Enhancement and stabilization of the proteolytic activity of protease

Abstract

The present invention relates to methods for enhancing and stabilizing the proteolytic activity of proteases, methods for producing compositions comprising proteases with enhanced and stabilized proteolytic activity, compositions comprising proteases with enhanced and stabilized proteolytic activity 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 and in cosmetic applications, and related kits.

Description

Technical Field

[0001] [Field of the Invention] The present invention relates to methods for enhancing and stabilizing the proteolytic activity of proteases, methods for producing compositions comprising proteases having enhanced and stabilized proteolytic activity, compositions comprising proteases having enhanced and stabilized proteolytic activity obtainable 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 and cosmetic applications, and related kits.

[0002] [Background Art] Papain and bromelain are proteases used in pharmaceutical products for debriding and in cosmetics / medicated cosmetics for keratolysis and skin lightening. The proteolytic activity of papain and bromelain results in such debriding, keratolysis, and skin lightening. However, neither papain preparations nor bromelain preparations can fully exert their potential because their proteolytic activity is unstable.

[0003] Considerable efforts have been made previously to develop stable compositions that promote wound healing through the removal of dead and damaged tissue as seen in wounds such as burns and chronic ulcers. Dead and dying tissue is an excellent medium for opportunistic infections, so effective debriding is essential. Sepsis resulting from infections is the main cause of death in severely burned patients.

[0004] One of the conventional approaches has been to use proteolytic enzymes such as papain, trypsin, and bromelain. In particular, NexoBrid™, a concentrate of proteolytic enzymes rich in bromelain, was approved in Europe in 2012 for the removal of eschar (i.e., debride) in adults with deep partial - thickness and full - thickness burns. However, the European Medicines Agency confirmed in section 2.2.3, page 14 of the "Assessment report - Nexobrid - Concentrate of proteolytic enzymes enriched in bromelain" on September 20, 2012, that a common problem with proteolytic enzyme compositions is their low stability of proteolytic activity, "New compatibility (in - use stability) tests were carried out at 25 °C and 37 °C, demonstrating that NexoBrid degrades within a few hours after mixing. Thus, the applicant's conclusion that the product should be used immediately after mixing is supported." as stated.

[0005] Therefore, NexoBrid™ is typically supplied as a lyophilized powder, which is reconstituted with a gel medium before use and must be used within 15 minutes after formulation. Therefore, it would be advantageous to provide a composition such as NexoBrid™ in a ready - to - use form without the need for reconstitution. However, to do so, it would be necessary to significantly enhance the stability of the wound - debriding activity to achieve an acceptable shelf - life.

[0006] Similarly, in cosmetics sold as having a keratolytic action and / or skin - lightening action and containing papain and / or bromelain, the keratolytic activity often significantly decreases in a short period due to the loss of activity of papain and / or bromelain in the cosmetics.

[0007] To address the problem of loss of proteolytic activity of proteases in pharmaceutical products, methods have previously been devised that involve storing the enzyme at a pH where loss of activity is low or absent (acidic pH in the case of papain), or in solid form. However, these treatments require processing by a skilled end-user to obtain a viable product. This limits the usefulness of such methods and compositions.

[0008] Another method that has been previously attempted is to immobilize the enzyme on a polymeric substrate to prevent mobility and self-reactivity. Examples of immobilized enzymes include PEG-papain and chitosan-papain. While such methods may improve the stability of proteolytic activity, the enzyme may undergo chemical changes in an irreversible manner and thus may not exhibit sufficient activity when applied to certain substrates, particularly complex substrates such as skin and cellular proteins. Furthermore, changes in the chemical structure of such enzymes can cause reactions that are harmful to the end-user, such as allergies and intolerances.

[0009] To address these problems, studies underlying the present invention verified the loss of proteolytic activity of proteases extracted from various sources such as the Carica papaya (papaya) plant (including papain) and the Ananas comosus (pineapple) plant (including bromelain). Based on these studies, methods were developed for enhancing and stabilizing the proteolytic activity of proteases, particularly in pharmaceutical and cosmetic / medicinal cosmetic compositions containing proteases derived from papaya and pineapple plants.

[0010] Surprisingly and unexpectedly, it has been found that some of the methods for enhancing the stability of proteolytic activity also significantly enhance the proteolytic activity itself.

[0011] [Summary of the Invention] The present invention teaches a new method for enhancing and stabilizing the proteolytic activity of proteases. In certain embodiments, the protease can be obtained from or is obtainable from fruits and / or vegetables. Thus, in some embodiments, the protease is obtained from fruits and / or vegetables, while in other embodiments, the protease is obtained from a recombinant expression system. In certain embodiments, the protease is a cysteine protease. In certain embodiments, the cysteine protease may be 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), ficain (EC 3.4.22.3), or actinidin (EC 3.4.22.14). In certain embodiments, the protease can be obtained from or is obtainable from the Carica papaya (papaya) plant or the Ananas comosus (pineapple) plant. In certain embodiments, the protease is papain or bromelain.

[0012] In a first aspect, the present invention provides a method for enhancing and / or stabilizing the proteolytic activity of a protease, comprising: (i) contacting the protease with a reducing agent such that the cysteine residues of the protease are maintained in a reduced state; and (ii) substantially removing all oxygen gas from the region surrounding the protease. In one embodiment, the oxygen gas is removed by degassing the preparation. In one embodiment, the protease is packaged in a substantially oxygen - free atmosphere.

[0013] In a related aspect, the present invention provides a method for enhancing and / or stabilizing the proteolytic activity of a protease, comprising: (i) providing a solution or gel comprising the protease; (ii) contacting the protease in the solution or gel with a reducing agent such that the cysteine residues of the protease are maintained in a reduced state; and (iii) substantially removing all oxygen gas from the solution or gel.

[0014] In a second aspect, the present invention provides a method for producing a composition comprising a protease having enhanced and / or stabilized proteolytic activity, the method comprising: (i) contacting the protease with a reducing agent such that the cysteine residues of the protease are maintained in a reduced state; and (ii) substantially removing all oxygen gas from the region surrounding the composition. In one embodiment, the oxygen gas is removed by degassing the composition. In one embodiment, the composition is placed in an atmosphere substantially free of oxygen.

[0015] In a related aspect, the present invention provides a method for producing a composition comprising a protease having enhanced and / or stabilized proteolytic activity, the method comprising: (i) providing a solution or gel comprising the protease; (ii) contacting the protease in the solution or gel with a reducing agent such that the cysteine residues of the protease are maintained in a reduced state; and (iii) substantially removing all oxygen gas from the solution or gel.

[0016] In a third aspect, the present invention provides a composition comprising one or more proteases having enhanced and / or stabilized proteolytic activity, the composition obtainable or obtainable by a method of a first aspect such as a method comprising: (i) contacting the protease with a reducing agent such that the cysteine residues of the protease are maintained in a reduced state; and (ii) substantially removing all oxygen gas from the region surrounding the composition. In one embodiment, the oxygen gas is removed by degassing the composition. In one embodiment, the composition is placed in an atmosphere substantially free of oxygen.

[0017] In a fourth aspect, the present invention provides a composition comprising one or more proteases and a reducing agent, wherein the cysteine residues of the protease are maintained in a reduced state and the composition is substantially free of oxygen. In one embodiment, oxygen gas is removed by degassing the composition. In one embodiment, the composition is placed in an atmosphere that is substantially free of oxygen.

[0018] According to the present invention, enhancement and / or stabilization of the proteolytic activity of a protease can also be achieved by immobilizing the protease on an anionic polymer matrix. The use of an anionic polymer allows the protease to be non-covalently bound to the matrix. This is in contrast to conventional methods in the art for immobilizing proteases by forming a covalent bond with a matrix material, for example, by reacting a portion of the primary amines of the protease with the carboxyl groups of a carbomer and cross-linking a portion of the remaining primary amines of the protease using an amine-reactive cross-linking reagent.

[0019] Thus, in a fifth aspect, the present invention provides a method for enhancing and / or stabilizing the proteolytic activity of a protease, the method comprising the step of combining the protease with an anionic polymer matrix such that the protease is non-covalently bound to the anionic polymer matrix. In one embodiment, the polymer is a carbomer. In one embodiment, the composition is placed in an atmosphere that is substantially free of oxygen.

[0020] In a sixth aspect, the present invention provides a method for producing a composition comprising a protease having enhanced and / or stabilized proteolytic activity, the method comprising the step of combining the protease with an anionic polymer matrix such that the protease is non-covalently bound to the anionic polymer matrix. In one embodiment, the polymer is a carbomer. In one embodiment, the composition is placed in an atmosphere that is substantially free of oxygen.

[0021] In a seventh aspect, the present invention provides a composition comprising one or more proteases having enhanced and / or stabilized proteolytic activity, the composition being obtainable or obtainable by a method comprising combining the protease with an anionic polymer matrix such that the protease binds non-covalently to the anionic polymer matrix. In one embodiment, the polymer is carbomer. In one embodiment, the composition is placed in a substantially oxygen-free atmosphere.

