Enhancement and stabilization of proteolytic activity of proteases

Abstract

The present invention relates to a method for the enhancement and stabilization of proteolytic activity of a protease, to a method for the production of a composition comprising a protease having enhanced and stabilized proteolytic activity, to a composition comprising a protease having enhanced and stabilized proteolytic activity obtained or obtainable by the above-mentioned method, to the use of such a composition in the manufacture of a medicament and a cosmetic, to the use of such a composition in the treatment of diseases and disorders, including wounds, and to the use of such a composition in cosmetics, and to related kits.

Description

TECHNICAL FIELD

[0001] The present invention relates to a method for the enhancement and stabilization of proteolytic activity of a protease, a method for the production of a composition comprising a protease having enhanced and stabilized proteolytic activity, a composition comprising a protease having enhanced and stabilized proteolytic activity obtained or obtainable by the above-mentioned method, the use of such composition in the manufacture of a medicament and a cosmetic, the use of such composition in the treatment of diseases and disorders, including wounds, and in cosmetics, and related kits. BACKGROUND

[0002] Papain and bromelain are proteases used in pharmaceutical products for debridement and in cosmetic / cosmeceutical products for exfoliation and skin lightening. It is the proteolytic activity of papain and bromelain that brings about such debridement, exfoliation and skin lightening effects. However, due to the instability of their proteolytic activity, neither papain nor bromelain preparations have realized their potential.

[0003] Significant efforts have been previously invested in developing stable compositions that promote wound healing by removing dead and damaged tissue, such as that found in wounds (e.g., burns and chronic ulcers). Effective debridement is critical because dead and dying tissue is an excellent medium for opportunistic infections. Infection-induced sepsis is a leading cause of death in severely burned patients.

[0004] One previous approach was to use proteolytic enzymes, such as papain, trypsin and bromelain. In particular, NexoBrid TM (An enriched proteolytic enzyme concentrate of bromelain) was approved in Europe in 2012 for the removal (i.e., debridement) of eschar in adults with deep partial- and full-thickness thermal burns. However, the European Medicines Agency confirmed in Section 2.2.3, page 14 of its “Assessment Report - Nexobrid - Enriched proteolytic enzyme concentrate of bromelain”, dated 20 September 2012, that a general problem of compositions of proteolytic enzymes is the poor stability of the proteolytic activity, setting forth:

[0005] “New compatibility (stability in use) studies at 25°C and 37°C have been performed, demonstrating degradation of NexoBrid within a few hours after mixing. The applicant’s conclusion that the product should be used immediately after mixing was therefore supported.”

[0006] Accordingly, NexoBridTM Typically supplied as a lyophilized powder, it is reconstituted with water using a gel carrier before use and must be used within 15 minutes of preparation. Therefore, it is often supplied in a ready-for-use form, as is the case with NexoBrid. TM It would be advantageous if the composition did not require rehydration. However, doing so would necessitate a significant enhancement in the stability of the debridement activity in order to achieve an acceptable shelf life.

[0007] Similarly, in cosmetic products containing papain and / or bromelain that are marketed as having epidermal exfoliation and / or skin brightening properties, the epidermal exfoliation activity is usually severely reduced in a short period of time due to the loss of activity of papain and / or bromelain in the cosmetic product.

[0008] Several methods have previously been envisioned to address the loss of proteolytic activity of proteases in pharmaceuticals, including storing the enzyme at a pH where activity loss is low or nonexistent (in the case of papain, an acidic pH has been used) or storing it in solid form. However, these processes require specialized end-user handling to obtain a viable product. This limits the usefulness of such methods and compositions.

[0009] Another previously attempted approach was the immobilization of enzymes on a polymer matrix to prevent mobility and self-reactivity. Examples of immobilized enzymes are PEG-papain and chitosan-papain. While the stability of proteolytic activity may be improved through this method, the enzyme may be irreversibly chemically altered and, consequently, may not exhibit sufficient activity on certain substrates, especially when applied to complex substrates such as skin and cellular proteins. Furthermore, the altered chemical structure of such enzymes can cause adverse reactions in end-users, such as allergies and intolerances.

[0010] To address these issues, research supporting this invention investigated the loss of proteolytic activity of proteases extracted from various sources, such as papaya (Carica papaya) plants (including papain) and pineapple (Ananascomosus) plants (including bromelain). Based on these studies, methods for enhancing and stabilizing the proteolytic activity of proteases were developed, particularly in pharmaceutical and cosmetic / pharmacocosmetic compositions containing proteases derived from papaya and pineapple plants.

[0011] Surprisingly and unexpectedly, it was found that some methods that enhance the stability of protein hydrolysis activity also significantly enhance the protein hydrolysis activity itself. Summary of the Invention

[0012] This invention teaches novel methods for enhancing and stabilizing the proteolytic activity of proteases. In specific embodiments, the protease is obtained from or can be obtained from fruits and / or vegetables. Accordingly, 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 specific embodiments, the protease is a cysteine ​​protease. In specific embodiments, the cysteine ​​protease may be papain (EC 3.4.22.2), papain chymopapain (EC 3.4.22.6), bromelain (stem bromelain - EC 3.4.22.32 and fruit bromelain - EC 3.4.22.33), fig protease (EC 3.4.22.3), or kiwi protease (EC 3.4.22.14). In specific embodiments, the protease is obtained from or can be obtained from the papaya (Caricapapaya) plant or the pineapple (Ananas comosus) plant. In specific embodiments, the protease is papain or bromelain.

[0013] In a first aspect, the present invention provides a method for enhancing and / or stabilizing the proteolytic activity of a protease, wherein the method comprises (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 from a region surrounding the protease. In one embodiment, the oxygen is removed by degassing the preparation. In one embodiment, the protease is packaged in a substantially anaerobic atmosphere.

[0014] In one related aspect, the present invention provides a method for enhancing and / or stabilizing the proteolytic activity of a protease, wherein the method comprises (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 from the solution or gel.

[0015] In a second aspect, the present invention provides a method for producing a composition comprising a protease with enhanced and / or stabilized proteolytic activity, wherein the method comprises (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 from a region surrounding the protease. In one embodiment, the oxygen is removed by degassing the composition. In one embodiment, the composition is packaged in a substantially anaerobic atmosphere.

[0016] In one related aspect, the present invention provides a method for producing a composition comprising a protease having enhanced and / or stable proteolytic activity, wherein the method comprises (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 from the solution or gel.

[0017] In a third aspect, the present invention provides a composition comprising one or more proteases with enhanced and / or stabilized proteolytic activity, wherein the composition is obtained or is available by the methods 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 from a region surrounding the protease. In one embodiment, the oxygen is removed by degassing the composition. In one embodiment, the composition is packaged in a substantially anaerobic atmosphere.

[0018] 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 proteases are maintained in a reduced state, and wherein the composition is substantially anaerobic. In one embodiment, oxygen is removed by degassing the composition. In one embodiment, the composition is packaged in a substantially anaerobic atmosphere.

[0019] According to the present invention, the enhancement and / or stabilization of the proteolytic activity of the protease can also be achieved by immobilizing the protease on an anionic polymer matrix. The use of anionic polymers makes it possible for the protease to non-covalently bind to the matrix. This contrasts with previous methods in the art for immobilizing proteases by forming covalent bonds with matrix materials, such as by reacting a portion of the primary amine of the protease with the carboxyl group of a carbomer, wherein an amine-based active crosslinking agent is used, and a portion of the remaining primary amine of the protease is crosslinked.

[0020] Accordingly, in a fifth aspect, the present invention provides a method for enhancing and / or stabilizing the proteolytic activity of a protease, wherein the method comprises binding a protease to 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 packaged in a substantially anaerobic atmosphere.

[0021] In a sixth aspect, the present invention provides a method for producing a composition comprising a protease with enhanced and / or stabilized proteolytic activity, wherein the method comprises binding the protease to 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 packaged in a substantially anaerobic atmosphere.

