows the SDS-PAGE Western blotting analysis of OPAL B filtrate treated with serine protease probes using various methods. SDS-PAGE Western blotting analysis of OPAL B filtrate treated with 1 μl probe (per 100 μl) without tris-HCl (lane 2), with 5 mg / ml tris-HCl (lane 4), with 10 mg / ml tris-HCl (lane 6), and with AEBSF, respectively (lanes 3, 5, and 7). OPAL B filtrate treated with 1 μl probe and 10 mg / ml tris-HCl, along with E-64 (lane 6) and with E-64 and AEBSF (lane 7), shows no background disturbance. [Figure 12] This figure shows the differential digestion (activity percentage) of AWE substrates by OPAL A filtrate compared to latex-papain, demonstrating that OPAL A is superior in digesting collagen and fibrin, while control latex-papain is superior in digesting elastin. Results are expressed as mean ± SD for n=3 experiments. Structural analysis was performed using Student's t-test (**p<0.01, ***p<0.001). [Figure 13]This figure shows the differential digestion (activity percentage) of AWE substrates by dialyzed OPAL A and OPAL A + 10% urea compared to natural OPAL A filtrate. Dialyzed OPAL A exhibited enhanced debride activity against both fibrin and elastin, but not against collagen, which correlates with the lower activity of OPAL A against collagen. The addition of urea to OPAL A inhibited the enzyme activity against fibrin and elastin in the AWE assay. Results are expressed as mean ± SD for experiments with n=3. Structural analysis was performed using Student's t-test (**p<0.01, ***p<0.001). [Figure 14] This is a diagram from an in vivo case study on the management of severe pressure ulcers using Opal B, showing the condition before the initiation of Opal B treatment. It depicts the central area of ​​necrosis. The diagram shows a failed attempt to reduce ulcer size using a romboid flap. The diagram also shows the darker distal portion of the flap. Note: Graphic image. [Figure 15] This is a diagram from an in vivo case study on the management of severe pressure ulcers using OPAL B, showing the condition on day 2 of treatment. It depicts the transition from viable to necrotic areas. The pink areas indicate the wound margins and good blood flow to the skin flap. Note: Graphic image. [Figure 16] This is a diagram from an in vivo case study on the management of severe pressure ulcers using OPAL B, showing the condition on day 4 of treatment. A clear boundary is visible between viable and non-viable tissue. The viable tissue appears healthy and pink. The skin flap appears sufficiently viable. Note: Graphic image. [Figure 17] This is a figure from an in vivo case study on the management of severe pressure ulcers using OPAL B, taken on day 16 of treatment. The wound was surgically debriden, sutures were removed, and the skin flap was excised. Clear early granulation tissue formation is evident on the clean wound surface. The wound margin is pink, suggesting new skin growth (exposure adjusted to match the skin color of previous images). Note - Graphic image. [Figure 18]This is a figure from an in vivo case study on the management of severe pressure ulcers using OPAL B, taken on day 19 of treatment. Skin and wound color, particularly the edges of the wound, are darkened. The wound remains clean (exposure adjusted to match skin color in earlier images). Caution - Graphic image. [Modes for carrying out the invention]

[0026] definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art in which the invention pertains. Any methods and materials similar to or equivalent to those described herein may be used in the practice or testing of the invention, but preferred methods and materials are described. For the purposes of the invention, the following terms are defined below.

[0027] "Approximately" means a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length that is referenced and varies by approximately 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1%.

[0028] "Active protease" refers to a protease that is active in the process of protein degradation (proteolytic activity).

[0029] Throughout this specification, unless otherwise required by context, the terms “comprise,” “comprises,” and “comprising” shall be understood to mean the inclusion of the specified step or element or group of steps or elements, but not the exclusion of any other step or element or group of steps or elements. Thus, the use of terms such as “comprises” indicates that the listed steps or elements are required or essential, while other steps or elements are optional and may or may not be present.

[0030] The phrase "consists of" means that anything following the phrase is included and limited to it. Therefore, the phrase "consists of" indicates that the enumerated elements are required or essential, and other elements cannot be present. The phrase "essentially consists of" means that it includes any elements enumerated after the phrase, and is limited to other elements that do not interfere with or contribute to the activity or effect specified in the disclosure for the enumerated elements. Therefore, the phrase "essentially consists of" indicates that the enumerated elements are required or essential, but other elements are optional and may or may not be present, depending on whether or not they affect the activity or effect of the enumerated elements.

[0031] The term "debridement" refers to the removal of dead and damaged tissue from a wound.

[0032] The term "derivable" may be used interchangeably with the term "obtainable."

[0033] The term "derived from" may be used interchangeably with the term "acquired."

[0034] The term "isolated" means a material that substantially or essentially does not contain some or all of the components that would normally be present in the material in its natural state. For example, as used herein, "isolated protease" refers to the in vitro isolation and / or partial purification of a peptide or polypeptide protease molecule from its natural cellular environment and from its binding to some or all of the components of the cell.

[0035] When used in connection with the proteins or compositions of the present invention, the term "obtainable" includes not only proteins or compositions produced by specific designated methods, but also, however, the same proteins or compositions produced by recombinant DNA technology or other genetic engineering methods, for example, by supplying proteases derived from fruits or vegetables, or by using recombinant expression systems.

[0036] The terms “patient,” “subject,” and “individual” are used interchangeably and refer to human or other animal patients, subjects, and individuals, including any disease, disorder, or condition for which it is desirable to treat, prevent, improve, or reduce the severity using the present invention. However, it will be understood that “patient” does not imply the presence of symptoms. Suitable animals that fall within the scope of the present invention include, but are not limited to, primates (e.g., humans, chimpanzees), domestic animals (e.g., sheep, cattle, horses, donkeys, pigs), laboratory test animals (e.g., rabbits, mice, rats, guinea pigs, hamsters), companion animals (e.g., cats, dogs), and captive wild animals (e.g., foxes, deer, dingoes).

[0037] The terms “wild-type” and “naturally occurring” are used interchangeably to refer to proteins that possess the characteristics of a protein when isolated from a naturally occurring source. A wild-type protein (e.g., polypeptide) is the most frequently observed form in a population and is therefore arbitrarily referred to as the “normal” or “wild-type” form of that protein.

[0038] The term "wound" means injury to living tissue in which the skin is cut or severed, and includes skin ulcers and burns. Skin ulcers may include diabetic ulcers, pressure ulcers, venous (or varicose vein) ulcers, and arterial ulcers.

[0039] Any reference to prior art in this specification should not be construed as an acknowledgment or any form of suggestion that the prior art forms part of the common general knowledge of those skilled in the art.

[0040] The entire contents of all publications, patents, patent applications, and other materials cited herein are incorporated herein by reference. Detailed description of the invention

[0041] Composition containing serine protease The present invention relates to a composition comprising at least one active serine protease that can be extracted from or otherwise derived from the ripe fruit of the Carica papaya plant, such as after pulping the ripe fruit of the Carica papaya plant with an alkali such as sodium bicarbonate.

[0042] The term "active serine protease" should be understood to refer to one or more active serine proteases.

[0043] In one embodiment, the active serine protease has an amino acid sequence that includes one or more of the sequences shown in SEQ ID NOs: 2 to 5, for example, the sequences shown in SEQ ID NOs: 2, 3, and 4, or all four sequences shown in SEQ ID NOs: 2, 3, 4, and 5, respectively.

[0044] At least one active serine protease typically contains a continuous sequence having at least 95% sequence homology (preferably sequence identity), for example 96, 97, 98, or 99% sequence homology (preferably sequence identity), with amino acids 142-618 of Sequence ID No. 1, which is the catalytic domain of the protein containing the three main catalytic residues Asp, His, and Ser (shown at residues 151, 221, and 558, respectively, in Sequence ID No. 1), as well as the conserved substrate binding site Asn at residue 326.

[0045] Sequence ID 1 (shown in bold, peptides His221 and Sequence IDs 2-19) JPEG2026062669000001.jpg100149 Amino acids 1-25 are the putative signal sequence, and amino acids 26-112 are the putative pro-region, which is cleaved to form the mature protein.

[0046] Sequence IDs 2 through 5 are peptide fragments of Sequence ID 7, Sequence ID 12, Sequence ID 14, and Sequence ID 15, respectively. JPEG2026062669000002.jpg113149

[0047] In one embodiment, at least one active serine protease of the present invention comprises an amino acid sequence derived from amino acids 118-763 of SEQ ID NO: 1, having up to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, or more, amino acid modifications (deletions, insertions, and / or substitutions), such as up to 3 or 5, provided that the protein retains serine protease activity.

[0048] Polypeptides can be modified by a variety of means, including amino acid substitution, deletion, excision, and insertion. Methods for such operations are generally known in the art.

[0049] The serine proteases of the present invention are their active forms, i.e., not as inactive enzyme precursors.

