WO2021130675A1 - Composition and methods of manufacture - Google Patents
Composition and methods of manufacture Download PDFInfo
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- WO2021130675A1 WO2021130675A1 PCT/IB2020/062349 IB2020062349W WO2021130675A1 WO 2021130675 A1 WO2021130675 A1 WO 2021130675A1 IB 2020062349 W IB2020062349 W IB 2020062349W WO 2021130675 A1 WO2021130675 A1 WO 2021130675A1
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- protease
- serine protease
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Definitions
- the present invention relates to compositions comprising at least one protease identified in the Carica papaya plant, methods for producing the compositions from the Carica papaya plant, compositions comprising the at least one protease obtained or obtainable by the aforementioned methods, the use of such compositions in the manufacture of medicaments and cosmetics, the use of such compositions in the treatment of diseases and disorders including wounds, the use of such compositions in cosmetic applications, and associated kits for carrying out a method or use of the invention.
- proteases such as papain and bromelain for wound debridement.
- the present invention provides a composition comprising an active serine protease extracted from the ripe fruit of the Carica papaya plant, such as an active serine protease extracted from alkai-treated, pulped ripe Carica papaya fruit.
- the active serine protease is substantially free of insoluble Carica papaya- derived material.
- the serine protease comprises a contiguous amino acid sequence having at least 95, 96, 97, 98 or 99% homology to (such as sequence identity with) amino acids 113 to 771 of SEQ ID NO:1.
- the serine protease has a molecular weight of from 65 to 75 kDa, such as about 70 kDa.
- the serine protease has a molecular weight of from 45 to 55 kDa and comprises a contiguous amino acid sequence having at least 95% homology to amino acids 142 to 618 of SEQ ID NO:1.
- the present invention provides a composition comprising an isolated, active serine protease having a molecular weight of from 45 to 55 kDa and comprising a contiguous amino acid sequence having at least 95% homology to amino acids 142 to 618 of SEQ ID NO:1.
- the present invention also provides a composition
- a composition comprising an isolated, active serine protease substantially free of insoluble Carica papaya- derived material, wherein the serine protease has a molecular weight of from 45 to 55 kDa and comprises a contiguous amino acid sequence having at least 95% homology to amino acids 142 to 618 of SEQ ID NO:1.
- the present invention provides a composition comprising a mixture of one or more active serine proteases derived or derivable from Carica papaya and one or more active cysteine proteases derived or derivable from Carica papaya.
- the one or more active proteases are extracted from ripe Carica papaya.
- the one or more active proteases are produced using a recombinant expression system.
- the present invention also relates to methods of preparing compositions comprising at least one active serine protease by extracting the protease from ripe Carica payaya.
- the present invention provides a process for preparing a composition comprising an active serine protease, which method comprises treating pulped ripe Carica papaya fruit with an alkali without subjecting the pulp to a heating step.
- the alkali is a weak alkali, preferably sodium bicarbonate.
- the compositions made by this process may further comprise one or more cysteine protease derived or derivable from Carica papaya.
- At least one soluble protease is separated from insoluble plant material after the alkali treatment.
- composition may be further treated to increase the concentration of the protease or proteases, for example, using freeze drying, dialysis, size exclusion chromatography or a combination thereof.
- the present invention further provides a composition comprising a serine protease obtained or obtainable by the methods of the invention.
- the present invention provides a pharmaceutical composition comprising an active Carica papaya serine protease together with a pharmaceutically acceptable carrier or diluent, as well as a cosmetic composition comprising an active Carica papaya serine protease together with a cosmetically acceptable carrier or diluent.
- a pharmaceutical composition comprising an active Carica papaya serine protease together with a pharmaceutically acceptable carrier or diluent
- a cosmetic composition comprising an active Carica papaya serine protease together with a cosmetically acceptable carrier or diluent.
- the present invention also provides a method of treating a patient suffering from a skin condition, the method comprising administering a composition of the invention to the affected area of the patient.
- the composition is used for the treatment of diseases and disorders including wounds.
- the composition is used for debridement.
- the composition is used for treating burns.
- the composition is used for treating ulcers.
- the composition is used for treating gangrene.
- the composition is applied topically.
- the present invention further provides in a seventh aspect a method of achieving a desired cosmetic outcome, such as smoothing and renewal of the epidermis of an individual, the method comprising applying a composition of the invention to the affected area of the patient.
- the present invention also provides a cosmetic method using the compositions of the invention.
- the composition is used for exfoliation, lightening skin, or for applying to wrinkles, skin blemishes, freckles, pimples, acne, rosacea, sun spots, scars or varicose veins, or for applying to dry, aged or damaged skin.
- the present invention provides use of the compositions of the first, second, fourth or fifth aspects in the manufacture of a medicament.
- the medicament is for the treatment of diseases and disorders including wounds.
- the medicament is for debridement.
- the medicament is for treating burns.
- the medicament is for treating ulcers.
- the medicament is for treating gangrene.
- the present invention provides use of the compositions of the first, second, fourth or fifth aspects in the manufacture of a cosmetic.
- the cosmetic is for exfoliation.
- the present invention provides the compositions of the first, second, fourth or fifth aspects, for use in the treatment of diseases and disorders including wounds.
- the treatment is debridement.
- the treatment is for ulcers.
- the composition is applied topically.
- the present invention provides the compositions of the first, second, fourth or fifth aspects, for use in exfoliation.
- kits comprising the compositions of the first, second, fourth or fifth aspects.
- the kits are used for performing the methods of the present invention.
- compositions of the present invention can be formulated, for example, as capsules, tablets, creams, ointments, solutions, pastes, drops, sprays, aerosols, vapours, wipes, patches, gauzes, gels or liquids. Accordingly, in one embodiment, the compositions of the present invention are provided or packaged as capsules, tablets, creams, ointments, solutions, pastes, drops, sprays, aerosols, vapours, wipes, patches, gauzes, gels or liquids and do not need to be reconstituted prior to use.
- active protease is meant a protease that is proteolytically active.
- fixation refers to the removal of dead and damaged tissue from a wound.
- derivable may be used interchangeably with the term “obtainable”.
- isolated is meant material that is substantially or essentially free from some or all of the components that normally accompany it in its native state.
- an “isolated rotease,” as used herein, refers to in vitro isolation and/or partial purification of a peptide or polypeptide protease molecule from its natural cellular environment, and from association with some or all of the components of the cell.
- proteins or compositions of the present invention includes proteins or compositions produced not only by a particular specified method, but also the same proteins or compositions however produced, for example, by sourcing proteases from fruits or vegetables, or by recombinant DNA technology or other genetic engineering methods, for example, by using a recombinant expression system.
- Suitable animals such as mammals, that fall within the scope of the invention include, but are not restricted 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).
- 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
- captive wild animals e.g. foxes, deer, dingoes.
- wild-type and “naturally occurring” are used interchangeably to refer to a protein that has the characteristics of that protein when isolated from a naturally occurring source.
- a wild type protein e.g., a polypeptide
- wound means an injury to living tissue in which the skin is cut or broken and includes dermal ulcers and burns.
- Dermal ulcers may include diabetic ulcers, pressure ulcers, venous (or varicose) ulcers and arterial ulcers.
