[0001 ] This application claims priority from Australian provisional patent application number 2014901282 filed on 8 April 2014, the content of which is to be taken as incorporated herein by this reference.

FIELD OF THE INVENTION

[0002] The present invention relates to a liposomal compound comprising a plant protein with peroxidase activity and use of the liposomal compound to produce a cosmetic effect by promoting production of one or more proteins in skin. The present invention also relates to a cosmetic composition comprising the liposomal compound.

BACKGROUND OF THE INVENTION

[0003] The skin is the largest organ in the human body and is one of the first lines of defence from external factors. For example, the skin plays a role in protecting the body against pathogens, such as bacteria and viruses, and chemical substances, as well as protecting against excessive water loss.

[0004] Mammalian skin contains two primary layers - the epidermis and the dermis. The dermis is connected to the epidermis through a basement membrane. Below the dermis is the hypodermis, which is not considered to form part of the skin.

[0005] The epidermis is the outermost layer and plays a major role in the skin's protective function. The epidermis is a stratified squamous epithelium. Starting at the outermost layer, the epidermis can be divided into the following layers or strata: stratum corneum, stratum lucidum, stratum granulosum, stratum spinosum and stratum germinativum (or stratum basale).

[0006] The stratum corneum plays a key role in the skin's protective function and can prevent the transcutaneous absorption of harmful agents, as well as more beneficial agents through the skin. The stratum corneum consists of terminally differentiated keratinocytes, referred to as corneocytes. Proliferating keratinocytes that form in the stratum basale undergo multiple stages of cell differentiation and migrate through the epidermis towards the surface of the skin where they become corneocytes forming part of the stratum corneum. The corneocytes are continually shed from the surface of the skin via desquamation or through rubbing, skin washing and the like.

[0007] The dermis is the layer of skin underneath the epidermis. The dermis contains mechanoreceptors (nerve endings), hair follicles, sweat glands, sebaceous glands, apocrine glands, lymphatic vessels and blood vessels. The dermis provides tensile strength and elasticity to the skin through an extracellular matrix (herein referred to as "ECM"). The ECM is composed of complex mixtures of structural proteins (such as collagen and elastin), specialized proteins (such as fibronectin and laminin) and, proteoglycans. The major fibrillar structural proteins, collagen and elastin, are responsible for tissue strength and resilience and play a dynamic role in promoting cell growth and differentiation.

[0008] As the skin ages, components of the ECM may be damaged or lost. Similarly, exposure to environmental factors such as UV, malnutrition and fatigue can also damage components of the ECM. This damage may affect the visual appearance of the skin. Some of the more obvious effects may be a loss of elasticity, increased sagging, loss of firmness, volume depletion and soft-tissue contour defects, wrinkles, crow's feet, nasolabial folds, marionette grooves and the like.

[0009] A number of treatments have been disclosed for the treatment of ECM loss. For example, collagen replacement therapy can be used to treat conditions associated with the breakdown or loss of collagen. Collagen replacement therapy can involve injecting purified collagen into the dermis to improve the appearance of the skin and treat lines and wrinkles. This type of treatment has its disadvantages. For example, collagen replacement therapy may involve the use of bovine collagen, which can cause allergic reactions and disease. The injection itself can also be difficult for the practitioner and confronting for the subject. Instead of injecting purified collagen into the dermis, collagen may also be topically applied to skin. However, this may not be very effective in treating lines and wrinkles if conventional forms of collagen do not penetrate through the stratum corneum.

[0010] Various methods for promoting transcutaneous absorption of compounds have been disclosed. For example, liposomes have been used to carry both hydrophilic and lipophilic agents across the stratum corneum. Liposomes are vesicles with a lipid bilayer that encloses an aqueous cavity. Liposomes can be a multilamellar vesicle, a small unilamellar vesicle or a large unilamellar vesicle etc. It has been suggested that the lipid bilayer of the liposome can merge with cell membranes, allowing delivery of the liposome contents to the cell. The use of liposomes as a vehicle for delivery of drugs and other active agents is not without its problems. For instance, some liposomes may have difficulty penetrating through the stratum corneum and thus may not always be effective in delivering an agent to the desired site of action. In addition, liposomes may be sensitive to temperature and may be unstable when stored. Some of the components used in the manufacture of liposomes may also be labile and expensive to purify or synthesize.

[001 1 ] Therefore, there is a need to identify compounds and compositions that are effective in providing a skin care benefit, to help maintain normal skin appearance and help restore aged skin to a youthful appearance.

[0012] The discussion of documents, acts, materials, devices, articles and the like is included in this specification solely for the purpose of providing a context for the present invention. It is not suggested or represented that any or all of these matters formed part of the prior art base or were common general knowledge in the field relevant to the present invention as it existed before the priority date of each claim of this application.

SUMMARY OF THE INVENTION

[0013] The present invention is based on the finding that the production of selected proteins in the skin can be stimulated by plant proteins with peroxidase activity that are delivered to the skin. In particular, the plant proteins have been found to be useful in stimulating the production of ECM components, in particular collagen, and filaggrin and/or profilaggrin. The plant proteins are delivered to the skin in a liposomal compound. The invention may therefore be useful in providing a skin care benefit in a subject.

[0014] In one aspect, the present invention provides a liposomal compound comprising: a plant protein with peroxidase activity; and a lipid bilayer, wherein the lipid bilayer comprises polyglycerol oleic acid ester and phospholipid.

[0015] In one embodiment, the plant protein with peroxidase activity is selected from the group consisting of horseradish peroxidase and soybean peroxidase and mixtures thereof. In some embodiments, the plant protein with peroxidase activity is horseradish peroxidase.

[0016] In some embodiments, the liposomal compound comprises a plant extract comprising the plant protein with peroxidase activity. In one embodiment, the plant extract is present in the liposomal compound in an amount from about 0.1 % to about 5% by volume of the compound. In some embodiments, the plant extract is present in the liposomal compound at about 1 % by volume of the compound. In some embodiments, the polyglycerol oleic acid ester is selected from the group consisting of diglycerol ester of monooleic acid, diglycerol ester of dioleic acid, triglycerol ester of monooleic acid, triglycerol ester of dioleic acid, tetraglycerol ester of monooleic acid, tetraglycerol ester of dioleic acid, diglycerol ester of trioleic acid, triglycerol ester of trioleic acid, triglycerol ester of tetraoleic acid, tetraglycerol ester of trioleic acid, tetraglycerol ester of tetraoleic acid, pentaglycerol ester of monooleic acid, pentaglycerol ester of dioleic acid, pentaglycerol ester of trioleic acid, pentaglycerol ester of tetraoleic acid, hexaglycerol ester of monooleic acid, hexaglycerol ester of dioleic acid, hexaglycerol ester of trioleic acid, hexaglycerol ester of tetraoleic acid and mixtures thereof. In some embodiments, the average number of glycerol residues per polyglycerol molecule in the polyglycerol oleic acid ester is 2 to 6. In some embodiments, the average number of glycerol residues per polyglycerol molecule in the polyglycerol oleic acid ester is 2. In some embodiments, there are not more than 4 oleic acid residues in the polyglycerol oleic acid ester. In some embodiments, there is 1 oleic acid residue in the polyglycerol oleic acid ester. In some embodiments, the polyglycerol oleic acid ester is present in the liposomal compound in an amount from about 1 % to about 5% by volume of the compound. In some embodiments, the polyglycerol oleic acid ester is present in the liposomal compound at about 1.9% by volume of the compound. In some embodiments, the polyglycerol oleic acid ester is a diglycerol monooleate ester.

[0017] In some embodiments, the phospholipid is selected from the group consisting of lecithin, phosphatidylcholine, phosphatidylglycerol, phosphatidyl-ethanolamine, phosphatidylinositol, phosphatidylserine, phosphatidic acid and mixtures thereof. In some embodiments, the phospholipid is present in the liposomal compound in an amount from about 0.01 % to about 1 % by volume of the compound. In some embodiments, the phospholipid is present in the liposomal compound at about 0.1 % by volume of the compound. In some embodiments, the lipid bilayer comprises phospholipid and polyglcerol oleic acid ester in a ratio of phospholipid to polyglcerol oleic acid ester (ratio by mass) of between 1 :5 to 1 :30. In some embodiments, the lipid bilayer comprises phospholipid and polyglcerol oleic acid ester in a ratio of phospholipid to polyglcerol oleic acid ester (ratio by mass) of between 1 :17 to 1 :22. In some embodiments, the phospholipid is lecithin.