[0022] In an eighth aspect, the present invention provides a composition comprising one or more proteases and an anionic polymer matrix, wherein the protease is non-covalently bound to the anionic polymer matrix. In one embodiment, the polymer is carbomer. In one embodiment, the composition is placed in a substantially oxygen-free atmosphere.

[0023] The methods taught by the present invention can also be combined. For example, the method of the first aspect can be combined with the method of the fifth aspect, or the method of the second aspect can be combined with the method of the sixth aspect.

[0024] Accordingly, in a ninth aspect, the present invention provides a method for enhancing and / or stabilizing the proteolytic activity of a protease, the method comprising: (i) contacting the protease with a reducing agent such that the cysteine residues of the protease are maintained in a reduced state; (ii) substantially removing all oxygen gas from the region surrounding the protease; and (iii) combining the protease with an anionic polymer matrix such that the protease binds non-covalently to the anionic polymer matrix. In one embodiment, the oxygen gas is removed by degassing the preparation. In one embodiment, the polymer is carbomer. In one embodiment, the protease is placed in a substantially oxygen-free atmosphere.

[0025] In one embodiment, step (iii) is performed before steps (i) and (ii). In another embodiment, steps (i) to (iii) are performed in this order.

[0026] In a related aspect, the present invention provides a method for enhancing and / or stabilizing the proteolytic activity of a protease, comprising: (i) preparing a solution containing the protease; (ii) contacting the protease in the solution with a reducing agent such that the cysteine residues of the protease are maintained in a reduced state; (iii) removing substantially all oxygen gas from the solution; and (iv) combining the protease with an anionic polymer matrix such that the protease binds non-covalently to the anionic polymer matrix.

[0027] In one embodiment, step (iv) is performed before steps (i) to (iii). In another embodiment, steps (i) to (iv) are performed in this order.

[0028] In a tenth aspect, the present invention provides a method for producing a composition comprising a protease having enhanced and / or stabilized proteolytic activity, comprising: (i) contacting the protease with a reducing agent such that the cysteine residues of the protease are maintained in a reduced state; (ii) removing substantially all oxygen gas from the region surrounding the protease; and (iii) combining the protease with an anionic polymer matrix such that the protease binds non-covalently to the anionic polymer matrix. In one embodiment, the oxygen gas is removed by degassing the preparation. In one embodiment, the polymer is carbomer. In one embodiment, the composition is placed in a substantially oxygen-free atmosphere.

[0029] In a related aspect, the present invention provides a method for producing a composition comprising a protease having enhanced and / or stabilized proteolytic activity, the method comprising: (i) preparing a solution or gel comprising a protease; (ii) contacting the protease in the solution or gel with a reducing agent such that the cysteine residues of the protease are maintained in a reduced state; (iii) substantially removing all oxygen gas from the solution or gel; and (iv) combining the protease with an anionic polymer matrix such that the protease binds non-covalently to the anionic polymer matrix.

[0030] In an eleventh aspect, the present invention provides a composition comprising one or more proteases having enhanced and / or stabilized proteolytic activity, the composition being obtainable or obtainable by a method according to the ninth aspect, such as a method comprising: (i) contacting the protease with a reducing agent such that the cysteine residues of the protease are maintained in a reduced state; (ii) substantially removing all oxygen gas from the region surrounding the protease; and (iii) combining the protease with an anionic polymer matrix such that the protease binds non-covalently to the anionic polymer matrix. In one embodiment, the oxygen gas is removed by degassing the preparation. In one embodiment, the polymer is carbomer. In one embodiment, the composition is placed in a substantially oxygen-free atmosphere.

[0031] In a twelfth aspect, the present invention provides a composition comprising one or more proteases, a reducing agent, and an anionic polymer matrix, wherein the cysteine residues of the protease are maintained in a reduced state, the composition is substantially oxygen-free, and the protease binds non-covalently to the anionic polymer matrix. In one embodiment, the oxygen gas is removed by degassing the composition. In one embodiment, the polymer is carbomer. In one embodiment, the composition is placed in a substantially oxygen-free atmosphere.

[0032] In one embodiment, the composition is in the form of a gel.

[0033] In a 13th aspect, the present invention provides the use of the composition of the 3rd, 4th, 7th, 8th, 11th, and / or 12th aspects in the manufacture of a medicament. In some embodiments, the medicament is for the treatment of diseases and disorders including wounds. In some embodiments, the medicament is for debriding. In some embodiments, the medicament is for treating burns. In some embodiments, the medicament is for treating ulcers. In some embodiments, the medicament is for treating gangrene.

[0034] In a 14th aspect, the present invention provides the use of the composition of the 3rd, 4th, 7th, 8th, 11th, and / or 12th aspects in the manufacture of a cosmetic. In some embodiments, the cosmetic is for keratolysis, skin lightening, or for application to wrinkles, skin spots, freckles, eruptions, acne, rosacea, solar lentigines, scars, or varicose veins, or for application to dry skin, aging skin, or damaged skin.

[0035] In a 15th aspect, the present invention provides a pharmaceutical composition comprising the composition of the 3rd, 4th, 7th, 8th, 11th, and / or 12th aspects together with a pharmaceutically acceptable carrier, diluent, excipient, surfactant, and / or adjuvant.

[0036] In a 16th aspect, the present invention provides a cosmetic composition comprising the composition of the 3rd, 4th, 7th, 8th, 11th, and / or 12th aspects together with a cosmetically acceptable carrier, diluent, excipient, surfactant, and / or adjuvant.

[0037] In a 17th aspect, the present invention provides a composition of the 3rd, 4th, 7th, 8th, 11th, and / or 12th aspect, or a pharmaceutical composition of the 15th aspect for use in treating diseases and disorders including wounds. In some embodiments, the treatment is debridement. In some embodiments, the treatment is for burns. In some embodiments, the treatment is for ulcers. In some embodiments, the treatment is for gangrene. In some embodiments, the composition is applied topically.

[0038] In an 18th aspect, the present invention provides a method for treating diseases and disorders including wounds, the method comprising the step of administering to a subject a composition of the 3rd, 4th, 7th, 8th, 11th, and / or 12th aspect, or a pharmaceutical composition of the 15th aspect. In some embodiments, the treatment is debridement. In some embodiments, the treatment is for burns. In some embodiments, the treatment is for ulcers. In some embodiments, the treatment is for gangrene. In some embodiments, the composition is applied topically.

[0039] In a 19th aspect, the present invention provides a composition of the 3rd, 4th, 7th, 8th, 11th, and / or 12th aspect, or a cosmetic composition of the 16th aspect for use in skin lightning, for keratolysis, or for application to wrinkles, skin spots, freckles, eruptions, acne, rosacea, solar lentigines, scars, or varicose veins, or for application to dry skin, aging skin, or damaged skin.

[0040] In a 20th aspect, the present invention provides a kit comprising a composition of the 3rd, 4th, 7th, 8th, 11th, and / or 12th aspect, a pharmaceutical composition of the 15th aspect, or a cosmetic composition of the 16th aspect. In one embodiment, the kit is used for carrying out the method of the 18th aspect or for the uses of the 17th and 19th aspects.

[0041] In contrast to conventionally available compositions, the compositions of the present invention do not need to be lyophilized to be sufficiently stable for pharmaceutical or cosmetic use. Thus, such compositions can be formulated, for example, as capsules, tablets, creams, ointments, solutions, pastes, drops, sprays, aerosols, vapors, wipes, patches, gauzes, gels, or liquids. Thus, in one embodiment, the compositions of the present invention are provided or packaged as capsules, tablets, creams, ointments, solutions, pastes, drops, sprays, aerosols, vapors, wipes, patches, gauzes, gels, or liquids and do not need to be reconstituted before use. In a preferred embodiment, the compositions of the present invention are provided or packaged in the form of a gel or a liquid and do not need to be reconstituted before use.

Brief Description of the Drawings

[0042]

Figure 1

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[0043] Definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are described. For the purposes of the present invention, the following terms are defined as follows.

[0044] As used herein, the articles "a" and "an" are used to refer to the grammatical object of one or more (i.e., at least one) articles. By way of example, "an element" means one element or more than one element.

[0045] "About" means an amount, level, value, number, frequency, percentage, dimension, size, amount, weight, or length that varies by 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1% relative to a reference amount, level, value, number, frequency, percentage, dimension, size, amount, weight, or length.

