Enhancement and stabilisation of proteolytic activity of proteases

Abstract

To provide new methods, compositions, and uses of compositions for the enhancement and stabilization of the proteolytic activity of proteases.SOLUTION: A method for the enhancement and / or stabilization of the proteolytic activity of one or more cysteine proteases, the method comprising (i) a step of contacting the one or more cysteine proteases with a reducing agent such that the active site cysteine residue of the one or more cysteine proteases is maintained in a reduced state, (ii) a step of removing substantially all oxygen gas from the region surrounding the one or more cysteine proteases, and (iii) a step of 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.SELECTED DRAWING: Figure 1

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 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.

[0002] [Background technology] Papain and bromelain are proteases used in pharmaceutical products for debridement and in cosmetics / cosmeceuticals for exfoliation and skin lightening. The proteolytic activity of papain and bromelain provides such debridement, exfoliation, and skin lightening. However, both papain and bromelain preparations have not achieved their full potential due to the unstable proteolytic activity.

[0003] Considerable effort has been expended in the past to develop stable compositions that promote wound healing through the removal of dead and damaged tissue, such as that found in wounds such as burns and chronic ulcers. Effective debridement is essential because dead and dying tissue provides an excellent medium for opportunistic infections. Sepsis resulting from infection is the leading cause of death in severely burned patients.

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

[0005] Thus, Nexobrid™ is typically supplied as a lyophilized powder that must be reconstituted with a gel vehicle prior to use and used within 15 minutes of 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, this would require significantly enhanced stability of the debriding activity to achieve an acceptable shelf life.

[0006] Similarly, cosmetic products containing papain and / or bromelain that are marketed as having exfoliating and / or skin lightening properties often experience a significant decrease in exfoliating activity over a short period of time due to loss of papain and / or bromelain activity in the cosmetic product.

[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 there is low or no loss of activity (in the case of papain, acidic pH has been used) or in solid form. However, these treatments require processing by skilled end users to obtain a viable product. This limits the usefulness of such methods and compositions.

[0008] Another approach that has been tried previously is to immobilize enzymes in polymer matrices 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, they may chemically alter the enzyme in an irreversible manner and therefore may not exhibit sufficient activity when applied to certain substrates, especially complex substrates such as skin and cellular proteins. Furthermore, such changes in the enzyme's chemical structure may cause adverse reactions for end users, such as allergies and intolerances.

[0009] To address these issues, studies supporting the present invention examined the loss of proteolytic activity of proteases extracted from various sources, including the Carica papaya (papaya) plant (which contains papain) and the Ananas comosus (pineapple) plant (which contains bromelain). Based on these studies, methods were developed for enhancing and stabilizing the proteolytic activity of proteases, particularly in pharmaceutical and cosmetic / cosmeceutical compositions containing proteases from the papaya and pineapple plants.

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

[0011] [Summary of the Invention] The present invention teaches new methods for enhancing and stabilizing the proteolytic activity of proteases. In certain embodiments, the proteases are obtained or obtainable from fruits and / or vegetables. Thus, in some embodiments, the proteases are obtained from fruits and / or vegetables, while in other embodiments, the proteases are obtained from recombinant expression systems. In certain embodiments, the proteases are cysteine ​​proteases. In certain embodiments, the cysteine ​​proteases 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 actinidyin (EC 3.4.22.14). In certain embodiments, the protease is obtained or obtainable from a Carica papaya (papaya) plant or an Ananas comosus (pineapple) plant, hi 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, the method comprising the steps of (i) contacting the protease with a reducing agent, wherein cysteine ​​residues of the protease are maintained in a reduced state, and (ii) removing substantially all oxygen gas from the area surrounding the protease. In one embodiment, 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 invention provides a method for enhancing and / or stabilizing the proteolytic activity of a protease, the method comprising the steps of: (i) providing a solution or gel comprising the protease; (ii) contacting the protease in the solution or gel with a reducing agent, wherein the cysteine ​​residues of the protease are maintained in a reduced state; and (iii) removing substantially 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 with enhanced and / or stabilized proteolytic activity, the method comprising the steps of: (i) contacting the protease with a reducing agent, wherein cysteine ​​residues of the protease are maintained in a reduced state; and (ii) removing substantially all oxygen gas from an area surrounding the composition. In one embodiment, oxygen gas is removed by degassing the composition. In one embodiment, the composition is placed in a substantially oxygen-free atmosphere.

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

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

[0017] In a fourth aspect, the present invention provides a composition comprising one or more proteases and a reducing agent, wherein cysteine ​​residues of the proteases are maintained in a reduced state, and wherein 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 a substantially oxygen-free atmosphere.

[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 in 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 covalent bonds with the matrix material, for example, by reacting some of the primary amines of the protease with the carboxyl groups of a carbomer and cross-linking some 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 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 a substantially oxygen-free atmosphere.

[0020] In a sixth aspect, the present invention provides a method for producing a composition comprising a protease with enhanced and / or stabilized proteolytic activity, the method comprising 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 a substantially oxygen-free atmosphere.