- the reference to any prior art in this specification is not, and should not be taken as an acknowledgement or any form of suggestion that prior art forms part of the common general knowledge of the person skilled in the art.
- compositions Comprisina Serine Proteases
- the present invention relates to compositions comprising at least one active serine protease that can be extracted from or otherwise derived from the ripe fruit of the Carica papaya plant, such as following treatment of the pulped ripe fruit of the Carica papaya plant with an alkali, such as sodium bicarbonate.
- active serine proeteases refers to one or to more than one active serine protease.
- the active serine protease has an amino acid sequence comprising one or more of the sequences shown in SEQ ID NOs: 2 to 5, such as SEQ ID NOs: 2, 3 and 4, or all four sequences shown in SEQ ID NOs: 2, 3, 4 and 5, respectively.
- the at least one active serine protease typically comprises a contiguous sequence having at least 95% sequence homology (preferably sequence identity), such as 96, 97, 98 or 99% sequence homology (preferably sequence identity) with amino acids 142 to 618 of SEQ ID NO: 1 , which is the catalytic domain of the protein containing the key catalytic triad residues Asp, His and Ser (shown in SEQ ID NO: 1 at residues 151, 221 and 558 respectively) and the conserved substrate-binding site Asn at residue 326.
- sequence identity preferably sequence identity
- amino acids 142 to 618 of SEQ ID NO: 1 which is the catalytic domain of the protein containing the key catalytic triad residues Asp, His and Ser (shown in SEQ ID NO: 1 at residues 151, 221 and 558 respectively) and the conserved substrate-binding site Asn at residue 326.
- Amino acids 1-25 are a putative signal sequence and amino acids 26 to 112 are a putative pro region that is cleaved to form the mature protein.
- SEQ ID NO 2 to SEQ ID NO 5 are peptide fragments of SEQ ID NO 7.
- TLPTKPAPVM AAFSSKGPNI VTPEILKPDIT APGWVIAA YTRAQGPTNQ NFDR SEQ ID NO: 14 VQFNSVSGTS MSCPHVSGIV GLLKTLYPSWS PAAIR SEQ ID NO: 15 ATPFSYGAGH VQPNQAMNPG LVYDLNTK SEQ ID NO: 16 TLISIFSNDK FNCPRTNISL ADFNYPSITV PELK SEQ ID NO: 17 GISVTVKPK SEQ ID NO: 18
- the at least one active serine protease of the invention comprises the amino acid sequence from amino acids 118 to 763 of SEQ ID NO: 1 , with up to 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more, such as up to 3 or 5, amino acid modifications (deletions, insertions and/or substitutions) provided the protein retains serine protease activity.
- a polypeptide may be modified in various ways including amino acid substitutions, deletions, truncations, and insertions. Methods for such manipulations are generally known in the art.
- the serine proteases of the invention are in their active form i.e. not as inactive zymogens.
- the experimental results herein suggest that a protein isolated from Carica papaya and having an apparent molecular weight of 50 kDa was present as an active serine protease.
- at least one active serine protease of the invention may have a molecular weight of from 45 to 55 kDa, such as from 48 to 52 kDa, for example about 50 kDa.
- the molecular weight of the at least one protease of the invention may be about 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54 or 55 kDa.
- the molecular weight may be assessed by SDS PAGE, thus the protease has an average molecular weight as measured by SDS PAGE of from 45 to 55 kDa, such as from 48 to 52 kDa, for example about 50 kDa.
- the molecular weight can be calculated on the basis of the primary amino acid sequence.
- the active serine protease(s) is(are) typically in substantially isolated form. This means that they have been at least partially purified from their Carica papaya source material or recombinantly produced.
- the isolated active serine proteases are substantially free of insoluble plant materials such as cellulose, lignin and the like. This will typically be as a result of one or more purification steps to remove insoluble materials leaving the soluble active serine proteases in solution. This will be described further below. “Substantially free of insoluble plant materials” means that less than 2% w/w, such as less than 1% w/w or 0.5% w/w, of the composition contains insoluble plant materials derived from the source material for the protease.
- compositions Comprising Mixtures of Serine Proteases and Cysteine Proteases
- 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 that involves a nucleophilic cysteine thiol in a catalytic triad or dyad. They occur in a 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 kiwifruit ( Actinidia chinensis). In one embodiment, the cysteine protease is derived from a fruit.
- cysteine proteases include papain (EC 3.4.22.2), chymopapain (EC 3.4.22.6), bromelain (stem bromelain - EC 3.4.22.32 and fruit bromelain - EC 3.4.22.33), ficain (EC 3.4.22.3) and actinidain (EC 3.4.22.14).
- active cysteine proteases refers to one or to more than one active cysteine protease.
- One or more active serine proteases derived from Carica papaya may be present in a mixture containing one or more active cysteine proteases, such as one or more of the cysteine proteases referred to above that are derived from Carica papaya.
- one source of the active cysteine proteases can be the same as for the active serine proteases, i.e. the fruit of the Carica papaya plant.
- the papaya is ripe.
- ripe papaya as the source material instead of unripe papaya (which is the current commercial source of papain) is that the resulting composition comprising the cysteine protease has much lower levels of latex than typical commercial sources of papain. Latex can cause allergic reactions.
- the active cysteine proteases are typically in substantially isolated form as described above for the active serine proteases. This means that they have been at least partially purified from its biological source material such as Carica papaya or recombinantly produced.
- the isolated cysteine proteases are substantially free of insoluble plant materials such as cellulose, lignin and the like. This will typically be as a result of one or more purification steps to remove insoluble materials to leave the soluble active protease in solution.
- a process of the invention can be used to isolate at least one active serine protease of the invention from ripe papaya fruit.
- Ripe papaya fruit is typically yellow over most of the exterior of the fruit. Unripe papaya (from the skin of which latex is harvested to produce papain) is green. Ripe papaya should not be hard when pressed but should give slightly and retain slight indentations. If it is very soft when pressed then it is overripe.
- papaya flesh is separated from the skin and seeds.
- the entire fruit is used including the skin and seeds. The fruit, such as the flesh, is then mechanically disrupted to form a pulp (for example in a blender until a smooth pulp is obtained).
- the resulting pulp is then treated with an alkali.
- Suitable alkalis include strong bases such as NaOH and weak bases such as sodium bicarbonate.
- the alkali can be added as dry powder or in aqueous solution.
- the alkali is sodium bicarbonate and is typically added as a powder in an amount of at least 3% w/w, such as at least 5% w/w.
- the amount is typically less than 15% w/w, such as less than 12% w/w. In a particular embodiment the amount added is from 5 to 12% w/w.
- the alkali may have a pKa of less than 11.
- the alkali may be a bicarbonate or a carbonate or a combination thereof.
- the alkali may be a water-soluble alkali metal bicarbonate salt or a water-soluble alkali metal carbonate salt or a combination thereof.
- the amount of alkali added may be from about 1 % w/w to about 40% w/w, from about 1 % w/w to about 35% w/w, from about 1 % w/w to about 30% w/w, from about 1 % w/w to about 25% w/w, from about 1 % w/w to about 20% w/w, from about 1% w/w to about 15% w/w, from about 1% w/w to about 10% w/w, from about 2% w/w to about 40% w/w, from about 2% w/w to about 35% w/w, from about 2% w/w to about 30% w/w, from about 2% w/w to about 25% w/w, from about 2% w/w to about 20% w/w, from about 2% w/w to about 15% w/w, from about 2% w/w to about 10% w/w, from about 3% w/w to about
- the amount of alkali added may be from about 1% w/w to about 20% w/w. In a preferred embodiment, the amount of alkali added may be from about 1% w/w to about 15% w/w. In a particularly preferred embodiment, the amount of alkali added may be from about 4% w/w to about 15% w/w.