[0046] Throughout this specification, unless the context requires otherwise, the words "comprise", "comprises" and "comprising" will be understood to mean including the stated step or element or group of steps or elements but not excluding any other step or element or group of steps or elements. Thus, the use of terms such as "comprising" indicates that the listed elements are essential or mandatory, but other elements are optional and may or may not be present. "Consisting of" means including and limited to all of the following the phrase "consisting of". Thus, the phrase "consisting of" indicates that the listed elements are essential or mandatory and that no other elements may be present. "Consisting essentially of" means including any elements listed after the phrase and limited to other elements that do not interfere with or contribute to the activity or action specified in the disclosure for the listed elements. Thus, the phrase "consisting essentially of" indicates that the listed elements are essential or mandatory, but other elements are optional and may or may not be present depending on whether they affect the activity or action of the listed elements.

[0047] The term "debride man" refers to the removal of dead and damaged tissue from a wound.

[0048] The term "obtainable" when used in connection with the compositions of the present invention includes not only compositions produced by a particular defined method, but also the same compositions produced by any method, for example, by obtaining protease from fruits or vegetables, or by using recombinant DNA technology or other genetic engineering methods, such as using a recombinant expression system.

[0049] "Isolated" means a material that substantially or essentially does not contain components that are normally associated with the material in its natural state. For example, as used herein, an "isolated protease" refers to the in vitro isolation and / or purification of a peptidic or polypeptide protease molecule from its natural cellular environment and from association with other components of the cell, i.e., it is not associated with in vivo substances.

[0050] The term "Opal" refers to a papain-containing composition obtained from the fruit of the papaya (excluding the latex of the pericarp). Opal can be prepared, for example, by the method disclosed in International Patent Application No. PCT / AU2003 / 000931, published as International Publication No. WO 2004 / 008887, the entire content of which is incorporated herein by reference.

[0051] The terms "patient", "subject", and "individual" are used interchangeably and refer to a human or other mammalian patient, subject, and individual, including any who it is desired to treat, prevent, improve, or reduce the severity of a disease, disorder, or condition using the present invention. However, it will be understood that "patient" does not imply the presence of symptoms. Suitable mammals within the scope of the present invention include, but are not limited to, primates (e.g., humans, chimpanzees), domestic animals (e.g., sheep, cows, 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).

[0052] Throughout this specification, the phrase "substantially oxygen-free" includes concentrations of less than 1 ppm.

[0053] As used herein, the phrases "enhancement and / or stabilization of the proteolytic activity of a protease" and "enhanced and / or stabilized proteolytic activity" and equivalent phrases refer to methods for enhancing the proteolytic activity of a protease and methods for stabilizing the proteolytic activity of a protease, such as by increasing, preserving, extending, or delaying a decrease in the ability of the protease to perform its normal enzymatic function, relative to a given unit of time or relative to a reference level of activity. Thus, such methods may result in a protease composition that performs its enzymatic function in a shorter time than would be required in the absence of such method. Alternatively, the method may result in the protease performing its enzymatic function at a higher activity level than the activity level in the absence of the method, when measured at a particular point in time or multiple points in time after the method has been carried out. The shorter time or higher activity level may be measured in relation to various other factors, including, but not limited to, exposure to heat and sterilizing radiation, and the storage time and storage conditions of the protease composition, and further including any transport conditions such as temperature, humidity, and atmospheric pressure.

[0054] Enhancement and / or stabilization of the proteolytic activity of a protease can be measured or confirmed, for example, by comparing the enzyme activity of a protease (e.g., "Sample A") that has been subjected to the method of the present invention with the stability of a protease that has not been subjected to the method of the present invention (e.g., a "control sample", which may be, for example, a wild-type or naturally occurring protease). Comparison between such samples can be carried out, for example, at a particular time after the method of the present invention has been carried out, to confirm an increase in the stability and / or activity of the protease of "Sample A" relative to the control sample.

[0055] The terms "wild-type" and "naturally occurring" are used interchangeably to refer to the gene product (e.g., a polypeptide such as a protease) that is most frequently observed in a population, and are thus optionally referred to as the "normal" or "wild-type" form of the gene.

[0056] The term "trauma" means an injury to living tissue where the skin has been cut or damaged, including skin ulcers and burns. Skin ulcers can include diabetic ulcers, pressure ulcers, venous (or varicose) ulcers, and arterial ulcers.

[0057] References to prior art in this specification are not to be taken as an admission that the prior art forms part of the common general knowledge of a person skilled in the art, nor as an indication in any way, and should not be construed as an admission or indication in any way.

[0058] The entire contents of all publications, patents, patent applications, and other materials cited in this specification are hereby incorporated by reference into this specification.

[0059] [Detailed Description of the Invention] Cysteine proteases, also known as thiol proteases and cysteine endopeptidases (EC 3.4.22), are enzymes that break down proteins. Cysteine proteases share a common catalytic mechanism in which a nucleophilic cysteine thiol is involved in the catalytic triad or dyad. Cysteine proteases are present in various organisms. In particular, cysteine proteases are commonly found in fruits such as papaya (Carica papaya and Vasconcellea cundianmarcensus), pineapple (Ananas comosus), fig (Ficus carica), and kiwifruit (Actinidia chinensis), but may also be found in other various fruits and vegetables. Cysteine proteases can be obtained by various methods including extraction from biological materials such as fruit extracts from papaya pulp, or by recombinant expression in suitable host cells. In one embodiment, the protease present in the composition of the present invention or subjected to the method of the present invention is a protease prepared from ripe pulp of papaya by a method such as that described in International Publication No. 2004 / 008887. In another embodiment, the protease is bromelain (EC 3.4.22.33).

[0060] Stabilization procedure In one aspect of the present invention, a composition containing a cysteine protease is subjected to treatment with a reducing agent that maintains the active site cysteine amino acid residue of the protease in a reduced state. When the cysteine residue is oxidized, a disulfide bridge is formed, which can thereby inhibit enzyme activity. The reducing agent can keep the active site residue in the reduced form and can also convert already oxidized cysteine residues to the reduced form. Suitable reducing agents are known in the art and contain cysteine. The amount of the reducing agent added should generally be sufficient to regenerate all or most of the active site cysteine amino acid residues of the protease in the solution and maintain them in the reduced form. In this case, typically, the reducing agent is added in excess. In one embodiment, the concentration of the reducing agent such as cysteine is typically 10 to 200 mM, for example 50 to 150 mM. In certain embodiments, the concentration of the reducing agent such as cysteine is 60 to 140 mM, 70 to 130 mM, 80 to 120 mM, 90 to 100 mM, 92 to 108 mM, 94 to 106 mM, 96 to 104 mM, or 98 to 102 mM. In one embodiment, the concentration of the reducing agent such as cysteine is about 100 mM or 100 mM.

[0061] The composition may be in the form of a liquid or a gel containing, for example, a protease. In one embodiment, the protease is pre-bound to an anionic polymer as described below prior to the above-described treatment step with the reducing agent.

[0062] This composition can also be subjected to the step of substantially removing all of the oxygen present in the region surrounding the protease, for example, the step of substantially removing all of the oxygen present in a liquid or gel such as a solution containing the protease. This can be achieved, for example, by degassing the composition, for example, by flushing the composition with an inert gas such as nitrogen or argon. For example, by purging nitrogen or argon at a flow rate of 25 mL / s for 20 to 40 minutes, the residual dissolved oxygen can be brought to about 0.2 to 0.4 ppm. Other methods for substantially removing all of the oxygen present in the composition can also be used, for example, but not limited to, heating under atmospheric pressure or reduced pressure, or ultrasonic treatment under atmospheric pressure or reduced pressure, etc., which are methods known to those skilled in the art. The step of removing oxygen can be carried out before the addition of the reducing agent, simultaneously with the addition, or immediately after the addition.

[0063] The resulting composition contains a cysteine protease and a reducing agent that reduces one or more active site cysteine residues of the cysteine protease, where the composition is substantially free of oxygen. At least a portion of the reducing agent may be in an oxidized state as a result of reacting with the cysteine protease and / or other components in the composition.

[0064] During storage, in order to reduce contact with oxygen, the composition can be packaged to reduce or prevent oxygen absorption. For example, the composition may be packaged in an atmosphere substantially free of oxygen, for example, in a container filled with an inert gas such as nitrogen or argon. Alternatively, the composition may be vacuum packaged.

[0065] In another aspect, which can also be combined with the foregoing aspects of the present invention, the anionic polymer matrix can be combined with a cysteine protease such that the cysteine protease binds non-covalently to the anionic polymer matrix. This is intended to suppress autolysis and enzyme inactivation by separating the enzyme molecules in a non-covalent manner. The selection of the anionic polymer is generally based on the high isoelectric point (pI) of papain and related proteases, i.e., papain and related proteases are positively charged at neutral pH.

[0066] The anionic polymer can also be selected to form a gel at a pH between, for example, 6.5 and 8, or at an alkaline pH.