[0022] In a seventh aspect, the present invention provides a composition comprising one or more proteases with enhanced and / or stabilized proteolytic activity, wherein the composition is obtained or is available by a method comprising binding the protease to 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 packaged in a substantially anaerobic atmosphere.

[0023] 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 a carbomer. In one embodiment, the composition is packaged in a substantially oxygen-free atmosphere.

[0024] 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.

[0025] Accordingly, in a ninth aspect, the present invention provides a method for enhancing and / or stabilizing the proteolytic activity of a protease, wherein the method comprises (i) contacting the protease with a reducing agent, wherein the cysteine ​​residues of the protease are maintained in a reduced state, (ii) removing substantially all oxygen from a region surrounding the protease, and (iii) binding the protease to an anionic polymer matrix such that the protease is non-covalently bound to the anionic polymer matrix. In one embodiment, the oxygen is removed by degassing the formulation. In one embodiment, the polymer is carbomer. In one embodiment, the protease is packaged in a substantially anaerobic atmosphere.

[0026] In one embodiment, step (iii) is performed before steps (i) and (ii). In another embodiment, steps (i) through (iii) are performed in this order.

[0027] In one related aspect, the present invention provides a method for enhancing and / or stabilizing the proteolytic activity of a protease, wherein the method comprises (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 from the solution; and (iv) binding the protease to an anionic polymer matrix such that the protease is non-covalently bound to the anionic polymer matrix.

[0028] In one embodiment, step (iv) is performed before steps (i) to (iii). In another embodiment, steps (i) to (iv) are performed in this order.

[0029] In a tenth aspect, the present invention provides a method for producing a composition comprising a protease having enhanced and / or stabilized proteolytic activity, wherein the method comprises (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 from a region surrounding the protease, and (iii) binding the protease to an anionic polymer matrix such that the protease is non-covalently bound to the anionic polymer matrix. In one embodiment, the oxygen is removed by degassing the formulation. In one embodiment, the polymer is a carbomer. In one embodiment, the composition is packaged in a substantially oxygen-free atmosphere.

[0030] In one related aspect, the present invention provides a method for producing a composition comprising a protease with enhanced and / or stabilized proteolytic activity, wherein the method comprises (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 from the solution or gel; and (iv) binding the protease to an anionic polymer matrix such that the protease is non-covalently bound to the anionic polymer matrix.

[0031] In an eleventh aspect, the present invention provides a composition comprising one or more proteases with enhanced and / or stabilized proteolytic activity, wherein the composition is obtained or is available by the method of the ninth 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, (ii) removing substantially all oxygen from a region surrounding the protease, and (iii) binding the protease to an anionic polymer matrix such that the protease is non-covalently bound to the anionic polymer matrix. In one embodiment, the oxygen is removed by degassing the formulation. In one embodiment, the polymer is a carbomer. In one embodiment, the composition is packaged in a substantially oxygen-free atmosphere.

[0032] 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 protease are maintained in a reduced state, the composition is substantially anaerobic, and the protease is non-covalently bound to the anionic polymer matrix. In one embodiment, oxygen is removed by degassing the composition. In one embodiment, the polymer is a carbomer. In one embodiment, the composition is packaged in a substantially anaerobic atmosphere.

[0033] In one embodiment, the composition is in the form of a gel.

[0034] In a thirteenth aspect, the present invention provides the use of the compositions of the third, fourth, seventh, eighth, eleventh, and / or twelfth aspects in the manufacture of a pharmaceutical agent. In some embodiments, the pharmaceutical agent is used for the treatment of diseases and disorders, including wounds. In some embodiments, the pharmaceutical agent is for debridement. In some embodiments, the pharmaceutical agent is for the treatment of burns. In some embodiments, the pharmaceutical agent is for the treatment of ulcers. In some embodiments, the pharmaceutical agent is for the treatment of gangrene.

[0035] In a fourteenth aspect, the present invention provides the use of the compositions of the third, fourth, seventh, eighth, eleventh, and / or twelfth aspects in the manufacture of cosmetics. In some embodiments, the cosmetic is for exfoliation, skin brightening, or application to wrinkles, skin blemishes, freckles, papules, acne, rosacea, sunspots, scars, or varicose veins, or for application to dry, aging, or damaged skin.

[0036] In a fifteenth aspect, the present invention provides a pharmaceutical composition comprising the compositions of the third, fourth, seventh, eighth, eleventh and / or twelfth aspects, and a pharmaceutically acceptable carrier, diluent, excipient, surfactant and / or adjuvant.

[0037] In a sixteenth aspect, the present invention provides a cosmetic composition comprising the compositions of the third, fourth, seventh, eighth, eleventh and / or twelfth aspects, and cosmetically acceptable carriers, diluents, excipients, surfactants and / or adjuvants.

[0038] In a seventeenth aspect, the present invention provides compositions of the third, fourth, seventh, eighth, eleventh, and / or twelfth aspects, or pharmaceutical compositions of the fifteenth aspect, for the treatment of 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 for topical application.

[0039] In an eighteenth aspect, the present invention provides a method for treating diseases and disorders, including wounds, wherein the method comprises administering to a subject a composition of the third, fourth, seventh, eighth, eleventh, and / or twelfth aspects, 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 for topical application.

[0040] In the nineteenth aspect, the present invention provides compositions of the third, fourth, seventh, eighth, eleventh and / or twelfth aspects, or cosmetic compositions of the sixteenth aspect, for use in skin brightening, epidermal exfoliation, or for application to wrinkles, skin blemishes, freckles, papules, acne, rosacea, sunspots, scars or varicose veins, or for application to dry, aging or damaged skin.

[0041] In a twentieth aspect, the present invention provides a kit comprising the compositions 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 the method of the eighteenth aspect or for the uses of the seventeenth and nineteenth aspects.

[0042] Compared to previously available compositions, the compositions of the present invention do not require lyophilization to ensure sufficient stability for pharmaceutical and / or cosmetic applications. Therefore, such compositions can be formulated as, for example, capsules, tablets, creams, ointments, solutions, pastes, drops, sprays, aerosols, steam, wipes, patches, gauze, gels, or liquids. Accordingly, in one embodiment, the compositions of the present invention are provided or packaged as capsules, tablets, creams, ointments, emulsions, solutions, pastes, drops, sprays, aerosols, steam, wipes, patches, gauze, gels, or liquids, and do not require rehydration 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 require rehydration before use. Attached Figure Description

[0043] Figure 1 Stability of Opal treated with X+Y, Z, and X+Y+Z. Total proteolytic activity as measured by the BApNA assay after X+Y, Z, and X+Y+Z treatments. Ratio to initial Opal activity (2E3 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 Opal.

[0044] Figure 2 Stability of papain treated with X+Y, Z, and X+Y+Z. Total proteolytic activity, as measured by the BApNA assay, is shown after X+Y, Z, and X+Y+Z treatments. The ratio to initial P activity (1E4 USP units / mL). Activity at t=0 reflects the immediate enhancement of proteolytic activity after X+Y, Z, and X+Y+Z treatments. All treatments were performed on the same batch of papain.

[0045] Figure 3 Stability of bromelain treated with X+Y, Z, and X+Y+Z. Total proteolytic activity, as measured by the BAPNA assay, is shown after X+Y, Z, and X+Y+Z treatments. The ratio 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. Detailed Implementation

[0046] Definitions

[0047] 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 pertains. While any methods and materials similar to or equivalent to those described herein may be used in the practice or testing of this invention, preferred methods and materials are described. For the purposes of this invention, the following terms are defined as follows.

[0048] The articles “a” and “an” are used in this text to refer to one or more of the grammatical objects of the article (i.e., at least one). For example, “an element” refers to one element or more elements.

[0049] "About" refers to a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length that varies by up to 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1% relative to that of the reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length.