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

[0023] The methods taught in this 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] Thus, in a ninth aspect, the present invention provides a method for enhancing and / or stabilizing the proteolytic activity of a protease, the method comprising the steps of: (i) contacting the protease with a reducing agent, wherein cysteine ​​residues of the protease are maintained in a reduced state; (ii) removing substantially all oxygen gas from the area 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. In one embodiment, oxygen gas is removed by degassing the preparation. In one embodiment, the polymer is a 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) through (iii) are performed in this order.

[0026] In a related aspect, the invention provides a method for enhancing and / or stabilizing the proteolytic activity of a protease, the method comprising the steps of: (i) providing a solution comprising the protease; (ii) contacting the protease in the solution with a reducing agent, wherein 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 is non-covalently bound to the anionic polymer matrix.

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

[0028] In a tenth aspect, the present invention provides a method for producing a composition comprising a protease with enhanced and / or stabilized proteolytic activity, the method comprising the steps of: (i) contacting the protease with a reducing agent, whereby cysteine ​​residues of the protease are maintained in a reduced state; (ii) removing substantially all oxygen gas from the area 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. In one embodiment, oxygen gas is removed by degassing the preparation. In one embodiment, the polymer is a carbomer. In one embodiment, the composition is placed in a substantially oxygen-free atmosphere.

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

[0030] In an eleventh aspect, the present invention provides a composition comprising one or more proteases with enhanced and / or stabilized proteolytic activity, the composition being obtainable or obtainable by a method of the ninth aspect, such as a method comprising the steps of: (i) contacting the protease with a reducing agent, whereby cysteine ​​residues of the protease are maintained in a reduced state; (ii) removing substantially all oxygen gas from an area 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. In one embodiment, oxygen gas is removed by degassing the preparation. In one embodiment, the polymer is a 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 cysteine ​​residues of the proteases are maintained in a reduced state, the composition is substantially free of oxygen, and the proteases are non-covalently bound to the anionic polymer matrix. In one embodiment, oxygen gas is removed by degassing the composition. In one embodiment, the polymer is a 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 thirteenth aspect, the present invention provides use of a composition of the third, fourth, seventh, eighth, eleventh, and / or twelfth aspects in the manufacture of a medicament. In some embodiments, the medicament is for the treatment of diseases and disorders involving wounds. In some embodiments, the medicament is for debridement. In some embodiments, the medicament is for the treatment of burns. In some embodiments, the medicament is for the treatment of ulcers. In some embodiments, the medicament is for the treatment of gangrene.

[0034] In a fourteenth aspect, the invention provides use of the composition of the third, fourth, seventh, eighth, eleventh, and / or twelfth aspects in the manufacture of a cosmetic product. In some embodiments, the cosmetic product is for exfoliating, lightening the skin, or for application to wrinkles, skin blemishes, freckles, pimples, acne, rosacea, sun spots, scars, or varicose veins, or for application to dry, aging, or damaged skin.

[0035] In a fifteenth aspect, the present invention provides a pharmaceutical composition comprising a composition of the third, fourth, seventh, eighth, eleventh, and / or twelfth aspects together with a pharmaceutically acceptable carrier, diluent, excipient, surfactant, and / or adjuvant.

[0036] In a sixteenth aspect, the present invention provides a cosmetic composition comprising a composition of the third, fourth, seventh, eighth, eleventh, and / or twelfth aspects together with a cosmetically acceptable carrier, diluent, excipient, surfactant, and / or adjuvant.

[0037] In a seventeenth aspect, the present invention provides a composition of the third, fourth, seventh, eighth, eleventh, and / or twelfth aspect, or a pharmaceutical composition of the fifteenth aspect, for use in treating diseases and disorders involving 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 eighteenth aspect, the present invention provides a method for the treatment of diseases and disorders involving wounds, the method comprising administering to a subject a composition of the third, fourth, seventh, eighth, eleventh, and / or twelfth aspect, or a pharmaceutical composition of the fifteenth 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 nineteenth aspect, the present invention provides a composition of the third, fourth, seventh, eighth, eleventh and / or twelfth aspect, or a cosmetic composition of the sixteenth aspect, for use in skin lightening, exfoliation, or for application to wrinkles, skin blemishes, freckles, pimples, acne, rosacea, sun spots, scars, or varicose veins, or for application to dry, aged, or damaged skin.

[0040] In a twentieth aspect, the present invention provides a kit comprising the composition of the third, fourth, seventh, eighth, eleventh, and / or twelfth aspects, the pharmaceutical composition of the fifteenth aspect, or the cosmetic composition of the sixteenth aspect. In one embodiment, the kit is used for carrying out the method of the eighteenth aspect or for the uses of the seventeenth and nineteenth aspects.