[0050] In one embodiment, the experimental results described herein suggest that a protein isolated from Carica papaya and having an apparent molecular weight of 50 kDa was present as an active serine protease. Therefore, in one embodiment, at least one active serine protease of the present invention may have a molecular weight of 45–55 kDa, e.g., 48–52 kDa, e.g., about 50 kDa. The molecular weight of at least one protease of the present invention may be about 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, or 55 kDa. The molecular weight can be assessed by SDS-PAGE, and thus the protease has an average molecular weight of 45–55 kDa, e.g., 48–52 kDa, e.g., about 50 kDa, as measured by SDS-PAGE. Alternatively, the molecular weight can be calculated based on the primary amino acid sequence.

[0051] Active serine proteases are typically in a substantially isolated form. Being substantially isolated means that the active serine proteases are at least partially purified or recombinantly produced from the Carica papaya source material for the active serine proteases. In one embodiment, the isolated active serine proteases are substantially free of insoluble plant material such as cellulose and lignin. Being substantially free of insoluble plant material would typically be the result of one or more purification steps that remove insoluble material, leaving soluble active serine proteases in solution. These purification steps will be further described below. “Substantially free of insoluble plant material” means that the composition contains less than 2% w / w, e.g., less than 1% w / w or 0.5% w / w, of insoluble plant material derived from the source material for the proteases.

[0052] A composition comprising a mixture of serine protease and cysteine ​​protease. Cysteine ​​proteases (EC 3.4.22), also known as thiol proteases and cysteine ​​endopeptidases, are enzymes that break down proteins. Cysteine ​​proteases share a common catalytic mechanism involving nucleophilic cysteinethiols in three or two catalytic residues. Cysteine ​​proteases are found in a variety of organisms. In particular, cysteine ​​proteases are commonly found in fruits including papaya (Carica papaya and Vasconcellea cundianmarcensus), pineapple (Ananas comosus), fig (Ficus carica), and kiwifruit (Actinidia chinensis). In one embodiment, cysteine ​​proteases originate from fruits. Therefore, cysteine ​​proteases include papain (EC 3.4.22.2), chymopapain (EC 3.4.22.6), bromelain (stem bromelain - EC 3.4.22.32 and fruit bromelain - EC 3.4.22.33), ficaine (EC 3.4.22.3), and actinidine (EC 3.4.22.14).

[0053] The term "active cysteine ​​protease" should be understood to refer to one or more active cysteine ​​proteases.

[0054] One or more active serine proteases derived from Carica papaya (produced by extraction or recombinant synthesis) may be present in mixtures containing one or more active cysteine ​​proteases, such as one or more of the cysteine ​​proteases derived from Carica papaya mentioned above. The methods of the present invention, further described below, result in one such mixture.

[0055] Therefore, one source of active cysteine ​​protease can be the same as for active serine protease, namely the fruit of the Carica papaya plant. Preferably, the papaya is ripe. One advantage of ripe papaya as a source material instead of unripe papaya (the current commercial source of papain) is that the resulting composition containing cysteine ​​protease has a much lower level of latex than typical commercial sources of papain. Latex can cause allergic reactions.

[0056] Active cysteine ​​proteases are typically in a substantially isolated form, as described above with respect to active serine proteases. Being in a substantially isolated form means that the active cysteine ​​protease has been at least partially purified or recombinantly produced from a biological source material of active cysteine ​​protease, such as Carica papaya. In one embodiment, the isolated cysteine ​​protease is substantially free of insoluble plant material such as cellulose and lignin. Being substantially free of insoluble plant material would typically be the result of one or more purification steps that remove the insoluble material, leaving a soluble active protease in solution.

[0057] Method for obtaining serine protease and cysteine ​​protease Using the method of the present invention, at least one active serine protease of the present invention can be isolated from ripe papaya fruit. Ripe papaya fruit is typically mostly yellow on the outside. Unripe papayas (from which latex is harvested to produce papain) are green. Ripe papayas should not be hard when pressed, but should give a slight indentation and retain a slight indentation. If the papaya is very soft when pressed, it is overripe. In one embodiment, the papaya pulp is separated from the peel and seeds. In another embodiment, the whole fruit, including the peel and seeds, is used. The fruit, such as the pulp, is then mechanically torn to form pulp (for example, in a blender until smooth pulp is obtained).

[0058] The resulting pulp is then treated with an alkali. Suitable alkalis include strong bases such as NaOH and weak bases such as sodium bicarbonate. The alkali may be added as a dry powder or in aqueous solution. In one embodiment, the alkali is sodium bicarbonate, and is typically added as a powder in an amount of at least 3% w / w, e.g., at least 5% w / w. The amount is typically less than 15% w / w, e.g., less than 12% w / w. In a particular embodiment, the amount added is 5-12% w / w.

[0059] In one embodiment, the alkali may have a pKa of less than 11. In some embodiments, the alkali may be a bicarbonate or carbonate, or a combination thereof. In some embodiments, the alkali may be a water-soluble alkali metal bicarbonate or water-soluble alkali metal carbonate, or a combination thereof. In some embodiments, the amount of alkali added may be about 1% w / w to about 40% w / w, about 1% w / w to about 35% w / w, about 1% w / w to about 30% w / w, about 1% w / w to about 25% w / w, about 1% w / w to about 20% w / w, about 1% w / w to about 15% w / w, about 1% w / w to about 10% w / w, or about 2% w / w to about 4% 0% w / w, approximately 2% w / w to approximately 35% w / w, approximately 2% w / w to approximately 30% w / w, approximately 2% w / w to approximately 25% w / w, approximately 2% w / w to approximately 20% w / w, approximately 2% w / w to approximately 15% w / w, approximately 2% w / w to approximately 10% w / w, approximately 3% w / w to approximately 40% w / w, approximately 3% w / w to approximately 35% w / w, approximately 3% w / w to approximately 30% w / w Approximately 3% w / w to approximately 25% w / w, approximately 3% w / w to approximately 20% w / w, approximately 3% w / w to approximately 15% w / w, approximately 3% w / w to approximately 10% w / w, approximately 4% w / w to approximately 40% w / w, approximately 4% w / w to approximately 35% w / w, approximately 4% w / w to approximately 30% w / w, approximately 4% w / w to approximately 25% w / w, approximately 4% w / w to approximately 20% w / w, approximately 4% w The amount of alkali added may be approximately 1% w / w to 15% w / w, approximately 4% w / w to 10% w / w, approximately 5% w / w to 40% w / w, approximately 5% w / w to 35% w / w, approximately 5% w / w to 30% w / w, approximately 5% w / w to 25% w / w, approximately 5% w / w to 20% w / w, approximately 5% w / w to 15% w / w, or approximately 5% w / w to 10% w / w. In one integration, the amount of alkali added may be approximately 1% w / w to 20% w / w. In a preferred integration, the amount of alkali added may be approximately 1% w / w to 15% w / w. In a particularly preferred integration, the amount of alkali added may be approximately 4% w / w to 15% w / w.

[0060] In one embodiment, the alkali is added in such an amount that the final pH of the composition after the alkali has reacted with the pulp is at least 7.5, for example, pH 7.5 to pH 11.5. In some embodiments, the composition is approximately 7.0 to 14.0, approximately 7.0 to 13.5, approximately 7.0 to 13.0, approximately 7.0 to 12.5, approximately 7.0 to 12.0, approximately 7.0 to 11.5, approximately 7.0 to 11.0, approximately 7.0 to 10.5, approximately 7.0 to 10.0, approximately 7.0 to 9.5, approximately 7.0 to 9.0, approximately 7.0 to 8.5, approximately 7.0 to 8.0, approximately 7.0 to 7.5, approximately 7.1 to 14.0, approximately 7.1 to 13.5, approximately 7.1 to 13.0, approximately 7.1 to 12.5, approximately 7.1 to 12.0, approximately 7.1 to 11. 0.5, approx. 7.1~approx. 11.0, approx. 7.1~approx. 10.5, approx. 7.1~approx. 10.0, approx. 7.1~approx. 9.5, approx. 7.1~approx. 9.0, approx. 7.1~approx. 8.5, approx. 7.1~approx. 8.0, approx. 7.1~approx. 7.5, approx. 7.2~approx. 14.0, approx. 7.2~approx. 13.5, approx. 7.2~approx. 13.0, approx. 7.2~approx. 12.5, approx. 7.2~approx. 12.0, approx. 7.2~approx. 11.5, approx. 7.2~approx. 11.0, approx. 7.2~approx. 10.5, approx. 7.2~approx. 10.0, approx. 7.2~approx. 9.5, approx. 7.2~approx. 9.0, approx. 7.2~approx. 8.5, approx. 7.2~approx. 8.0, approx. 7.2~ Approximately 7.5, approximately 7.3-14.0, approximately 7.3-13.5, approximately 7.3-13.0, approximately 7.3-12.5, approximately 7.3-12.0, approximately 7.3-11.5, approximately 7.3-11.0, approximately 7.3-10.5, approximately 7.3-10.0, approximately 7.3-9.5, approximately 7.3-9.0, approximately 7.3-8.5, approximately 7.3-8.0, approximately 7.3-7.5, approximately 7.4-14.0, approximately 7.4-13.5, approximately 7.4-13.0, approximately 7.4-12.5, approximately 7.4-12.0, approximately 7.4-11.5, approximately 7.4-11.0 The final pH may be in the range of approximately 7.4 to 10.5, approximately 7.4 to 10.0, approximately 7.4 to 9.5, approximately 7.4 to 9.0, approximately 7.4 to 8.5, approximately 7.4 to 8.0, approximately 7.4 to 7.5, approximately 7.5 to 14.0, approximately 7.5 to 13.5, approximately 7.5 to 13.0, approximately 7.5 to 12.5, approximately 7.5 to 12.0, approximately 7.5 to 11.5, approximately 7.5 to 11.0, approximately 7.5 to 10.5, approximately 7.5 to 10.0, approximately 7.5 to 9.5, approximately 7.5 to 9.0, approximately 7.5 to 8.5, or approximately 7.5 to 8.0.In a particularly preferred embodiment, the composition may have a final pH in the range of about 7.5 to about 9.5.