[0050] Throughout this specification, unless the context otherwise requires, the terms “comprise,” “comprises,” and “comprising” will be understood as referring to the inclusion of the stated steps or elements or groups of steps or elements, but not excluding any other steps or elements or groups of steps or elements. Therefore, the use of the term “comprising” and similar terms indicates that the listed elements are necessary or mandatory, but other elements are optional and may or may not be present. “consisting of” means including and limited to anything between the phrase “consisting of.” Therefore, the phrase “consisting of” indicates that the listed elements are necessary or mandatory, and other elements may not be present. “consisting essentially of” means including any elements listed between the phrases, and limited to other elements that do not impede or contribute to the specific activity or function of the listed elements in the disclosure. Therefore, the phrase “consisting essentially of” indicates that the listed elements are necessary or mandatory, but other elements are optional and their presence or absence depends on whether they affect the activity or function of the listed elements.

[0051] The term "debridement" refers to the removal of dead and damaged tissue from a wound.

[0052] When used in relation to the compositions of the present invention, the term "obtainable" includes not only compositions produced by a specific method, but also the same compositions produced by any method, such as by proteases obtained from fruits or vegetables, or by recombinant DNA technology or other genetic engineering methods (e.g., by using a recombinant expression system).

[0053] "Isolated" means material that is substantially or substantially free from the limitations of the components that normally accompany it in its natural state. For example, as used herein, "isolated protease" refers to a peptide or polypeptide protease molecule that has been isolated and / or purified in vitro from its native cellular environment and from its association with other components of the cell, i.e., it is not associated with substances in vivo.

[0054] The term "Opal" refers to a composition containing papain obtained from papaya fruit (excluding the latex in the peel). For example, Opal may be prepared by the method disclosed in International Patent Application No. PCT / AU2003 / 000931 (Publication No. WO2004 / 008887), the entire contents of which are incorporated herein by reference.

[0055] The terms “patient,” “subject,” and “individual” are used interchangeably to refer to patients, subjects, and individuals of humans or other mammals, and include anyone who desires to use the present invention to treat, prevent, improve, or reduce the severity of a disease, disorder, or symptom. However, it will be understood that “patient” does not imply the presence of symptoms. Suitable mammals falling within the scope of the present invention include, but are not limited to, primates (e.g., humans, chimpanzees), livestock (e.g., sheep, cattle, horses, donkeys, pigs), laboratory test animals (e.g., rabbits, mice, rats, guinea pigs, hamsters), companion animals (e.g., cats, dogs), and captive wild animals (e.g., foxes, deer, dingoes).

[0056] Throughout the instruction manual, the phrase "substantially oxygen-free" includes concentrations of less than 1 ppm.

[0057] 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 and stabilizing the proteolytic activity of a protease, such as by increasing, maintaining, prolonging, or slowing down the protease’s ability to perform its usual enzymatic function relative to a given unit of time or relative to a reference activity level. Such methods may therefore result in a protease composition that takes less time to perform its enzymatic function than would take without such methods. Alternatively, such methods may result in a higher level of activity of the protease performing its enzymatic function than would be without such methods, for example, when measured at a specific time point or multiple time points after such methods have been performed. Less time spent or higher levels of activity may also be measured relative to a range of other factors, including but not limited to exposure to heat and sterilization radiation, storage time and storage conditions of the protease composition, and in addition, any transport conditions (including temperature, humidity and atmospheric pressure).

[0058] The enhancement and / or stability of the proteolytic activity of the protease can be measured or confirmed, for example, by comparing the enzymatic activity of the protease affected by the method of the present invention (e.g., “Sample A”) with the stability of the protease not affected by the method of the present invention (e.g., “Control Sample”, which may be, for example, a wild-type or naturally occurring protease). Such comparisons between samples can be performed, for example, at a specific time after the method of the present invention has been performed, in order to determine the increase in stability and / or activity of the “Sample A” protease relative to the control sample.

[0059] The terms “wild-type” and “naturally occurring” are used interchangeably to refer to the gene product (e.g., polypeptides such as proteases) most commonly observed in a population, and are therefore arbitrarily designated as the “normal” or “wild-type” form of that gene.

[0060] The term "wound" refers to injury to living tissue where the skin is cut or broken, and includes skin ulcers and burns. Skin ulcers may include diabetic ulcers, pressure ulcers, venous (or varicose) ulcers, and arterial ulcers.

[0061] Any reference to prior art in this specification is not and should not be construed as an admission or in any way an implication that the prior art constitutes common general knowledge to those skilled in the art.

[0062] All publications, patents, patent applications and other materials listed in this specification are incorporated herein by reference in their entirety.

[0063] Detailed description of the invention

[0064] Cysteine ​​proteases, also known as thiol proteases and cysteine ​​endopeptidases (EC 3.4.22), are enzymes that degrade proteins. They share a common catalytic mechanism, requiring the catalysis of nucleophilic cysteine ​​thiols in a triplet or dummy. They are found in a wide variety of organisms. In particular, they are commonly found in fruits, including papaya (Carica papaya and Vasconcellea cundianmarcensus), pineapple (Ananas comosus), fig (Ficus carica), and kiwi (Actinidia chinensis), but may also be found in a variety of other fruits and vegetables. Cysteine ​​proteases can be obtained by various methods, including extraction from biological materials (e.g., fruit extracts, such as from papaya pulp) or by recombinant expression in suitable host cells. In one embodiment, the protease present in the compositions of the present invention or based on the methods of the present invention is a protease obtained from the ripe pulp of papaya, for example by the method described in WO2004 / 008887. In another embodiment, the protease is bromelain (EC 3.4.22.33).

[0065] Stability program

[0066] In one aspect of the invention, compositions comprising cysteine ​​proteases are treated with a reducing agent, which keeps the cysteine ​​amino acid residues at the active site of the protease in a reduced state. When oxidized, cysteine ​​residues can form disulfide bridges that disrupt enzymatic activity. The reducing agent can help keep the active site residues in their reduced form or convert oxidized cysteine ​​residues to their reduced form. Suitable reducing agents are those 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 ​​residues of the dissolved protease and keep them in their reduced form. This typically requires an excess of reducing agent. In one embodiment, the concentration of the reducing agent (e.g., cysteine) is typically 10 to 200 mM, for example 50 to 150 mM. In specific embodiments, the concentration of the reducing agent (e.g., 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 specific embodiment, the concentration of the reducing agent (e.g., cysteine) is approximately 100 mM.

[0067] The composition may be, for example, in the form of a liquid or gel comprising the protease. In one embodiment, the protease has been previously bound to an anionic polymer, as described below, prior to the treatment steps with a reducing agent.

[0068] The composition may also be based on a step of removing substantially all oxygen present in the region surrounding the protease, for example, substantially all oxygen present in the liquid or gel (e.g., solution) containing the protease. This can be achieved, for example, by degassing the composition, such as flushing the composition with an inert gas (e.g., nitrogen or argon). For example, purging with nitrogen or argon at a flow rate of 25 mL / s for 20-40 min can achieve approximately 0.2-0.4 ppm of residual dissolved oxygen. Other methods for removing substantially all oxygen present in the composition may also be utilized and will be known to those skilled in the art, such as, but not limited to, heating at atmospheric or reduced pressure, or sonication at atmospheric or reduced pressure. The oxygen removal step can be performed before, simultaneously with, or immediately after the addition of a reducing agent.

[0069] The resulting composition comprises a cysteine ​​protease and a reducing agent that reduces one or more cysteine ​​residues at the active site of the cysteine ​​protease, wherein the composition is substantially anaerobic. As a result of the reaction with the cysteine ​​protease and / or other components in the composition, at least a portion of the reducing agent may be in an oxidized state.

[0070] To reduce exposure to oxygen during storage, the composition may be packaged to reduce or prevent oxygen absorption. For example, the composition may be packaged in a substantially oxygen-free atmosphere, such as in a container with an inert gas (e.g., nitrogen or argon). Alternatively, the composition may be vacuum-packed.