[0041] In contrast to previously 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, gauze, 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, gauze, 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 liquid, and do not need to be reconstituted before use. [Brief explanation of the drawings]

[0042] [Figure 1] Figure 1 shows the stability of Opal treated with X+Y, Z, and X+Y+Z. The total proteolytic activity measured by the BApNA assay after X+Y, Z, and X+Y+Z treatments is shown as a percentage of the initial Opal activity (2E3 USP units / mL). The activity at t=0 reflects the immediate enhancement of proteolytic activity by X+Y, Z, and X+Y+Z treatments. All treatments were performed on the same batch of Opal. [Figure 2] Figure 2 shows the stability of papain treated with X+Y, Z, and X+Y+Z. The total proteolytic activity measured by the BApNA assay after X+Y, Z, and X+Y+Z treatments is shown. The ratio of the initial P activity (1E4 USP units / mL) is shown. The activity at t=0 reflects the immediate enhancement of proteolytic activity by X+Y, Z, and X+Y+Z treatments. All treatments were performed on the same batch of papain. [Figure 3]Figure 3 shows the stability of bromelain subjected to X+Y, Z, and X+Y+Z treatments. Total proteolytic activity measured by BApNA assay after X+Y, Z, and X+Y+Z treatments is shown. Relative to initial B activity (1.5E4 USP units / mL). Activity at t=0 reflects the immediate enhancement of proteolytic activity by X+Y, Z, and X+Y+Z treatments. All treatments were performed on the same batch of bromelain.

[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, preferred methods and materials are described. For 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 one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element.

[0045] "About" means a quantity, 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 quantity, 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 the inclusion of the stated 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, use of the term "comprising," etc., indicates that the listed elements are required or mandatory, but that other elements are optional and may or may not be present. "Consisting of" means including and limited to everything that follows the phrase "consisting of." Thus, the phrase "consisting of" indicates that the listed elements are required or mandatory, and that other elements must not be present. "Consisting essentially of" means including any elements listed after the phrase, but is limited to other elements that do not interfere with or contribute to the disclosure-defined activity or function of the listed elements. Thus, the phrase "consisting essentially of" indicates that the listed elements are required or mandatory, but other elements are optional and may or may not be present depending on whether they affect the activity or function of the listed elements.

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

[0048] The term "derivable," 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 in any manner, for example, by obtaining the protease from a fruit or vegetable, or by using recombinant DNA technology or other genetic engineering methods, such as recombinant expression systems.

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

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

[0051] The terms "patient," "subject," and "individual" are used interchangeably and refer to human or other mammalian patients, subjects, and individuals, including any for whom it is desired to use the present invention to treat, prevent, ameliorate, or reduce the severity of a disease, disorder, or condition. 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), livestock 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 "enhancing and / or stabilizing 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 for stabilizing the proteolytic activity of a protease, such as by increasing, preserving, extending, or delaying the decline in the ability of a protease to perform its normal enzymatic function over a given time unit or relative to a baseline 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 to perform that enzymatic function in the absence of the method. Alternatively, the methods may result in a protease performing its enzymatic function at a higher activity level than would be the activity level in the absence of the method, e.g., when measured at a particular time point or time points after the method is performed. The shorter time or higher activity levels required can also be measured in relation to a variety of other factors, including, but not limited to, exposure to heat and sterilizing radiation, and storage time and conditions of the protease composition, as well as any shipping 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 enzymatic activity of a protease that has been subjected to a method of the invention (e.g., "Sample A") with the stability of a protease that has not been subjected to a method of the invention (e.g., a "control sample," which may be, for example, a wild-type or naturally occurring protease). Such a comparison between samples can be performed, for example, at a particular time after performing a method of the invention, to confirm increased stability and / or activity of the protease in "Sample A" relative to the control sample.

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

[0056] The term "wound" means an injury to living tissue in which the skin is cut or damaged, and includes skin ulcers and burns. Skin ulcers may include diabetic ulcers, pressure ulcers, venous (or varicose) ulcers, and arterial ulcers.

[0057] Reference to prior art in this specification is not, and should not be construed as, an acknowledgment or in any way suggestion that prior art forms part of the common general knowledge of those skilled in the art.

[0058] The entire contents of all publications, patents, patent applications and other materials mentioned herein are hereby incorporated by reference.

[0059] [Detailed Description of the Invention] Cysteine ​​proteases, also known as thiol proteases and cysteine ​​endopeptidases (EC 3.4.22), are enzymes that degrade proteins. Cysteine ​​proteases share a common catalytic mechanism involving a nucleophilic cysteine ​​thiol in the catalytic triad or dyad. Cysteine ​​proteases are present in a variety of 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 can also be found in a variety of other fruits and vegetables. Cysteine ​​proteases can be obtained by a variety of methods, including extraction from biological material, such as fruit extracts from papaya pulp, or by recombinant expression in a suitable host cell. In one embodiment, the protease present in the composition or subjected to the method of the invention is a protease prepared from the ripe pulp of papaya, such as by the method described in WO 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 treated with a reducing agent that maintains the active site cysteine ​​amino acid residue of the protease in a reduced state. Cysteine ​​residues, when oxidized, can form disulfide bridges, which can inhibit enzymatic activity. The reducing agent can maintain the active site residue in a reduced form and can also convert previously oxidized cysteine ​​residues to the reduced form. Suitable reducing agents are known in the art and include cysteine. The amount of reducing agent added should generally be sufficient to regenerate all or most of the active site cysteine ​​amino acid residues of the protease in solution and maintain them in the reduced form. In this case, the reducing agent is typically added in excess. In one embodiment, the concentration of the reducing agent, such as cysteine, is typically 10 to 200 mM, e.g., 50 to 150 mM. In certain embodiments, the concentration of the reducing agent, such as cysteine, is 60-140 mM, 70-130 mM, 80-120 mM, 90-100 mM, 92-108 mM, 94-106 mM, 96-104 mM, or 98-102 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, for example, in the form of a liquid or gel containing the protease. In one embodiment, the protease is pre-bound to an anionic polymer, as described below, prior to the above treatment step with a reducing agent.