[0061] The reaction is preferably carried out using the pulp at a temperature of 30°C or below, without subjecting the pulp to any heating step. Therefore, the reaction may be carried out at room temperature, for example, at standard room temperature and pressure. The reaction can also be carried out at lower temperatures, such as about 4°C. Accordingly, in some embodiments, the reaction is carried out in the range of about 4°C to about 25°C, or about 4°C to about 22°C, or about 4°C to about 20°C.

[0062] After sufficient time for the alkali to react with the pulp, the treated pulp is then subjected to a separation step to remove insoluble plant material (in the case of sodium bicarbonate, the separation step may be carried out when the treated pulp no longer produces bubbles). For example, the treated pulp may be centrifuged and / or filtered. Centrifugation may be carried out, for example, at about 6000 to 18000 g. In one embodiment, the treated pulp is centrifuged and the resulting supernatant is then filtered through a 0.22 μm filter.

[0063] The resulting composition may be subjected to one or more purification steps to concentrate and / or further separate the proteases of the present invention from other cellular components. Suitable techniques include (i) gel filtration (size exclusion) chromatography for specific separation of proteins based on molecular weight, for example using Sephadex G-100, as described in the Examples; (ii) ion exchange chromatography; (iii) dialysis; (iv) ammonium acetate precipitation; and / or (v) lyophilization. The composition / fraction obtained from chromatography may be tested to confirm the continued presence of protease activity using, for example, the assay described in the Examples (L-BapNA, optionally used with a serine protease inhibitor or cysteine ​​protease inhibitor added during the assay to determine whether the protease activity is due to serine protease or cysteine ​​protease activity). The presence and amount of at least one serine protease can also be confirmed, for example by SDS-PAGE, using the known molecular weights of at least one serine protease of the present invention.

[0064] The purification process can also be conveniently used to adjust the pH and / or salt concentration to levels suitable for formulation of the composition into pharmaceutical or cosmetic products.

[0065] In another embodiment, the method may further include the step of beating the mixture after the addition of alkali. In a further embodiment, the method may further include the step of filtering the mixture to obtain a filtrate. In a further embodiment, the method may further include the step of filtering the mixture to obtain a residue. In a further embodiment, the method may further include the step of freezing and thawing the composition. The obtained mixture may be frozen and thawed before filtration.

[0066] The final composition preferably contains substantially no insoluble plant material, for example, about 2%, 1%, 0.5%, or less than 0.1% w / w of insoluble material. In one embodiment, the weight percentage of protease relative to total protein is in the range of about 0.01% to 5%. The final composition may contain serine protease activity of about 1 to 100 IU / ml. In some embodiments, the final composition contains approximately 1 to 10 IU / ml, approximately 1 to 20 IU / ml, approximately 1 to 30 IU / ml, approximately 1 to 40 IU / ml, approximately 1 to 50 IU / ml, approximately 1 to 60 IU / ml, approximately 1 to 70 IU / ml, approximately 1 to 80 IU / ml, approximately 1 to 90 IU / ml, approximately 10 to 100 IU / ml, approximately 20 to 100 IU / ml, approximately 30 to 100 IU / ml, approximately 40 to 100 IU / ml, approximately 50 to 100 IU / ml, and approximately 60 to 100 IU / ml. l, about 70 to about 100 IU / ml, about 80 to about 100 IU / ml, about 90 to about 100 IU / ml, about 1 to about 90 IU / ml, about 10 to about 90 IU / ml, about 20 to about 90 IU / ml, about 30 to about 90 IU / ml, about 40 to about 90 IU / ml, about 50 to about 90 IU / ml, about 60 to about 90 IU / ml, about 70 to about 90 IU / ml, about 80 to about 90 IU / ml, about 1 to about 80 IU / ml, about 10 to about 80 IU / ml, about 20 to about 80 IU / ml, about 30 to about 80 IU / ml , about 40 to about 80 IU / ml, about 50 to about 80 IU / ml, about 60 to about 80 IU / ml, about 70 to about 80 IU / ml, about 1 to about 70 IU / ml, about 10 to about 70 IU / ml, about 20 to about 70 IU / ml, about 30 to about 70 IU / ml, Approximately 40 to approximately 70 IU / ml, approximately 50 to approximately 70 IU / ml, approximately 60 to approximately 70 IU / ml, approximately 1 to approximately 60 IU / ml, approximately 10 to approximately 60 IU / ml, approximately 20 to approximately 60 IU / ml, approximately 30 to approximately 60 IU / ml, approximately 40 to approximately 60 IU / ml, approximately 5 It may contain serine protease activity of 0 to approximately 60 IU / ml, approximately 1 to approximately 50 IU / ml, approximately 10 to approximately 50 IU / ml, approximately 20 to approximately 50 IU / ml, approximately 30 to approximately 50 IU / ml, approximately 40 to approximately 50 IU / ml, approximately 1 to approximately 40 IU / ml, approximately 10 to approximately 40 IU / ml, approximately 20 to approximately 40 IU / ml, approximately 30 to approximately 40 IU / ml, approximately 1 to approximately 30 IU / ml, approximately 10 to approximately 30 IU / ml, approximately 20 to approximately 30 IU / ml, approximately 1 to approximately 20 IU / ml, or approximately 10 to approximately 20 IU / ml.

[0067] Formulation of pharmaceutical compositions A pharmaceutical composition may be formed by combining a composition comprising at least one serine protease of the present invention, such as a protease derived from or derived from Carica papaya, and optionally a cysteine ​​protease, with a pharmaceutically acceptable carrier or diluent. When used herein, the pharmaceutical compositions of the present invention will typically contain water, however, the term "pharmaceutically acceptable carrier or diluent" excludes water.

[0068] The carriers and diluents described in the following sections relating to cosmetic compositions can also typically be used in pharmaceutical compositions. Such compositions may be in the form of, for example, creams; lotions; serums; ointments; or gels. Compositions may also be applied to / impregnated in solid forms such as wound dressings, for example, gauze pads.

[0069] Formulation of cosmetic compositions Cosmetic compositions can also be formed by combining a composition comprising at least one serine protease of the present invention, such as a protease derived from or derived therefrom of Carica papaya, and optionally also comprising a cysteine ​​protease, with a cosmetically acceptable carrier or diluent. When used herein, the cosmetic compositions of the present invention will typically contain water, however, the term “cosmetically acceptable carrier or diluent” excludes water.

[0070] Cosmetic compositions according to the present invention may appropriately contain all conventionally known active materials and appropriate components for such formulations. Such components include, for example, gelling agents, anionic polymers, thickeners, surfactants, hydrates, emollients, chelating agents, antioxidants, preservatives, buffering compounds, fragrances, fillers, colorants, volatile or non-volatile substances, modified or unmodified silicones, and reducing agents. The relative ratios of the various components are appropriately those used in conventional cosmetic formulations. It is assumed that those skilled in the art will, of course, select existing materials at their discretion and incorporate these materials into compositions according to the present invention in such a way that the advantageous properties related to the desired enzyme activity are not significantly altered or removed. It is further assumed that certain components may satisfy more than one criterion; for example, glycerin is known to act as both a solvent and a humectant.

[0071] As mentioned above, a variety of components may be present in the composition of the present invention; the most prominent of these components is probably water as a solvent. The amount of water may range from about 1% to about 95% by weight, such as about 25% to about 90% by weight of the composition. Other suitable solvents include glycerin and benzyl alcohol.

[0072] The cosmetic composition of the present invention may contain one or more emulsifiers, for example, when the composition is in the form of a lotion, cream, gel, or serum. Suitable emulsifiers that can be used in the present invention include, for example, one or more alkoxylated fatty alcohols, C 14-22 Alcohol, alkyl polyglycoside, C 14-20 This includes alkyl glucosides, saponifying agents, alkyl sulfates, monoalkyl and dialkyl phosphates, alkyl sulfonates, acyl isothionates, cetyl alcohols, stearyl alcohols, sorbitan, stearic acid, glyceryl stearate, or any combination thereof.

[0073] A particular oil-in-water emulsion base composition of the present invention may also contain one or more stabilizers for stabilizing the emulsion. Suitable stabilizers include, for example, alcohols, alkoxylated alcohols, fatty alcohols, glyceryl esters such as glyceryl stearate, rubber, soap, synthetic polymers, waxes, or any combination thereof. Particularly suitable emulsion stabilizers include stearyl alcohol or glyceryl stearate.