[0018] In some embodiments, the lipid bilayer further comprises a phytosterol. In some embodiments, the ratio of phytosterol to polyglcerol oleic acid ester (ratio by mass) in the lipid bilayer is between 1 :5 to 1 :30. In some embodiments, the ratio of phytosterol to polyglcerol oleic acid ester (ratio by mass) in the lipid bilayer is between 1 :17 to 1 :22. In some embodiments, the phytosterol is selected from the group consisting of campesterol, sitosterol, and stigmasterol, and mixtures thereof. [0019] In some embodiments, the liposomal compound further comprises an additional agent selected from the group consisting of Morus bombycis root extract, Rosmarinus officinalis (rosemary) leaf extract, Mentha piperita (peppermint) leaf extract, glycogen, and mixtures thereof.

[0020] In some embodiments, the liposomal compound has an average diameter in the range of from about 20 nm to 200 nm. In some embodiments, the liposomal compound has an average diameter in the range of from about 60 nm to 100 nm. In some embodiments, the liposomal compound has an average diameter of about 80 nm.

[0021 ] In another aspect, the present invention provides a liposomal compound comprising: horseradish peroxidase; and a lipid bilayer, wherein the lipid bilayer comprises diglycerol monooleate ester and lecithin. In another aspect, the present invention provides a liposomal compound comprising: a plant extract comprising a plant protein with peroxidase activity; and a lipid bilayer, wherein the lipid bilayer comprises diglycerol monooleate ester and lecithin. In one embodiment, the plant extract is present in the liposomal compound at about 1 % by volume of the compound, the diglycerol monooleate ester is present in the liposomal compound at about 1 .9% by volume of the compound, and the lecithin is present in the liposomal compound at about 0.1 % by volume of the compound.

[0022] In another aspect, the present invention provides a cosmetic composition for topical application to the skin, wherein the composition comprises a liposomal compound of any one of the aforementioned aspects and a cosmetically acceptable carrier or vehicle. In some embodiments, the cosmetic composition may be in the form of a solution, spray, lotion, cream, emulsion, oil, gel, essence, powder, paste, serum, or ointment.

[0023] In another aspect, the present invention provides a method for promoting production of collagen I and/or collagen IV in skin, the method comprising applying to skin an effective amount of the liposomal compound or cosmetic composition of any one of the aforementioned aspects.

[0024] In another aspect, the present invention provides a method for promoting production of profilaggrin and/or filaggrin in skin, the method comprising applying to skin an effective amount of the liposomal compound or cosmetic composition of any one of the aforementioned aspects. [0025] In another aspect, the present invention provides a method for providing a skin care benefit, the method comprising topically applying the liposomal compound or cosmetic composition of any one of the aforementioned aspects to the skin of a subject.

[0026] In another aspect, the present invention provides use of an effective amount of the liposomal compound of any one of the aforementioned aspects in the manufacture of a composition for application to the skin of a subject to promote the production of collagen I and/or collagen IV in the skin.

[0027] In another aspect, the present invention provides use of an effective amount of the liposomal compound of any one of the aforementioned aspects in the manufacture of a composition for application to the skin of a subject to promote the production of profilaggrin and/or filaggrin in the skin.

[0028] In another aspect, the present invention provides use of an effective amount of the liposomal compound of any one of the aforementioned aspects in the manufacture of a composition for application to the skin of a subject to provide a skin care benefit to the subject.

BRIEF DESCRIPTION OF THE FIGURE

[0029] For a further understanding of the aspects and advantages of the present invention, reference should be made to the following detailed description and Examples, taken in conjunction with the accompanying Figure.

[0030] FIGURE 1 - Western blots showing the depth of penetration of the liposomal compound of an embodiment of the present invention into the skin. Liposomal compound formulated in serum (A) compared to a control vehicle (B). Liposomal compound formulated in water (C) compared to a control vehicle (D). E: epidermis; SC: stratum corneum. Note: the comparison was made 1 hour after application of the liposomal compound and control vehicle.

DESCRIPTION OF THE INVENTION

[0031 ] Various terms that will be used throughout the specification have meanings that will be well understood by a skilled addressee. However, for ease of reference, some of these terms will now be defined. [0032] The term "subject" as used throughout the specification is to be understood to mean a mammalian subject, in particular a human subject. In one set of embodiments, a subject in need of such treatment may be a subject in need of a skin care benefit. In some embodiments, a subject in need of treatment may be a subject suffering from, or susceptible to, a condition for example arising from or leading to ECM loss or absence, such as a subject having wrinkles, crow's feet, nasolabial folds and marionette grooves. In some embodiments, a subject in need of treatment may be a subject suffering from, or susceptible to, a reduction in filaggrin and/or profilaggrin levels. In some embodiments, the reduction in filaggrin and/or profilaggrin levels may be in the skin.

[0033] The term "promote" as used throughout the specification is to be understood to mean an increase in the progress of a process, including any one or more of the start, rate, probability, continuation or termination of a process and the like. For example, to "promote" the production of a protein is intended to mean a 1 %, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 2-fold, 5-fold, 10-fold, 20 fold, 50-fold, or 100-fold increase in the level and/or activity of a protein in the skin compared to control "untreated" skin, i.e. skin in which the level and/or activity of the protein has not been promoted.

[0034] The term "cosmetically acceptable" as used throughout the specification is to be understood to mean that the compositions or components thereof so described are suitable for use in cosmetics such as creams, lotions, serums and the like without undue toxicity, incompatibility, instability, allergic response, and the like.

[0035] The term "cosmetic composition" as used throughout the specification is to be understood to mean that the compositions so described are suitable for use as a cosmetic such as creams, lotions, serums, and the like without undue toxicity, incompatibility, instability, allergic response, and the like.

[0036] The terms "protein" and "polypeptide" are used synonymously throughout the specification and are to be understood to mean any peptide-linked chain of amino acids, regardless of length or post-translational modification, e.g. glycosylation or phosphorylation. A "purified" protein is one that has been substantially separated or isolated away from other components in a cell or organism in which the protein naturally occurs (e.g. 90, 95, 98, 99, 100% free of contaminants). When referring to a protein or polypeptide, the term "native" refers to a naturally-occurring protein or polypeptide. Liposomal compound

[0037] As discussed above, in one aspect the present invention provides a liposomal compound comprising: a plant protein with peroxidase activity; and a lipid bilayer, wherein the lipid bilayer comprises polyglycerol oleic acid ester and phospholipid.

[0038] Advantageously, the liposomal compound of the present invention is useful for delivering a plant protein with peroxidase activity to, and into, the skin. Without wishing to be bound by theory, the liposomal compound is thought to carry the plant protein with peroxidase activity into the skin, and in particular through the stratum corneum to the lower epidermal layers of the skin, where the plant protein with peroxidase activity can help to promote production of proteins in the skin. In some embodiments, the plant protein with peroxidase activity may help promote production of collagen, such as collagen I and/or collagen IV, in skin. The liposomal compound of the present invention may therefore be used to provide a skin care benefit by promoting collagen production. In some embodiments, the plant protein with peroxidase activity may help promote production of profilaggrin and/or filaggrin in skin. The liposomal compound of the present invention may therefore be used to provide a skin care benefit by promoting production of profilaggrin and/or filaggrin.

[0039] The liposomal compound according to the present invention is in the form of a vesicle having one or more lipid bilayers surrounding an inner cavity which is generally hydrophilic. Each lipid bilayer is a membrane composed of two layers of lipid molecules which are assembled so that the hydrophilic head groups of the lipid molecules point "out" and are exposed to a hydrophilic environment, while the hydrophobic tails of the lipid molecules point "in" to the core of the bilayer. In one form, a lipid bilayer forms a sphere which entirely encloses an inner, hydrophilic cavity.