[0062] The composition may also be subjected to a step of removing substantially all oxygen from the area surrounding the protease, e.g., removing substantially all oxygen from a liquid or gel, such as a solution containing the protease. This can be accomplished, for example, by degassing the composition, e.g., by flushing the composition with an inert gas such as nitrogen or argon. For example, purging with nitrogen or argon at a flow rate of 25 mL / s for 20-40 minutes can result in a residual dissolved oxygen concentration of approximately 0.2-0.4 ppm. Other methods of removing substantially all oxygen from the composition may also be used, such as, but not limited to, heating at atmospheric or reduced pressure or sonication at atmospheric or reduced pressure, and would be known to those skilled in the art. The oxygen removal step may be performed before, simultaneously with, or immediately after the addition of the reducing agent.

[0063] The resulting composition comprises a cysteine ​​protease and a reducing agent that reduces one or more active site cysteine ​​residues of the cysteine ​​protease, wherein the composition is substantially free of oxygen, and 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] To reduce exposure to oxygen during storage, the composition can be packaged to reduce or prevent oxygen absorption. For example, the composition may be packaged in a container with a substantially oxygen-free atmosphere, for example, an inert gas such as nitrogen or argon. Alternatively, the composition may be vacuum packaged.

[0065] In another embodiment, which can be combined with the previous embodiment of the invention, the anionic polymer matrix can be combined with a cysteine ​​protease such that the cysteine ​​protease is non-covalently bound to the anionic polymer matrix. This is intended to prevent autolysis and enzyme inactivation by non-covalently separating the enzyme molecules. The selection of the anionic polymer is generally based on the high isoelectric point (pI) of papain and related proteases, i.e., the fact that papain and related proteases are positively charged at neutral pH.

[0066] The anionic polymer may 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. Anionic polymers typically have high molecular weights. 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 polymers of acrylic acid and C10-C30 alkyl acrylates crosslinked with, for example, 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] The anionic polymer can be combined with a protease to form a liquid suspension. The concentration of the anionic polymer can be 0.01-3% w / w or more, e.g., 0.1-2% w / w. When combined with another aspect of the present invention, a reducing agent can be added at this stage, if not already done, and oxygen can be removed by nitrogen flushing or the like. Subsequent addition of an alkali to adjust the pH, e.g., to 7.5-8, can induce gelation of the liquid suspension, resulting in 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, followed by the addition of the reducing agent / removal of oxygen, in either order. Alternatively, the addition of the reducing agent / removal of oxygen can be performed, in either order, before the combination with the anionic polymer. It would also be possible to perform the combination with the anionic polymer between the reducing agent step and the oxygen removal step. The various steps can be performed sequentially or simultaneously (or with some overlap).

[0070] The compositions of the present invention typically exhibit greater stability of proteolytic activity than corresponding untreated protease control samples, i.e., those not non-covalently bound to anionic polymers and stored under normal oxidizing conditions, e.g., atmospheric oxygen levels, without added reducing agents. For example, some protease compositions of the present invention, particularly papain (P) and papaya extracts (e.g., opal), ... The protease compositions may exhibit 0, 85, 90, or 95% proteolytic activity, while other protease compositions, particularly bromelain (B), may exhibit at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, or 95% proteolytic activity after storage at room temperature and 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 with 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 an untreated protease composition, such as having at least 1.5-fold greater activity than the untreated protease, e.g., 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-fold greater activity than 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 reaction buffer (100 mM potassium phosphate, 116 mM potassium chloride, and 3 mM EDTA, pH 6). A premix can be made with 2 parts water, 2 parts reaction buffer, and 1 part v / v BApNA. A spectrophotometer (e.g., a Jasco V-630 UV-Vis spectrophotometer) can be blanked at 410 nm with the premix solution. 1 / 5 v / v water can then be added to both the control and positive samples, and readings can be taken immediately thereafter. Relative enzyme activity can be measured as the slope of the resulting line.

[0073] composition In contrast to previously 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, gauze, 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, gauze, 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 gel or liquid form, and do not need to be reconstituted before use.

[0074] In certain aspects of the present invention, the composition may be in the form of a gel or liquid. The increased stability of the proteolytic activity of the composition allows it to be provided in liquid or gel form, which means it is ready to use and does not need to be reconstituted immediately before use. This can be very beneficial in administering the composition and can be useful in situations where it is difficult to reconstitute a lyophilized composition, for example, outside of a clinical setting where a wound, such as a burn, requires immediate treatment.

[0075] The compositions can also be absorbed / adsorbed onto solid materials, such as wound dressings. The compositions can be combined with pharmaceutical and / or cosmetic carriers, diluents, excipients, surfactants, and / or adjuvants to provide the pharmaceutical or cosmetic compositions of the present invention. Thus, the compositions of the present invention can be formulated to include one or more additional ingredients.

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

[0077] The compositions 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 of being compatible with the other components of the composition and not deleterious to the formation of a composition that can be stored for extended periods of time, if desired. Such carriers, excipients, and diluents can be used to further enhance the integrity and shelf life of the compositions of the present invention.