[0074] The composition of the present invention may further contain one or more emollients that soften and smooth the skin. Suitable emollients include, for example, one or more fats and oils (hydrogenated or unhydrogenated) such as vegetable oils, castor oil, coconut oil, and cocoa butter, shea butter, as well as esters such as tricapryl citrate and isononyl isonanoate, or any combination thereof.

[0075] The compositions of the present invention may contain one or more humectants. A humectant is a component that absorbs or retains moisture. Suitable humectants that can be used in the compositions include, for example, urea, pyroglutamic acid, amino acids (e.g., glutamic acid), polyols or other compounds having hygroscopic properties, or any combination thereof. Typically, the humectant is a polyol such as sorbitol. In the compositions of the present invention in the form of a serum, the main humectants present include methyl gluceth-20 and propylene glycol.

[0076] Suitable preservatives for use in the compositions of the present invention include, for example, one or more alkanols such as phenoxyethanol, ethylenediaminetetraacetic acid (EDTA) salts, EDTA fatty acid conjugates, isothiazolinone, parabens such as methylparaben and propylparaben, propylene glycol, sorbate, urea derivatives such as diazolidinylurea and imidazolidinylurea, quaternary ammonium salts, or any combination thereof.

[0077] Suitable chelating agents to be included in the composition of the present invention include, for example, EDTA derivatives or any combination thereof.

[0078] The composition may contain one or more buffering compounds that adjust and maintain the pH of the composition. Suitable buffering agents include, for example, one or more adipic acids, glycine, citric acid, calcium hydroxide, magnesium aluminometasilicate, triethanolamine, or any combination thereof.

[0079] If present, buffers should be present at an appropriate level to ensure a stable pH in the composition over the normal shelf life and period of use of the composition.

[0080] The cosmetic composition may also contain one or more surfactants. Surfactants are particularly important in the compositions of the present invention that are for the purpose of cleansing the skin, such as soaps or facial cleansers. Suitable surfactants include, for example, anionic, nonionic, cationic, amphoteric, or any combination thereof. Preferably, one or more surfactants are nonionic surfactants. Suitable nonionic surfactants include, for example, one or more alkoxylated alcohols, ethoxylated alcohols, propoxylated alcohols, inter-dispersed ethoxylated-propoxylated alcohols, copolymers, fatty acids, alkylphenols, polyglycosides, polyglucosides, n-alkylpyrrolidones, block copolymers, or any combination thereof.

[0081] Typically, the cosmetic compositions of the present invention are in the form of creams; lotions; serums; facial cleansers; shampoos; foams; skin cleansers; skin tonics; ointments; or gels. However, those skilled in the art will understand that any composition suitable for administering the active enzymes of the present invention to the epidermis may be used.

[0082] The composition of the present invention should contain sufficient proteolytic activity to allow a total serine protease activity of about 0.1 IU / ml to about 100 IU / ml to be applied to the skin with each administration, and the administration is assumed to be in the amount of product of about 2 ml to about 10 ml. In some embodiments, the compositions of the present invention are approximately 1 to approximately 10 IU / ml, approximately 1 to approximately 20 IU / ml, approximately 1 to approximately 30 IU / ml, approximately 1 to approximately 40 IU / ml, approximately 1 to approximately 50 IU / ml, approximately 1 to approximately 60 IU / ml, approximately 1 to approximately 70 IU / ml, approximately 1 to approximately 80 IU / ml, approximately 1 to approximately 90 IU / ml, approximately 10 to approximately 100 IU / ml, approximately 20 to approximately 100 IU / ml, approximately 30 to approximately 100 IU / ml, approximately 40 to approximately 100 IU / ml, approximately 50 to approximately 100 IU / ml, approximately 60 to approximately 100 IU / ml, and approximately 70 to approximately 10 0IU / ml, about 80 to about 100IU / ml, about 90 to about 100IU / ml, about 1 to about 90IU / ml, about 10 to about 90IU / ml, about 20 to about 90IU / ml, about 30 to about 90IU / ml, about 40 to about 90IU / ml, about 50 to about 90IU / m l, about 60 to about 90 IU / ml, about 70 to about 90 IU / ml, about 80 to about 90 IU / ml, about 1 to about 80 IU / ml, about 10 to about 80 IU / ml, about 20 to about 80 IU / ml, about 30 to about 80 IU / ml, about 40 to about 80 IU / ml, about 50 to about 80 IU / ml, about 60 to about 80 IU / ml, about 70 to about 80 IU / ml, about 1 to about 70 IU / ml, about 10 to about 70 IU / ml, about 20 to about 70 IU / ml, about 30 to about 70 IU / ml, about 40 to about 70 IU / ml, about 50 to about 70 IU / ml , about 60 to about 70 IU / ml, about 1 to about 60 IU / ml, about 10 to about 60 IU / ml, about 20 to about 60 IU / ml, about 30 to about 60 IU / ml, about 40 to about 60 IU / ml, about 50 to about 60 IU / ml, about 1 to about 50 IU / ml, about 10 to about 50 The total serine protease activity of approximately IU / ml, about 20-50 IU / ml, about 30-50 IU / ml, about 40-50 IU / ml, about 1-40 IU / ml, about 10-40 IU / ml, about 20-40 IU / ml, about 30-40 IU / ml, about 1-30 IU / ml, about 10-30 IU / ml, about 20-30 IU / ml, about 1-20 IU / ml, or about 10-20 IU / ml should contain sufficient proteolytic activity to allow the total serine protease activity to be applied to the skin with each dose, and the dose should beApproximately 2ml to approximately 3ml, approximately 2ml to approximately 4ml, approximately 2ml to approximately 5ml, approximately 2ml to approximately 6ml, approximately 2ml to approximately 7ml, approximately 2ml to approximately 8ml, approximately 2ml to approximately 9ml, approximately 3ml to approximately 4ml, approximately 3ml to approximately 5ml, approximately 3ml to about 6ml, about 3ml to about 7ml, about 3ml to about 8ml, about 3ml to about 9ml, about 3ml to about 10ml, about 4ml to about 5ml, about 4ml to about 6ml, about 4ml to about 7ml, about 4ml to about 8ml, about 4 The product comes in quantities of approximately 10 ml to 9 ml, 4 ml to 10 ml, 5 ml to 6 ml, 5 ml to 7 ml, 5 ml to 8 ml, 5 ml to 9 ml, 5 ml to 10 ml, 6 ml to 7 ml, 6 ml to 8 ml, 6 ml to 9 ml, 6 ml to 10 ml, 7 ml to 8 ml, 7 ml to 9 ml, 7 ml to 10 ml, 8 ml to 9 ml, 8 ml to 10 ml, or 9 ml to 10 ml.

[0083] Serine protease activity can be measured by the L-BApNA assay described in the examples involving the use of a serine protease inhibitor during the assay to determine the serine protease components. Alternatively, the benzoyl-L-arginine ethyl ester (BAEE) assay described in UK Patent No. 2,440,117 may be used. In one embodiment, serine protease activity is in the range of about 5 to about 60 units per applied dose.

[0084] The compositions of the present invention may also contain similar levels of cysteine ​​protease activity. Cysteine ​​protease activity can be measured by the L-BApNA assay described in the examples, which involves the use of a cysteine ​​protease inhibitor during the assay to determine the cysteine ​​protease components.

[0085] A combination of serine protease and cysteine ​​protease derived from Aspergillus melleus in the form of a cosmetic serum has been shown to improve skin smoothness and reduce wrinkles over a period of 14 to 28 days, as determined using skin surface microtopography measurements, in a group of 40 female volunteers (UK Patent No. 2,440,117). The composition of the present invention can also be used in a variety of cosmetic applications, such as cosmetic methods for smoothing and promoting the regeneration of the epidermis of an individual.

[0086] Use of the composition Typically, the cosmetic compositions of the present invention are applied to the face, hands, and neck; however, other skin surfaces may also be subjected to cosmetic treatment. The only contraindication is that particularly sensitive areas of the skin, such as inflamed skin or skin near the eyes, should be avoided. The compositions of the present invention are typically applied directly to the skin surface by the user by hand, as in the conventional method.

[0087] Therefore, the method of the present invention includes the step of applying the cosmetic composition of the present invention to the epidermis of an individual.

[0088] The pharmaceutical compositions of the present invention can be used in a variety of pharmaceutical applications related to the treatment of diseases, disorders, and conditions, including skin conditions and wounds. The compositions of the present invention can also be used in a variety of cosmetic applications.

[0089] Accordingly, the present invention further provides a method for debridement of a wound, including topical application of the composition of the present invention; a composition of the present invention for use in a method for treating a wound; a method for treating an individual suffering from a burn, the method comprising the step of administering the preparation of the present invention topically to a lesion area of ​​the individual or by other routes of administration; a composition of the present invention for use in or when used in a method for treating a wound; a method for enhancing wound healing, comprising the step of administering the composition of the present invention topically to a wound or by other routes of administration; a method for exfoliating or brightening skin, comprising the step of applying the cosmetic composition of the present invention to the skin; the use of the cosmetic composition of the present invention for exfoliating or brightening skin; a method for treating dry, aged or damaged skin, comprising the step of applying the cosmetic composition of the present invention to the skin; and the use of the cosmetic composition of the present invention for treating dry, aged or damaged skin.