[0067] In one embodiment, the anionic polymer matrix is a polyacrylic acid such as a homopolymer, copolymer, or interpolymer of acrylic acid. The anionic polymer typically has a high molecular weight. Examples of homopolymers include polymers of acrylic acid crosslinked with any of several polyhydric alcohol allyl ethers (e.g., allyl ether pentaerythritol, allyl ether of sucrose, or allyl ether of propylene). Examples of copolymers include, for example, polymers of acrylic acid and C10-C30 alkyl acrylates crosslinked with allyl pentaerythritol. Specific examples of suitable anionic polymers include Carbopol, Carbopol Ultrez, Carbomer 910, Carbomer 934, Carbomer 934p, Carbomer 940, and Carbomer 941 (available from Lubrizol).

[0068] An anionic polymer can be combined with a protease to obtain a liquid suspension. The concentration of the anionic polymer may be 0.01 to 3% w / w or more, for example 0.1 to 2% w / w. When combined with another aspect of the present invention, if not already performed, a reducing agent can be added at this stage, and oxygen can be removed by nitrogen flushing or the like. Then an alkali is added to cause gelation of the liquid suspension, for example by adjusting the pH to 7.5 to 8, to obtain a viscous gel.

[0069] The three different processing steps can be performed in different orders. For example, the combination with the anionic polymer can be performed first, and then the addition of the reducing agent / removal of oxygen can be performed in either order. Alternatively, the addition of the reducing agent / removal of oxygen can be performed in either order first, and then the combination with the anionic polymer can be performed. It may also be possible to perform the combination with the anionic polymer between the step of the reducing agent and the step of removing oxygen. The various steps may be performed sequentially or simultaneously (or to some extent overlapping).

[0070] The compositions of the present invention typically show greater stability of proteolytic activity than the corresponding untreated protease control samples, i.e., samples that are not non-covalently bound to an anionic polymer and are stored under normal oxidation conditions, such as atmospheric oxygen levels, without the addition of a reducing agent. For example, some protease compositions of the present invention, particularly papain (P) and papaya extracts (such as Opal), can show at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, or 95% proteolytic activity after being stored at room temperature and atmospheric pressure for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, or 60 days or more, while other protease compositions, particularly bromelain (B), can show at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, or 95% proteolytic activity after being stored at room temperature and atmospheric pressure for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days or more. The measured activity can be compared to the activity immediately after treatment by the process of the present invention.

[0071] In some embodiments, the initial proteolytic activity of the protease compositions of the present invention is greater than that of the untreated protease compositions, such as having at least 1.5 times the activity, for example at least 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, or 10 times the activity, compared to the untreated protease. For comparison purposes, the initial activity can be measured immediately after performing the treatment step.

[0072] Activity can be measured using a BApNA spectrophotometric assay: A BApNA solution (10 mM in DMSO) can be prepared with a reaction buffer (100 mM potassium phosphate, 116 mM potassium chloride, and 3 mM EDTA, pH 6). The premix can be made with 2 parts water, 2 parts reaction buffer, and 1 part v / v of BApNA. A spectrophotometer (e.g., Jasco V-630 UV-Vis spectrophotometer) can be blanked at 410 nm with the premix solution. Then 1 / 5 v / v of water can be added to both the control sample and the positive sample, and readings can be started immediately thereafter. Relative enzyme activity can be measured as the slope of the resulting straight line.

[0073] Composition In contrast to conventionally available compositions, the compositions of the present invention do not need to be lyophilized to be sufficiently stable for pharmaceutical and / or cosmetic use. Thus, such compositions can be formulated, for example, as capsules, tablets, creams, ointments, solutions, pastes, drops, sprays, aerosols, vapors, wipes, patches, gauzes, gels, or liquids. Thus, in one embodiment, the compositions of the present invention are provided or packaged as capsules, tablets, creams, ointments, emulsions, solutions, pastes, drops, sprays, aerosols, vapors, wipes, patches, gauzes, gels, or liquids and do not need to be reconstituted prior to use. In a preferred embodiment, the compositions of the present invention are provided or packaged in the form of a gel or a liquid and do not need to be reconstituted prior to use.

[0074] In certain aspects of the present invention, the composition may be in the form of a gel or a liquid. The improved stability of the proteolytic activity of the composition allows it to be provided in liquid or gel form, which means it can be used immediately and does not need to be reconstituted immediately before use. This is very beneficial in the administration of the composition and may be useful in situations where it is difficult to reconstitute a lyophilized composition, such as in clinical settings outside of wounds like burns that require immediate treatment.

[0075] The composition can also be absorbed / adsorbed onto solid materials such as wound dressings. The composition can be combined with a pharmaceutical and / or cosmetic carrier, diluent, excipient, surfactant, and / or adjuvant to obtain the pharmaceutical composition or cosmetic composition of the present invention. Thus, the composition of the present invention can be formulated to contain one or more additional components.

[0076] For example, in order to improve the physical properties of the composition, a surfactant can be used as part of the protease composition. The presence of the surfactant does not affect the efficacy of the composition. Thus, the composition can also incorporate any suitable surfactant, such as an anionic, cationic, or nonionic surfactant such as sorbitan esters or their polyoxyethylene derivatives. Suitable surfactants may also include sodium dodecyl sulfate (SDS), ammonium lauryl sulfate, sodium laureth sulfate, and sodium myreth sulfate. Surfactants are commonly used to reduce non-specific adsorption and require careful selection and optimization. It may also contain suspending agents such as natural gums, cellulose derivatives, or inorganic materials such as siliceous silica, and other components such as lanolin.

[0077] The composition can be prepared according to methods known to those skilled in the art and can also contain additional carriers, excipients, or diluents. The carriers, excipients, and diluents must be "acceptable" in the sense that they are compatible with the other components of the composition and must not be harmful to the formation of a composition that can be stored for a longer period if necessary. Such carriers, excipients, and diluents can be used to further enhance the integrity and half-life of the composition of the present invention.

[0078] Further examples of acceptable carriers or diluents include deionized or sterilized water; physiological saline; vegetable oils such as peanut oil, safflower oil, olive oil, cottonseed oil, corn oil, sesame oil, soybean oil, or coconut oil; silicone oils such as polysiloxanes like methylpolysiloxane, phenylpolysiloxane, methylphenylpolysolpoxane; volatile silicones; mineral oils such as liquid paraffin, soft paraffin, or squalane; cellulose derivatives such as methylcellulose, ethylcellulose, carboxymethylcellulose, sodium carboxymethylcellulose, or hydroxypropylmethyl - cellulose; lower alkanols such as ethanol or isopropanol; lower aralkanols; lower polyalkylene glycols or lower alkylene glycols such as polyethylene glycol, polypropylene glycol, ethylene glycol, propylene glycol, 1,3 - butylene glycol, or glycerin; fatty acid esters such as isopropyl palmitate, isopropyl myristate, or ethyl oleate; polyvinylpyrrolidone; agar; tragacanth gum or gum acacia, and petrolatum. Carriers such as polyvinylpyrrolidone (PVP), carboxymethylcellulose, polyvinyl alcohol, and polyethylene oxide can also be used.

[0079] Additional carriers that can be included in the compositions of the present invention include non - reducing sugars such as sucrose and reducing sugars such as lactulose. Such carriers, as well as sugar alcohols such as mannitol, xylitol, glycerol, and sorbitol, can also act as antioxidants and potential stabilizers and are thus useful for inclusion in the compositions of the present invention.

[0080] Methods for preparing the administrable compositions will be apparent to those skilled in the art and are more particularly described, for example, in Remington’s Pharmaceutical Science, 15th Edition, Mack Publishing Company, Easton, Pa., which is incorporated herein by reference.

[0081] Use The compositions of the present invention can be used for various 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 for various cosmetic applications.

[0082] Accordingly, the present invention provides a method of debriding a wound comprising topical application of a composition of the present invention, a composition of the present invention for use in a method of treating a wound, a method of treating an individual suffering from a burn, the method comprising administering a preparation of the present invention topically or by another route of administration to the affected area of the individual, a composition of the present invention for use in a method of treating a wound or when used, a method of promoting wound healing comprising administering a composition of the present invention topically or by another route of administration to a wound, a method of keratolysis or skin lightening comprising applying a cosmetic composition of the present invention to the skin, use of a cosmetic composition of the present invention for keratolysis or skin lightening, a method of treating dry skin, aged skin, or damaged skin comprising applying a cosmetic composition of the present invention to the skin, and use of a cosmetic composition of the present invention for treating dry skin, aged skin, or damaged skin.

[0083] The protease compositions of the present invention can be used, inter alia, for preventing, treating, reducing, or improving various skin conditions including wounds, including chronic wounds such as vascular / compressive skin ulcers, burns, and other skin conditions including, but not limited to, eczema, psoriasis, acne, rosacea, ichthyosis, vitiligo vulgaris, urticaria, seborrheic dermatitis.

[0084] The composition of the present invention can be administered therapeutically or cosmetically. In such uses, the composition can be administered to a subject already suffering from a condition in an amount sufficient to cure or at least partially arrest the condition and its complications. The amount of the composition should be sufficient to effectively treat the patient.