[0078] Further examples of acceptable carriers or diluents include demineralized or sterile water; saline solution; vegetable oils such as peanut oil, safflower oil, olive oil, cottonseed oil, corn oil, sesame oil, peanut oil, or coconut oil; methylpolysiloxane, phenylpolysiloxane, methylphenylpolysorboxane, Examples of suitable carriers include silicone oils such as polysiloxanes (e.g., polysolpoxane); volatile silicones; mineral oils such as liquid paraffin, soft paraffin, or squalane; cellulose derivatives such as methylcellulose, ethylcellulose, carboxymethylcellulose, sodium carboxymethylcellulose, or hydroxypropylmethylcellulose; 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; gum tragacanth or acacia, and petrolatum. Other carriers that can be used include polyvinylpyrrolidone (PVP), carboxymethylcellulose, polyvinyl alcohol, and polyethylene oxide.

[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 therefore useful for inclusion in the compositions of the present invention.

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

[0081] use The 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.

[0082] Thus, the present invention further provides a method of wound debridement 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 comprising administering a preparation of the present invention topically or by other route of administration to the affected area of ​​the individual, a composition of the present invention for or when used in a method of treating a wound, a method of promoting wound healing comprising administering a composition of the present invention to a wound topically or by other route of administration, a method of exfoliating or skin lightening comprising applying a cosmetic composition of the present invention to skin, use of the cosmetic composition of the present invention for exfoliating or lightening skin, a method of treating dry, aged or damaged skin comprising applying a cosmetic composition of the present invention to skin, and use of the cosmetic composition of the present invention for treating dry, aged or damaged skin.

[0083] The protease compositions of the present invention can be used to prevent, treat, reduce, or ameliorate a variety of skin conditions, including wounds, including chronic wounds such as vascular / pressure skin ulcers, burns, and other skin conditions, including, but not limited to, eczema, psoriasis, acne, rosacea, ichthyosis, vitiligo, urticaria, and seborrheic dermatitis, among others.

[0084] The compositions of the present invention can be administered therapeutically or cosmetically. In such applications, the compositions can be administered to a subject already suffering from a condition in an amount sufficient to cure or at least partially prevent the condition and 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, or may be formed by mono- or multi-lamellar 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 the form of liposomes may contain stabilizers, preservatives, and excipients. Preferred lipids include both natural and synthetic phospholipids and phosphatidylcholines (lecithins). Methods for producing liposomes are known in the art, and in this regard, specific reference is made to Prescott (ed.), Methods in Cell Biology, Vol. 14, Academic Press, New York, NY (1976), pp. 33 et seq., the contents of which are incorporated herein by reference.

[0086] Dosage The "therapeutically effective" dose level for any particular patient will depend on a variety of factors, such as the condition being treated and the severity of the condition, the activity of the composition used, the patient's age, weight, general health, sex, and diet, the time of administration, the route of administration, the duration of treatment, and any drugs used in combination or concomitantly with the treatment, as well as other relevant factors well known in the art. Thus, one of ordinary skill in the art will be able, by routine experimentation, to determine an effective, 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 amount and interval of individual doses of the composition will be determined by the nature and extent of the condition to be treated, the form, route and site of administration, and the nature of the particular individual to be treated, and such optimal conditions can be determined by conventional techniques.

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

[0089] Administration route The compositions of the present invention can be administered by standard routes. Generally, the compositions can be administered by topical routes. Typically, the compositions of the present invention are administered locally to the affected area of ​​an individual.

[0090] In other embodiments, the compositions can be administered by other enteral / enteral routes, such as rectally, sublingually, or sublabially, or via the central nervous system, such as through epidural, intracerebral, or intraventricular routes. Other locations of administration may include via epicutaneous, transdermal, intradermal, nasal, intra-arterial, intracardiac, intraosseous, intrathecal, intraperitoneal, intravesical, intravitreal, intracavernous, intravaginal, or intrauterine routes.

[0091] Timing of treatment Typically, in therapeutic applications, treatment is for the duration 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 thereafter, for example, as a follow-up or intensification treatment to complement currently available therapies 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 as a prophylactic treatment in subjects with a genetic or environmental predisposition to developing such diseases.

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

[0094] kit The kits of the present invention facilitate the implementation of the methods and uses of the present invention. Typically, kits for implementing the methods or uses of the present invention contain all the reagents and means necessary to implement 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 point-of-care methods.

[0095] Typically, the kit described herein also includes one or more containers.In the context of the present invention, a compartmentalized kit includes any kit in which compositions are contained in separate containers, and may include small glass containers, plastic containers, or plastic or paper strips.Such containers can allow the composition to be efficiently transferred from one compartment to another, while avoiding cross-contamination of the compositions, and the drug or solution in each container can be added quantitatively from one compartment to another.

[0096] Typically, the kits of the present invention also include instructions for using the kit to carry out 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, equine, bovine, ovine, caprine, avian, feline and canine species, etc. Thus, for application to different species, a single kit of the present invention may be applicable, or different kits may be required, for example, containing compositions specific to each individual species.

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

[0099] Enumerated Embodiments 1. A method for enhancing and / or stabilizing the proteolytic activity of a protease, the method comprising the steps of: (i) contacting the protease with a reducing agent, wherein cysteine ​​residues of the protease are maintained in a reduced state; (ii) removing substantially all oxygen gas from the area 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.