- the alkali is added in an amount such that the final pH of the composition after the alkali has reacted with the pulp is at least 7.5, such as from pH 7.5 to pH 11.5.
- the composition may have a final pH in the range of from about 7.0 to about 14.0, from about 7.0 to about 13.5, from about 7.0 to about 13.0, from about 7.0 to about
- the composition may have a final pH in the range of from about 7.5 to about 9.5.
- the reaction is performed with the pulp at a temperature of 30°C or less, preferably without subjecting the pulp to any heating step.
- the reaction may therefore for example be performed at room temperature, such as standard room temperature and pressure. It can also be carried out at lower temperatures, such as about 4°C. Accordingly, in some embodiments, the reaction is performed in a range of about 4°C to about 25°C, or about 4°C to about 22°C, or about 4°C to about 20°C.
- the treated pulp is then subjected to a separation step to remove insoluble plant materials (in the case of sodium bicarbonate, the separation step can be performed once the treated pulp is no longer effervescing).
- the treated pulp can be centrifuged and/or filtered. Centrifugation can be performed at for example between about 6000 and 18000 g. In one embodiment the treated pulp is centrifuged and the resulting supernatent is then filtered through a 0.22 pm filter.
- the resulting composition can be subject to one or more purification steps to concentrate and/or further separate the proteases of the invention from other cellular components.
- Suitable techniques include (i) gel filtration (size exclusion) chromatography to separate the proteins specifically on the basis of molecular weight e.g. using Sephadex G-100 as described in the examples; (ii) ionic exchange chromatography; (iii) dialysis; (iv) ammonium acetate precipitation and/or (v) freeze-drying.
- compositions/fractions from chromatography can be tested to confirm the continued presence of protease activity using, for example, the assays described in the examples (L-BapNA, optionally with a serine protease inhibitor or a cysteine protease inhibitor added during the assay to determine that the protease activity is due to serine protease or cysteine protease activity).
- the known molecular weight of the at least one serine protease of the invention can also be used to confirm the presence and amount of the at least one serine protease e.g. by SDS-PAGE.
- Purification processes may also conveniently be used to adjust the pH and/or salt concentration to levels suitable for the formulation of the composition into a pharmaceutical or cosmetic product.
- the process may further comprise a step of beating the mixture following addition of the alkali.
- the process may further comprise a step of filtering the mixture to obtain a filtrate.
- the process may further comprise a step of filtering the mixture to obtain a residue.
- the process may further comprise a step of freezing and thawing the composition. The mixture obtained may be frozen and thawed prior to filtering.
- the final composition is preferably substantially free of insoluble plant materials, for example containing less than about 2%, 1%, 0.5% or 0.1% w/w insoluble material.
- the percentage of protease by weight of total protein is in the range of about 0.01% to 5%.
- the final composition may comprise from about 1 to 100 lU/ml of serine protease activity.
- final composition may comprise from about 1 to about 10 lU/ml, about 1 to about 20 lU/ml, about 1 to about 30 lU/ml, about 1 to about 40 lU/ml, about 1 to about 50 lU/ml, about 1 to about 60 lU/ml, about 1 to about 70 lU/ml, about 1 to about 80 lU/ml, about 1 to about 90 lU/ml, about 10 to about 100 lU/ml, about 20 to about 100 lU/ml, about 30 to about 100 lU/ml, about 40 to about 100 lU/ml, about 50 to about 100 lU/ml, about 60 to about 100 lU/ml, about 70 to about 100 lU/ml, about 80 to about 100 lU/ml, about 90 to about 100 lU/ml, about 1 to about 90 lU/ml, about 10 to about 90 lU/ml, about 20 to about 90
- compositions comprising at least one serine protease of the invention, and optionally cysteine proteases, such as proteases derived from or derivable from Carica papaya, can be combined with pharmaceutically acceptable carriers or diluents to form a pharmaceutical composition.
- pharmaceutically acceptable carriers or diluents excludes water.
- compositions can typically also be used for pharmaceutical compositions.
- Such compositions can for example, be in the form of a cream; a lotion; a serum; an ointment; or a gel.
- the compositions can also be applied onto/impregnated into solid forms such as a wound dressing, for example a gauze pad and the like.
- compositions comprising the at least one serine protease of the invention, and optionally also comprising cysteine proteases, such as proteases derived from or derivable from Carica papaya, can also be combined with cosmetically acceptable carriers or diluents to form a cosmetic composition.
- cosmetically acceptable carriers or diluents excludes water.
- Cosmetic compositions according to the present invention can suitably include all active materials and appropriate components conventionally known for such formulations.
- Such components include, for instance, gelling agents, anionic polymers, thickeners, surfactants, hydrating agents, emollients, chelating agents, antioxidants, preservatives, buffering compounds, perfumes, fillers, colourings, volatile or non-volatile, modified or non-modified silicones and reducing agents.
- the relative proportions of the different components are suitably those that are used in conventional cosmetic formulations. It is envisaged that the person skilled in the art will naturally select the optionally present materials and incorporate these into the composition according to the invention in such a manner that the advantageous properties associated with the desirous enzyme activity are not significantly modified or eliminated. It is further envisaged that certain components may fulfil more than one criteria, for instance, glycerine is known to act as both a solvent and a humectant.
- compositions of the present invention may be present in the compositions of the present invention; foremost of these components is likely to be water, as a solvent. Amounts of water may range from about 1% to about 95%, such as from about 25% to about 90%, by weight of the composition.
- suitable solvents include glycerine and benzyl alcohol.
- the cosmetic compositions of the present invention may include one or more emulsifiers, for example, if the composition is in the form of a lotion, cream, gel or serum.
- Suitable emulsifiers that can be used in the present invention include, for example, one or more alkoxylated fatty alcohols, CM-22 alcohols, alkylpolyglycosides, Ci4-2o alkylglucoside, saponifiers, alkyl sulfates, monoalkyl and dialkyl phosphates, alkyl sulphonates, acyl isothionates, cetyl alcohol, stearyl alcohol, sorbitans, stearic acid, glyceryl stearate or any combinations thereof.
- Certain oil-in-water emulsion-based compositions of the invention may also include one or more stabilisers to stabilise the emulsion.
- Suitable stabilisers include, for example, alcohols, alkoxylated alcohols, fatty alcohols, glyceryl esters, such as, glyceryl stearate, gums, soaps, synthetic polymers, waxes, or any combinations thereof.
- Particularly suitable emulsion stabilisers include a stearyl alcohol or glyceryl stearate.
- compositions of the invention may further include one or more emollients to soften and soothe the skin.
- emollients comprise, for example, one or more fats and oils (hydrogenated or non-hydrogenated), such as, vegetable oil, castor oil, coconut oil and cocoa butter, shea butter, as well as esters, such as, tricapryl citrate and isononyl isonanoate, or any combinations thereof.