Plant protein with peroxidase activity

[0040] The liposomal compound comprises a plant protein with peroxidase activity. The plant protein with peroxidase activity may be encapsulated in the hydrophilic inner cavity of the compound or it may be associated with the lipid bilayer of the liposomal compound, for example, by partitioning in the lipid bilayer or attached to the lipid bilayer surface, e.g. by electrostatic attachment, or any combination of the above. The plant protein with peroxidase activity may be present within an extract from the plant, or may be present in an isolated and/or purified form from the plant (or extract thereof). Accordingly, for the purposes of the following description, reference to a "plant protein with peroxidase activity" includes reference to the plant protein present within an extract from the plant or the plant protein in an isolated and/or purified form. [0041 ] The term "plant protein with peroxidase activity" in the various embodiments of the present invention is a protein from a plant source having an activity which catalyses a reaction of the form:

ROOR' + electron donor (2e-) + 2H+→ROH + R'OH.

[0042] A plant protein with peroxidase activity useful for the liposomal compound of the present invention may be a polypeptide with peroxidase activity, such as a peroxidase enzyme, a fragment of the polypeptide, and a natural or synthetic variant of the polypeptide. In some embodiments, a plant protein with peroxidase activity may be a plant protein which has peroxidase activity but which is not classified as a peroxidase enzyme, such as for example cytochrome P450 (CYP). CYP also has the ability to catalyse oxidation/reduction reactions and is considered to be a protein with peroxidase activity.

[0043] A plant protein with peroxidase activity may contain a heme cofactor in their active sites, or redox-active cysteine or selenocysteine residues. In this regard, it will be appreciated that the peroxidases in the various embodiments of the present invention also may be polypeptides that utilise a metal substitution of the heme group, or which are heme- independent.

[0044] The term "variant" as used throughout the specification is to be understood to mean an amino acid sequence of a polypeptide that is altered by one or more amino acids. The variant may have "conservative" changes, wherein a substituted amino acid has similar structural or chemical properties to the replaced amino acid (e.g. replacement of leucine with isoleucine) as described in further detail below. A variant may also have "non-conservative" changes (e.g. replacement of a glycine with a tryptophan) or a deletion and/or insertion of one or more amino acids. A variant may also be a form of the polypeptide that has one or more deleted amino acids (e.g. a truncated form of the enzyme or protein), and/or a form of the polypeptide that has one or more additional exogenous amino acids (e.g. a form of the enzyme or protein fused to another polypeptide sequence). It will be appreciated that a variant will therefore have within its scope a fragment of a polypeptide. It is to be understood that the variant is a functional variant, that is, the variant retains the functional ability of the progenitor plant protein with peroxidase activity.

[0045] Conservative substitutions may be substitutions within the following groups: glycine and alanine; valine, isoleucine, and leucine; aspartic acid and glutamic acid; asparagine and glutamine; serine and threonine; lysine and arginine; and phenylalanine and tyrosine. Under some circumstances, substitutions within the aliphatic group alanine, valine, leucine and isoleucine are also considered as conservative. Sometimes substitution of glycine for one of these can also be considered conservative. Other conservative interchanges are those within the aliphatic group aspartate and glutamate; within the amide group asparagine and glutamine; within the hydroxyl group serine and threonine; within the aromatic group phenylalanine, tyrosine and tryptophan; within the basic group lysine, arginine and histidine; and within the sulfur-containing group methionine and cysteine. Sometimes substitution within the group methionine and leucine can also be considered conservative. Sometimes substitution within the group serine and cysteine can also be considered conservative.

[0046] The term "fragment" as used throughout the specification is to be understood to mean a shorter segment of the polypeptide. As such, a fragment may be a form of the polypeptide that has one or more amino acids deleted from the N- and/or C-terminus (e.g. a truncated form of the protein) and/or a form of the polypeptide that has one of more amino acids deleted internally. The fragment will be a functional fragment, that is, a fragment that retains the functional ability of the progenitor plant protein with peroxidase activity.

[0047] A plant protein with peroxidase activity may be a peroxidase enzyme. A peroxidase enzyme is classified as an oxidoreductase and has an EC number (Enzyme commission number) of EC 1.1 1.1 . Peroxidases are widely distributed in nature and have been isolated from plants such as horseradish (Cochlearia armoracia) and soybean (Glycine max).

[0048] A peroxidase enzyme may be a polypeptide with an amino acid sequence as provided by the polypeptides defined by an EC number selected from the following group consisting of EC 1.1 1.1.1 ; EC 1.1 1.1.2; EC 1.1 1.1 .3; 1.13.1 1.1 1 ; EC 1.1 1.1 .5; EC 1 .1 1.1.6; EC 1 .1 1 .1.7; EC 1 .1 1.1.8; EC 1.1 1.1.9; EC 1.1 1.1 .10; EC 1.1 1.1.12; EC 1 .1 1 .1.13; EC 1.1 1.1.14; EC 1.1 1 .1.15; EC 1.1 1.1.16; EC 1 .1 1 .1.17, EC 1.1 1.1.18, EC 1 .1 1 .1.19, EC 1.1 1.1.20, EC 1.1 1.1.21 , EC 1.1 1.1 .22, EC 1.1 1.1.23, or a fragment or variant of any of the aforementioned.

[0049] Details of polypeptides defined by the above EC numbers are as described in Enzyme Nomenclature 1992 [Academic Press, San Diego, California, ISBN 0-12-227164-5 (hardback), 0-12-227165-3 (paperback)] with Supplement 1 (1993), Supplement 2 (1994), Supplement 3 (1995), Supplement 4 (1997) and Supplement 5 (in Eur. J. Biochem. 1994, 223, 1-5; Eur. J. Biochem. 1995, 232, 1 -6; Eur. J. Biochem. 1996, 237, 1 -5; Eur. J. Biochem. 1997, 250; 1 -6, and Eur. J. Biochem. 1999, 264, 610-650; respectively). Details are also available from the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology (http://www.chem.qmul.ac.Uk/iubmb/enzyme/EC1/1 1/1/), and ExPASy (http://enzyme.expasy.Org/EC/1.1 1.1.-), amongst others.

[0050] The amino acid sequences of the relevant polypeptides can be readily obtained by a person skilled in the art. For example, the sequences are available from UniProt (http://www.uniprot.org/uniprot/?query=ec:1.1 1.1.-%20reviewed:yes).

[0051 ] In some embodiments, the plant protein with peroxidase activity is selected from the group consisting of horseradish peroxidase and soybean peroxidase, and mixtures thereof. In one embodiment, the plant protein with peroxidase activity is horseradish peroxidase.

[0052] Methods are known in the art for producing a plant protein with peroxidase activity. For example, it will be appreciated that the plant protein with peroxidase activity can be in the form of a substantially pure plant protein, or as part of a mixture with one or more components. For example, a plant protein with peroxidase activity can be contained in a fluid having one or more proteins with the activity.

[0053] In some embodiments, the liposomal compound may comprise a plant extract comprising a plant protein with peroxidase activity. Plant proteins with peroxidase activity can be extracted and/or isolated from plants, such as horseradish and soybean, by conventional methods known to a person skilled in the art.