[0085] The composition can also be administered in the form of liposomes. Liposomes may be derived from phospholipids or other lipid substances and may be formed by single-layer or multi-layer lamellar hydrated liquid crystals dispersed in an aqueous medium. Any non-toxic, physiologically acceptable, and metabolizable lipid capable of forming liposomes can be used. The composition in the form of liposomes may contain stabilizers, preservatives, and excipients. Preferred lipids include both natural and synthetic phospholipids and phosphatidylcholine (lecithin). Methods for producing liposomes are known in the art, and specific reference is made in this regard to Prescott, Methods in Cell Biology, Volume 14, Academic Press, New York, N.Y. (1976), pages 33 and below, the content of which is incorporated herein by reference.

[0086] Dosage The "therapeutically effective" dosage level for any particular patient depends on various factors such as the condition being treated and its severity, the activity of the composition being used, the patient's age, weight, general health status, gender and diet, time of administration, route of administration, duration of treatment, and any drugs used in combination with or concurrently with the treatment, as well as other relevant factors well known in the art. Accordingly, one of ordinary skill in the art will be able to determine, by routine experimentation, the effective and non-toxic amount of the composition required to treat the applicable condition.

[0087] Furthermore, it will be apparent to those skilled in the art that the optimal amounts and intervals of individual dosages of the composition will be determined by the nature and extent of the condition being treated, the form, route and site of administration, and the nature of the particular individual being treated. Such optimal conditions can also be determined by the prior art.

[0088] It will also be apparent to those skilled in the art that an optimal course of treatment, such as the number of administrations of the composition given per day for a defined number of days, can be ascertained by those skilled in the art using conventional course-of-treatment determination tests.

[0089] Route of administration The compositions of the present invention can be administered by standard routes. Generally, the compositions can be administered by local routes. Typically, the compositions of the present invention are administered locally to the affected part of the individual.

[0090] In other embodiments, the compositions can be administered via other enteral / enteric routes such as rectal, sublingual, or sublabial, or via the central nervous system through routes such as epidural, intracerebral, or intraventricular. Other sites of administration can include those via the skin, transdermal, intradermal, nasal, intraarterial, intracardiac, intraosseous, intrathecal, intraperitoneal, intravesical, intravitreal, intracorporeal, vaginal, or intrauterine routes.

[0091] Timing of treatment Typically, for therapeutic use, the treatment is over the course of the disease state.

[0092] Those skilled in the art will appreciate that the compositions disclosed herein can be administered at the time of diagnosis or subsequently, for example, as a follow-up treatment or booster treatment as a complement to currently available treatments for such treatment, either as a single agent or as part of a combination treatment approach to the methods disclosed herein. The compositions disclosed herein can also be used for the prophylactic treatment of subjects having a genetic or environmental predisposition to develop such diseases.

[0093] The composition can be administered periodically for a necessary period, such as until an improvement in the condition is observed. Thus, the composition can be administered hourly, multiple times a day, daily, multiple times a week, weekly, monthly, or at a frequency deemed appropriate.

[0094] Kit The kits of the present invention facilitate the implementation of the methods and uses of the present invention. Typically, a kit for implementing the method or use of the present invention includes all of the reagents and means necessary for implementing the method. For example, in one embodiment, the kit can include the composition of the present invention and, optionally, means for administering the composition, such as a device for a point-of-care method.

[0095] Typically, the kits described herein also include one or more containers. In the context of the present invention, a compartmentalized kit includes any kit in which the composition is contained in separate containers, which may include small glass containers, plastic containers, or plastic or paper strips. Such containers can enable the efficient transfer of the composition from one compartment to another while avoiding cross-contamination of the composition, and the addition of the drug or solution of each container from one compartment to another in a quantitative manner.

[0096] Typically, the kits of the present invention also include instructions for using the kit to perform the appropriate methods and uses.

[0097] The methods, uses, compositions, and kits of the present invention are equally applicable to any animal, including humans, such as non-human primates, horses, cows, sheep, goats, rabbits, birds, cats, and dog species. Thus, to apply to different species, a single kit of the present invention can be applied, or, for example, different kits containing compositions specific to each individual species may be required.

[0098] Those skilled in the art will understand and appreciate that different features disclosed herein can be combined to form combinations of features within the scope of the present invention.

[0099] Enumerated embodiments 1. A method for enhancing and / or stabilizing the proteolytic activity of a protease, comprising: (i) contacting the protease with a reducing agent such that the cysteine residues of the protease are maintained in a reduced state; (ii) substantially removing all oxygen gas from the region surrounding the protease; and (iii) combining the protease with an anionic polymer matrix such that the protease binds non-covalently to the anionic polymer matrix.

[0100] 2. An embodiment wherein the protease is placed in a substantially oxygen-free atmosphere. 1 The method according to item 1.

[0101] 3. A method for producing a composition comprising a protease having enhanced and / or stabilized proteolytic activity, comprising: (i) contacting the protease with a reducing agent such that the cysteine residues of the protease are maintained in a reduced state; (ii) substantially removing all oxygen gas from the region surrounding the protease; and (iii) combining the protease with an anionic polymer matrix such that the protease binds non-covalently to the anionic polymer matrix.

[0102] 4. An embodiment 3 wherein the composition is placed in a substantially oxygen-free atmosphere.

[0103] 5. The method according to any one of embodiments 1 to 4, wherein the oxygen gas is removed by degassing the preparation.

[0104] 6. The method according to any one of embodiments 1 to 5, wherein the polymer is carbomer.

[0105] 7. A composition comprising one or more proteases having enhanced and / or stabilized proteolytic activity, obtained or obtainable by a method comprising: (i) contacting the protease with a reducing agent such that the cysteine residues of the protease are maintained in a reduced state; (ii) substantially removing all oxygen gas from the region surrounding the protease; and (iii) combining the protease with an anionic polymer matrix such that the protease is non-covalently bound to the anionic polymer matrix.

[0106] 8. A composition comprising one or more proteases, a reducing agent, and an anionic polymer matrix, wherein the cysteine residues of the protease are maintained in a reduced state, the composition is substantially oxygen-free, and the protease is non-covalently bound to the anionic polymer matrix.

[0107] 9. The composition according to embodiment 7 or 8, wherein the oxygen gas is removed by degassing the composition.

[0108] 10. The composition according to any one of embodiments 7 to 9, wherein the polymer is carbomer.

[0109] 11. The composition according to any one of embodiments 7 to 10, wherein the composition is placed in an atmosphere substantially free of oxygen.

[0110] 12. A method for enhancing and / or stabilizing the proteolytic activity of a protease, comprising: (i) contacting the protease with a reducing agent such that the cysteine residues of the protease are maintained in a reduced state; and (ii) substantially removing all oxygen gas from the region surrounding the protease.

[0111] 13. The method according to embodiment 12, wherein the protease is placed in an atmosphere substantially free of oxygen.

[0112] 14. A method for producing a composition comprising a protease having enhanced and / or stabilized proteolytic activity, the method comprising: (i) contacting the protease with a reducing agent such that cysteine residues of the protease are maintained in a reduced state; and (ii) removing substantially all oxygen gas from the region surrounding the composition.

[0113] 15. The method according to any one of embodiments 12 to 14, wherein the oxygen gas is removed by degassing the composition.

[0114] 16. The method according to embodiment 14 or 15, wherein the composition is placed in an atmosphere substantially free of oxygen.

[0115] 17. A composition comprising one or more proteases having enhanced and / or stabilized proteolytic activity, the composition obtainable or obtainable by a method comprising: (i) contacting the protease with a reducing agent such that cysteine residues of the protease are maintained in a reduced state; and (ii) removing substantially all oxygen gas from the region surrounding the composition.

[0116] 18. A composition comprising one or more proteases and a reducing agent, wherein cysteine residues of the protease are maintained in a reduced state and the composition is substantially free of oxygen.

[0117] 19. The composition according to embodiment 17 or 18, wherein the oxygen gas is removed by degassing the composition.

[0118] 20. The composition according to any one of embodiments 17 to 19, wherein the composition is placed in an atmosphere substantially free of oxygen.

[0119] 21. A method for enhancing and / or stabilizing the proteolytic activity of a protease, the method comprising combining the protease with an anionic polymer matrix such that the protease binds non-covalently to the anionic polymer matrix.

[0120] 22. A method for producing a composition comprising a protease having enhanced and / or stabilized proteolytic activity, the method comprising the step of combining a protease with an anionic polymer matrix such that the protease binds non-covalently to the anionic polymer matrix.

[0121] 23. The method according to embodiment 21 or 22, wherein the polymer is carbomer.

[0122] 24. The method according to any one of embodiments 21 to 23, wherein the composition is placed in a substantially oxygen-free atmosphere.

[0123] 25. A composition comprising one or more proteases having enhanced and / or stabilized proteolytic activity, the composition being obtainable or obtainable by a method comprising the step of combining a protease with an anionic polymer matrix such that the protease binds non-covalently to the anionic polymer matrix.