[0100] 2. The method of embodiment 2, wherein the protease is placed in a substantially oxygen-free atmosphere.

[0101] 3. A method for producing a composition comprising a protease with enhanced and / or stabilized proteolytic activity, the method comprising the steps of: (i) contacting the protease with a reducing agent, wherein cysteine ​​residues of the protease are maintained in a reduced state; (ii) removing substantially all oxygen gas from the area 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.

[0102] 4. The method of embodiment 3, wherein the composition is placed in a substantially oxygen-free atmosphere.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

[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 is non-covalently bound 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 combining the protease with an anionic polymer matrix such that the protease is non-covalently bound to the anionic polymer matrix.

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

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

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

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

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

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

[0127] 29. Use of a 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 debridement, or for treating burns, ulcers, or gangrene.

[0128] 30. Use of a composition according to any one of embodiments 7 to 11, 17 to 20, or 25 to 28 in the manufacture of a cosmetic product for skin lightening, exfoliation, or application to wrinkles, skin blemishes, freckles, pimples, acne, rosacea, sun spots, scars, or varicose veins, or for application to dry, aged, or damaged skin.

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

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

[0131] 33. The composition of any one of embodiments 7 to 11, 17 to 20, or 25 to 28, or the pharmaceutical composition of embodiment 31, for use in the treatment of diseases and disorders involving wounds, for debridement, or for treating burns, ulcers, or gangrene.

[0132] 34. The composition of embodiment 33, wherein the composition is applied topically.

[0133] 35. A method for the treatment of diseases and disorders involving wounds, for debridement, or for treating burns, ulcers, or gangrene, comprising administering to a subject a composition according to any one of embodiments 7-11, 17-20, or 25-28, or a pharmaceutical composition according to embodiment 31.

[0134] 36. The method of embodiment 35, wherein the composition is applied topically.

[0135] 37. The composition of any one of embodiments 7-11, 17-20, or 25-28, or the cosmetic composition of embodiment 32, for use in skin lightening, exfoliation, or for application to wrinkles, skin blemishes, freckles, pimples, acne, rosacea, sun spots, scars, or varicose veins, or for application to dry, aging, or damaged skin.

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

[0137] 39. The kit of embodiment 38 for use in, or when used for, the treatment of diseases and disorders including wounds, for debridement, or for treating burns, ulcers, or gangrene, or for use in skin lightening, exfoliation, or for application to wrinkles, skin blemishes, freckles, pimples, acne, rosacea, sun spots, scars, or varicose veins, or for application to dry, aging, or damaged skin.

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

[0139] [Example] Cysteine ​​proteases are a family of proteases characterized by the presence of the amino acid cysteine ​​in the active site, which, together with an attached histidine residue, is responsible for the proteolytic activity by attacking the amide carbonyl of the peptide bond to form a thioester, which is subsequently attacked by water to regenerate the cysteine ​​and release a carboxylic acid.

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

[0141] However, cysteine ​​proteases have a significant weakness that limits their usefulness. Cysteine ​​has a relatively strong reduction potential of the thiol group (E 0 =-0.34V), cysteine ​​is particularly sensitive to oxidation. Thus, in the presence of oxygen or an oxidizing agent, thiols undergo a series of oxidation steps, ultimately producing sulfonic acid groups (-SO3-). Oxidation can inactivate cysteine ​​proteases, as neither of these oxidized species retains catalytic activity for peptide bonds.

[0142] Another reason for the low proteolytic stability of proteases is autolysis. Because proteases are proteins themselves, they can be degraded by active proteases, resulting in the loss of proteolytic activity. This is true for all proteases, including cysteine ​​proteases, which do not require external substances such as oxygen. Without wishing to be bound by theory, the present invention can therefore prevent or inhibit autolysis.

[0143] In this study, we identified and validated a combination of methods that have been shown to enhance and stabilize the proteolytic activity of proteases, thus providing a solution to the problem of protease inactivation due to oxidative damage and autolysis.

[0144] The methods of the invention, in one aspect, involve 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 area surrounding the protease, for example, by flushing the solution with a stable halogen gas such as nitrogen or argon.

[0145] In additional or alternative embodiments, the addition of anionic polymers (such as cross-linked polyacrylates) has also been explored as a means of inhibiting autolysis by non-covalently sequestering the protease. The selection of anionic polymers based on the high pI of papain and related proteases means that papain and related proteases are positively charged at neutral pH. Thus, the present invention potentially allows the protease to bind to the polymer through reversible electrostatic interactions.

[0146] Materials and Methods Papain and bromelain (technical grade) derived from plant milk resin were obtained from Sigma Aldrich. Papain-containing compositions were obtained from papaya fruit (without the milk resin of the peel). Such papain-containing compositions, such as opal, can be prepared, for example, by the method disclosed in International Patent Application No. PCT / AU2003 / 000931, published as WO 2004 / 008887, the entire contents of which are incorporated herein by reference.