[0054] In some embodiments, the plant extract may be prepared by extracting plant material with a solvent. The plant material may be a whole plant or part thereof (e.g. roots, stems, leaves, bark, fruit and the like). Fresh plant material is suitable as the starting material, although dried plant material may be used. The plant material may be reduced in size before extraction. Any size reduction methods known to those skilled in the art, for example crushing in a mortar or slicing and dicing, may be used. In some embodiments, the extraction is performed by contacting the plant material with solvent for a time and at a temperature suitable to extract the plant protein with peroxidase activity from the plant material and obtain a plant extract comprising the plant protein with peroxidase activity. In some embodiments, the solvent for the extraction process is selected from the group consisting of organic solvents, water and mixtures thereof. In some embodiments, the organic solvent is selected from the group consisting of low molecular weight alcohols, esters, hydrocarbons, ketones or halogenated hydrocarbons. In some embodiments, the solvent for extraction may be selected from the group consisting of water, ethanol, glycerol, propylene glycols, polyethylene glycols, ethyl acetate and mixtures thereof. It would be understood by a person skilled in the art that a selected solvent should be suitable for topical application or the plant extract able to be processed such that the solvent used for the extraction process is exchanged for a solvent that is suitable for topical application. The extraction process may be carried out at 10 to 50 , preferably at 15 to 30 , and more preferably at 20 to 25Ό. The extraction times can be determined by a person skilled in the art based upon the starting plant material, the extraction process, the extraction temperature and the ratio of solvent to raw plant material, etc. In some embodiments, the extraction is carried out for 1 to 4 days, preferably for 2 to 3 days. Optionally, the obtained plant extract may be subjected to other steps, such as for example filtration, purification, concentration and/or decolouration to remove undesired components or impurities. In some embodiments, the plant extract is filtered.

[0055] The plant extracts can be crude or enriched for the protein with peroxidase activity. In some embodiments, the plant protein with peroxidase activity is contained in a plant extract such as horseradish extract or soybean extract.

[0056] The plant extract comprising the plant protein with peroxidase activity and/or the plant protein with peroxidase activity may be concentrated if desired by a method known to a person skilled in the art.

[0057] In some embodiments, the plant extract comprising the plant protein with peroxidase activity is present in the liposomal compound in an amount from about 0.01 % to about 30% by weight of the compound. In some embodiments, the plant extract comprising the plant protein with peroxidase activity is present in the liposomal compound in an amount from about 0.1 % to about 5% by weight of the compound. For example, the plant extract comprising the plant protein with peroxidase activity is present in an amount from about 0.1 % to about 1 %, about 0.1 % to about 0.5%, about 0.5% to about 5%, about 0.5% to about 1 %, about 1 % to about 5%, about 1 % to about 4.5%, about 1 % to about 4%, about 1 % to about 3.5%, about 1 % to about 3%, about 1 % to about 2.5%, about 1 % to about 2%, about 1 % to about 1 .5%, about 1.5% to about 5%, about 1 .5% to about 4.5%, about 1.5% to about 4%, about 1.5% to about 3.5%, about 1.5% to about 3%, about 1.5% to about 2.5%, about 1 .5% to about 2%, about 2% to about 5%, about 2% to about 4.5%, about 2% to about 4%, about 2% to about 3.5%, about 2% to about 3%, about 2% to about 2.5%, about 2.5% to about 5%, about 2.5% to about 4.5%, about 2.5% to about 4%, about 2.5% to about 3.5%, about 2.5% to about 3%, about 3% to about 5%, about 3% to about 4.5%, about 3% to about 4%, about 3% to about 3.5%, about 3.5% to about 5%, about 3.5% to about 4.5%, about 3.5% to about 4%, about 4% to about 5%, about 4% to about 4.5%, or about 4.5% to about 5%, by weight of the compound. In some embodiments, the plant extract comprising the plant protein with peroxidase activity is present in the liposomal compound at about 1 % by weight of the compound.

[0058] In some embodiments, the plant protein with peroxidase activity is present in the liposomal compound in an amount from about 0.1 μg/ml to about 20 μg/ml. For example, the plant protein with peroxidase activity is present in an amount from about 0.1 μg/ml to about 15 μg/ml, about 0.1 μg/ml to about 12.5 μg/ml, about 0.1 μg/ml to about 10 μg/ml, about 0.1 μg/ml to about 5 μg/ml, about 0.1 μg/ml to about 1 μg/ml, about 0.2 μg/ml to about 20 μg/ml, about 0.2 μg/ml to about 15 μg/ml, about 0.2 μg/ml to about 12.5 μg/ml, about 0.2 μg/ml to about 10 μg/ml, about 0.2 μg/ml to about 5 μg/ml, about 0.2 μg/ml to about 1 μg/ml, about 0.3 μg/ml to about 20 μg/ml, about 0.3 μg/ml to about 15 μg/ml, about 0.3 μg/ml to about 12.5 μg/ml, about 0.3 μg/ml to about 10 μg/ml, about 0.3 μg/ml to about 5 μg/ml, about 0.3 μg/ml to about 1 μg/ml, about 0.4 μg/ml to about 20 μg/ml, about 0.4 μg/ml to about 15 μg/ml, about 0.4 μg/ml to about 12.5 μg/ml, about 0.4 μg/ml to about 10 μg/ml, about 0.4 μg/ml to about 5 μg/ml, about 0.4 μg/ml to about 1 μg/ml, about 1 μg/ml to about 20 μg/ml, about 1 μg/ml to about 15 μg/ml, about 1 μg/ml to about 10 μg/ml, about 1 μg/ml to about 5 μg/ml, about 5 μg/ml to about 20 μg/ml, about 5 μg/ml to about 15 μg/ml, about 5 μg/ml to about 10 μg/ml, about 10 μg/ml to about 20 μg/ml, about 10 μg/ml to about 15 μg/ml, or about 15 μg/ml to about 20 μg/ml. In some embodiments, the plant protein with peroxidase activity is present in the liposomal compound at about 0.4 μg/ml to about 12.5 μg/ml.

[0059] Plant proteins with peroxidase activity can also be purchased commercially from general chemical suppliers such as Sigma-Aldrich (Castle Hill, NSW, Australia) or from specialised companies such as Biozyme Laboratories (San Diego, USA).

[0060] Methods for identifying plant proteins with peroxidase activity, and for determining their activity, are known in the art. For example, the peroxidase activity of a protein, whether in its isolated form or within a plant extract, may be measured using an enzyme substrate assay such as those offered by Sigma-Aldrich (Castle Hill, NSW, Australia)(see for example http://www.sigmaaldrich.com/content/dam/sigma- aldrich/docs/Sigma/General_lnformation/2/biofiles_issue3_4.p df). Specific exemplary assays include the SIGMAFAST™ OPD detection system and the ISOPAC ^® detection system.

[0061 ] The plant protein with peroxidase activity is present in the liposomal compound at a suitable concentration to allow the plant protein with peroxidase activity to have the desired effect. In this regard, the "effective amount" of the plant protein with peroxidase activity to be administered to the subject is not particularly limited, so long as it is within such an amount and in such a form that generally exhibits a useful, beneficial or cosmetic effect. The amount to be administered will depend on the particular characteristics of the treatment, the mode of administration, and the characteristics of the subject, such as age of the subject, skin damage and the like. A person skilled in the art will be able to determine appropriate effective amounts depending on these and other factors.

[0062] In some embodiments, the liposomal compound may, in addition to the plant protein with peroxidase activity, also comprise additional agents which provide skin care benefits. For example, the liposomal compound may comprise sunscreens, skin-protectant agents, skin-soothing agents, moisturizers, skin-lightening agents, skin tanning agents and the like. In some embodiments, the additional agent is preferably obtained from natural sources, such as for example plant sources.