- compositions of the invention may include one or more humectants.
- a humectant is a component that absorbs or retains moisture. Suitable humectants that can be used in the present composition include, for example, urea, pyroglutamic acid, amino acids (e.g. glutamic acid), polyols or other compounds with hygroscopic properties, or any combinations thereof.
- the humectant is a polyol, such as a sorbitol.
- the primary humectants present include methyl-gluceth-20 and propylene glycol.
- Suitable preservatives for use in the compositions of the present invention include, for example, one or more alkanols such as phenoxy ethanol, ethylenediaminetetraacetic acid (EDTA) salts, EDTA fatty acid conjugates, isothiazolinone, parabens such as methylparaben and propylparaben, propylene glycols, sorbates, urea derivatives such as diazolidinyl urea and imidazolidinyl urea, quaternary ammonium salts, or any combinations thereof.
- alkanols such as phenoxy ethanol, ethylenediaminetetraacetic acid (EDTA) salts, EDTA fatty acid conjugates, isothiazolinone, parabens such as methylparaben and propylparaben, propylene glycols, sorbates, urea derivatives such as diazolidinyl urea and imidazolidinyl urea,
- Suitable chelating agents for inclusion in the compositions of the invention may include, for example EDTA derivatives, or any combinations thereof.
- the composition may include one or more buffering compounds that serve to adjust and maintain the pH of the composition.
- Suitable buffering agents include, for example, one or more adipic acids, glycines, citric acids, calcium hydroxides, magnesium aluminometa- silicates, triethanolamine, or any combinations thereof.
- the buffering agent should be present at a level suitable to ensure stable pH in the composition throughout its normal shelf life and period of use.
- the cosmetic compositions may also include one or more surfactants.
- Surfactants are of particular importance in compositions of the invention that are for the purposes of skin washes, such as soaps or face washes.
- Suitable surfactants include, for example, anionic, non-ionic, cationic, amphoteric, or any combinations thereof.
- the one or more surfactants are non-ionic surfactants.
- Suitable nonionic surfactants include, for example, one or more alkoxylated alcohols, ethoxylated alcohols, propoxylated alcohols, inter-dispersed ethoxylated-propoxylated alcohols, copolymers, fatty acids, alkyl phenols, polyglycosides, polyglucosides, n-alkylpyrrolidones, block copolymers, or any combinations thereof.
- the cosmetic compositions of the invention are in the form of a cream; a lotion; a serum; a face wash; a shampoo; a foam; a skin wash; a skin tonic; an ointment; or a gel.
- any composition suitable for administering the active enzymes of the invention to the epidermis can be used.
- compositions of the invention should contain sufficient proteolytic activity to enable from about 0.1 lU/ml to about 100 lU/ml of total serine protease activity to be applied to the skin per dose, wherein a dose is an amount of between about 2 ml and about 10 ml of product.
- compositions of the invention should contain sufficient proteolytic activity to enable from about 1 to about 10 lU/ml, about 1 to about 20 lU/ml, about 1 to about 30 lU/ml, about 1 to about 40 lU/ml, about 1 to about 50 lU/ml, about 1 to about 60 lU/ml, about 1 to about 70 lU/ml, about 1 to about 80 lU/ml, about 1 to about 90 lU/ml, about 10 to about 100 lU/ml, about 20 to about 100 lU/ml, about 30 to about 100 lU/ml, about 40 to about 100 lll/ml, about 50 to about 100 lll/ml, about 60 to about 100 lll/ml, about 70 to about 100 lll/ml, about 80 to about 100 lll/ml, about 90 to about 100 lll/ml, about 1 to about 90 lll/ml, about 10 to about 90 l, about 10 to
- Serine protease activity can be measured by the L-BApNA assay described in the examples with the use of a serine protease inhibitor during the assay to determine the serine protease component.
- the benzoyl-L-arginine ethyl ester (BAEE) assay described in GB 2,440,117 may be used.
- the serine protease activity is in the range of from about 5 units to about 60 units per application dose.
- compositions of the invention may also contain a similar level of cysteine protease activity.
- Cysteine protease activity can be measured by the L-BApNA assay described in the examples with the use of a cysteine protease inhibitor during the assay to determine the cysteine protease component.
- compositions of the present invention can also be used in various cosmetic applications such as a cosmetic method for promoting smoothing and renewal of the epidermis of an individual.
- the cosmetic compositions of the invention are applied to the skin of the face, hands and neck, however other skin surfaces may be subjected to the cosmetic treatment.
- the only contraindication is that particularly sensitive areas of skin, such as inflamed skin or the skin close to the eyes be avoided.
- the compositions of the invention are usually applied directly to the skin surface manually by the user as is conventional.
- the methods of the invention therefore include applying a cosmetic composition of the invention to the epidermis of the individual.
- 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.
- compositions of the present invention can also be used in a variety of cosmetic applications.
- the present invention further provides methods of debridement of wounds comprising the topical application of a composition of the invention, compositions of the present invention for use in a method of treating wounds, methods of treating an individual suffering from burns, wherein the method comprises administering topically, or by other routes of administration, to an affected area of the individual a preparation of the invention, compositions of the invention for use or when used in a method of treating wounds, methods of enhancing wound healing, comprising administering topically, or by other routes of administration, to a wound a composition of the invention, methods of exfoliating or lightening skin comprising applying to the skin a cosmetic composition of the invention, uses of a cosmetic composition of the invention to exfoliate or lighten skin, methods of treating dry, aged or damaged skin comprising applying to the skin a cosmetic composition of the invention, and uses of a cosmetic composition of the invention to treat dry, aged or damaged skin.
- the protease compositions of the present invention may in particular be used to prevent, treat, reduce or ameliorate a variety of skin conditions including wounds, including chronic wounds such as diabetic ulcers, pressure ulcers, venous ulcers and arterial ulcers, and other skin conditions including but not limited to, eczema, psoriasis, acne, rosacea, ichthyosis, vitiligo, hives, seborrheic dermatitis.
- the pharmaceutical compositions can be used to debride wounds.
- Such wounds will typically contain necrotic tissue and include diabetic ulcers, pressure ulcers, venous ulcers, arterial ulcers and the like. Thus such wounds may be chronic wounds.
- the compositions or dressings etc. that are impregnated with a composition
- the compositions are applied to the affected area. This will typically be carried out at least once daily, such as twice daily. The amount of composition and frequency of application can be determined by a physician.
- compositions of the present invention may be administered therapeutically or cosmetically.
- compositions may be administered to a subject already suffering from a condition, in an amount sufficient to cure or at least partially arrest the condition and any complications.
- the quantity of the composition should be sufficient to effectively treat the patient.
- compositions may also be administered in the form of liposomes.
- Liposomes may be derived from phospholipids or other lipid substances, and may be formed by mono- or multi- lamellar hydrated liquid crystals dispersed in an aqueous medium. Any non-toxic, physiologically acceptable and metabolisable lipid capable of forming liposomes may be used.
- the compositions in liposome form may contain stabilisers, preservatives and excipients.
- Preferred lipids include phospholipids and phosphatidyl cholines (lecithins), both natural and synthetic.
- composition of the present invention may be used in the manufacture of a medicament for preventing, treating, reducing or ameliorating a skin condition of a subject.