[0071] In another aspect (which can also be combined with the foregoing aspects of the invention), the anionic polymer matrix can be bound to a cysteine ​​protease so that the cysteine ​​protease binds non-covalently to the anionic polymer matrix. This aims to isolate the enzyme molecule in a non-covalent manner and thus limit autolysis and enzyme inactivation. The choice of anionic polymer is generally based on the high isoelectric point (pI) of papain and related proteases, meaning that they are positively charged at neutral pH.

[0072] The anionic polymer may also be selected such that it forms a gel, for example at a pH between 6.5 and 8 or at an alkaline pH.

[0073] In one embodiment, the anionic polymer matrix is ​​polyacrylic acid, such as a homopolymer, copolymer, or interpolymer of acrylic acid. The anionic polymer typically has a high molecular weight. Examples of homopolymers are polymers of acrylic acid crosslinked with any one of the allyl ethers of several polyols (e.g., pentaerythritol allyl ether, sucrose allyl ether, or propylene allyl ether). Examples of copolymers are polymers of acrylic acid and C10-C30 alkyl acrylates crosslinked with, for example, allyl pentaerythritol ether. Specific examples of suitable anionic polymers are Carbopol, Carbopol Ultraz, Carbomer 910, Carbomer 934, Carbomer 934p, Carbomer 940, and Carbomer 941 (available from Lubrizol).

[0074] The anionic polymer can bind to the protease to produce a liquid suspension. The concentration of the anionic polymer may be from 0.01 to 3% w / w or more, for example, from 0.1 to 2% w / w. If in conjunction with other aspects of the invention, and if not already performed, the reducing agent may be added at this stage and oxygen may be removed, for example, by rinsing with nitrogen. A base may then be added to induce gelation of the liquid suspension to produce a viscous gel, for example, by adjusting the pH to 7.5 to 8.

[0075] These three different processing steps can be performed in different orders. For example, the binding with the anionic polymer may be performed first, followed by the addition of the reducing agent / removal of oxygen in any order. Alternatively, the addition of the reducing agent / removal of oxygen may be performed in any order, followed by the binding with the anionic polymer. It is also possible to perform the binding with the anionic polymer between the reducing agent and oxygen removal steps. The steps may be performed sequentially or simultaneously (or with some degree of overlap).

[0076] The compositions of the present invention typically exhibit greater stability of proteolytic activity than corresponding untreated protease control samples (i.e., not non-covalently bound to anionic polymers and stored under normal oxidative conditions, such as at atmospheric oxygen levels and without the addition of reducing agents). For example, some protease compositions of the present invention, particularly papain (P) and papaya extracts (e.g., Opal), may exhibit at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55 or 60 days or longer of storage at room temperature and pressure. 5, 60, 65, 70, 75, 80, 85, 90, or 95% proteolytic activity; while others, 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 longer. The measured activity may be compared with the activity immediately following treatment according to the procedure of the invention.

[0077] In some embodiments, the initial proteolytic activity of the protease composition of the present invention is greater than that of an untreated protease composition, for example, having at least 1.5 times the activity compared to an untreated protease, such as 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. For comparative purposes, the initial activity can be measured immediately after the treatment step has been performed.

[0078] Activity can be measured spectrophotometrically using BApNA: A BApNA solution (10 mM, in DMSO) can be prepared simultaneously with a reaction buffer (100 mM potassium dihydrogen phosphate, 116 mM potassium chloride, and 3 mM EDTA, pH 6). The premix can be prepared 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 used at 410 nm, with the premix as a blank. Then, 1 / 5 v / v water is added to both the control and positive samples, and readings are taken immediately thereafter. Relative enzyme activity can be measured using the slope of the resulting line.

[0079] Composition

[0080] Compared to previously available compositions, the compositions of the present invention do not require lyophilization to ensure sufficient stability for pharmaceutical and / or cosmetic applications. Therefore, such compositions can be formulated as, for example, capsules, tablets, creams, ointments, solutions, pastes, drops, sprays, aerosols, steam, wipes, patches, gauze, gels, or liquids. Accordingly, in one embodiment, the compositions of the present invention are provided or packaged as capsules, tablets, creams, ointments, emulsions, solutions, pastes, drops, sprays, aerosols, steam, wipes, patches, gauze, gels, or liquids, and do not require rehydration 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 require rehydration before use.

[0081] In a specific aspect of the invention, the composition may be in the form of a gel or a liquid. The enhanced stability of the composition's proteolytic activity allows it to be provided in liquid or gel form, meaning it is ready to use and does not require immediate rehydration before use. This is highly advantageous for the application of the composition and may be useful in situations where rehydration of lyophilized compositions is difficult, such as outside of clinical settings where immediate treatment of wounds (e.g., burns) is required.

[0082] The composition may also be absorbed into / onto a solid material, such as a wound dressing. The composition may also be pharmaceutically and / or cosmetically combined with a carrier, diluent, excipient, surfactant, and / or adjuvant to obtain the pharmaceutical or cosmetic composition of the present invention. Therefore, the compositions of the present invention may be formulated to include one or more additional ingredients.

[0083] For example, surfactants may 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 may also incorporate any suitable surfactant, such as anionic, cationic, or nonionic surfactants, such as sorbitol esters or their polyoxyethylene derivatives. Suitable surfactants may also include sodium lauryl sulfate (SDS), ammonium lauryl sulfate, sodium lauryl sulfate, and sodium myristyl ether sulfate. Surfactants are generally used to reduce nonspecific adsorption and require careful selection and optimization. Suspensions (e.g., natural gums, cellulose derivatives, or inorganic materials (e.g., silica-containing silicas) and other ingredients (e.g., lanolin) may also be included.

[0084] The composition may be prepared according to methods known to those skilled in the art and may also include additional carriers, excipients, or diluents. The carriers, excipients, and diluents must be "acceptable" in terms of compatibility with the other components of the composition and must be non-adverse to the formation of the composition, allowing for longer storage periods if desired. Such carriers, excipients, and diluents may be used to further enhance the integrity and half-life of the compositions of the present invention.

[0085] Further examples of acceptable carriers or diluents are demineralized or distilled water; salt solutions; vegetable-based oils such as peanut oil, safflower oil, olive oil, cottonseed oil, corn oil, sesame oil, peanut oil, or coconut oil; and silicone oils, including polysiloxanes such as methylpolysiloxane, phenylpolysiloxane, and methylphenylpolysiloxane. Polysolpoxane; volatile silicones; mineral oils, such as liquid paraffin, soft paraffin, or squalane; cellulose derivatives, such as methylcellulose, ethylcellulose, carboxymethylcellulose, sodium carboxymethylcellulose, or hydroxypropyl methylcellulose; 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-butanediol, or glycerin; fatty acid esters, such as isopropyl palmitate, isopropyl myristate, or ethyl oleate; polyvinylpyrrolidone; agar; gum arabic or gum arabic; and petroleum gels. Carriers (such as polyvinylpyrrolidone (PVP), carboxymethylcellulose, polyvinyl alcohol, and polyethylene oxide) may also be used.

[0086] Additional carriers that may be included in the compositions of the present invention include non-reducing sugars (e.g., sucrose) and reducing sugars (e.g., lactulose). Such carriers, as well as sugar alcohols (e.g., mannitol, xylitol, glycerol, and sorbitol), are also useful for inclusion in the compositions of the present invention because they can act as antioxidants and potential stabilizers.

[0087] Methods for preparing the applicable compositions are readily apparent to those skilled in the art and are described in detail, for example, in Remington's Pharmaceutical Science, 15th ed., Mack Publishing Company, Easton, Pa., which are incorporated herein by reference.

[0088] Use

[0089] The compositions of the present invention can be used in various pharmaceutical applications related to the treatment of diseases, disorders, and conditions, including dermatological and wound treatments. The compositions of the present invention can also be used in various cosmetic applications.