[0063] In some embodiments, the liposomal compound may comprise an additional agent which is a plant extract. The plant extract may be selected from the group consisting of Akebia extract, Thujopsis dolobrata extract, asparagus extract, avocado extract, sweet hydrangea leaf extract, almond extract, arnica extract, aloe extract, apricot extract, gingko extract, fennel extract, rose fruit extract, Isodon japonicus extract, Scutellaria root extract, Phellodendron bark extract, Coptis japonica extract, Panax ginseng extract, Hypericum erectum extract, Lamium album var. barbatum extract, orange extract, Pyracantha fortuneana extract, Pueraria root extract, camomile extract, carrot extract, Artemisia capillaris extract, liquorice extract, kiwifruit extract, cucumber extract, guava extract, Sophora root extract, Gardenia jasminoides extract, Sasa veitchii extract, Sophora flavescens extract, walnut extract, grapefruit extract, black rice extract, Chlorella extract, mulberry extract, Alpinia zerumbet extract, gentian extract, Geranium thunbergii extract, Indian tea extract, Arctium lappa extract, rice extract, fermented rice extract, fermented rice bran extract, rice germ oil, Vaccinium vitis-idaea extract, salvia extract, Saponaria officinalis extract, reed extract, hawthorn extract, Coriandrum sativum extract, Zanthoxylum piperitum extract, shiitake extract, Lithospermum root extract, Perilla frutescens var. crispa extract, Tilia japonica extract, Filipendula multijuga extract, Paeonia lactiflora extract, ginger extract, Calamus extract, Betula platyphylla var. japonica extract, Equisetum arvense extract, Stevia extract, fermented Stevia, Hedera helix extract, Sambucus nigra extract, peppermint extract, sage extract, Malva sylvestris var. mauritiana extract, Cnidium officinale extract, Swertia japonica extract, mulberry root bark extract, rhubarb extract, soybean extract, jujube extract, thyme extract, dandelion extract, tea extract, clove extract, Aurantii nobilis pericarpium extract, blackberry leaf tea extract, capsicum extract, Angelica acutiloba extract, Calendula officinalis extract, peach kernel extract, spruce extract, Houttuynia cordata extract, tomato extract, natto extract, garlic extract, carrot extract, eglantine extract, hibiscus extract, Ophiopogon tuber extract, lotus extract, parsley extract, birch extract, Hamamelis virginiana extract, Isodon japonicus extract, Chamaecyparis obtusa extract, loquat extract, Tussilago farfara extract, Petasites japonicus extract, Poria sclerotum extract, Ruscus aculeatus extract, grape extract, grape seed extract, Luffa cylindrica extract, safflower extract, peppermint extract, Tilia miqueliana extract, Paeonia suffruticosa extract, hop extract, pine extract, horse chestnut extract, Lysichiton americanus extract, Sapindus mukurossi extract, Melissa officinalis extract, Cladosiphon okamuranus extract, peach extract, Centaurea cyanus extract, eucalyptus extract, Saxifraga extract, Citrus junos extract, Lilium sp. extract, Coicis semen extract, Artemisia vulgaris indica extract, lavender extract, green tea extract, apple extract, rooibos tea extract, lychee extract, lettuce extract, lemon extract, Forsythia fructus extract, Astragalus sinicus extract, rose extract, rosemary extract, Anthemis nobilis extract, royal jelly extract and Sanguisorba officinalis extract, Morus bombycis extract, Tremella fuciformis extract, Glycyrrhiza glabra extract, and the like.

[0064] In some embodiments, the liposomal compound may comprise at least one additional agent selected from the group consisting of Morus bombycis root extract, Rosmarinus officinalis (rosemary) leaf extract, Mentha Piperita (peppermint) leaf extract, glycogen and mixtures thereof. In some embodiments, the liposomal compound comprises a mixture of Morus bombycis root extract, Rosmarinus officinalis (rosemary) leaf extract, Mentha piperita (peppermint) leaf extract and glycogen.

[0065] The additional agent should be suitable for topical application to the skin - that is when they are present in the liposomal compound they should be suitable for use in contact with skin without undue toxicity, incompatibility, instability, allergic reaction and the like. The additional agent may be encapsulated in the hydrophilic inner cavity of the liposomal compound or it may be associated with the lipid bilayer of the liposomal compound, for example, by partitioning in the lipid bilayer or attached to the lipid bilayer surface, e.g. by electrostatic attachment, or any combination of the above. It may be desirable to have an additional agent, preferably a plant extract, in the liposomal compound to provide a further effect, such as an anti-inflammatory effect, a skin lightening effect, inhibition of the activity of tyrosinase, proliferation of skin cells, promotion of blood circulation, a moisturizing effect, or help prevent age spots, and the like. It would be understood by a person skilled in the art that a selected additional agent may provide more than one skin care benefit. Lipid bilayer

[0066] In addition to the plant protein with peroxidase activity, the liposomal compound of the present invention also comprises a lipid bilayer comprising polyglycerol oleic acid ester and phospholipid. As discussed above, the lipid bilayer may surround and encapsulate the plant protein with peroxidase activity, facilitating transport of the plant protein with peroxidase activity across the stratum corneum and into deeper layers of the epidermis.

[0067] Polyglycerol oleic acid esters employed in the lipid bilayer of the liposomal compound of the invention are obtainable by esterification of a polyglycerol mixture with oleic acid. The degree of polymerisation of the polyglycerol varies, and is specified by a number that is related to the average number of glycerol residues per polyglycerol molecule.

[0068] In some embodiments, the average number of glycerol residues per polyglycerol molecule in the polyglycerol oleic acid ester is 2 to 6, for example 2 to 3, 2 to 4, 2 to 5, 3 to 4, 3 to 5, 3 to 6, 4 to 5, 4 to 6, or 5 to 6. In some embodiments, the average number of glycerol residues per polyglycerol molecule in the polyglycerol oleic acid ester is 2.

[0069] In some embodiments, there are not more than 4 oleic acid residues in the polyglycerol oleic acid ester. In some embodiments, there are not more than 3 oleic acid residues, or not more than 2 oleic acid residues in the polyglycerol oleic acid ester. In some embodiments, there is 1 oleic acid residue in the polyglycerol oleic acid ester. However, it is undesirable that the oleic acid completely esterifies the hydroxyl groups of the polyglycerol because this nullifies the surfactant function.

[0070] In some embodiments, the polyglycerol oleic acid ester is present in the liposomal compound in an amount from about 0.01 % to about 20% by weight of the compound. In some embodiments, the polyglycerol oleic acid ester is present in the liposomal compound in an amount from about 1 % to about 5% by weight of the compound. For example, the polyglycerol oleic acid ester may be present in an amount from about 1 % to about 4%, about 1 % to about 3%, about 1 % to about 2%, about 2% to about 5%, about 2% to about 4%, about 2% to about 3%, about 3% to about 5%, about 3% to about 4%, or about 4% to about 5%, by weight of the compound. In some embodiment, the olyglycerol oleic acid ester is present in the liposomal compound at about 1.9% by weight of the compound.

[0071 ] In some embodiments, the polyglycerol oleic acid ester is selected from the group consisting of diglycerol ester of monooleic acid, diglycerol ester of dioleic acid, triglycerol ester of monooleic acid, triglycerol ester of dioleic acid, tetraglycerol ester of monooleic acid, tetraglycerol ester of dioleic acid, diglycerol ester of trioleic acid, triglycerol ester of trioleic acid, triglycerol ester of tetraoleic acid, tetraglycerol ester of trioleic acid, tetraglycerol ester of tetraoleic acid, pentaglycerol ester of monooleic acid, pentaglycerol ester of dioleic acid, pentaglycerol ester of trioleic acid, pentaglycerol ester of tetraoleic acid, hexaglycerol ester of monooleic acid, hexaglycerol ester of dioleic acid, hexaglycerol ester of trioleic acid, hexaglycerol ester of tetraoleic acid and mixtures thereof. In one embodiment, the polyglycerol oleic acid ester is diglycerol ester of monooleic acid.

[0072] Phospholipid employed in the lipid bilayer of the liposomal compound of the invention may, in some embodiments, be selected from the group consisting of lecithin, phosphatidylcholine, phosphatidylglycerol, phosphatidyl-ethanolamine, phosphatidylinositol, phosphatidylserine, phosphatidic acid and mixtures thereof. Lecithin may be desirable as it may enhance the stability of the liposomal compound containing polyglycerol esters of oleic acid. In some embodiments, the lecithin is a hydrogenated lecithin. The phospholipids can be obtained from natural sources, such as soybeans, rapeseed, cottonseed, and sunflower. In some embodiments, the natural source of lecithin is soybean, that is, the phospholipid is soybean lecithin. Phospholipids may contain added water. In some embodiments, the phospholipid may be chemically synthesised.

[0073] In some embodiments, the phospholipid is present in the liposomal compound in an amount from about 0.001 % to about 3% by weight of the compound. In some embodiments, the phospholipid is present in the liposomal compound in an amount from about 0.01 % to about 1 % by weight of the compound. For example, the phospholipid may be present in an amount from about 0.01 % to about 0.5%, about 0.01 % to about 0.1 %, about 0.01 % to about 0.05%, about 0.05% to about 1 %, about 0.05% to about 0.5%, about 0.05% to about 0.1 %, about 0.1 % to about 1 %, about 0.1 % to about 0.5%, or about 0.5% to about 1 %, by weight of the compound. In some embodiments, the phospholipid is present in the liposomal compound at about 0.1 % by weight of the compound.