- the composition of the present invention may be also used in the manufacture of a cosmetic for preventing, treating, reducing or ameliorating a skin condition of a subject.
- the “therapeutically effective” dose level for any particular patient will depend upon a variety of factors including the condition being treated and the severity of the condition, the activity of the composition employed, the age, body weight, general health, sex and diet of the patient, the time of administration, the route of administration, the duration of the treatment, and any drugs used in combination or coincidental with the treatment, together with other related factors well known in the art.
- One skilled in the art would therefore be able, by routine experimentation, to determine an effective, non-toxic amount of the composition which would be required to treat applicable conditions.
- compositions of the present invention can be administered by standard routes.
- the compositions may be administered by topical routes.
- compositions of the present invention are administered topically to the affected area of an individual.
- compositions may be administered by other enteral/enteric routes, such as rectal, sublingual or sublabial, or via the central nervous system, such as through epidural, intracerebral or intracerebroventricular routes.
- Other locations for administration may include via epicutaneous, transdermal, intradermal, nasal, intraarterial, intracardiac, intraosseus, intrathecal, intraperitoneal, intravesical, intravitreal, intracavernous, intravaginal or intrauterine routes.
- the treatment would be for the duration of the disease state.
- compositions disclosed herein may be administered as a single agent or as part of a combination therapy approach to the methods disclosed herein, either at diagnosis or subsequently thereafter, for example, as follow-up treatment or consolidation therapy as a compliment to currently available therapies for such treatments.
- the compositions disclosed herein may also be used as preventative therapies for subjects who are genetically or environmentally predisposed to developing such diseases.
- compositions may be administered regularly for as long as is needed, such as until an improvement in the condition is seen. Therefore they may be administered hourly, multiple times a day, daily, multiple times a week, weekly, monthly or at whatever frequency is deemed appropriate.
- kits of the present invention facilitate the employment of the methods and uses of the present invention.
- kits for carrying out a method or use of the invention contain all the necessary reagents and means to carry out the method.
- the kit may comprise a composition of the present invention and, optionally, means to administer the composition such as devices for point of care methods.
- kits described herein will also comprise one or more containers.
- a compartmentalised kit includes any kit in which compositions are contained in separate containers, and may include small glass containers, plastic containers or strips of plastic or paper. Such containers may allow the efficient transfer of compositions from one compartment to another compartment whilst avoiding cross-contamination of compositions, and the addition of agents or solutions of each container from one compartment to another in a quantitative fashion.
- kits of the present invention will also include instructions for using the kit to perform the appropriate methods and uses.
- Methods, uses, compositions and kits of the present invention are equally applicable to any animal, including humans, for example including non-human primate, equine, bovine, ovine, caprine, leporine, avian, feline and canine species. Accordingly, for application to different species, a single kit of the invention may be applicable, or alternatively different kits, for example containing compositions specific for each individual species, may be required.
- Figure 1 Overlay-zymography schematic representation. Overlay-zymography process and subsequent excision of bands from enzyme activity spot to process on SDS PAGE gels.
- Figure 2 Representation of debriding assay using Franz cell diffusion system (Shi, Ermis et al. 2009).
- FIG. 3 Bar chart plot of the L-BApNA proteolytic activity of OPAL A and OPAL B filtrates.
- Figure 4 Spectrophotometric plot of enzymatic hydrolysis of L-BApNA by OPAL B filtrate and with addition of E-64. Representative plot showing enzymatic hydrolysis of the substrate L-BAPNA by OPAL B filtrate, and partial activity inhibition by cysteine protease inhibitor E-64, suggesting the presence of other proteases in addition to cysteine proteases.
- Figure 5 Spectrophotometric plot of the effect of protease inhibitor cocktail (PIC) on the proteolytic activity of OPAL B filtrate. Representative trace showing enzymatic hydrolysis of the substrate L-BAPNA by OPAL B filtrate, and its complete inhibition by the PIC.
- Figure 6 Bar chart plot of effect of different protease inhibitor classes on the proteolytic activity of OPAL B filtrate.
- PIC protease inhibitor cocktail
- Figure 7 Spectrophotometric plot of the effect of E-64+AEBSF on the proteolytic activity of OPAL B filtrate. Representative trace showing the complete inhibition of L-BAPNA activity in OPAL B filtrate by the combination of cysteine protease inhibitor E-64 and serine protease inhibitor AEBSF.
- Figure 8 Overlay zymography analysis of OPAL B filtrate.
- Figure 9 Overlay zymography analysis of OPAL B filtrate and effect on using inhibitors. Native PAGE gel bands in OPAL B samples were obtained and the protein bands were subsequently transferred from gel to nitrocellulose membrane. Zymography analysis shows the two enzymatic activities in control and complete inhibition of upper band (— ) with cysteine protease inhibitor E-64 and inhibition of lower band ( — ) with serine protease inhibitor AEBSF.
- Figure 10 Silver staining of SDS PAGE gel run with elutions 2-10.
- FIG 11 SDS PAGE western blotting analysis of OPAL B filtrate treated with serine protease probe using various treatments.
- FIG 13 Differential digestion of AWE substrate (percentage activity) by dialyzed OPAL A and OPAL A+10% urea as compared to native OPAL A filtrate.
- Dialyzed OPAL A had enhanced debriding activity on both fibrin and elastin but not collagen which correlates with the lower activity of OPAL A towards collagen.
- Figure 14 In vivo case study regarding management of severe pressure ulcer with OPAL B, pre-commencement of treatment with Opal B. Central area of necrosis. Failed attempt to reduce the size of the ulcer with a romboid flap. Distal part of the flap dusky. Warning - graphic images.
- Figure 15 In vivo case study regarding management of severe pressure ulcer with OPAL B, treatment day 2. Delineation of area that will become necrotic from that which will be viable. Wound edge pink and better blood flow to the skin flap. Warning - graphic images.
- Figure 16 In vivo case study regarding management of severe pressure ulcer with OPAL B, treatment day 4. Clear demarcation between viable and non-viable tissue. Viable tissue looks clean and pink. Skin flap looks fully viable. Warning - graphic images.
- Figure 17 In vivo case study regarding management of severe pressure ulcer with OPAL B, treatment day 16. Wound debrided surgically, sutures removed and flap excised. Early granulation of the clean wound surface is evident. Wound edge is pink and suggestion of new skin growth (exposure adjusted to match skin colour of previous images). Warning - graphic images.
- Figure 18 In vivo case study regarding management of severe pressure ulcer with OPAL B, treatment day 19. Skin and wound colour becoming dusky, particularly wound edges. Wound remains clean (exposure adjusted to match skin colour of earlier images). Warning - graphic images.
- OPAL A filtrate was performed in sterile conditions.
- the ends of the ripe fruit of the Carica papaya plant were trimmed and the skin was peeled.
- the flesh of the fruit was quartered and seeds were then excised.
- the papaya pieces were mixed in a blender until a smooth pulp was obtained.
- the pulp was placed in a beaker, which was inside a water bath set at 80°C and stirred continuously until the temperature of the pulp was 55°C (measured using a glass thermometer). 10% by weight dry sodium bicarbonate was added to the beaker.
- the beaker was then placed in the water bath, set at 55°C and stirred slowly for 5 minutes.