[0124] 26. A composition comprising one or more proteases and an anionic polymer matrix, wherein the protease binds non-covalently to the anionic polymer matrix.

[0125] 27. The composition according to embodiment 25 or 26, wherein the polymer is carbomer.

[0126] 28. The composition according to any one of embodiments 25 to 27, wherein the composition is placed in a substantially oxygen-free atmosphere.

[0127] 29. Use of the composition according to any one of embodiments 7 to 11, 17 to 20, or 25 to 28 in the manufacture of a medicament for the treatment of diseases and disorders involving wounds, for debriding, or for treating burns, ulcers, or gangrene.

[0128] Use of the composition according to any one of Embodiments 7 to 11, 17 to 20, or 25 to 28 in the manufacture of a cosmetic for skin lightning, exfoliation, or for application to wrinkles, skin spots, freckles, acne, pimples, age spots, sun spots, scars, or varicose veins, or for application to dry skin, aging skin, or damaged skin.

[0129] 31. A pharmaceutical composition comprising the composition according to any one of Embodiments 7 to 11, 17 to 20, or 25 to 28 together with a pharmaceutically acceptable carrier, diluent, excipient, surfactant, and / or adjuvant.

[0130] 32. A cosmetic composition comprising the composition according to any one of Embodiments 7 to 11, 17 to 20, or 25 to 28 together with a cosmetically acceptable carrier, diluent, excipient, surfactant, and / or adjuvant.

[0131] 33. The composition according to any one of Embodiments 7 to 11, 17 to 20, or 25 to 28, or the pharmaceutical composition according to Embodiment 31, for use in the treatment of diseases and disorders including wounds, for debriding, or for treating burns, ulcers, or gangrene.

[0132] 34. The composition according to Embodiment 33, wherein the composition is applied topically.

[0133] 35. A method for the treatment of diseases and disorders including wounds, for debriding, or for treating burns, ulcers, or gangrene, the method comprising the step of administering the composition according to any one of Embodiments 7 to 11, 17 to 20, or 25 to 28 or the pharmaceutical composition according to Embodiment 31 to a subject.

[0134] 36. The method according to Embodiment 35, wherein the composition is applied topically.

[0135] For skin lightning, for use in exfoliation, or for application to wrinkles, skin spots, freckles, pimples, acne, rosacea, age spots, scars, or varicose veins, or for application to dry skin, aging skin, or damaged skin, the composition according to any one of Embodiments 7 to 11, 17 to 20, or 25 to 28, or the cosmetic composition according to Embodiment 32.

[0136] 38. A kit comprising the composition according to any one of Embodiments 7 to 11, 17 to 20, or 25 to 28, the pharmaceutical composition according to Embodiment 31, or the cosmetic composition according to Embodiment 32.

[0137] 39. For the treatment of diseases and disorders including wounds, for debriding, or for treating burns, ulcers, or gangrene, or for use in skin lightning, exfoliation, or for application to wrinkles, skin spots, freckles, pimples, acne, rosacea, age spots, scars, or varicose veins, or for application to dry skin, aging skin, or damaged skin, or for use when using, the kit according to Embodiment 38.

[0138] Next, the present invention will be further described with reference to the following examples, which are merely illustrative and non-limiting.

[0139] [Examples] Cysteine proteases are a family of proteases characterized by the presence of the amino acid cysteine at the active site. This sulfur-containing amino acid, together with the attached histidine residue, attacks the amide carbonyl of the peptide bond to form a thioester, which is later attacked by water to regenerate cysteine and release a carboxylic acid, thereby playing a role in proteolytic activity.

[0140] Cysteine proteases are abundantly contained in the latex of many plant species such as Carica papaya (papain, chymopapain, caricain, glycyl endopeptidase), genus Ficus (ficin), and some members of the Bromeliaceae family such as Ananas comosus (bromelain).

[0141] However, cysteine proteases have a major weakness that limits their usefulness. Since cysteine has a relatively strong reduction potential of the thiol group (E 0 =-0.34 V), cysteine is particularly sensitive to oxidation. Therefore, in the presence of oxygen or an oxidizing agent, the thiol undergoes a series of oxidation steps and finally generates a sulfonic acid group (-SO3-). Since none of these oxidized species retain catalytic activity towards peptide bonds, oxidation may inactivate cysteine proteases.

[0142] Another reason for the low proteolytic stability of proteases is autolysis. Since proteases themselves are proteins, they may be degraded by active proteases and lose their proteolytic activity. This applies to all proteases including cysteine proteases and does not require external substances such as oxygen. Without wishing to be bound by theory, the present invention can therefore prevent or suppress autolysis.

[0143] In this study, combinations of methods shown to enhance and stabilize the proteolytic activity of proteases were identified and verified. Therefore, these methods provide solutions to the problems of protease inactivation due to oxidative damage and autolysis.

[0144] In one aspect, the method of the present invention includes the addition of cysteine as a reducing agent to maintain the active site cysteine residue of the protease in a reduced state, and the substantial removal of oxygen gas from the region surrounding the protease by flushing the solution with a stable Inactive gas such as nitrogen or argon.

[0145] In an additional or alternative embodiment, the addition of an anionic polymer (such as a cross-linked polyacrylate) has also been demonstrated as a means to inhibit self-degradation by non-covalently separating proteases. The selection of an anionic polymer based on the high pI of papain and related proteases means that papain and related proteases are positively charged at neutral pH. Thus, according to the present invention, there is a possibility that proteases may bind to the polymer by reversible electrostatic interactions.

[0146] Materials and methods Papain and bromelain (industrial grade) derived from vegetable milk resin were obtained from Sigma Aldrich. Papain-containing compositions were obtained from the fruit of the papaya (excluding the milk resin of the peel). Such papain-containing compositions, such as Opal, can be prepared by the method disclosed in International Patent Application No. PCT / AU2003 / 000931, published as International Publication No. WO 2004 / 008887, the entire content of which is incorporated herein by reference.

[0147] Nitrogen gas was supplied by BOC Gases (UK), and Carbopol Ultrez was supplied by Lubrizol Inc (OH, USA). All other chemicals were purchased from Sigma Aldrich. Spectrophotometric readings were taken on a Jasco V-630 UV-Vis spectrophotometer.

[0148] Cysteine addition: Cysteine was added to each sample to a final concentration of 100 mM.

[0149] Nitrogen flushing: Samples were placed in plastic vials and nitrogen was bubbled through the solution for 5 minutes at room temperature. Each vial was immediately closed to prevent oxygen ingress.

[0150] Carbopol addition: Carbopol Ultrez was added to each solution to a final concentration of 0.25% to obtain a liquid suspension. For samples without oxygen, nitrogen flushing was performed at this stage. Subsequently, 10 M NaOH was added (1:1000, final 10 mM), and the solution immediately gelled to obtain a viscous gel.

[0151] BApNA spectrophotometric assay: A BApNA solution (10 mM in DMSO) was prepared together with a reaction buffer (100 mM potassium phosphate, 116 mM potassium chloride, and 3 mM EDTA, pH 6).

[0152] The premix was made with 2 parts water, 2 parts reaction buffer, and 1 part v / v BApNA. The spectrophotometer was blanked at 410 nm using this solution. Subsequently, 1 / 5 v / v water was added to both the control sample and the positive sample, and readings were started immediately thereafter. The relative enzyme activity was measured as the slope of the resulting straight line.

[0153] Key Opal (or O)-papaya extract; P-papain; B-bromelain, respectively Untreated (O, P, or B), Treated with Z-Carbopol (0.25%), Treated with the addition of XY-cysteine (X) and degassing with nitrogen (Y), or Treated with the addition of XYZ-cysteine (X) and degassing with nitrogen (Y) and Carbopol (0.25%) evaluated.

[0154] Opal Stabilization: Freshly prepared opal (16 mL) was divided into 2 × 8 mL aliquots. Each aliquot was then further divided into two, resulting in 4 × 4 mL samples (opal, opal + XY, opal + Z, opal + XY + Z). For the X sample, cysteine was added (12 mg / mL, 100 mM). For the Z sample, carbopol was added (0.25%). The XY sample was degassed under nitrogen for 5 minutes. Next, 10 M NaOH was added to the opal + XY + Z sample at a ratio of 1:1000 and the final pH was adjusted to 7.5 under an N2 flush. Then, 10 M NaOH was added to the opal + Z sample at a ratio of 1:1000 and the final pH was adjusted to 7.5.

[0155] Papain Stabilization: Papain at 1 mg / mL (16 mL) was divided into 2 × 8 mL aliquots. Each aliquot was then further divided into two, resulting in 4 × 4 mL samples (P, P + XY, P + Z, P + XY + Z). For the X sample, cysteine was added (12 mg / mL, 100 mM). For the Z sample, carbopol was added (0.25%). The XY sample was degassed under nitrogen for 5 minutes. Next, 10 M NaOH was added to the P + XY + Z sample at a ratio of 1:1000 and the final pH was adjusted to 7.5 under an N2 flush. Then, 10 M NaOH was added to the P + Z sample at a ratio of 1:1000 and the final pH was adjusted to 7.5.