[0074] In some embodiments, the lipid bilayer of the liposomal compound of the invention comprises phospholipid and polyglcerol ester of oleic acid in a ratio of phospholipid to polyglcerol ester of oleic acid (ratio by mass) of between 1 :5 to 1 :30, for example between 1 :5 to 1 :25, 1 :5 to 1 :20, 1 :5 to 1 :15, 1 :5 to 1 :10, 1 :10 to 1 :25, 1 :10 to 1 : 20, 1 :10 to 1 :15, 1 :15 to 1 :25, 1 :15 to 1 :20, or 1 :20 to 1 :25. In one embodiment, the lipid bilayer of the liposomal compound of the invention comprises phospholipid and polyglcerol ester of oleic acid in a ratio of phospholipid to polyglcerol ester of oleic acid (ratio by mass) of between 1 :17 to 1 :22. [0075] In some embodiments, the lipid bilayer of the liposomal compound may also comprise non-phospholipid surfactants, charge producing agents, steroids such as cholesterol, phytosterols and/or targeting molecules.

[0076] Phytosterols may be plant sterols and stands. The inclusion of phytosterols may be advantageous in some embodiments to enhance the stability of the liposomal compound and enhance transcutaneous absorption by regularly aligning the polyglycerol esters of oleic acid and phospholipids in the lipid bilayer of the compound.

[0077] Phytosterols may be used if they are generally classified as phytosterols (vegetable sterols). Preferred phytosterols may be campesterol, sitosterol and stigmasterol and so forth as constituent components. Such components may be obtained by extraction by means of organic solvents from the germ of cereals and by discarding the water-soluble portion. Alternatively, commercially available phytosterols may be purchased and used. Such commercial products include, for example, Phytosterol S (manufactured by Seikagaku Corporation), and the like.

[0078] In embodiments where the lipid bilayer also comprises phytosterols, the phytosterols are present in the liposomal compound in an amount from about 0.001 % to about 3% by weight of the compound. In some embodiments, the phytosterols are present in the liposomal compound in an amount from about 0.01 % to about 1 % by weight of the compound. For example, the phytosterols may be present in an amount from about 0.01 % to about 0.5%, about 0.01 % to about 0.1 %, about 0.01 % to about 0.05%, about 0.05% to about 1 %, about 0.05% to about 0.5%, about 0.05% to about 0.1 %, about 0.1 % to about 1 %, about 0.1 % to about 0.5%, or about 0.5% to about 1 %, by weight of the compound. In some embodiments, the phytosterols are present in the liposomal compound at about 0.1 % by weight of the compound.

[0079] In embodiments where the lipid bilayer also comprises phytosterols, the ratio of the phytosterols to the polyglcerol esters of oleic acid (ratio by mass) is between 1 :5 to 1 :30, for example between 1 :5 to 1 :25, 1 :5 to 1 :20, 1 :5 to 1 :15, 1 :5 to 1 :10, 1 :10 to 1 :25, 1 :10 to 1 : 20, 1 :10 to 1 :15, 1 :15 to 1 :25, 1 :15 to 1 :20, or 1 :20 to 1 :25. In one embodiment, the ratio of the phytosterols to the polyglcerol esters of oleic acid (ratio by mass) is between 1 :17 to 1 :22.

Preparation of the liposomal compound

[0080] Methods for manufacturing liposomes have been described in the art and the liposomal compound according to the present invention may be made using conventional techniques known to a person skilled in the art (see, e.g. Williams, A. P., "Liposomes: A Practical Approach", 2nd Edition, Oxford Univ. Press (2003); Lasic, D. D.; Gregoriadis, G. (ed), "Liposome Technology", CRC Press Inc., Boca Raton, Fla., Vol. I, II & III (1984)).

[0081 ] In some embodiments, the liposomal compound according to the present invention may be prepared by forming an aqueous dispersion comprising a lipophilic phase and an aqueous phase. The lipophilic phase comprises the lipid bilayer components, while the aqueous phase comprises the plant protein with peroxidase activity. The aqueous dispersion may be prepared by forming a lipid mixture containing the lipid bilayer components. The lipid mixture is heated and blended to form a homogeneous lipophilic phase. The lipophilic phase is then blended with the aqueous phase comprising a plant protein with peroxidase activity. The aqueous phase may optionally also contain buffer components, electrolytes and/or active agents. The lipophilic phase and aqueous phase may be blended under shear mixing conditions to form the aqueous dispersion. The aqueous dispersion will contain the liposomal compound of the invention. To prepare the aqueous dispersion, the temperature of the lipophilic phase may be elevated to make it flowable, followed by carrying out the shear mixing between the lipophilic phase and the aqueous phase at a temperature such that both phases are liquids. While it is often desirable to use the same temperature for both phases, this is not always necessary.

[0082] In some embodiments, the aqueous dispersion may be formulated to comprise the plant protein with peroxidase activity in an amount described in paragraph [0058] above. For example, the aqueous dispersion may be formulated to comprise an amount of plant protein with peroxidase activity from about 0.1 μg/ml to about 20 μg/ml. The plant protein present in the aqueous dispersion is believed to be incorporated in the liposomal compound contained in the dispersion.

[0083] In some embodiments, the aqueous dispersion comprises an aqueous phase comprising a plant extract comprising a plant protein with peroxidase activity. The plant extract may be incorporated in the liposomal compound contained in the aqueous dispersion to thereby provide the plant protein with peroxidase activity in the liposomal compound.

[0084] In some embodiments, the aqueous dispersion may be formulated to comprise the plant extract comprising the plant protein with peroxidase activity in an amount described in paragraph [0057] above. For example, the aqueous dispersion may be formulated to comprise an amount of plant extract comprising the plant protein with peroxidase activity from about 0.01 % to about 30% by weight of the aqueous dispersion. In some embodiments, the plant extract comprising the plant protein with peroxidase activity is present in the aqueous dispersion in an amount from about 0.1 % to about 5% by weight of the aqueous dispersion. For example, the plant extract comprising the plant protein with peroxidase activity is present in the aqueous dispersion at about 1 % by weight of the aqueous dispersion. Preferably, the plant extract is selected from the group consisting of horseradish extract and soybean extract and mixtures thereof, preferably horseradish extract.

[0085] In some embodiments, the aqueous dispersion may be formulated to comprise polyglycerol esters of oleic acid in an amount described in paragraph [0070] above. For example, the aqueous dispersion may be formulated to comprise polyglycerol esters of oleic acid in an amount in the range of from about 0.01 % to about 20% by weight of the aqueous dispersion. In some embodiments, the aqueous dispersion may be formulated to comprise polyglycerol esters of oleic acid in an amount in the range of from about 1 % to about 5% by weight of the compound. For example, the aqueous dispersion may comprise an amount of olyglycerol oleic acid ester at about 1.9% by weight of the aqueous dispersion.

[0086] In some embodiments, the aqueous dispersion may be formulated to comprise phospholipid in an amount described in paragraph [0073] above. For example, the aqueous dispersion may be formulated to comprise phospholipid in an amount in the range from about 0.001 % to about 3% by weight of the aqueous dispersion. In some embodiments, the aqueous dispersion may be formulated to comprise phospholipid in an amount in the range from about 0.01 % to about 1 % by weight of the aqueous dispersion. For example, the aqueous dispersion may comprise an amount of phospholipid at about 0.1 % by weight of the aqueous dispersion.

[0087] It will be appreciated that the polyglycerol esters of oleic acid and phospholipid present in the aqueous dispersion are lipid molecules and are believed to be incorporated in the lipid bilayer of the liposomal compound contained in the aqueous dispersion.