[0090] The protease composition of the present invention can be used in particular to prevent, treat, reduce, or improve a variety of skin conditions, including wounds such as chronic wounds including diabetic ulcers, pressure ulcers, venous ulcers, and arterial ulcers, as well as other skin conditions including but not limited to eczema, psoriasis, acne, rosacea, ichthyosis, vitiligo, urticaria, and seborrheic dermatitis.

[0091] In one preferred embodiment, the pharmaceutical composition may be used to debride a wound. Such wounds typically contain necrotic tissue and include diabetic ulcers, pressure ulcers, venous ulcers, arterial ulcers, etc. Thus, such wounds may be chronic wounds. In use, the composition (or a bandage impregnated with the composition, etc.) is applied to the lesion area. The application will typically be done at least once a day, for example, twice a day. The amount of the composition and the frequency of application may be determined by a physician.

[0092] The compositions of the present invention may be administered therapeutically or cosmetically. In such applications, the compositions may be administered to a subject already suffering from a disease in an amount sufficient to cure or at least partially halt the disease and any complications. The amount of the composition should be sufficient to effectively treat the patient.

[0093] The composition may also be administered in the form of liposomes. Liposomes may be derived from phospholipids or other lipid substances and may be formed by monolayers or multilayers of hydrated liquid crystals dispersed in an aqueous medium. Any non-toxic, physiologically acceptable, and metabolizable lipid capable of forming liposomes may be used. The composition in liposome form may contain stabilizers, preservatives, and excipients. Preferred lipids, both natural and synthetic, include phospholipids and phosphatidylcholine (lecithin). Methods for producing liposomes are known in the art, and specific references to these methods are made to Prescott, ed., Methods in Cell Biology, Vol. XIV, Academic Press, New York, NY (1976), p. 33, and the contents of the literature are incorporated herein by reference.

[0094] The compositions of the present invention may be used in the manufacture of pharmaceuticals for preventing, treating, reducing, or improving a target skin condition.

[0095] The compositions of the present invention may also be used in the manufacture of cosmetics for preventing, treating, reducing, or improving a target skin condition.

[0096] Dosage The “therapeutic” dose level for any particular patient will depend on a variety of factors, including the condition being treated and its severity, the activity of the composition employed, the patient’s age, weight, overall health, sex, and diet, along with other relevant factors well known in the art, as well as the time of administration, route of administration, duration of treatment, and any drugs used in combination with or concurrently with the treatment. Therefore, those skilled in the art will be able to determine, by routine experimentation, the effective, non-toxic amount of composition required to treat applicable conditions.

[0097] Furthermore, it will be apparent to those skilled in the art that the optimal dosage and interval of individual doses of the composition will be determined by the nature and severity of the medical condition being treated, the form, route, and site of administration, and the characteristics of the specific individual being treated. Such optimal conditions can also be determined by conventional techniques.

[0098] It will also be apparent to those skilled in the art that the optimal course of treatment, including the number of doses of the composition given per day over a predetermined number of days, can be determined by using a conventional course of treatment evaluation testing.

[0099] Route of administration The compositions of the present invention can be administered by standard routes. Generally, the compositions may be administered by topical routes. Typically, the compositions of the present invention are administered topically to the lesion area of ​​an individual.

[0100] In other embodiments, the composition may be administered via other enteral / intestinal routes such as the rectum, sublingual, or sublabial, or via the central nervous system such as through epidural, intracerebral, or intraventricular routes. Other sites of administration may include via the skin, percutaneous, intradermal, nasal, intraarterial, intracardiac, intraosseous, intrathecal, intraperitoneal, intravesical, intravitreous, intracavernosal, vaginal, or intrauterine routes.

[0101] Timing of therapy Typically, in therapeutic applications, treatment will be related to the duration of the disease state.

[0102] Those skilled in the art will understand that the compositions disclosed herein may be administered at or after diagnosis, for example, as monotherapy or as part of a combination therapy with the methods disclosed herein, as a complement to currently available therapies for such treatment, such as observational therapy or consolidation therapy. The compositions disclosed herein may also be used as prophylactic therapy for subjects genetically or environmentally susceptible to developing such diseases.

[0103] The composition may be administered regularly for as long as necessary, such as until improvement in the patient's condition is observed. Therefore, the composition may be administered once every hour, multiple times a day, daily, multiple times a week, once a week, once a month, or at any frequency deemed appropriate.

[0104] kit The kit of the present invention facilitates the adoption and use of the method of the present invention. Typically, a kit for carrying out the method or use of the present invention contains all the reagents and means necessary to carry out the method. For example, in one embodiment, the kit may include the composition of the present invention and, optionally, means for administering the composition, such as a device for the method in a clinical setting.

[0105] Typically, the kits described herein may also include one or more containers. In the context of the present invention, a segmented kit includes any kit in which the compositions are contained in separate containers, which may include small glass containers, plastic containers, or elongated pieces of plastic or paper. Such containers can enable the efficient transfer of compositions from one segment to the other while avoiding cross-contamination of the compositions, and the quantitative addition of the active substance or solution from one segment to the other in each container.

[0106] Typically, the kit of the present invention will also include instructions for using the kit in order to carry out appropriate methods and uses.

[0107] The methods, uses, compositions, and kits of the present invention are equally applicable to any animal, including humans, and for example, non-human primates, horses, cattle, sheep, goats, rabbits, birds, cattle, and dogs. Therefore, with respect to application to different species, a single kit of the present invention may be applicable, or alternatively, different kits containing, for example, compositions specific to each individual species may be required.

[0108] Those skilled in the art will understand and comprehend that various features disclosed herein can be combined to form combinations of features that fall within the scope of the present invention.

[0109] The present invention will be described herein with further reference to the following exemplary and non-limiting embodiments, which are shown in the following figures. [Examples]

[0110] Preparation of OPAL A and OPAL B The preparation process for OPAL A filtrate was carried out under sterile conditions. The ends of ripe Carica papaya fruits were trimmed and peeled. The fruit pulp was divided into four sections, and then the seeds were removed. The papaya pieces were mixed in a blender until a smooth pulp was obtained. The pulp was placed in a beaker inside a water bath set to 80°C and continuously stirred until the pulp temperature reached 55°C (measured using a glass thermometer). 10% by weight of sodium bicarbonate was added to the beaker. The beaker was then placed in the water bath, set to 55°C, and stirred slowly for 5 minutes. The pulp containing sodium bicarbonate was centrifuged at 12000 × g for 30 minutes, the supernatant was collected, and transferred to a sterile universal tube by filtration using a 0.22 μm filter (FDR-050-071N-Fisher Scientific). OPAL B filtrate was prepared with a one-step change in the preparation process, in that the heating step was omitted.

[0111] Gel filtration chromatography using Sephadex G-100 Superfine Proteins were specifically separated based on molecular weight using gel filtration chromatography. A 14 mL long chromatography column (C3919-1EA-sigma Aldrich) with Sephadex G-100 (G10050-Sigma) (separation between 4 kDa and 150 kDa) was packed and washed overnight with 100 mM Tris-HCl at pH 8 with 0.15 M NaCl. 1 mL of the required sample was passed through the column separately to separate proteins based on molecular weight. The required number of 0.5 mL fractions were collected, and A 280The following measurements were taken. Next, 50 μL of each fraction was placed in a 96-well plate, and 50 μL of 1.5 mM L-BApNA buffer (prepared with 10 mM Tris-HCl pH 8.5 buffer) was added. L-BApNA activity in A410 was then measured both immediately and 2 hours later using a Synergy HT microplate reader (catalog 12926527, Bio-Tek Instruments, Thermo Fisher Scientific, UK). The difference in values ​​was plotted, and fractions with higher L-BApNA activity were run through an SDS-PAGE gel, and the gel was then stained using Coomassie or silver staining.

[0112] L-BApNA (N-benzoyl-L-arginine 4-nitroanilide hydrochloride) assay 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 of dimethyl sulfoxide (DMSO) and then diluting it with water to 100 ml. Hydrolysis of L-BApNA at the binding between arginine and the p-nitroaniline moiety releases the chromophore p-nitroaniline, which can be detected by spectroscopy at 410 nm in international units (IU). IU is defined as the amount of enzyme that causes a 0.01 unit / min increase in absorbance under excess substrate conditions (zero-order kinetics). Specific activity of solids is expressed in IU / mg, while activity of liquid formulations is expressed in IU / mL.

[0113] To determine the optimal substrate concentration for maintaining zero-order reaction kinetics throughout the experiment, a series of titration runs were performed using a range of substrate concentrations. The minimum concentration found in this manner was 0.2 mM or a 1:6 dilution of 1.5 mM stock solution. Table 1 shows the reagent doses used in all kinetic experiments. Since phosphate buffer yielded the highest enzyme activity readout compared to other buffers, phosphate buffer at pH 6 was used in the L-BApNA assay. [Table 1]

[0114] To verify substrate purity, controlled total hydrolysis catalyzed by alkali was performed under the following conditions. [Table 2] Therefore, JPEG2026062669000005.jpg18149 The results confirmed the 99% purity of the substrate as stated by the manufacturer.