[0090] Accordingly, the present invention further provides methods for wound debridement, including topical application of the composition of the present invention, methods for treating wounds using the composition of the present invention, methods for treating an individual suffering from burns, wherein the methods include topical application of the preparation of the present invention to the affected area of ​​the individual or by other routes of application, methods for treating wounds using the composition of the present invention, methods for enhancing wound healing, including topical application of the composition of the present invention to the wound or by other routes of application, methods for epidermal exfoliation or skin brightening, including application of the cosmetic composition of the present invention to the skin, use of the cosmetic composition of the present invention for epidermal exfoliation or skin brightening, methods for treating dry, aging, or damaged skin, including application of the cosmetic composition of the present invention to the skin, and use of the cosmetic composition of the present invention for treating dry, aging, or damaged skin.

[0091] The protease compositions of the present invention may be particularly used to prevent, treat, reduce or improve various 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, seborrheic dermatitis).

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

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

[0094] Dosage

[0095] The therapeutically effective dose level for any specific patient will depend on a variety of factors, including the condition being treated and its severity, 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, any drugs used in combination with or by chance, and other relevant factors known in the art. Therefore, those skilled in the art will be able to determine, through routine experimental procedures, the effective, non-toxic amount of the composition required to treat the applicable condition.

[0096] Furthermore, it will be apparent to those skilled in the art that the optimal quantity and time interval of a single dose of the composition will be determined by the nature and extent of the condition being treated, the form, route and site of application, and the nature of the specific individual being treated. Moreover, such optimal conditions can be determined using conventional techniques.

[0097] It will also be apparent to those skilled in the art that they can use conventional course of treatment determination tests to determine the optimal course of treatment, such as the number of doses of the composition administered daily over a defined number of days.

[0098] Route of administration

[0099] The compositions of the present invention can be administered via standard routes. Generally, the compositions may be administered via a topical route. Typically, the compositions of the present invention are applied topically to the affected area of ​​an individual.

[0100] In other embodiments, the composition may be administered via other enteric routes (e.g., rectum, sublingual, or sublipal) or via the central nervous system (e.g., through the epidural, intracerebral, or ventricular pathways). Other sites of administration may include epipicutaneous, percutaneous, intradermal, nasal, intra-arterial, intracardiac, intraosseous, intrasheathic, intraperitoneal, intrabladder, intravitreal, intracavernous, intravaginal, or intrauterine routes.

[0101] Time of therapy

[0102] Typically, in therapeutic applications, treatment will be carried out over the duration of the disease state.

[0103] Those skilled in the art will understand that, at or after diagnosis, the compositions disclosed herein may be administered as a single agent or as part of a combination therapy of the methods disclosed herein, for example, as follow-up or consolidation therapy, as a supplement to currently available therapies of this kind. The compositions disclosed herein may also be used as a preventative therapy for subjects who are genetically or environmentally susceptible to this disease.

[0104] The composition may be applied regularly as needed, for example, until improvement in the symptoms is seen. Therefore, it may be applied hourly, multiple times a day, daily, multiple times a week, weekly, monthly, or at any frequency deemed appropriate.

[0105] Kit

[0106] The kit of the present invention facilitates the use of the methods and uses of the present invention. Typically, a kit for carrying out the methods or uses of the present invention contains all the necessary reagents and means for carrying out the method. For example, in one embodiment, the kit may include the composition of the present invention and, optionally, means for administering the composition, such as a device for a point-of-care approach.

[0107] Typically, the kits described herein will also include one or more containers. In the context of this invention, a compartmentalized kit includes any kit that uses individual containers to hold the composition, and may include small glass containers, plastic containers, or plastic or paper strips. Such containers may allow for efficient transfer of the composition from one compartment to another while avoiding cross-contamination of the composition, and allow reagents or solutions from each container to be added quantitatively from one compartment to another.

[0108] Typically, the kits of the present invention will also include instructions on using the kit for appropriate methods and purposes.

[0109] The methods, uses, compositions, and kits of this invention are equally applicable to any animal (including humans), such as non-human primates, horses, cattle, sheep, goats, hares, birds, felines, and canines. Accordingly, a single kit of this invention may be suitable for application to different species, or alternatively, different kits may be required, for example, containing specific ingredients for each individual species.

[0110] Those skilled in the art will understand and appreciate that the different features disclosed herein may be combined to form a combination of features within the scope of this invention.

[0111] Enumerated examples

[0112] 1. A method for enhancing and / or stabilizing the proteolytic activity of a protease, wherein the method comprises (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 from a region surrounding the protease, and (iii) binding the protease to an anionic polymer matrix such that the protease is non-covalently bound to the anionic polymer matrix.

[0113] 2. The method of Example 1, wherein the protease is packaged in a substantially anaerobic atmosphere.

[0114] 3. A method for producing a composition comprising a protease with enhanced and / or stabilized proteolytic activity, wherein the method comprises contacting the protease with a reducing agent, wherein cysteine ​​residues of the protease are maintained in a reduced state, (ii) removing substantially all oxygen from a region surrounding the protease, and (iii) binding the protease to an anionic polymer matrix such that the protease is non-covalently bound to the anionic polymer matrix.

[0115] 4. The method of Example 3, wherein the composition is packaged in a substantially oxygen-free atmosphere.

[0116] 5. The method of any one of Examples 1 to 4, wherein oxygen is removed by degassing the formulation.

[0117] 6. The method of any one of Examples 1 to 5, wherein the polymer is carbomer.

[0118] 7. A composition comprising one or more proteases with enhanced and / or stable proteolytic activity, wherein the composition is obtained or is available by a method comprising (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 from a region surrounding the protease, and (iii) binding the protease to an anionic polymer matrix such that the protease is non-covalently bound to the anionic polymer matrix.

[0119] 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 anaerobic, and the proteases are non-covalently bound to the anionic polymer matrix.

[0120] 9. The composition of any one of Examples 7 or 8, wherein oxygen is removed by degassing the composition.

[0121] 10. The composition of any one of Examples 7 to 9, wherein the polymer is carbomer.

[0122] 11. The composition of any one of Examples 7 to 10, wherein the composition is packaged in a substantially oxygen-free atmosphere.

[0123] 12. A method for enhancing and / or stabilizing the proteolytic activity of a protease, wherein the method comprises (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 from a region surrounding the protease.

[0124] 13. The method of Example 12, wherein the protease is packaged in a substantially anaerobic atmosphere.

[0125] 14. A method for producing a composition containing a protease with enhanced and / or stabilized proteolytic activity, wherein the method comprises (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 from a region surrounding the composition.

[0126] 15. The method of any one of Examples 12 to 14, wherein the oxygen is removed by degassing the composition.

[0127] 16. The method of any one of Examples 14 or 15, wherein the composition is packaged in a substantially oxygen-free atmosphere.

[0128] 17. A composition comprising one or more proteases with enhanced and / or stable proteolytic activity, wherein the composition is obtained or is available by 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 from a region surrounding the composition.

[0129] 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 anaerobic.

[0130] 19. The composition of any one of Examples 17 or 18, wherein oxygen is removed by degassing the composition.

[0131] 20. The composition of any one of Examples 17 to 19, wherein the composition is packaged in a substantially oxygen-free atmosphere.

[0132] 21. A method for enhancing and / or stabilizing the proteolytic activity of a protease, wherein the method comprises binding the protease to an anionic polymer matrix such that the protease is non-covalently bound to the anionic polymer matrix.

[0133] 22. A method for producing a composition of a protease with enhanced and / or stable proteolytic activity, wherein the method comprises binding the protease to an anionic polymer matrix such that the protease is non-covalently bound to the anionic polymer matrix.

[0134] 23. The method of any one of Examples 21 or 22, wherein the polymer is carbomer.

[0135] 24. The method of any one of Examples 21 to 23, wherein the composition is packaged in a substantially oxygen-free atmosphere.

[0136] 25. A composition comprising one or more proteases with enhanced and / or stable proteolytic activity, wherein the composition is obtained or is available by a method comprising binding the protease to an anionic polymer matrix such that the protease is non-covalently bound to the anionic polymer matrix.