[0088] In some embodiments it may be desirable for the lipid bilayer of the liposomal compound to also contain phytosterol. In order to form such a liposomal compound, phytosterol may be present in the lipophilic phase comprising the lipid bilayer components. When phytosterol is used, the aqueous dispersion may be formulated to comprise phytosterol in an amount described in paragraph [0078] above. For example, the aqueous dispersion may be formulated to comprise phytosterol in an amount from about 0.001 % to 3% by weight of the aqueous dispersion. In some embodiments, the aqueous dispersion may be formulated to comprise phytosterol in an amount from about 0.01 % to about 1 % by weight of the aqueous dispersion. For example, the phytosterols may be present at about 0.1 % by weight of the aqueous dispersion.

[0089] If desired, the aqueous dispersion may be further treated in order to isolate the liposomal compound and/or to produce liposomal compounds of a desired particle size. Alternatively, the aqueous dispersion may be used directly without further treatment and combined with other ingredients to form a skin care product, such as a cosmetic composition, containing the liposomal compound.

[0090] Any other method known to the skilled artisan can also be used. For example, the liposomal compound can be produced by, but not limited to, techniques such as extrusion, agitation, sonication, reverse phase evaporation, self-assembly in aqueous solution, electrode-based formation techniques, microfluidic directed formation techniques, and the like. In certain embodiments, methods can be used to produce liposomes that are multilamellar and/or unilamellar, which may be large unilamellar vesicles (LUV) and/or small unilamellar vesicles (SUV).

Properties of the liposomal compound

[0091 ] The liposomal compound of the invention may be spherical and be of a diameter that facilitates its transcutaneous penetration. Without wishing to be bound by theory, the diameter of the liposomal compound is thought to aid penetration of the liposomal compound into the deeper layers of the skin, such as the lower stratum corneum, and therefore aid transport of the plant protein with peroxidase activity to sites in the skin where it is able to exert a beneficial effect in stimulating the production of proteins in the skin, such as collagen, profilaggrin and/or filaggrin.

[0092] In some embodiments, the liposomal compound may have a diameter of about 20 nm to about 200 nm. For example, the diameter of the liposomal compound may be about 20 nm to about 150 nm, about 20 nm to about 100 nm, about 20 nm to about 50 nm, about 50 nm to about 200 nm, about 50 nm to about 150 nm, about 50 nm to about 100 nm, about 100 nm to about 200 nm, about 100 nm to about 150 nm, or about 150 nm to about 200 nm. In some embodiments, the liposomal compound has a diameter of about 80 nm. The diameter of the liposomal compound can be determined by techniques known to a person skilled in the art. For example, dynamic light scattering (also referred to as photon correlation spectroscopy or quasi-elastic light scattering). This technique provides the mean diameter and distribution of the particles. [0093] Advantageously, the liposomal compound of the present invention is also believed to possess excellent temperature stability, which may be of benefit when blending with one or more cosmetically acceptable ingredients to form lotions, emulsions, cleansers and other skin care products, in particular cosmetic compositions for topical application to the skin.

[0094] In some embodiments, the liposomal compound according to the present invention may be unilamellar or multilamellar. In some embodiments, the liposomal compound is multilamellar.

Cosmetic composition for topical application to the skin

[0095] The liposomal compound according to the present invention is highly absorbable transcutaneously, and hence desirable in a skin care product, preferably a cosmetic composition for topical application to the skin.

[0096] In another aspect, the present invention also provides a skin care product comprising the liposomal compound as described herein for providing a skin care benefit. Preferably the skin care product is a cosmetic composition for topical application to the skin. For topical application, the composition may be in the form of a solution, spray, lotion, cream (for example a non-ionic cream), emulsion, oil, gel, essence, powder, paste, serum, ointment, and the like.

[0097] Skin care benefits achieved following application of the cosmetic composition may be for example those selected from the group consisting of treating/reducing wrinkling, sagging, scarring, aged and/or photo-damaged skin; boosting collagen deposition in skin, enhancing tissue repair; improving skin texture, smoothness and/or firmness, or a combination thereof. These may be considered to be cosmetic skin benefits. Naturally, the amount of benefit achieved, for example the amount of reduction in the appearance of wrinkles, sagging, and so forth will depend on the nature of the treatment, the condition of the skin prior to treatment and the length of treatment. Measurement of a skin care benefit is somewhat subjective. In one embodiment, the reduction of wrinkling, sagging, etc. of a subject's skin is a reduction compared to the condition of the skin prior to initiating treatment and is apparent to the naked eye. A range of non-invasive methods can be utilised to measure the amount of benefit achieved, for example ultrasound can be used to measure skin thickness, with an increase in skin thickness representative of a beneficial effect. Silicone dental impression material can be used to take an impression of the skin before and after use of the composition and the impression analysed by a profilometer to assess the roughness of the skin surface with decreased roughness representative of a beneficial effect. A hand-held uniaxial extensometer can be used to assess the extensibility of skin with a greater resistance to skin stretching representative of a beneficial effect. Digital images can be captured of the skin being treated with the composition using a facial imaging system such as the Rapid Evaluation of Anti-aging Leads (REAL; Proctor and Gamble Co, Cincinnati, OH USA). The captured images can be analysed to measure the wrinkle and depression area of selected facial regions with a reduction of the area representative of a beneficial effect. Transepidermal water loss (TEWL) can also be measured using a Dermalab® TEWL instrument (Cortex Technology, Hadsund, Denmark) or similar apparatus with a decrease in TEWL representative of a beneficial effect on the skin barrier.

[0098] In some embodiments, the cosmetic composition contains the liposomal compound in a range from about 0.01 % to about 20% by weight of the composition. In some embodiments, the cosmetic composition contains the liposomal compound in a range from about 0.01 % to about 20%, about 0.1 % to 10%, about 1 % to about 10% and about 1 % to about 5% by weight of the composition. For example, the cosmetic composition contains the liposomal compound at about 5% by weight of the composition.

[0099] In one embodiment, the cosmetic composition comprises a cosmetically acceptable carrier or vehicle. The cosmetically acceptable carrier or vehicle may act as a diluent, dispersant or carrier for the liposomal compound and/or other active agents. The vehicle may also contain materials commonly used in skin care products and can be in a wide variety of forms. For example, the carrier or vehicle may be water, liquid or solid emollients, silicone oils, emulsifiers, surfactants, solvents, humectants, thickeners, powders, propellants and the like.

[0100] The carrier or vehicle will generally form from about 1 % to about 10% by weight of the cosmetic composition. In some embodiments, the carrier or vehicle will form from about 1 % to about 8%, about 1 % to about 6%, about 1 % to about 4%, about 1 % to about 2%, about 2% to about 10%, about 2% to about 8%, about 2% to about 6%, about 2% to about 4%, about 4% to about 10%, about 4% to about 8%, about 4% to about 6%, about 6% to about 10%, about 6% to about 8%, or about 8% to about 10% by weight of the cosmetic composition. The carrier or vehicle can, in the absence of other cosmetic adjuncts, form the balance of the composition.

[0101 ] In some embodiments, the cosmetic composition may also comprise one or more agents which provide additional skin care benefits. For example, the cosmetic composition may comprise sunscreens, skin-protectant agents, skin-soothing agents, moisturizers, skin- lightening agents, skin tanning agents and the like. It would be understood by a person skilled in the art that a selected agent may provide more than one skin care benefit. In some embodiments, the agent is preferably obtained from natural sources, such as for example from plant sources. In some embodiments, the cosmetic composition may comprise a plant extract as described above. In some embodiments, the cosmetic composition may also comprise horseradish peroxidase and/or horseradish extract, in addition to the horseradish peroxidase and/or horseradish extract present in the liposomal compound.

[0102] The cosmetic composition may also further contain adjuncts such as binders, antioxidants, perfumes, stabilizers, penetration enhancers, lubricants, anti-microbial agents, solubilizing agents, suspending agents, coating agents, opacifiers, thickeners, acids, bases, salts, chelants, gums, alcohols, polyols, preservatives, colourants, buffers and the like. Furthermore, the composition may also contain other natural or nutraceutical products.