[0115] inhibitors E-64 (IUPAC name: (1S,2S)-2-(((S)-1-((4-guanidinobutyl)amino)-4-methyl-1-oxopentan-2-yl)carbamoyl)cyclopropanecarboxylic 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.

[0116] 2,2'-Dipyridyl disulfide (2DPS) is used as a thiol-specific reversible inhibitor against cysteine ​​proteases, including papain, bromelain, and ficin.

[0117] The protease inhibitor cocktail (PIC) was formulated as shown in Table 3. [Table 3]

[0118] Overlay zymography Overlay zymography was performed following the procedure of Vinokurov et al. (2005) with some modifications (Figure 1). Briefly, a nitrocellulose membrane was immersed in a 1.2 mg / mL L-BApNA solution in water. The membrane was then left to air dry for 5 minutes and then overlaid on top of a native PAGE gel after the samples had been run, with the membrane briefly immersed in the running buffer of each sample. Two additional experiments were performed by immersing the membrane in 35M β-alanine, 0.14M acetic acid, pH 4.3, with either 10 mM DTT or 25 mM cysteine. All zymographs were then incubated in a sealed chamber at 37°C for 30–60 minutes. The membrane was removed, left to dry again for 5 minutes, and p-nitroaniline was visualized by diazotization.

[0119] Diazotization was carried out by following the protocol detailed by Hosseininaveh et al. (2009). Briefly, the membrane was sequentially immersed in sodium nitrite solution (1 mg / mL in 1 M HCl) for 5 minutes, then in ammonium sulfamate solution (5 mg / mL in 1 M HCl), and finally in 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 / band.

[0120] mass spectrometry Protein bands identified by colloidal Coomassie staining were analyzed by MALDI-Mass fingerprinting at the Department of Biochemistry, PNAC Facility, University of Cambridge. Gel bands were excised and subjected to the following treatments in 200 μL of 100 mM ammonium bicarbonate / 50% acetonitrile at 20°C for 30 minutes per step: 1) reduction with 5 mM Tris(2-carboxyethyl)phosphine; 2) alkylation by addition of iodoacetamide (final concentration 25 mM); and 3) removal of the liquid and subsequent washing.

[0121] After washing, the gel samples were dried under vacuum for 10 minutes, and then 25 μl of 100 mM ammonium bicarbonate containing 5 μg / mL modified trypsin (Promega) was added. Digestion was carried out at 32°C for 17 hours. The peptides were recovered, desalted using μC18 ZipTip (Millipore), and eluted onto a Maldi target plate using 1-2 μL of alpha-cyano-4-hydroxycinnamic acid matrix (Sigma) in 50% acetonitrile / 0.1% trifluoroacetic acid. Peptide mass was determined using a Bruker ultrafleXtreme Maldi mass spectrometer in a reflectron-like manner, and ms / ms fragmentation was performed in a LIFT-like manner (peroformed). Data analysis was initiated using FlexAnalysis, BioTools, and ProteinScape software (Bruker). Database searches of combined mass fingerprint data were performed using Mascot (Matrix Science). Where necessary, additional maneuvers were performed using Protein Prospector. Debride assay The AWE debride assay is an in vitro surrogate for wound necrotic tissue proteolytic activity developed by Health Point (Shi, Ermis et al., 2009). The AWE debride assay has been shown to be highly comparable to in vivo animal data. The AWE substrate consists of a pellet of three wound-associated extracellular matrix proteins (collagen, elastin, and fibrin), each tagged with a different fluorophore. Stepwise degradation of this matrix can be measured by a gradual increase in fluorescence intensity in a Franz-type diffusion cell setting (Figure 2). The final readout for the experiment is obtained at 24 hours.

[0122] The materials used for the debride 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 along with 100 mM NaCl and 10 mM CaCl2, all from Sigma, magnetic stirrer bar (Z328936), L-cysteine ​​(W326305), elastin-bovine (E1625), 7-amino-4-methylcoumarin (A9891), and papain (P3375) derived from papaya latex.

[0123] Preparation of fibrin-coumarin: Fibrinogen was labeled with 7-amino-4-methylcoumarin by mixing it with a coumarin solution (0.02 mg / mL in Tris buffer) to a final fibrinogen concentration of 10 mg / mL (in Tris buffer). The mixture was incubated at room temperature for 1 hour using a rotary shaker. Thrombin solution (2.5 units / mL) was added to the fibrinogen-coumarin solution, and coumarin-labeled fibrin was obtained by coagulation for 2 hours.

[0124] The formed solid was washed three times with water and left to stand overnight in distilled water and methanol (1:1) to remove excess pigment. The solid was then transferred to a glass container lined with clear, non-stick filter paper and dried for three days. The dried fibrin-coumarin was ground into a fine powder using a mortar and pestle.

[0125] Preparation of artificial wound eschar (AWE) matrix Collagen-FITC, elastin-rhodamine, and fibrin-coumarin were mixed according to the compositions mentioned in Table 4. All materials except fibrinogen were weighed into 50 mL conical centrifuge tubes. The materials were homogenized in 10 mL Tris buffer for 3–5 minutes using a tearer. 10 mL of 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 for about 2 minutes using a tearer. Thrombin solution (50 U / mL) was added and mixed rapidly, and the solution was then poured into a Petri dish containing a 90 mm non-reactive membrane and allowed to solidify for 1 hour. The solidified substrate was then rinsed with water three times (5 minutes each) to remove thrombin. After washing, excess water was removed using tissue paper and the mixture was stored at 4°C until further use.

[0126] Franz-type diffusion cell system Using the AWE substrate still adhering to the membrane, a 9 mm diameter hole was punched using a biopsy punch. The 9 mm AWE substrate was placed on top of a non-reactive nitrocellulose membrane and placed in a Franz-type diffusion cell between two chambers, with the lower chamber (receptor cell) filled with Tris buffer containing 1% (v / v) penicillin-streptomycin (Figure 2). A sample holder was placed on top, and the entire assembly was attached to the receptor cell using screws and surrounded by a 35°C water bath. The sample was then loaded into the upper chamber of the sample holder and covered with perforated Parafilm. The solution was mixed throughout the experiment using a magnetic stirrer at 500 rpm. 100 μL of the solution was sampled from the receptor cell at zero and at regular intervals up to 24 hours. The collected samples were loaded into a 96-well microplate for immediate fluorescence measurement using a fluorescence plate reader (Synergy HT KC4 v3.4). After measurement, 100 μL of the sample was returned to the receptor cell.

[0127] The total cumulative digestion of all three protein components in the substrate was determined by applying the following equation. CD n =In×V セル In the formula, n = time in time, CD n And In are the cumulative digestion parameter and fluorescence intensity at time n, and V セル This is the volume of the cell (5.1 mL). [Table 4]

[0128] Example 1 - Variants of OPAL B filtrate and OPAL A filtrate, and proteolytic activity of OPAL B filtrate Carica papaya belongs to a small family of the Caricaceae family and is one of the main fruits cultivated in tropical and subtropical regions for its edible fruit and latex. The fruit used in these examples was ripe papaya fruit mainly obtained from Brazil and Jamaica, purchased from a local UK-based supermarket. Most of the well-characterized and intensively studied cysteine ​​proteases are members of the papain family. Papain, chymopapain A and B, chymopapain M, and calicaine have all been extracted from Carica papaya, but have never been characterized from ripe fruit.

[0129] The inventors have previously conducted mass spectrometry work (Richard J. Lipscombe - Proteomics International laboratory, University of Western Australia) and shown that OPAL A filtrate derived from ripe fruit extract of Carica papaya contains cysteine ​​(thiol-dependent) proteases. These cysteine ​​proteases are traditionally extracted only from papaya latex or papaya peel (Proteomics International, personal communications). Spectrophotometric assays were performed to functionally identify the cysteine ​​proteases in the OPAL A filtrate. These experiments involved monitoring the catalytic hydrolysis of the chromogenic substrate L-BApNA (Nα-benzoyl-L-arginine 4-nitroanilide hydrochloride) by the OPAL A filtrate.

[0130] Hydrolysis of L-BApNA in the presence of a protease at the binding between arginine and the p-nitroaniline moiety releases the chromophore p-nitroaniline, which can then be detected by spectroscopy at a wavelength of 410 nm. The procedure for determining L-BApNA activity is described above. Under the optimized conditions employed, excess substrate leads to zero-order reaction kinetics, which are evident as a straight line, and the slope of the line is proportional to the enzyme activity.

[0131] The inventors have previously demonstrated that OPAL A possesses cysteine ​​protease activity that is essentially neutralized by the addition of E-64, an inhibitor that specifically inhibits cysteine ​​protease (unpublished data). Therefore, the inventors have previously concluded that the protease activity in OPAL A is essentially attributable solely to the presence of active cysteine ​​protease. To determine the effect of omitting the heat treatment step, protease activity in both OPAL A and OPAL B filtrates was tested using an L-BApNA assay. After 10 minutes, the E-64 inhibitor was added.