[0147] Nitrogen gas was supplied by BOC Gases (UK) and Carbopol Ultrez by Lubrizol Inc (OH, USA). All other chemicals were purchased from Sigma Aldrich. Spectrophotometric readings were performed 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: The 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 oxygen-free samples, nitrogen flushing was performed at this stage. Subsequent addition of 10 M NaOH (1:1000, 10 mM final) caused the solution to gel immediately, resulting in a viscous gel.

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

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

[0153] key Opal (or O) - papaya extract; P - papain; B - bromelain, respectively No treatment (O, P, or B), Treated Z-Carbopol (0.25%), Treatment XY - Addition of cysteine ​​(X) and degassing with nitrogen (Y), or Addition of processed XYZ-cysteine ​​(X) and degassing with nitrogen (Y) and carbopol (0.25%) Evaluated by.

[0154] Opal stabilization: Freshly prepared opal (16 mL) was divided into 2 x 8 mL aliquots. Each aliquot was then further divided into 2 x 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. The opal + XY + Z sample was then added 1:1000 with 10 M NaOH to bring the final pH to 7.5 under N2 flush. The opal + Z sample was then added 1:1000 with 10 M NaOH to bring the final pH to 7.5.

[0155] Papain stabilization: Papain at 1 mg / mL (16 mL) was divided into 2 x 8 mL aliquots. Each aliquot was then further divided into 2 x 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. The P + XY + Z sample was then added with 10 M NaOH at 1:1000 to bring the final pH to 7.5 under N2 flush. The P + Z sample was then added with 10 M NaOH at 1:1000 to bring the final pH to 7.5.

[0156] Bromelain stabilization: 16 mL of 1 mg / mL bromelain was divided into 2 x 8 mL aliquots. Each aliquot was further divided into 2 x 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. The B+XY+Z sample then received 1:1000 10 M NaOH to bring the final pH to 7.5 under N2 flush. The B+Z sample received 1:1000 10 M NaOH to bring the final pH to 7.5.

[0158] Results and Discussion 1. Stabilization of proteolytic activity of proteases In summary, treatment with XY and XYZ was found to stabilize the proteolytic activity of the proteases for the opal and papain samples compared to the untreated opal and papain samples. Treatment with Z also stabilized the proteolytic activity of the proteases for the papain samples, as can be seen in Figures 1 and 2. Treatment with XY and XYZ was shown to less stabilize the proteolytic activity of the proteases for the bromelain samples compared to the untreated bromelain samples, as can be seen in Figure 3. These results are discussed in more detail below.

[0159] Papaya extract (opal) The stability of the papaya extract stabilized solution was evaluated weekly for two months by the BApNA assay. The measured activity was then 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] The stabilizing effect of the cysteine / nitrogen flushing combination resulted in no detectable loss of activity over the period studied (at least up to day 58). This could be explained by the absence of oxygen keeping the reducing agent cysteine ​​in a reduced state, which in turn keeps the active cysteine ​​in the cysteine ​​protease active site reduced and prevents oxidation. Similarly, the stabilizing effect of carbopol, although shorter-lasting than the cysteine / nitrogen flushing combination treatment, was still significant (at least up to day 12).

[0161] Furthermore, we also considered that the enzyme activity increases over time in the XY+Z sample (described later in Results Part 2).

[0162] 1.1 Papain (P) The results for the commercial milk resin papain solution are shown in Figure 2. Observations are similar to the opal sample in that the XY combination and Z (Carbopol) treatment both stabilized the enzyme solution at day 64. The XYZ treatment of papain also showed stabilization of the enzyme solution at day 64.

[0163] 1.2 Bromelain (B) The results observed for bromelain are similar to those for opal and papain, with regard to the increase in activity immediately after treatment in the XY and XYZ treatments. The immediate effect of cysteine / nitrogen + carbopol was highly significant, reaching a more than 10-fold increase compared to untreated bromelain (Figure 3). Furthermore, stabilization was achieved by day 14 in the Z treatment.

[0164] Although the stabilization of bromelain proteolytic activity decreased over the test period, treatment with carbopol (0.25%) alone (Z), both cysteine ​​and nitrogen degassing (XY), and all of cysteine, nitrogen degassing, and carbopol (0.25%) (XYZ) ultimately maintained stabilization of bromelain proteolytic activity compared to untreated samples at both 7 and 14 days.

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

[0166] These results suggest immediate activation of the proenzyme (i.e., 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 reversibly oxidized species. The unexpected carbopol effect could be interpreted as the prevention of autolysis by the polymer of enzyme aggregates.

[0167] 2.1 Papaya extract (opal) Table 1 shows that treatment with carbopol (0.25%) alone (Z) resulted in an immediate (day 0) 3-fold increase in proteolytic activity compared to untreated samples. The increase in activity peaked on day 12, after which activity declined to basal levels for the remainder of the study 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 untreated samples. This increase in activity was maintained at approximately this level throughout the study, up to day 58. The maximum increase in activity was observed on day 12 (3.59-fold increase), and the minimum increase in activity was observed on day 30 (3-fold increase).

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

[0170] [Table 1]

[0171] The enhanced proteolytic activity of opal observed with the various treatments appears to persist for a considerable period of time: treatment with carbopol (0.25%) alone (Z) showed enhanced proteolytic activity for at least 12 days, while treatment with both cysteine ​​and nitrogen degassing (XY), and with cysteine, nitrogen degassing, and carbopol (0.25%) all together (XYZ) showed 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 untreated samples. This increase was largely maintained (4.1-fold increase) until at least day 26, after which activity declined to approximately a 2.6- to 2.7-fold increase for the remainder of the study period, until 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 then gradually decreased over the study period, with a 1.905-fold increase in activity observed on day 26 and a 1.667-fold increase on days 51 and 64.