- L-BApNA activity at A410 was then measured both immediately and then after 2 hours using a Synergy HT microplate reader (cat. 12926527, Bio-Tek Instruments, Thermo Fisher Scientific, UK). The difference in values was plotted and the fractions with higher L-BApNA activity were run through SDS PAGE gels, which were then stained using Coomassie or silver staining.
- a 1.5 mM stock solution of L-BApNA (B-3133 sigma) was prepared by dissolving 63 mg of L-BApNA in 1.5 ml dimethyl sulfoxide (DMSO), and then made up to 100 ml with water. Hydrolysis of the L-BApNA at the bond between the arginine and the p-nitroaniline moieties releases the chromophore p-nitroaniline, which can be detected by spectroscopy at an absorbance of 410 nm in International Units (IU). IU is defined as the amount of enzyme that causes an increase in absorbance of 0.01 units/min under excess substrate conditions (zero order kinetics).
- Solids specific activity is expressed in lU/mg, while in the case of liquid formulations, the activity is expressed in lU/mL.
- a series of titration runs were carried out with a range of substrate concentrations. The minimum concentration thus found is 0.2 mM or 1:6 dilution of the stock 1.5 mM solution.
- Table 1 shows reagent doses used for all kinetic experiments. Phosphate buffer at pH 6 was used in the L-BApNA assays because it gave the highest enzymatic activity reading compared to other buffers.
- E-64 (l U PAC name: (1S,2S)-2-(((S)-1-((4-Guanidinobutyl)amino)-4-methyl-1-oxopentan- 2-yl) carbamoyl) cyclopropane carboxylic acid) is a strong and irreversible specific inhibitor of cysteine proteases.
- the trans-epoxysuccinyl group (active moiety) of E-64 irreversibly binds to the active thiol group of cysteine proteases, thereby inhibiting them.
- E-64 does not react with non-protease enzymes and does not inhibit serine proteases.
- 2,2’-dipyridyldisulfide (2DPS) is used as a thiol-specific reversible inhibitor for cysteine proteases, including papain, bromelain, and ficin.
- PICs protease inhibitor cocktail
- Overlay zvmoaraohv Overlay-zymography was carried out according to the procedure in Vinokurov et al. (2005) with some modifications ( Figure 1). Briefly, a nitrocellulose membrane was soaked in a 1.2 mg/ml_ L-BApNA solution in water. The membrane was subsequently left to air dry for 5 minutes and then laid on top of the native PAGE gel after running the samples through, which were slightly soaked in their respective running buffers. Two additional experiments were carried out in which membranes were soaked in 35M b-alanine, 0.14 M acetic acid, pH 4.3, with either 10 mM DTT or 25mM cysteine. All zymographies were then incubated in a closed chamber at 37°C for 30-60 min. The membrane was removed and again left to dry for 5 minutes and the p-nitroaniline was visualized by diazotization.
- Diazotization was performed by following the protocol detailed in Hosseininaveh et al. (2009). Briefly, the membrane was soaked sequentially for 5 minutes in a sodium nitrite solution (1 mg/ml_ in 1 M HCI), then an ammonium sulfamate solution (5 mg/ml_ in 1M HCI) and finally a NNED solution (N-(l-naphthyl)-ethylenediamine dihydrochloride) (0.5 mg/ml_ in 48% v/v ethanol/water), for about 30 seconds to 1 minute until any diazotized p-nitroaniline became clearly visible as a purple smear/bands.
- a sodium nitrite solution (1 mg/ml_ in 1 M HCI)
- an ammonium sulfamate solution 5 mg/ml_ in 1M HCI
- a NNED solution N-(l-naphthyl)-ethylenediamine dihydrochloride
- Protein bands identified with colloidal-coomassie staining were analyzed by MALDI-Mass fingerprinting at the PNAC Facility, Department of Biochemistry at the University of Cambridge. The gel bands were excised and subjected to the following treatment - 30 min per step, 20°C, in 200 pL 100 mM ammonium bicarbonate/50% acetonitrile: 1) Reduction with 5 mM Tris (2-carboxyethyl) phosphine; 2) Alkylation by addition of iodoacetamide (25 mM final concentration); and 3) Removal of liquid and then wash.
- the gel pieces were dried in vacuo for 10 min and then 25mI 100mM ammonium bicarbonate containing 5 pg/mL modified trypsin (Promega) was added. Digestion was performed for 17 h at 32°C. Peptides were recovered and desalted using pC18 ZipTip (Millipore) and eluted to a maldi target plate using 1-2 mI_ alpha-cyano-4- hydroxycinnamic acid matrix (Sigma) in 50% acetonitrile/0.1% trifluoroacetic acid.
- Peptide masses were determined using a Bruker ultrafleXtreme Maldi mass spectrometer in reflectron mode and ms/ms fragmentation peroformed in LIFT mode. Data analysis was undertaken with FlexAnalysis, BioTools and ProteinScape software (Bruker). Database searches of the combined mass fingerprint data were performed using Mascot (Matrix Science). Where required, additional manipulation was performed through Protein Prospector.
- the AWE debriding assay is an in vitro surrogate of wound necrotic tissue proteolysis activity developed by Health Point (Shi, Ermis et al. 2009). It has been shown to compare well to in vivo animal data.
- the AWE substrate consists of a pellet of three wound related extra cellular matrix proteins (collagen, elastin and fibrin), each tagged with a different fluorophore. Gradual degradation of this matrix can be measured by progressive increase in fluorescence intensity in a Franz diffusion cell setup ( Figure 2). The final readings for the experiments are taken at 24 hours.
- FITC Fluorescein Isothiocyanate
- R144 Elastin - Rhodamine
- Thrombin and fibrinogen (605157, 341573) from Merck chemicals limited.
- Fibrinogen was labelled with 7-amino-4-methyl coumarin by mixing fibrinogen in a coumarin solution (0.02 mg/ml_ in Tris buffer) with a final fibrinogen concentration of 10 mg/ml_ (in Tris buffer). The mixture was incubated at room temperature with rotary shaking for 1 hour. The coumarin-labelled fibrin was derived by adding a thrombin solution (2.5 units/mL) to the fibrinogen-coumarin solution and allowed to clot for 2 hours.
- the formed clots were washed 3 times with water and allowed to stand overnight in distilled water and methanol (1:1) to remove excess dye. The clots were then transferred to a glass container with transparent non-sticky filter paper and allowed to dry for 3 days. The dried fibrin-coumarin was ground into fine powder using a mortar and pestle.
- Collagen-FITC, elastin-rhodamine and fibrin-coumarin were mixed according to the composition referred to in Table 4. Except fibrinogen, all the materials were weighed into a 50 ml_ conical centrifuge tube. Using a tissue tearer the materials were homogenised in 10 ml_ Tris buffer for 3-5 minutes. A 10 ml_, 15-mg/mL fibrinogen solution was prepared in Tris buffer pH 6.8 in a separate tube. The two solutions were combined and thoroughly mixed by using the tissue tearer for about 2 minutes. Thrombin solution (50 U/mL) was added and quickly mixed, and the solution was then poured into a petri dish containing a 90 mm non reactive membrane and allowed to clot for 1 hour. The clotted substrate was then rinsed with water 3 times (5 minutes each) to remove thrombin. After washing, excess water was removed by using tissue paper and stored at 4°C until further use.