[0137] 26. A composition comprising one or more proteases and an anionic polymer matrix, wherein the protease is non-covalently bound to the anionic polymer matrix.

[0138] 27. The composition of any one of Examples 25 or 26, wherein the polymer is carbomer.

[0139] 28. The composition of any one of Examples 25 to 27, wherein the composition is packaged in a substantially oxygen-free atmosphere.

[0140] 29. Use of the composition of any one of Examples 7 to 11, 17 to 20 or 25 to 28 in the manufacture of an agent for the treatment of diseases and disorders, for debridement, or for the treatment of burns, ulcers or gangrene, wherein the diseases and disorders include wounds.

[0141] 30. The composition of any one of Examples 7 to 11, 17 to 20 or 25 to 28 may be used in the manufacture of cosmetics for skin brightening, epidermal exfoliation, or for application to wrinkles, skin blemishes, freckles, papules, acne, rosacea, sunspots, scars or varicose veins, or for application to dry, aged or damaged skin.

[0142] 31. A pharmaceutical composition comprising the composition of any one of Examples 7 to 11, 17 to 20 or 25 to 28, and a pharmaceutically acceptable carrier, diluent, excipient, surfactant and / or adjuvant.

[0143] 32. A cosmetic composition comprising the composition of any one of Examples 7 to 11, 17 to 20 or 25 to 28, and a cosmetically acceptable carrier, diluent, excipient, surfactant and / or adjuvant.

[0144] 33. The composition of any one of Examples 7 to 11, 17 to 20 or 25 to 28 or the pharmaceutical composition of Example 31, for the treatment of diseases and disorders, for debridement, or for the treatment of burns, ulcers or gangrene, said diseases and disorders including wounds.

[0145] 34. The composition described in Example 33, wherein the composition is for topical application.

[0146] 35. A method for treating diseases and disorders, for debridement, or for treating burns, ulcers, or gangrene, wherein the method comprises administering to a subject a composition of any one of Examples 7 to 11, 17 to 20, or 25 to 28, or a pharmaceutical composition of Example 31, wherein the diseases and disorders include wounds.

[0147] 36. The method of Example 35, wherein the composition is applied topically.

[0148] 37. The composition of any one of Examples 7 to 11, 17 to 20 or 25 to 28 or the cosmetic composition of Example 32, for use in brightening skin, exfoliating epidermis, or for application to wrinkles, skin blemishes, freckles, papules, acne, rosacea, sunspots, scars or varicose veins, or for application to dry, aging or damaged skin.

[0149] 38. A kit comprising the composition of any one of Examples 7 to 11, 17 to 20 or 25 to 28, the pharmaceutical composition of Example 31 or the cosmetic composition of Example 32.

[0150] 39. The kit described in Example 38 is used or is intended for the treatment of diseases and disorders, for debridement, or for the treatment of burns, ulcers or gangrene, or for skin brightening, epidermal exfoliation, or for application to wrinkles, skin blemishes, freckles, papules, acne, rosacea, sunspots, scars or varicose veins, or for application to dry, aging or damaged skin, said diseases and disorders including wounds.

[0151] The invention will now be further described with reference to the following examples, which are merely illustrative and not limiting.

[0152] Examples

[0153] Cysteine ​​proteases are a family of proteases characterized by the presence of the amino acid cysteine ​​at their active site. This sulfur-containing amino acid contributes to proteolytic activity by attacking the amide carbonyl group of the peptide bond with an accessory histidine residue to form a thioester. The thioester is then attacked by water to regenerate cysteine ​​and release the carboxylic acid.

[0154] Cysteine ​​proteases are found in large quantities in the latex of a large number of plant species, such as papaya (papain, papain chymopapain, papain, aminoacetyl endopeptidase), fig (figase), and several members of the bromeliad family, such as pineapple (Ananas comosus) (bromelain).

[0155] However, cysteine ​​proteases suffer from a major weakness that limits their usefulness: cysteine ​​is particularly sensitive to oxidation due to the relatively strong reduction potential (E) of the thiol group. 0 = -0.34V). Therefore, in the presence of oxygen or an oxidizing agent, thiols will undergo a series of oxidation steps, ultimately producing sulfonic acid groups (-SO3-). Since none of these oxidized species retain catalytic activity for peptide bonds, oxidation can inactivate cysteine ​​proteases.

[0156] Another reason for the poor proteolytic stability of proteases is autolysis. Because proteases are proteins, they can be degraded by active proteases, resulting in a loss of proteolytic activity. This applies to all proteases (including cysteine ​​proteases) and requires no external reagent, such as oxygen. While not wishing to be bound by theory, the present invention may therefore prevent or inhibit autolysis.

[0157] Current research has identified and tested combinations of methods that demonstrate enhanced and stable proteolytic activity of proteases. Therefore, these methods offer solutions to the problem of protease inactivation caused by oxidative damage and autolysis.

[0158] In one aspect, the method of the present invention involves the addition of cysteine ​​as a reducing agent to maintain the cysteine ​​residues at the active site of the protease in a reduced state and to substantially remove oxygen from the region surrounding the protease, for example, by rinsing the solution with a stable gas (e.g., nitrogen or argon).

[0159] In an additional or alternative aspect, the addition of anionic polymers (e.g., cross-linked polyacrylates) was also tested as a means of non-covalently isolating the protease and thus limiting autolysis. The choice of anionic polymers with high pI based on papain and related proteases implies that they are positively charged at neutral pH. Accordingly, the present invention could lead to the binding of the protease to the polymer via reversible electrostatic interactions.

[0160] Materials and methods

[0161] Papain and bromelain (industrial grade) from plant latex were obtained from Sigma Aldrich. Compositions containing papain were obtained from papaya fruit (latex excluding the peel). Such papain-containing compositions (e.g., Opal) may be prepared, for example, by the method disclosed in International Patent Application PCT / AU2003 / 000931 (Publication No. WO2004 / 008887), the entire contents of which are incorporated herein by reference.

[0162] Nitrogen was obtained from BOC Gases (UK) and Carbopol Ultrez from Lubrizol Inc (OH, USA). All other chemicals were purchased from Sigma Aldrich. Spectrophotometer readings were obtained using a Jasco V-630UV-Vis spectrophotometer.

[0163] Cysteine ​​addition: Cysteine ​​was added to each sample to a final concentration of 100 mM.

[0164] Nitrogen rinsing: Place the sample in a plastic vial and purge the solution with nitrogen at room temperature for 5 minutes. Each vial should be immediately closed to prevent oxygen from entering.

[0165] Addition of Carbopol: Carbopol Ultrez was added to each solution to a final concentration of 0.25%, producing a liquid suspension. For anaerobic samples, nitrogen rinsing was performed at this stage. Subsequent addition of 10M NaOH (1:1000, final concentration 10mM) caused immediate gelation of the solution, producing a viscous gel.

[0166] BApNA spectrophotometric method: BApNA solution (10 mM in DMSO) and reaction buffer (100 mM potassium phosphate, 116 mM potassium chloride and 3 mM EDTA, pH 6) are prepared simultaneously.

[0167] A premix was prepared using 2 parts water, 2 parts reaction buffer, and 1 part v / v BApNA. This solution was used as a blank on the spectrophotometer at 410 nm. Then, 1 / 5 v / v water was added to the control and positive samples, and readings were taken immediately. Relative enzyme activity was measured as the slope of the resulting line.

[0168] Key

[0169] Opal (or O) – papaya extract; P – papain; B – bromelain, each determined using the following assays:

[0170] • No processing (O, P, or B) was performed.

[0171] • Z-treatment – ​​Carbopol (0.25%)

[0172] • XY treatment – ​​addition of cysteine ​​(X) and degassing with nitrogen (Y), or

[0173] • XYZ treatment – ​​addition of cysteine ​​(X) and nitrogen degassing (Y) and Carbopol (0.25%).