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

[0175] [Table 2]

[0176] The enhanced proteolytic activity of papain observed with the various treatments also appears to persist for a considerable period of time: treatment with carbopol (0.25%) alone (Z), with both cysteine ​​and nitrogen degassing (XY), and with cysteine, nitrogen degassing, and carbopol (0.25%) all together (XYZ) each showed enhanced proteolytic activity for at least 64 days (the entire test period).

[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 untreated samples. This increase was largely maintained (1.5-fold increase) through at least day 14, increasing activity to a 3.0-fold increase by 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 declined over the study period, with a 0.6-fold increase in activity observed at day 7 and a 0.65-fold increase in activity observed at day 14.

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

[0180] [Table 3]

[0181] The immediate increase in bromelain proteolytic activity observed with the various treatments is consistent with the same trend observed with both opal and papain. Although the increase in activity for bromelain decayed more quickly than for opal and papain, treatment with carbopol (0.25%) alone (Z), both cysteine ​​and nitrogen degassing (XY), and all of cysteine, nitrogen degassing, and carbopol (0.25%) (XYZ) still maintained a net increase in bromelain proteolytic activity compared to untreated samples over the entire test period.

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

[0183] Surprisingly and unexpectedly, it has been found that the method for enhancing the stability of 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 proteases. In particular, the enhancement of stabilization of proteolytic activity by treatment with XYZ is very significant.

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

[0185] The processes disclosed herein involve reagents that are safe and do not involve covalent chemical modification of enzymes, thus preventing consumer safety issues such as allergies and regulatory challenges.

[0186] While the present invention has been disclosed with reference to particular embodiments, it will be apparent that other embodiments and modifications of the present invention may be devised by those skilled in the art without departing from the true spirit and scope of the invention. Features and embodiments of different sections may be combined mutatis mutandis.

Claims

1. 1. A method for enhancing and / or stabilizing the proteolytic activity of a protease, the method comprising the steps of: (i) contacting the protease with a reducing agent, wherein cysteine ​​residues of the protease are maintained in a reduced state; (ii) removing substantially all oxygen gas from an area 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.

2. 3. The method of claim 2, wherein the protease is placed in a substantially oxygen-free atmosphere.

3. A method for producing a composition comprising a protease with enhanced and / or stabilized proteolytic activity, the method comprising the steps of: (i) contacting the protease with a reducing agent, wherein cysteine ​​residues of the protease are maintained in a reduced state; (ii) removing substantially all oxygen gas from an area 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.

4. The method of 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. The method of any one of claims 1 to 5, wherein the polymer is a carbomer.

7. 1. A composition comprising one or more proteases having enhanced and / or stabilized proteolytic activity, the composition being obtained or obtainable by a method comprising the steps of: (i) contacting the protease with a reducing agent, wherein cysteine ​​residues of the protease are maintained in a reduced state; (ii) removing substantially all oxygen gas from the area 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.

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

9. 9. The composition of claim 7 or 8, wherein the oxygen gas is removed by degassing the composition.

10. The composition of any one of claims 7 to 9, wherein the polymer is a carbomer.

11. The composition of any one of claims 7 to 10, wherein the composition is placed in a substantially oxygen-free atmosphere.

12. 12. Use of a composition according to any one of claims 7 to 11 in the manufacture of a medicament for the treatment of diseases and disorders involving wounds, for debridement, or for treating burns, ulcers, or gangrene.

13. 12. Use of a composition according to any one of claims 7 to 11 in the manufacture of a cosmetic product for skin lightening, exfoliation, or for application to wrinkles, skin blemishes, freckles, pimples, acne, rosacea, sun spots, scars or varicose veins, or for application to dry, aged or damaged skin.

14. A pharmaceutical composition comprising the composition of any one of claims 7 to 11 together with a pharmaceutically acceptable carrier, diluent, excipient, surfactant, and / or adjuvant.

15. A cosmetic composition comprising the composition according to any one of claims 7 to 11 together with a cosmetically acceptable carrier, diluent, excipient, surfactant and / or adjuvant.

16. A composition according to any one of claims 7 to 11 or a pharmaceutical composition according to claim 14 for use in the treatment of diseases and disorders involving wounds, for debridement or for treating burns, ulcers or gangrene.

17. The composition of claim 16, wherein the composition is applied topically.

18. A method for the treatment of diseases and disorders involving wounds, for debridement, or for treating burns, ulcers, or gangrene, comprising the step of administering to a subject a composition according to any one of claims 7 to 11 or a pharmaceutical composition according to claim 14.

19. 20. The method of claim 18, wherein the composition is applied topically.

20. 16. A composition according to any one of claims 7 to 11 or a cosmetic composition according to claim 15 for use in skin lightening, exfoliating, or for application to wrinkles, skin blemishes, freckles, pimples, acne, rosacea, sun spots, scars or varicose veins, or for application to dry, aged or damaged skin.

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