[0156] Bromelain Stabilization: Bromelain at 1 mg / mL (16 mL) was divided into 2 × 8 mL aliquots. Each aliquot was further divided into two, resulting in 4 × 4 mL samples (B, B + XY, B + Z, B + XY + Z).

[0157] For the X sample, cysteine was added (12 mg / mL, 100 mM). For the Z sample, carbopol was added (0.25%). The XY sample was degassed under nitrogen for 5 minutes. Next, 10 M NaOH was added to the B + XY + Z sample at a ratio of 1:1000 and the final pH was adjusted to 7.5 under an N2 flush. 10 M NaOH was added to the B + Z sample at a ratio of 1:1000 and the final pH was adjusted to 7.5.

[0158] Results and discussion 1. Stabilization of the proteolytic activity of proteases In summary, when treated with XY and XYZ, it was found that for opal and papain samples, the proteolytic activity of the protease was stabilized compared to the untreated opal and papain samples. Even when treated with Z, the proteolytic activity of the protease was stabilized for the papain sample. This can be seen from Figures 1 and 2. When treated with XY and XYZ, it was shown that for the bromelain sample, the proteolytic activity of the protease was not much stabilized compared to the untreated bromelain sample. This can be seen from Figure 3. These results will be discussed in more detail below.

[0159] Papaya extract (opal) The stability of the papaya extract stabilizing solution was evaluated weekly for 2 months by the BApNA assay. Next, the measured activity was normalized to the activity of the opal (untreated) sample at the start of the experiment and set to 1. The results are shown in Figure 1.

[0160] Due to the stabilizing effect of the cysteine / nitrogen flushing combination, no loss of activity was detected over the study period (up to at least Day 58). This can probably be explained by the fact that in the absence of oxygen, the reducing agent cysteine is kept in a reduced state, which in turn keeps the active cysteine of the cysteine protease active site in a reduced state and prevents oxidation. Similarly, the stabilizing effect of carbopol, although of shorter duration than treatment with the cysteine / nitrogen flushing combination, was still significant (up to at least Day 12).

[0161] Furthermore, the enhancement of enzyme activity over time in the XY+Z sample was also considered (described later in Part 2 of the results).

[0162] 1.1 Papain (P) The results of the commercially available milk resin papain solution are shown in Figure 2. The observation results are similar to those of the opal sample in that both the combination of XY and the treatment with Z (carbopol) stabilized the enzyme solution on Day 64. The XYZ treatment of papain also showed stabilization of the enzyme solution on Day 64.

[0163] 1.2 Bromelain (B) The observation results of bromelain are also the same as those of opal and papain regarding the increase in activity immediately after treatment in the XY and XYZ treatments. The immediacy of cysteine / nitrogen + carbopol is extremely remarkable, reaching more than a 10-fold amplification compared to untreated bromelain (Figure 3). Furthermore, stabilization was achieved on the 14th day with the Z treatment.

[0164] The stabilization of the proteolytic activity of bromelain decreased during the test period. Nevertheless, when treated with carbopol (0.25%) alone (Z), with both degassing using cysteine and nitrogen (XY), and with all of cysteine, degassing using nitrogen, and carbopol (0.25%) (XYZ), the proteolytic activity of bromelain was finally maintained in stabilization on both the 7th and 14th days compared to the untreated samples.

[0165] 2. Enhancement of the proteolytic activity of proteases Surprisingly and unexpectedly, it was found that the method of enhancing the stability of proteolytic activity also significantly enhanced the proteolytic activity itself. In summary, when treated with Z, XY, and XYZ, it was found that for each sample of opal, papain, and bromelain, the proteolytic activity of the protease was enhanced compared to the untreated samples. This can be seen from Tables 1, 2, and 3 below, where "Average" refers to the average relative enzyme activity, "SD" refers to the standard deviation, "Z" refers to the treatment with carbopol (0.25%), "XY" refers to the treatment with cysteine (X) and degassing using nitrogen (Y), and "XYZ" refers to the treatment with cysteine (X), degassing using nitrogen (Y), and carbopol (0.25%) (Z). The same batch of the same protease was used for the sample treatments shown in each table.

[0166] These results suggest the immediate activation of the proenzyme (i.e., the conversion of the proenzyme to the enzyme). The cysteine effect may be due to the reducing / activating effect of cysteine on the putative proenzyme and the reversibly oxidized species. The unexpected carbopol effect could perhaps be interpreted as interference with the autolysis of the enzyme aggregates by the polymer.

[0167] 2.1 Papaya extract (opal) Table 1 shows that treatment with carbopol (0.25%) alone (Z) resulted in an immediate (day 0) three-fold increase in proteolytic activity compared to the untreated sample. The increase in activity reached a peak on day 12, after which the activity decreased to the base level for the remainder of the test period.

[0168] Treatment with both cysteine and nitrogen degassing (XY) resulted in an immediate (day 0) 3.3-fold increase in proteolytic activity compared to the untreated sample. This increase in activity was maintained at approximately this level throughout the test period until day 58. The maximum increase in activity was observed on day 12 (a 3.59-fold increase), and the minimum increase in activity was observed on day 30 (a three-fold increase).

[0169] Treatment with all of cysteine, nitrogen degassing, and carbopol (0.25%) (XYZ) resulted in an immediate (day 0) 12-fold increase in proteolytic activity compared to the untreated sample. Importantly, this enhancement of proteolytic activity further increased over time, with a 13.8-fold increase observed on day 5, a 14.3-fold increase observed on day 12, a 16.6-fold increase observed on day 30, a 15.3-fold increase observed on day 45, and a 17.9-fold increase observed on day 58. Thus, the active enzyme (i.e., protease) associated with the proenzyme and / or other inactive forms of the enzyme appears to increase over time.

[0170]

Table 1

[0171] The enhanced proteolytic activity of opal observed in various treatments appears to persist for a considerable period. Treatment with carbopol (0.25%) alone (Z) showed an enhanced proteolytic activity for at least 12 days, while treatment with both cysteine and nitrogen degassing (XY), as well as all of cysteine, nitrogen degassing, and carbopol (0.25%) (XYZ), showed an enhanced proteolytic activity for at least 58 days (the entire test period).

[0172] 2.2 Papain (P) Table 2 shows that treatment with carbopol (0.25%) alone (Z) resulted in an immediate (day 0) 4.6-fold increase in proteolytic activity compared to the untreated sample. This increase was maintained at approximately 4.1-fold until at least day 26, after which the activity decreased to an increase of about 2.6 - 2.7-fold for the remainder of the test period up to at least day 64.

[0173] Treatment with both cysteine and nitrogen degassing (XY) resulted in an immediate (day 0) 2.1-fold increase in proteolytic activity compared to the untreated sample. This increase in activity gradually decreased throughout the test period, with an increase in activity observed as 1.905-fold at day 26, and 1.667-fold at days 51 and 64.

[0174] Treatment with all of cysteine, nitrogen degassing, and carbopol (0.25%) (XYZ) resulted in an immediate (day 0) 5.2-fold increase in proteolytic activity compared to the untreated sample. Importantly, this enhancement of proteolytic activity showed a further net increase over time, with an 8.0-fold increase observed at day 26, a 5.7-fold increase observed at day 51, and a 6.1-fold increase observed at day 64.

[0175]

Table 2

[0176] The enhancement of papain proteolytic activity observed in various treatments also seems to persist for a fairly long period. Treatment with carbopol (0.25%) alone (Z), with both cysteine and nitrogen degassing (XY), and with all of cysteine, nitrogen degassing, and carbopol (0.25%) (XYZ) showed an enhancement of proteolytic activity for at least 64 days (the entire test period), respectively.

[0177] 2.3 Bromelain (B) Table 3 shows that treatment with carbopol (0.25%) alone (Z) resulted in an immediate (day 0) 1.6-fold increase in proteolytic activity compared to the untreated sample. This increase was maintained at approximately 1.5-fold increase until at least day 14, and the activity increased up to 3.0-fold increase on day 7.

[0178] Treatment with both cysteine and nitrogen degassing (XY) resulted in an immediate (day 0) 6.3-fold increase in proteolytic activity compared to the untreated sample. This increase in activity then decreased throughout the test period, with an increase in activity observed as 0.6-fold increase on day 7 and 0.65-fold increase on day 14.

[0179] Treatment with all of cysteine, nitrogen degassing, and carbopol (0.25%) (XYZ) resulted in an immediate (day 0) 11-fold increase in proteolytic activity compared to the untreated sample. This enhancement of proteolytic activity then decreased over time, with an increase of 4.6-fold observed on day 7 and 1.3-fold increase observed on day 14.