[0174] Stability of Opal: Freshly prepared Opal (16 mL) was aliquoted into 2 x 8 mL aliquots. Each aliquot was then further divided into two 4 x 4 mL samples (Opal, Opal+XY, Opal+Z, Opal+XY+Z). For sample X, cysteine ​​was added (12 mg / mL, 100 mM). For sample Z, Carbopol was added (0.25%). Sample XY was degassed under nitrogen for 5 min. Then, under N2 rinsing, 10 M NaOH was added to the Opal+XY+Z sample at a ratio of 1:1000 until the final pH was 7.5. Then, 10 M NaOH was added to the Opal+Z sample at a ratio of 1:1000 until the final pH was 7.5.

[0175] Stability of Papain: Papain 1 mg / mL (16 mL) was aliquoted into 2 x 8 mL aliquots. Each aliquot was then further divided into two 4 x 4 mL samples (P, P+XY, P+Z, P+XY+Z). For sample X, cysteine ​​(12 mg / mL, 100 mM) was added. For sample Z, Carbopol (0.25%) was added. Samples XY were degassed under nitrogen for 5 min. Then, 10 M NaOH was added to samples P+XY+Z at a ratio of 1:1000 under N2 rinsing until the final pH was 7.5. Then, 10 M NaOH was added to samples P+Z at a ratio of 1:1000 until the final pH was 7.5.

[0176] Stability of bromelain: 1 mg / mL (16 mL) of bromelain was divided into 2 x 8 mL aliquots. Each aliquot was then further divided into two portions to form 4 x 4 mL samples (B, B+XY, B+Z, B+XY+Z).

[0177] For sample X, cysteine ​​was added (12 mg / mL, 100 mM). For sample Z, Carbopol was added (0.25%). Samples X and Y were degassed under nitrogen for 5 minutes. Then, under N2 rinsing, 10 M NaOH was added to samples B+XY+Z at a ratio of 1:1000 until the final pH was 7.5. Then, 10 M NaOH was added to samples B+Z at a ratio of 1:1000 until the final pH was 7.5.

[0178] Results and discussion

[0179] 1. Stabilization of proteolytic activity of proteases

[0180] In summary, treatment with XY and XYZ resulted in stable proteolytic activity of the proteases in both Opal and papain samples compared to untreated samples. Treatment with Z also stabilized the proteolytic activity of the papain samples. This can be seen from... Figure 1 and Figure 2 This can be seen from the results. Compared to the untreated bromelain sample, the bromelain samples treated with XY and XYZ showed lower stability in the proteolytic activity of the bromelain. This can be seen from... Figure 3 As can be seen from this. These results are discussed in further detail below.

[0181] Opal

[0182] The stability of the papaya extract stabilized solution was determined weekly for two months using the BAPNA assay. The measured activities were then normalized to the activity of the Opal (untreated) sample at the start of the experiment and set to 1. Results were obtained on... Figure 1 Presented.

[0183] The stabilizing effect of the cysteine / nitrogen flushing combination resulted in an undetectable loss of activity during the considered period (up to at least day 58). This can be explained by the oxygen deficiency keeping the reducing agent cysteine ​​in its reduced state, thus allowing it to continue to keep the active cysteine ​​at the active site of the cysteine ​​protease in its reduced state and preventing oxidation. Similarly, the stabilizing effect of Carbopol, although lasting for a shorter period than the treatment with the cysteine / nitrogen flushing combination, was still significant (up to at least day 12).

[0184] An additional consideration is that the enzyme activity of the XY+Z samples increased over time (discussed in Part 2 of the results below).

[0185] 1.1 Papain (P)

[0186] Results of commercial emulsion papain solution Figure 2 The results are similar to those observed with the Opal sample; both the XY combination and the Z (Carbopol) treatment resulted in stability of the enzyme solution after 64 days. XYZ treatment of papain also showed stability of the enzyme solution after 64 days.

[0187] 1.2 Bromelain (B)

[0188] The results observed for bromelain also followed those for Opal and papain regarding the immediate increase in activity following XY and XYZ treatment. The immediate effect of cysteine / nitrogen combined with Carbopol was extremely pronounced, achieving an increase of more than 10-fold compared to untreated bromelain. Figure 3 Furthermore, stability was achieved using Z-processing in 14 days.

[0189] Although the stability of the proteolytic activity of bromelain decreased during the testing period, it maintained net stability compared to untreated samples at days 7 and 14 when treated with Carbopol (0.25%) alone (Z), with both cysteine ​​and nitrogen degassing (XY), and with cysteine, nitrogen degassing, and Carbopol (0.25%) alone (XYZ).

[0190] 2. Enhancement of proteolytic activity of proteases

[0191] Surprisingly and unexpectedly, it was also found that methods for enhancing the stability of proteolytic activity also significantly enhanced the proteolytic activity itself. In summary, treatments with Z, XY, and XYZ resulted in enhanced proteolytic activity in each Opal, papain, and bromelain sample compared to the untreated sample. This can be seen in Tables 1, 2, and 3 below, where “mean” refers to relative enzyme activity, “SD” refers to 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). The sample treatments shown in each table used the same batch of the same protease.

[0192] These results indicate immediate activation of the zymogen (i.e., conversion of the zymogen into an enzyme). The role of cysteine ​​may be due to its reductive / activating effect on the putative zymogen and the reversibly oxidized species. The unexpected Carbopol effect can be explained by the disruption of autolysis by the polymer of the enzyme aggregate.

[0193] 2.1 Opal

[0194] 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 the untreated sample. The increase in activity peaked on day 12, and then decreased to baseline levels for the remainder of the experimental period.

[0195] Compared to untreated samples, treatment with cysteine ​​and nitrogen degassing (XY) resulted in an immediate (day 0) 3.3-fold increase in proteolytic activity. This increase in activity persisted throughout the assay, up to day 58. The maximum increase in activity was observed on day 12 (3.59-fold), and the minimum increase was observed on day 30 (3-fold).

[0196] Compared to the untreated sample, treatment with cysteine, nitrogen degassing, and Carbopol (0.25%) (XYZ) resulted in an immediate (day 0) 12-fold increase in proteolytic activity. Notably, this increase 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. The activity of enzymes associated with zymogens and / or other inactive forms (i.e., proteases) thus exhibited an increase over time.

[0197] Table 1 - Enhanced proteolytic activity of Opal (O)

[0198] Day 0 Day 5 Day 12 Day 30 Day 45 Day 58 Untreated (mean) 1.000 0.718 0.103 0.000 0.000 0.000 Untreated (SD) 0.050 0.035 0.005 0.001 0.001 0.001 Z (mean) 3.077 2.821 3.333 0.000 0.000 0.000 Z (SD) 0.153 0.060 0.060 0.001 0.001 0.001 XY (mean) 3.333 3.333 3.590 3.077 3.333 3.333 XY (SD) 0.166 0.166 0.179 0.153 0.166 0.166 XYZ (mean) 12.051 13.846 14.359 16.667 15.385 17.949 XYZ (SD) 0.602 0.692 0.717 0.833 0.769 0.040

[0199] The enhanced proteolytic activity of Opal observed with various treatments persisted for a considerable period of time. Carbopol (0.25%) treatment alone (Z) showed enhanced proteolytic activity lasting at least 12 days, while treatment with both cysteine ​​and nitrogen degassing (XY), and treatment with cysteine, nitrogen degassing, and Carbopol (0.25%) alone (XYZ) showed enhanced proteolytic activity lasting at least 58 days (the entire experimental period).

[0200] 2.2 Papain (P)

[0201] 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 until at least day 26 (4.1-fold increase), after which the activity decreased to approximately a 2.6-2.7-fold increase for the remainder of the experimental period until at least day 64.

[0202] Compared to untreated samples, treatment with cysteine ​​and nitrogen degassing (XY) resulted in an immediate (day 0) 2.1-fold increase in proteolytic activity. This increase in activity gradually decreased throughout the test period, with a 1.905-fold increase observed on day 26 and 1.667-fold increases observed on days 51 and 64.