[0132] Compared with OPAL A, OPAL B appeared to exhibit approximately eight-fold increased catalytic activity (linear increase in substrate release over time of A 410 as shown in Figure 3). Interestingly, the addition of an excess of E-64 to the OPAL B filtrate did not result in complete inhibition of activity as was the case with OPAL A. Therefore, and without wishing to be bound by theory, the increase in A 410 values with substrate could have been due to a non-thiol-dependent (non-cysteine protease) catalyst. The fact that treatment with the cysteine protease inhibitor E-64 did not completely abolish protease activity suggests, as shown in Figure 4, that both cysteine and non-cysteine proteases were present in the OPAL B filtrate.

[0133] Example 2 - Identification of cysteine ​​and serine proteases in OPAL B filtrate by enzyme reaction kinetics. To better understand the non-thiol-dependent or non-cysteine protease in the OPAL B filtrate, an enzyme assay was first performed using a protease inhibitor cocktail (PIC) (sigma P2714) containing all essential protease inhibitors (see Table 3). As shown in Figure 5, complete inhibition of protease activity was achieved by adding 20 μL of PIC to the OPAL B filtrate.

[0134] Based on these data, the inventors then examined the effect of each activity inhibitor by individually testing each component of the PIC that may be involved in the enhanced activity of OPAL B. The components of the protease inhibitors present in the cocktail are shown in Table 3.

[0135] After testing each inhibitor present in the PIC using the OPAL B filtrate, only two inhibitors were able to interrupt proteolytic activity. These two inhibitors were E-64 and AEBSF, which are cysteine protease and serine protease inhibitors, respectively, as shown in Figure 6.

[0136] The combination of both inhibitors completely eliminated enzyme activity, as shown in Figure 7. This suggests that the enzyme activity in the OPAL B filtrate consists solely of cysteine ​​and serine proteases.

[0137] Example 3 - Zymography assay for functional identification of proteases determined by enzyme reaction kinetics The OPAL B filtrate appears to be a complex mixture of many proteins. Therefore, to individually visualize the protease activity detected by spectrophotometric assay, the inventors used a technique called superposition zymography. In this technique, proteins are first separated on a standard native PAGE gel that preserves enzyme activity. A membrane containing a substrate solution (1.2 mg / mL L-BApNA) is then superimposed on the gel and developed using a color enhancer. This technique allows for the direct visualization of active proteases on the membrane.

[0138] As shown in Figure 8, the colloidal blue-stained native PAGE gel revealed a band in the OPAL B filtrate during the unidirectional (anode-side) phase. Protein migration from the gel to the nitrocellulose membrane, and subsequent zymography analysis, showed a clear increase in enzyme activity (demonstrating activity of two enzymes).

[0139] The inventors further characterized the two zymography bands obtained in Figure 8 to verify that the two observed signals or bands were cysteine ​​and serine proteases, respectively. The method was modified by including specific inhibitors (E-64 for cysteine ​​proteases and AEBSF for serine proteases) in the nitrocellulose membrane before placing the membrane on top of the native PAGE gel. The obtained data clearly revealed that the bands were indeed cysteine ​​proteases and serine proteases in the OPAL B concentrated filtrate. As shown in Figure 9, the cysteine ​​proteases correspond to the upper bands and the serine proteases correspond to the lower bands.

[0140] Example 4 - Size exclusion chromatography for identifying serine proteases To purify serine proteases, OPAL B was concentrated and purified by size exclusion chromatography using a sephadex G-100 column to separate proteins in the 4kDa–150kDa range. Dialyzed OPAL B, pretreated with E-64 and 100mM L-arginine (incubated for 2 hours), was run through a 14 mL sephadex G-100 column. Twenty-four eluted fractions were collected and a 96-well plate L-BApNA assay was performed (data not shown). The fraction containing the highest L-BApNA activity was selected onto an SDS-PAGE gel, which was then silver-stained as shown in Figure 10. Bands in the fraction whose abundance correlated with L-BApNA activity were selected and analyzed by LC / MS-MS (i.e., the bands of intensity present in fraction 6 at 80kDa, 50kDa, and 30kDa). These bands matched the Carica papaya sequence shown in Sequence ID No. 1, which had been presumably identified as a subtilase.

[0141] This result is consistent with the analysis of the complete OPAL B filtrate, which identified four fragments within Sequence ID No. 1, as shown below. JPEG2026062669000008.jpg144149

[0142] Example 5 - Reactive probe for identifying the active form of serine protease To determine which of the various bands corresponds to the proteolytically active form of subtilase in the OPAL B filtrate, the inventors used a reactive probe that specifically labels active serine protease via a biotin-linked fluorosulfonate group. This reactive probe can then be visualized on a standard SDS-PAGE gel by Western blotting using a streptavidin conjugate enzyme such as alkaline phosphatase. Using this technique, it was possible to visualize an active serine protease band inhibited by AEBSF treatment at approximately 50 kDa, as shown in Figure 11. Although this band corresponds to one of the bands observed from chromatographic experiments identified as papaya subtilase by mass spectrometry, the molecular weight of the band does not match the published values ​​for either the full-length protein or the proenzyme (70 and 85 kDa, respectively). Without wishing to be constrained by theory, the fact that the inventors observed several bands originating from the same protein suggests that various isoforms may exist, and that only the 50 kDa form of these isoforms is proteolytically active.

[0143] overall conclusion In International Publication No. 2004 / 008887, the inventors previously described a method for producing a composition known as OPAL A derived from the pulp of ripe papaya (Carica papaya). This method involves a heating step, followed by treatment with sodium bicarbonate, and then a filtration step. The inventors' previous analysis of OPAL A identified cysteine ​​protease activity. OPAL A has shown activity in treating a wide range of disorders. The inventors modified the OPAL A process to produce OPAL B by omitting the heating step. The inventors surprisingly and unexpectedly found that OPAL B contains additional protease activity unrelated to cysteine ​​protease. The inventors characterized this additional protease activity as being due to the presence of at least one serine protease that is not present in OPAL A. Although this protein appears to exist in more than one form, only the protein with an average molecular weight of 50 kDa, as determined by SDS-PAGE, exhibited serine protease activity. LC-MS / MS analysis indicated that this protein has the same sequence as the C. papaya sequence (Othman and Nuraziyan, 2010) that was presumably identified as a subtilase, but this was not confirmed by biochemical characterization. Furthermore, when expressed recombinantly, this subtilase has a molecular weight of approximately 70 kDa, compared to the 50 kDa molecular weight of the active serine proteases currently identified by the inventors.

[0144] Therefore, the inventors 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, while the only known active protease recognized to date in Carica papaya is a cysteine ​​protease.

[0145] Example 6 - Quantification of the debridement efficacy of OPAL A and OPAL B filtrates in vitro. Debridement is the removal of necrotic (dead) tissue and foreign material from a wound to expose the underlying living tissue. This process promotes and accelerates wound healing. The inventors investigated whether the cysteine ​​protease identified in OPAL A filtrate would be active in debridement as measured using an artificial wound scab (AWE) debridement assay.

[0146] The AWE debride assay is an in vitro surrogate for wound necrotic tissue proteolytic activity developed by Health Point (Shi, Ermis et al., 2009). The AWE debride assay has been shown to be sufficiently comparable to in vivo animal data and was therefore used to assess the debride efficacy of OPAL A filtrate formulations. The AWE substrate consists of a pellet of three wound-associated extracellular matrix proteins—collagen, elastin, and fibrin—each tagged with a different fluorophore. Stepwise degradation of this matrix can be measured by a gradual increase in fluorescence intensity in a Franz-type diffusion cell setting. The final readout for the experiment is obtained at 24 hours.

[0147] In the first set of experiments, the proteolytic activity of natural OPAL A filtrate (0.7 mg / mL protein equivalent) was compared to that of papaya latex-derived crude papain (10 mg / mL, Sigma) using a Franz-type cell diffusion system. The results shown in Figure 12 clearly demonstrate that the digestion of collagen and fibrin in the OPAL A filtrate sample was consistently superior to that of latex papain (10 mg / mL), but weaker for elastin.

[0148] Example 7 - Concentration of OPAL A filtrate activity using dialysis to improve debridement efficacy The L-BApNA activity of the newly prepared OPAL A filtrate averaged 0.60 IU / mL. The protein content of the new OPAL A filtrate was approximately 0.7 mg / mL, corresponding to a specific solid activity of approximately 1 IU / mg. Compared to the activity of pure latex papain of approximately 10 IU / mg, approximately 1 IU / mg is close to the papain content of approximately 9% in the OPAL solid. Notably, the AWE debride assay performed in Example 6 revealed significantly higher activity of OPAL A than would be expected from its nominal papain content (0.6 IU / mL vs. 30 IU / mL compared to latex papain), as shown in Figure 12.