[0180]

Table 3

[0181] The immediate enhancement of the proteolytic activity of bromelain observed in various treatments is consistent with the same trend observed with both opal and papain. Bromelain had a more rapid decay of increased activity than opal and papain, but still, when treated with carbopol (0.25%) alone (Z), with both degassing using cysteine and nitrogen (XY), and with all of cysteine, degassing using nitrogen, and carbopol (0.25%) (XYZ), a net increase in the proteolytic activity of bromelain was maintained throughout the test period compared to the untreated sample.

[0182] Conclusion The data presented herein teach that the enhancement and stabilization of the proteolytic activity of proteases can be achieved using each of the methods disclosed herein, namely, treatment of the protease with carbopol (0.25%) alone (Z), treatment of the protease with both degassing using cysteine and nitrogen (XY), and treatment of the protease with all of cysteine, degassing using nitrogen, and carbopol (0.25%) (XYZ).

[0183] Surprisingly and unexpectedly, it has also been found that the method of enhancing the stability of the proteolytic activity also significantly enhances the proteolytic activity itself. In summary, it has been found that treatment with Z, XY, and XYZ all enhance the proteolytic activity of the protease. In particular, the enhancement of the stabilization of the proteolytic activity by XYZ treatment is very significant.

[0184] Therefore, the data presented herein teach that both the stabilization of the proteolytic activity of proteases and the enhancement of the proteolytic activity itself can be achieved using the methods disclosed herein.

[0185] The treatment methods disclosed herein are excellent in safety and contain reagents that do not involve chemical covalent modification of the enzyme, thus preventing problems related to consumer safety such as allergies and regulatory issues.

[0186] Although the present invention has been disclosed with reference to specific embodiments, it is apparent that other embodiments and modifications of the present invention can be devised by those skilled in the art without departing from the true spirit and scope of the present invention. The features and embodiments of different sections can be combined with necessary modifications. The present invention may be in the following aspects. [Item 1] A method for enhancing and / or stabilizing the proteolytic activity of a protease, comprising: (i) contacting the protease with a reducing agent such that the cysteine residues of the protease are maintained in a reduced state; (ii) substantially removing all oxygen gas from the region surrounding the protease; and (iii) combining the protease with an anionic polymer matrix such that the protease binds non-covalently to the anionic polymer matrix. [Item 2] The method according to item 1, wherein the protease is placed in an atmosphere substantially free of oxygen. [Item 3] A method for producing a composition comprising a protease having enhanced and / or stabilized proteolytic activity, comprising: (i) contacting the protease with a reducing agent such that the cysteine residues of the protease are maintained in a reduced state; (ii) substantially removing all oxygen gas from the region surrounding the protease; and (iii) combining the protease with an anionic polymer matrix such that the protease binds non-covalently to the anionic polymer matrix. [Item 4] The method according to item 3, wherein the composition is placed in an atmosphere substantially free of oxygen. [Item 5] The method according to any one of items 1 to 4, wherein the oxygen gas is removed by degassing the preparation. [Item 6] The method according to any one of items 1 to 5, wherein the polymer is carbomer. [Item 7] A composition comprising one or more proteases having enhanced and / or stabilized proteolytic activity, obtainable or obtained by a method comprising: (i) contacting the protease with a reducing agent such that the cysteine residues of the protease are maintained in a reduced state; (ii) substantially removing all oxygen gas from the region surrounding the protease; and (iii) combining the protease with an anionic polymer matrix such that the protease is non-covalently bound to the anionic polymer matrix. [Item 8] A composition comprising one or more proteases, a reducing agent, and an anionic polymer matrix, wherein the cysteine residues of the protease are maintained in a reduced state, the composition is substantially free of oxygen, and the protease is non-covalently bound to the anionic polymer matrix. [Item 9] The composition according to item 7 or 8, wherein the oxygen gas is removed by degassing the composition. [Item 10] The composition according to any one of items 7 to 9, wherein the polymer is carbomer. [Item 11] The composition according to any one of items 7 to 10, wherein the composition is placed in an atmosphere substantially free of oxygen. [Item 12] Use of the composition according to any one of items 7 to 11 in the manufacture of a medicament for the treatment of diseases and disorders including wounds, for debriding, or for treating burns, ulcers, or gangrene. [Item 13] Use of the composition according to any one of items 7 to 11 in the manufacture of a cosmetic for skin lightening, for keratolysis, or for application to wrinkles, skin spots, freckles, eruptions, acne, rosacea, sun spots, scars, or varicose veins, or for application to dry skin, aged skin, or damaged skin. [Item 14] A pharmaceutical composition comprising the composition according to any one of items 7 to 11 together with a pharmaceutically acceptable carrier, diluent, excipient, surfactant, and / or adjuvant. [Item 15] A cosmetic composition comprising the composition according to any one of items 7 to 11 together with a cosmetically acceptable carrier, diluent, excipient, surfactant, and / or adjuvant. [Item 16] The composition according to any one of items 7 to 11 or the pharmaceutical composition according to item 14 for use in the treatment of diseases and disorders including wounds, for debriding, or for treating burns, ulcers, or gangrene. [Item 17] The composition according to item 16, wherein the composition is applied locally. [Item 18] A method for treating diseases and disorders including wounds, for debriding, or for treating burns, ulcers, or gangrene, the method comprising the step of administering to a subject the composition according to any one of items 7 to 11 or the pharmaceutical composition according to item 14. [Item 19] The method according to item 18, wherein the composition is applied locally. [Item 20] The composition according to any one of items 7 to 11 or the cosmetic composition according to item 15 for use in skin lightning, for exfoliation, or for application to wrinkles, skin spots, freckles, pimples, acne, rosacea, solar lentigines, scars, or varicose veins, or for application to dry skin, aging skin, or damaged skin.

Claims

A method for enhancing and / or stabilizing the proteolytic activity of one or more cysteine proteases, comprising: (i) contacting the one or more cysteine proteases with cysteine, such that the cysteine residues of the one or more cysteine proteases are maintained in a reduced state; (ii) substantially removing all oxygen gas from the region surrounding the one or more cysteine proteases; and (iii) combining the one or more cysteine proteases with a carbomer such that the one or more cysteine proteases are non-covalently bound to the carbomer. The method according to claim 1, wherein the one or more cysteine proteases are placed in a substantially oxygen-free atmosphere.

3. A method for producing a composition comprising one or more cysteine proteases having enhanced and / or stabilized proteolytic activity, comprising: (i) contacting the one or more cysteine proteases with cysteine, such that the cysteine residues of the one or more cysteine proteases are maintained in a reduced state; (ii) substantially removing all oxygen gas from the region surrounding the one or more cysteine proteases; and (iii) combining the one or more cysteine proteases with a carbomer such that the one or more cysteine proteases are non-covalently bound to the carbomer.

4. The method according to claim 3, wherein the composition is placed in a substantially oxygen-free atmosphere.

5. The method according to any one of claims 1 to 4, wherein the oxygen gas is removed by degassing the preparation.

6. A composition comprising one or more cysteine proteases, cysteine, and a carbomer, wherein the cysteine residues of the one or more cysteine proteases are maintained in a reduced state, the composition is substantially oxygen-free, the proteolytic activity of the one or more cysteine proteases is enhanced or stabilized, and the one or more cysteine proteases are non-covalently bound to the carbomer. The composition according to claim 6, wherein the composition is substantially oxygen-free by degassing.

7. The composition according to claim 6 or 7, wherein the composition is placed in a substantially oxygen-free atmosphere.

9. Use of a composition according to any one of claims 6 to 8 in the manufacture of a medicament for the treatment of diseases and disorders including wounds, for debriding, or for treating burns, ulcers or gangrene.

10. Use of a composition according to any one of claims 6 to 8 in the manufacture of a cosmetic for skin lightening, for keratolysis, or for application to wrinkles, skin spots, freckles, blemishes, acne, rosacea, solar lentigines, scars or varicose veins, or for application to dry skin, aged skin or damaged skin.

11. A pharmaceutical composition comprising a composition according to any one of claims 6 to 8 together with a pharmaceutically acceptable carrier, diluent, excipient, surfactant and / or adjuvant.

12. A cosmetic composition comprising a composition according to any one of claims 6 to 8 together with a cosmetically acceptable carrier, diluent, excipient, surfactant and / or adjuvant.

13. A composition according to any one of claims 6 to 8 or a pharmaceutical composition according to claim 11 for use in the treatment of diseases and disorders including wounds, for debriding, or for treating burns, ulcers or gangrene.

14. The composition according to claim 13, wherein the composition is applied topically.

15. A composition according to any one of claims 6 to 8 or a cosmetic composition according to claim 12 for use in skin lightening, for keratolysis, or for application to wrinkles, skin spots, freckles, blemishes, acne, rosacea, solar lentigines, scars or varicose veins, or for application to dry skin, aged skin or damaged skin.

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