- the 9mm AWE substrate was placed on the top of a non-reactive nitrocellulose membrane and placed in the Franz diffusion cell between the two chambers, of which the lower (receptor cell) was filled with Tris buffer containing 1% (v/v) penicillin- streptomycin ( Figure 2).
- the sample holder was placed on the top and the whole assembly was fastened with a screw clamp to the receptor cell and surrounded with a 35°C water bath. The sample was then loaded in the upper chamber of the sample holder and covered with parafilm in which an air hole was made.
- the solution was mixed using a magnetic stirrer at 500 rpm throughout the experiment.
- CDn In x Vcell
- CDn and In are the cumulative digestion parameter and fluorescent intensity at hour n
- Vcell is the volume of the cell (5.1 ml_).
- Example 1 OPAL B filtrate a variant of OPAL A filtrate and its proteolytic activity
- Carica papaya belongs to the small family of Caricaceae and is one of the major fruits cultivated in tropical and sub tropical zones for its edible fruit and latex.
- the fruits used in these examples are ripe papaya fruits, mainly obtained from Brazil and Jamaica, purchased in local UK-based supermarkets.
- Most of the well-characterized and intensively studied cysteine proteases are members of the papain family.
- Papain, chymopapain A and B, chymopapain M, and caricain have all been extracted from the latex of Carica papaya but have never been characterized from the ripe fruit.
- OPAL B appeared to exhibit an almost 8-fold increased catalytic activity (linear increase in A410 substrate release over time), as compared to OPAL A ( Figure 3).
- addition of E-64 to OPAL B filtrate in excess did not result in complete inhibition of activity, as in OPAL A. Therefore, and without wishing to be bound by theory, the increase of A410 values with substrate could be due to non-thiol-dependent (non-cysteine proteases) catalysis.
- the fact that treatment with the cysteine protease inhibitor E-64 did not completely abolish protease activity suggests that both cysteine and non-cysteine proteases were present in the OPAL B filtrate, as shown in Figure 4.
- PIC Protease Inhibitor Cocktail
- Example 3 Zymography assays to functionally identify proteases determined by enzyme kinetics
- OPAL B filtrate appears to be a complex mixture of many proteins. Therefore, to individually visualize the protease activities that were detected by spectrophotometry assays, we used a technique called overlay zymography. In this technique, proteins are first separated on a standard native PAGE gel, which preserves enzymatic activity. A membrane containing substrate solution (L-BApNA at 1.2 mg/mL) is then overlaid onto the gel, and developed using a colour-enhancing agent. This enables active proteases to be directly visualized on the membrane.
- L-BApNA L-BApNA at 1.2 mg/mL
- OPAL B was concentrated and purified through size exclusion chromatography using a sephadex G-100 column that separates proteins in the range of 4 kDa to 150 kDa.
- the bands in the fractions were selected and analysed by LC/MS-MS analysis (namely the intensity bands present in the 6 th fraction at the 80 kDa, 50 kDa and 30 kDa bands) . These were matched to a Carica papaya sequence shown in SEQ ID NO: 1 that had been putatively identified as a subtilase.
- OPAL A a composition known as OPAL A from the flesh of ripe papaya ( Carica papaya). This process involves a heating step followed by treatment with sodium bicarbonate and then a filtration step.
- Our previous analysis of OPAL A identified cysteine protease activity.
- OPAL A has shown activity in treating a range of disorders.
- We have modified the OPAL A process by omitting the heating step to produce OPAL B.
- OPAL B contains additional protease activity that is not related to cysteine proteases. We have characterized this as being due to the presence of at least one serine protease which is not present in OPAL A.
- This protein appears to be present in more than one form, but only a protein having an average molecular weight of 50 kDa as determined by SDS-PAGE showed serine protease activity.
- LC-MS/MS analysis showed the proteins as having sequences identical to the sequence of a C. papaya sequence putatively identified as a subtilase (Othman and Nuraziyan, 2010), but this was not confirmed by biochemical characterization. Further, this subtilase when expressed recombinantly has a molecular weight of about 70 kDa compared to the 50 kDa molecular weight of the active serine protease that we have presently identified.
- OPAL B contains a mixture of both one or more proteolytically active serine proteases and one or more proteolytically active cysteine proteases, whereas until now the only known active proteases that have been recognised in Carica papaya have been cysteine proteases.
- Debridement is the removal of necrotic (dead) tissue and foreign material from wounds to expose underlying viable tissue. This process promotes and accelerates wound healing.
- cysteine proteases identified in the OPAL A filtrates would be active in debridement measured using an Artificial Wound Eschar (AWE) debriding assay.
- the AWE debriding assay is an in vitro surrogate of wound necrotic tissue proteolysis activity developed by Health Point (Shi, Ermis et ai. 2009). It has been shown to compare well to in vivo animal data and was therefore used to assess debriding efficacy of OPAL A filtrate formulations.
- the AWE substrate consists of a pellet of three wound-related extra cellular matrix proteins, collagen, elastin and fibrin, each tagged with a different fluorophore. Gradual degradation of this matrix can be measured by progressive increase in fluorescence intensity in a Franz diffusion cell setup. The final readings for the experiments are taken at 24 hours.
- Example 7 Concentrating OPAL A filtrate activity using dialysis to improve debriding efficacy
- the L-BApNA activity of freshly prepared OPAL A filtrate is on average 0.60 lU/mL.
- the protein content of fresh OPAL A filtrate measures around 0.7 mg/mL, corresponding to a specific solid activity of around 1 lU/mg. Compared to the activity of pure latex papain of around 10 lU/mg, this approximates to about 9% papain content of the OPAL solids.
- the AWE debriding assay performed in Example 6, as shown in Figure 12 reveals a significantly higher activity of OPAL A than would be expected from its nominal papain content (0.6 lll/mL versus 30 lll/mL for latex papain).
- Results in Table 5 show almost full retention of activity for either dialysis using cut-off sizes of 12 and 25 kDa and combined with a solid reduction of around 86%.
- Table 5 Specific activity and solid content of native OPAL A filtrate and dialyzed OPAL A filtrates with 12 kDa and 25 kDa cut-off molecular sizes.
- dialyzed OPAL A filtrate showed full retention of proteolytic activity in comparison with the native OPAL A filtrate, but a solid reduction of 86% less than control. This establishes that the majority of solids in OPAL A filtrate are non-proteolytic in nature, and exist as low molecular weight sugars that can be removed through dialysis. Dialyzed OPAL A filtrate had enhanced debriding activity on both fibrin and elastin as compared to latex papain and native OPAL A, but a marginally minor effect on collagen compared to native OPAL A.
- Example 8 In vivo case study regarding management of severe pressure ulcer with
- OPAL B therefore appears to provide a therapeutic benefit in the healing of severe ulcers through a multifactorial mode of action involving at least tissue regenerative and anti-necrotic / debriding factors.
- a vacuum dressing was applied in an attempt to clear excessive exudate produced by the ulcerated wound. Additional surgical intervention to reduce the size of the ulcer with a rhomboid flap was also attempted.
- the severity of the wound persisted ( Figure 14).
- OPAL B may have a number of modes of action: OPAL B appeared to localise tissue that was not viable, and to reverse marginal ischaemia, thereby reducing the amount of necrotic tissue. There is also evidence that OPAL B had begun to debride the slough in the ulcer.