[0149] The solid residue content of OPAL A was measured by freeze-drying the extract, yielding consistent values ​​across batches of 0.216 ± 0.005 g / mL. Therefore, it is clear that the vast majority of OPAL A filtrate solids are naturally potentially non-proteolytic. Thus, it may be possible to increase the specific activity of the extract by removing the solids. To investigate this, the inventors subjected fresh OPAL A filtrates to dialysis using membranes with cutoff sizes of 12 kDa and 25 kDa. Dialysis was performed at 4°C for 24 hours, and after dialysis, the residual proteolytic activity of the solution was measured by L-BApNA assay. Aliquots of the solution were also freeze-dried and their solid content measured. The treatment caused a small (20%) increase in osmotic volume, and this increase was taken into account in the calculations.

[0150] The results in Table 5 demonstrate near-complete retention of activity for either dialysis using cutoff sizes of 12 and 25 kDa, combined with approximately 86% solid reduction. [Table 5] This result is consistent with the generally accepted size of Carica papaya protease (23-30 kDa) and the fact that OPAL A filtrate contains a large amount of low molecular weight sugars that are removed upon dialyzing. The fact that the solid content does not change significantly with dialysis of 12 kDa-25 kDa indicates the absence of molecules with sizes intermediate between these two values ​​in the composition. Therefore, the inventors used a 12 kDa cutoff dialysis membrane for further experiments.

[0151] The debride strength of OPAL A filtrate versus natural Opal A was measured as in Example 6. The data in Figure 13 show that dialyzed OPAL A is superior to natural OPAL A in digesting fibrin and elastin, but inferior in digesting collagen.

[0152] In summary, dialyzed OPAL A filtrate showed complete retention of proteolytic activity compared to natural OPAL A filtrate, but with 86% less solid reduction than the control. This demonstrates that the majority of solids in OPAL A filtrate are naturally non-proteolytic and exist as low molecular weight sugars that can be removed by dialyzing. Dialyzed OPAL A filtrate had enhanced debride activity against both fibrin and elastin compared to latex papain and natural OPAL A, but had a slightly less effect on collagen compared to natural OPAL A.

[0153] Example 8 - In vivo case study on the management of severe pressure ulcers using OPAL B This case study discloses the rapid treatment of severe ulcers with tissue necrosis in elderly patients through topical application of OPAL B. Therefore, OPAL B appears to offer therapeutic benefits in the healing of severe ulcers through a multifactorial mechanism of action involving at least tissue regenerative and anti-necrotic / debridement factors.

[0154] A bedridden nursing home resident in her 70s with dementia developed a severe pressure ulcer. The ulcer extended laterally across a large portion of the patient's lower back and showed significant tissue necrosis. Upon transfer to the hospital, vacuum dressing was applied in an attempt to clear the excessive exudate from the ulcerated wound. Additional surgical intervention using a rhomboid flap to reduce the size of the ulcer was also attempted. Despite continuous treatment efforts by hospital staff, the severity of the wound persisted (Figure 14).

[0155] With the consent of the surgeon treating the patient, vacuum dressing and other surgical interventions were postponed, in favor of daily application of fresh, undiluted OPAL B directly to the wound surface, in conjunction with daily application of 30% diluted OPAL B aqueous cream to the skin immediately surrounding the wound.

[0156] By days 2–4 of Opal B treatment, the wound appeared cleaner and less inflamed (Figures 15 and 16). Although some necrotic tissue was still observed, it appeared to be localized to smaller areas of the wound. By days 16–19, further improvement was observed, accompanied by a decrease in wound size (Figures 17 and 18). Once stable, the wound was surgically debridified. The extremely rapid halt of exacerbation / necrosis and progression achieved in treating what had previously been an extremely severe and untreatable ulcer is remarkable and significant.

[0157] Without being constrained by theory, OPAL B appears to have several modes of action: it seemed to localize to unviable tissue, reverse peripheral ischemia, and thereby reduce the amount of necrotic tissue. There is also evidence that OPAL B was beginning to debride slough in ulcers.

[0158] The various features and embodiments of the present invention referred to in the individual sections above will be applied to other sections as necessary. As a result, features specified in one section may be combined with features specified in other sections as necessary.

[0159] All publications referenced in the above specification are incorporated herein by reference. All compositions and / or methods disclosed and asserted herein can be made and performed without excessive experimentation based on this disclosure. Although the compositions and methods of the present invention are described in terms of preferred embodiments, it will be apparent to those skilled in the art that variations may be applied to the compositions and / or methods described herein, and to the steps or arrangement of the steps, without departing from the concept, spirit, and scope of the present invention.

[0160] Example 9 - Proteome mapping of OPAL B In proteome mapping of OPAL B using Protein Pilot (trademark), which searches the UniProt Viridplantae database, the detected peptide fragments are shown in bold below.

[0161] Sequence ID 1 (shown in bold: three catalytic residues, Asn336, and peptides from Sequence IDs 2-5) JPEG2026062669000010.jpg100149

[0162] Amino acids 1-25 are the putative signal sequence, and amino acids 26-112 are the putative pro region, which is cleaved to form the mature protein. JPEG2026062669000011.jpg87149

[0163] References Arkin AP, YouvanDC. An algorithm for protein engineering: simulations of recursive ensemblemutagenesis, Proc Natl Acad SciU S A. 1992 Aug 15;89(16):7811-5. Delagrave S, Goldman ER, Youvan DC.Recursive ensemble mutagenesis. Protein Eng. 1993 Apr;6(3):327-31 Hosseininaveh V, Bandani A, HosseininavehF. Digestive proteolytic activity in the Sunn pest, Eurygaster integriceps. J Insect Sci. 2009;9:1-11. Kunkel TA. Rapid and efficientsite-specific mutagenesis without phenotypic selection. Proc Natl Acad Sci U S A. 1985 Jan;82(2):488-92. Kunkel TA, Roberts JD, Zakour RA. Rapidand efficient site-specific mutagenesiswithout phenotypic selection. Methods Enzymol. 1987;154:367-82. Othman and Nuraziyan, Fruit-specific expression of papayasubtilase gene. J. Plant Physiology 2010; 167(2):131-137. Prescott, Ed., Methods in Cell Biology,Volume XIV, Academic Press, New York, N.Y. (1976) Rosenberg AH, Goldman E, Dunn JJ, StudierFW, Zubay G. Effects of consecutive AGG codons on translation in Escherichiacoli, demonstrated with a versatile codon test system. J Bacteriol. 1993Feb;175(3):716-22. Shi L, Ermis R, Lam K, Cowart J, Attar P,Aust D. Study on the debridement efficacy of formulated enzymatic wound debriding agents by in vitroassessment using artificialwound eschar and by an in vivo pig model. Wound Repair Regen. 2009 Nov-Dec;17(6):853-62. van der Plas MJ, Andersen AS, Nazir S,van Tilburg NH, Oestergaard PR, Krogfelt KA, van Dissel JT, Hensbergen PJ, Bertina RM, NibberingPH. A novel serine proteasesecreted by medicinal maggots enhances plasminogen activator-induced fibrinolysis. PLoS One. 2014 Mar 19;9(3):e92096.Erratum in: PLoS One. 2014;9(6). Vinokurov KS, Oppert B, Elpidina EN. Anoverlay technique for postelectrophoreticanalysis of proteinase spectra in complex mixtures using p-nitroanilide substrates. Anal Biochem.2005 Feb 1;337(1):164-6.

Claims

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

2. A composition comprising an active serine protease having a continuous amino acid sequence having at least 95% homology to amino acids 118-763 of SEQ ID NO:

1.

3. A composition comprising an active serine protease having a continuous amino acid sequence having at least 95% homology to amino acids 142-618 of SEQ ID NO:

1.

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

5. A method for preparing a composition containing an active serine protease, comprising the step of treating the ripe fruit pulp of the Carica papaya plant with alkali without subjecting it to a heating step.

6. The method according to claim 5, wherein the alkali is weakly alkaline.

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

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

9. The method according to any one of claims 5 to 8, wherein the composition further comprises an active cysteine ​​protease.

10. The method according to any one of claims 5 to 9, wherein the soluble protease is separated from the insoluble plant material after the alkali treatment.

11. The method according to any one of claims 5 to 10, wherein the composition is further treated to increase the concentration of the protease.

12. A composition comprising an active serine protease obtainable by the method described in any one of claims 5 to 11.

13. A pharmaceutical composition comprising activated Carica papaya serine protease together with a pharmaceutically acceptable carrier or diluent.

14. A pharmaceutical composition comprising the composition according to any one of claims 1 to 4 and 12, together with a pharmaceutically acceptable carrier or diluent.

15. A cosmetic composition comprising activated Carica papaya serine protease together with a carrier or diluent acceptable for use in cosmetics.

16. A cosmetic composition comprising the composition according to any one of claims 1 to 4 and 12, together with a carrier or diluent that is acceptable as a cosmetic.

17. A pharmaceutical composition according to claim 13 or 14 for use in wound debridement.

18. Use of the composition according to claim 15 or 16 in skin exfoliation.

19. A method for preventing, treating, reducing, or improving a skin condition, comprising the step of administering a composition according to any one of claims 1 to 4 and 12 to 16.

20. A kit for use in or as to be used in preventing, treating, reducing, or improving a skin condition, comprising the composition according to any one of claims 1 to 4 and 12 to 16, and instructions for use.

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