[0203] Compared to the untreated sample, treatment with cysteine, nitrogen degassing, and Carbopol (0.25%) (XYZ) resulted in an immediate (day 0) 5.2-fold increase in proteolytic activity. Notably, 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.

[0204] Table 2 - Enhanced proteolytic activity of papain (P)

[0205] Day 0 Day 26 Day 51 Day 64 Untreated (mean) 1.000 0.129 0.048 0.000 Untreated (SD) 0.048 0.014 0.014 0.002 Z (mean) 4.667 4.143 2.619 2.762 Z (SD) 0.048 0.048 0.238 0.048 XY (mean) 2.190 1.905 1.667 1.667 XY (SD) 0.190 0.048 0.048 0.048 XYZ (mean) 5.238 8.095 5.714 6.190 XYZ (SD) 0.143 0.048 0.095 0.143

[0206] The enhanced proteolytic activity of papain observed with various treatments again showed a sustained duration of considerable time. Treatment with Carbopol (0.25%) alone (Z), treatment with both cysteine ​​and nitrogen degassing (XY), and treatment with cysteine, nitrogen degassing, and Carbopol (0.25%) alone (XYZ) each showed enhanced proteolytic activity lasting at least 64 days (the entire experimental period).

[0207] 2.3 Bromelain (B)

[0208] 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 until at least day 14 (1.5-fold increase), with the activity increasing to 3.0-fold by day 7.

[0209] Treatment with 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 a 0.6-fold increase observed on day 14 and a 0.65-fold increase observed on day 14.

[0210] Compared to the untreated sample, treatment with cysteine, nitrogen degassing, and Carbopol (0.25%) (XYZ) resulted in an immediate (day 0) 11-fold increase in proteolytic activity. This enhancement of proteolytic activity then decreased over time, with a 4.6-fold increase observed on day 7 and a 1.3-fold increase observed on day 14.

[0211] Table 3 – Enhanced proteolytic activity of bromelain (B)

[0212] Day 0 Day 7 Day 14 Untreated (mean) 1.000 0.110 0.000 Untreated (SD) 0.040 0.409 0.001 Z (mean) 1.600 3.000 1.500 Z (SD) 0.025 0.033 0.067 XY (mean) 6.300 0.600 0.650 XY (SD) 0.022 0.067 0.108 XYZ (mean) 11.000 4.600 1.300 XYZ (SD) 0.036 0.109 0.077

[0213] The immediate enhancement of bromelain proteolytic activity observed with various treatments was consistent with the same trend observed with both Opal and papain. Although the increase in bromelain activity was faster than the decrease in Opal and papain activity, the net increase in bromelain proteolytic activity remained compared with the untreated sample throughout the test period when treated with Carbopol (0.25%) alone (Z), with both cysteine ​​and nitrogen degassing (XY), and with all of them (XYZ) including cysteine, nitrogen degassing, and Carbopol (0.25%).

[0214] Conclusion

[0215] The data presented herein teach that, using each of the methods disclosed herein, enhanced and stable proteolytic activity of the protease can be achieved, namely, treatment of the protease alone with Carbopol (0.25%) (Z), treatment of the protease with both cysteine ​​and nitrogen degassing (XY), and treatment of the protease with cysteine, nitrogen degassing, and Carbopol (0.25%) (XYZ).

[0216] Surprisingly and unexpectedly, it was also 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 resulted in enhanced proteolytic activity of the proteases. In particular, the enhancement of proteolytic activity stability by XYZ treatment was highly significant.

[0217] Therefore, the data presented herein teach that, using the methods disclosed herein, both the stability of the proteolytic activity of a protease and the enhancement of the proteolytic activity itself can be achieved.

[0218] The treatment disclosed herein involves reagents with an excellent safety record and does not involve chemical covalent modification of enzymes, thereby preventing consumer safety issues such as allergies and regulatory challenges.

[0219] Although the invention has been disclosed with reference to specific aspects, it will be apparent to those skilled in the art that other aspects and variations of the invention may be conceived without departing from the true spirit and scope of the invention. Features and embodiments in different parts may be combined as necessary.

Claims

1. A method for enhancing and / or stabilizing the proteolytic activity of one or more cysteine ​​proteases, characterized in that, The method includes (i) contacting the one or more cysteine ​​proteases with cysteine ​​residues, wherein the cysteine ​​residues of the one or more cysteine ​​proteases are maintained in a reduced state, (ii) removing substantially all oxygen from the region surrounding the one or more cysteine ​​proteases, and (iii) binding the one or more cysteine ​​proteases to carbomer such that the one or more cysteine ​​proteases are non-covalently bound to the carbomer.

2. The method as described in claim 1, characterized in that, The one or more cysteine ​​proteases are packaged in a substantially anaerobic atmosphere.

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

4. The method as described in claim 3, characterized in that, The composition was packaged in a substantially oxygen-free atmosphere.

5. The method as described in claim 1 or 3, characterized in that, Oxygen is removed by degassing the formulation.

6. A composition comprising one or more cysteine ​​proteases having enhanced and / or stable proteolytic activity, wherein the composition is obtained or is available by a method comprising (i) contacting the one or more cysteine ​​proteases with cysteine ​​residues, wherein the cysteine ​​residues of the one or more cysteine ​​proteases are maintained in a reduced state, (ii) removing substantially all oxygen from a region surrounding the one or more cysteine ​​proteases, and (iii) binding the one or more cysteine ​​proteases to a carbomer such that the one or more cysteine ​​proteases are non-covalently bound to the carbomer.

7. A composition comprising one or more cysteine ​​proteases, cysteine, and carbomer, wherein the composition is substantially anaerobic, and the proteolytic activity of the one or more cysteine ​​proteases is enhanced or stabilized, wherein the composition is obtained or is available by a method comprising (i) contacting the one or more cysteine ​​proteases with the cysteine, wherein the cysteine ​​residues of the one or more cysteine ​​proteases are maintained in a reduced state, (ii) removing substantially all oxygen from the region surrounding the one or more cysteine ​​proteases, and (iii) binding the one or more cysteine ​​proteases to the carbomer such that the one or more cysteine ​​proteases are non-covalently bound to the carbomer.

8. The composition according to claim 6 or 7, characterized in that, Oxygen is removed by degassing the composition.

9. The composition according to claim 6 or 7, characterized in that, The composition was packaged in a substantially oxygen-free atmosphere.

10. The composition of claim 6 or 7, further comprising a pharmaceutically acceptable carrier.

11. The composition of claim 6 or 7, further comprising a pharmaceutically acceptable diluent, excipient, surfactant, and / or adjuvant.

12. The composition of claim 6 or 7 further comprises a cosmetically acceptable carrier.

13. The composition of claim 6 or 7 further comprises a cosmetically acceptable diluent, excipient, surfactant, and / or adjuvant.

14. The composition of claim 6, 7 or 10, used for the treatment of diseases and disorders, for debridement, or for the treatment of burns, ulcers or gangrene, wherein the diseases and disorders are wounds.

15. The composition according to claim 6 or 7, characterized in that, The composition is for topical application.

16. The composition of claim 6, 7 or 12, for use in skin brightening, for epidermal exfoliation, or for application to wrinkles, skin blemishes, freckles, papules, acne, sunspots or scars, or for application to dry, aged or damaged skin.

17. The composition of claim 6, 7 or 12, for application to erythematous acne.

18. Use of the composition of claim 6 or 7 in the manufacture of an agent for the treatment of diseases and disorders, for debridement, or for the treatment of burns, ulcers, or gangrene, wherein the diseases and disorders are wounds.

19. Use of the composition of claim 6 or 7 in the manufacture of cosmetics for skin brightening, epidermal exfoliation, or application to wrinkles, skin blemishes, freckles, papules, acne, sunspots, or scars, or for application to dry, aging, or damaged skin.

20. Use of the composition of claim 6 or 7 in the manufacture of a cosmetic for application to rosacea.

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