- SEQ ID NO: 1 catalytic triad, Asn 336 and peptides of SEQ ID NOs: 2 to 5 shown in bold
- Amino acids 1-25 are a putative signal sequence and amino acids 26 to 112 are a putative pro region that is cleaved to form the mature protein.
- Arkin AP Youvan DC. An algorithm for protein engineering: simulations of recursive ensemble mutagenesis, Proc Natl Acad Sci U S A. 1992 Aug 15;89(16):7811-5.
- Hosseininaveh V Bandani A
- Hosseininaveh F Digestive proteolytic activity in the Sunn pest, Eurygaster integriceps. J Insect Sci. 2009;9:1-11.
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BR112022012775A BR112022012775A2 (en) | 2019-12-27 | 2020-12-22 | COMPOSITION AND MANUFACTURING METHODS |
US17/788,986 US20230054563A1 (en) | 2019-12-27 | 2020-12-22 | Composition and methods of manufacture |
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WO2022140817A1 (en) * | 2020-12-31 | 2022-07-07 | Phoenix Eagle Company Pty Ltd | Anti-viral proteases and methods of use |
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US6187743B1 (en) | 1999-07-22 | 2001-02-13 | Deroyal Industries, Inc. | Composition and method for enhancing wound healing |
WO2004008887A1 (en) | 2002-07-23 | 2004-01-29 | Phoenix Eagle Company Pty Ltd | Fruit and/or vegetable derived composition |
GB2440117A (en) | 2006-07-19 | 2008-01-23 | Elemis Ltd | Skin care composition comprising a trienzyme activity |
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US20070275045A1 (en) * | 2006-02-28 | 2007-11-29 | Evans Robin D | Composition for the treatment of warts and molluscum contagiosum |
EP2226382A1 (en) * | 2009-03-03 | 2010-09-08 | B.R.A.I.N. Biotechnology Research and Information Network AG | Protease for wound conditioning and skin care |
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US6187743B1 (en) | 1999-07-22 | 2001-02-13 | Deroyal Industries, Inc. | Composition and method for enhancing wound healing |
WO2004008887A1 (en) | 2002-07-23 | 2004-01-29 | Phoenix Eagle Company Pty Ltd | Fruit and/or vegetable derived composition |
GB2440117A (en) | 2006-07-19 | 2008-01-23 | Elemis Ltd | Skin care composition comprising a trienzyme activity |
Non-Patent Citations (19)
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"Methods in Cell Biology", vol. XIV, 1976, ACADEMIC PRESS, pages: 33 |
ALARA, 0. R. : " Carica papaya: comprehensive overview of the nutritional values, phytochemicals and pharmacological activities", ADVANCES IN TRADITIONAL MEDICINE, 08-2020, pages 1 - 31, XP037711135 * |
ANONYMOUS: "Prep Up Your Skin With Papaya : 10 Amazing Masks For Glowing Skin", XP055899929, Retrieved from the Internet <URL:https://www.powerofpositivity.com/prep-up-your-skin-with-papaya/> [retrieved on 20220310] * |
ARKIN APYOUVAN DC: "An algorithm for protein engineering: simulations of recursive ensemble mutagenesis", PROC NATL ACAD SCI USA., vol. 89, no. 16, 15 August 1992 (1992-08-15), pages 7811 - 5, XP002989624, DOI: 10.1073/pnas.89.16.7811 |
DELAGRAVE SGOLDMAN ERYOUVAN DC: "Recursive ensemble mutagenesis", PROTEIN ENG, vol. 6, no. 3, 1993, pages 327 - 31, XP001109463 |
HAKIM RACHMI FANANI, FAKHRURRAZI, DINNI: "Effect of Carica papaya Extract toward Incised Wound Healing Process in Mice ( Mus musculus ) Clinically and Histologically", EVIDENCE-BASED COMPLEMENTARY AND ALTERNATIVE MEDICINE, OXFORD UNIVERSITY PRESS, US, vol. 2019, 19 November 2019 (2019-11-19), US , pages 1 - 5, XP055899933, ISSN: 1741-427X, DOI: 10.1155/2019/8306519 * |
HOSSEININAVEH V, BANDANI A, HOSSEININAVEH F.: "Eurygaster integriceps", J INSECT SCI., vol. 9, 2009, pages 1 - 11 |
IWEALA E E J, UHEGBU F O, OGU G N: "Preliminary in vitro antisickilng properties of crude juice extracts of Persia Americana, Citrus sinensis, Carica papaya and Ciklavit", AFR. J. TRAD. CAM, vol. 7, no. 2, 1 January 2010 (2010-01-01), pages 113 - 117, XP055899924 * |
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OTHMAN, R. ; NURAZIYAN, A.: "Fruit-specific expression of papaya subtilase gene", JOURNAL OF PLANT PHYSIOLOGY, ELSEVIER, AMSTERDAM, NL, vol. 167, no. 2, 15 January 2010 (2010-01-15), AMSTERDAM, NL, pages 131 - 137, XP026801232, ISSN: 0176-1617 * |
OTHMANNURAZIYAN: "Fruit-specific expression of papaya subtilase gene", J. PLANT PHYSIOLOGY, vol. 167, no. 2, 2010, pages 131 - 137, XP026801232 |
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SHI LERMIS RLAM KCOWART JATTAR PAUST D: "Study on the debridement efficacy of formulated enzymatic wound debriding agents by in vitro assessment using artificial wound eschar and by an in vivo pig model", WOUND REPAIR REGEN, vol. 17, no. 6, November 2009 (2009-11-01), pages 853 - 62 |
STARLEY I F, ET AL.: "The treatment of paediatric burns using topical papaya", BURNS., BUTTERWORTH HEINEMANN., GB, vol. 25, no. 7, 1 November 1999 (1999-11-01), GB , pages 636 - 639, XP002999003, ISSN: 0305-4179, DOI: 10.1016/S0305-4179(99)00056-X * |
VINOKUROV KSOPPERT BELPIDINA EN: "An overlay technique for postelectrophoretic analysis of proteinase spectra in complex mixtures using p-nitroanilide substrates", ANAL BIOCHEM., vol. 337, no. 1, 1 February 2005 (2005-02-01), pages 164 - 6, XP004707223, DOI: 10.1016/j.ab.2004.10.043 |
Cited By (1)
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---|---|---|---|---|
WO2022140817A1 (en) * | 2020-12-31 | 2022-07-07 | Phoenix Eagle Company Pty Ltd | Anti-viral proteases and methods of use |
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AU2020411053B2 (en) | 2022-08-18 |
AU2022268382A1 (en) | 2022-12-22 |
EP4081233A1 (en) | 2022-11-02 |
JP2023512422A (en) | 2023-03-27 |
CA3163074A1 (en) | 2021-07-01 |
CN115151269A (en) | 2022-10-04 |
BR112022012775A2 (en) | 2022-09-20 |
AU2020411053A1 (en) | 2022-07-14 |
GB201919385D0 (en) | 2020-02-05 |
KR20220123526A (en) | 2022-09-07 |
ZA202207539B (en) | 2023-12-20 |
EP4081233A4 (en) | 2024-01-10 |
US20230054563A1 (en) | 2023-02-23 |
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