[0103] In addition to the agents mentioned above, the cosmetic composition for topical application to the skin may comprise other components commonly used in cosmetic formulations. The additional components should be suitable for topical application to the skin, that is when they are present in the cosmetic composition they should be suitable for use in contact with skin without undue toxicity, incompatibility, instability, allergic reaction and the like. The CTFA International Cosmetic Ingredient Dictionary and Handbook, 12 ^th Edition (2008) describes a wide range of non-limiting cosmetic ingredients that can used in the skin care industry, which may be suitable for use with the cosmetic compositions described herein.

[0104] Advantageously, the cosmetic compositions of the present invention may have additional desirable properties. For example, in some embodiments the compositions are temperature stable, have a long shelf life, and have good aesthetics.

Product Preparation, Form, Use and Packaging

[0105] In some embodiments, the liposomal compound according to the present invention is mixed with other ingredients to produce a skin care product. Preferably the skin care product is a cosmetic composition for topical application to the skin.

[0106] To prepare a cosmetic composition according to the present invention, the usual manner for preparing skin care products may be employed although care must be taken to avoid conditions that may result in protein denaturation. Such methods generally involve mixing of the ingredients in one or more steps to a relatively uniform state, with or without heating, cooling, application of a vacuum, and the like. The active components can suitably first be dissolved or dispersed in a portion of the water or another solvent or liquid to be incorporated in the composition. Typical compositions are oil-in- water or water-in-oil or water- in-oil-in-water emulsions.

[0107] In some embodiments, the cosmetic composition may be a skin lotion, cream, gel, foam, ointment, paste, emulsion, spray, conditioner, tonic, or the like, as described above. In some embodiments, the composition may be in the form of a so-called "wash-off product, e.g. a bath, shower gel or lotion, possibly containing a delivery system for certain components to promote adherence to the skin during rinsing. In other embodiments, the product is a "leave- on" product - that is, a product to be applied to the skin without a deliberate rinsing step soon after its application to the skin.

[0108] The cosmetic composition may be packaged in any suitable manner such as in a jar, a bottle, tube, roll-ball, pump, spray or the like, in the conventional manner.

[0109] The cosmetic composition can be applied with the fingers or with an implement or device (e.g. pad, cotton ball, applicator pen, spray applicator, and the like). For example, the cosmetic composition can be applied using a material which is optionally pre-soaked in the liposomal compound or composition of the invention. In one embodiment the material can be a gauze, sponge or the like which can be used to apply the composition of the invention for any period of time.

[01 10] Application of the cosmetic composition can be carried out one or more times daily to the skin which requires treatment. In general, a small quantity of the composition, for example from 0.1 to 5 grams is applied to a suitable area of the skin from a suitable container or applicator and spread over and/or rubbed into the skin using the hands or fingers or a suitable device. A rinsing step may optionally follow depending on whether the composition is formulated as a "leave-on" or a "rinse-off product.

[01 1 1 ] It will be understood that the cosmetic compositions may be made available to the general public as "over the counter" or non-prescription products. In one embodiment the topical composition is suitable to be made available to the general public over the counter.

Method of treatment I uses

[01 12] In another aspect, the present invention provides a method for promoting production of a protein, including collagen, profilaggrin and/or filaggrin, in skin, the method comprising applying to skin an effective amount of the liposomal compound or the cosmetic composition described above.

[01 13] In another aspect, the present invention provides use of an effective amount of the liposomal compound as described above in the manufacture of a composition for application to the skin of a subject to promote the production of a protein, including collagen, profilaggrin and/or filaggrin, in the skin.

[01 14] Promoting the production of a protein in the skin involves topically applying to the epidermal tissue a safe and effective amount of a liposomal compound or cosmetic composition of the present invention. The amount of the liposomal compound or composition which is applied, the frequency of application and the period of use will vary widely depending upon the level of plant protein with peroxidase activity (and, when present, other skin care agents) of a given liposomal compound or composition and the level of regulation desired, e.g. in light of the level of epidermal tissue damage present or expected to occur. Any part of the external portion of the body can be treated, e.g. lips, under-eye area, eyelids, hands, neck, torso, etc.

[01 15] In another aspect, the present invention provides a method for promoting production of collagen, including collagen I and/or collagen IV, in skin, the method comprising applying to skin an effective amount of the liposomal compound or the cosmetic composition described above.

[01 16] In another aspect, the present invention also provides use of an effective amount of the liposomal compound as described above in the manufacture of a composition for application to the skin of a subject to promote the production of collagen, including collagen I and/or collagen IV, in the skin.

[01 17] Without wishing to be limited by theory, it is thought that the promotion of collagen IV production may be beneficial as it is thought that at the site of a wrinkle, the dermoepidermal junction contains less collagen IV. This lack of collagen IV is thought to contribute to the depth and magnitude of wrinkles. The application of the liposomal compound or a cosmetic composition incorporating a liposomal compound as defined herein to the skin of a subject may improve the appearance of skin by promoting the production of collagen, preferably collagen IV, in the skin and help reduce the depth and/or occurrence of wrinkles in skin. [01 18] In another aspect, the present invention provides a method for promoting production of profilaggrin and/or filaggrin in skin, the method comprising applying to skin an effective amount of the liposomal compound or the cosmetic composition described above.

[01 19] In another aspect, the present invention provides use of an effective amount of the liposomal compound as described above in the manufacture of a composition for application to the skin of a subject to promote the production of profilaggrin and/or filaggrin, in the skin.

[0120] The structural protein filaggrin is found in the stratum corneum and plays an important role in epidermal homeostasis. Filaggrin is synthesised as profilaggrin, a >400kDa precursor protein which is processed during the later stages of the terminal differentiation of keratinocytes to form filaggrin. Filaggrin interacts with intermediate filaments, such as keratin, causing their aggregation into macrofibrils. This process contributes to cellular compaction and the formation of a highly insoluble keratin matrix. The keratin matrix acts as an attachment point for cornefied envelope proteins and lipids which together form the stratum corneum. Within the stratum corneum, filaggrin may also be incorporated into the lipid envelope, which is responsible the skin barrier function. Filaggrin may also undergo further processing in the upper stratum corneum to release free amino acids which aid in water retention. The loss of profilaggrin or filaggrin can result in a poorly formed stratum corneum, which is also prone to water loss. Filaggrin levels are reduced at the bottom of a wrinkle.

[0121 ] Without wishing to be limited by theory, it is thought that the promotion of profilaggrin and/or filaggrin production may be beneficial as filaggrin levels and water retention have been found to be reduced at the bottom of a wrinkle. Moreover, it is thought that keratinization and desquamation are disrupted at the bottom of a wrinkle. The application of the liposomal compound or a cosmetic composition incorporating a liposomal compound as described above to the skin of a subject may improve the appearance of skin by promoting the production of profilaggrin and/or filaggrin in the skin, which in turn may reduce disruption to keratinization and desquamation at the bottom of a wrinkle.

[0122] In another aspect, the present invention provides a method for providing a skin care benefit, the method comprising topically applying the liposomal compound or the cosmetic composition described above to the skin of a subject. In some embodiments, a skin care benefit may be as defined herein. [0123] In another aspect, the present invention provides use of an effective amount of the liposomal compound as described above in the manufacture of a composition for application to the skin of a subject to provide a skin care benefit to the subject.

[0124] It is to be noted that where a range of values is expressed, it will be clearly understood that this range encompasses the upper and lower limits of the range, and all values in between these limits.

[0125] The term "about" as used in the specification means approximately or nearly and in the context of a numerical value or range set forth herein is meant to encompass variations of +/- 10% or less, +/- 5% or less, +/- 1 % or less, or +/- 0.1 % or less of and from the numerical value or range recited or claimed.

[0126] Throughout the description and claims of this specification, the word "comprise" and variations of the word, such as "comprising" and "comprises", is not intended to exclude other additives, components, integers or steps.

[0127] It will be apparent to the person skilled in the art that while the invention has been described in some detail for the purposes of clarity and understanding, various modifications and alterations to the embodiments and methods described herein may be made without departing from the scope of the inventive concept disclosed in this specification.

[0128] Reference will now be made to experiments that embody the above general principles of the present invention. The following examples are for the purpose of describing particular embodiments only and are not intended to be limiting with respect to the above description.

EXAMPLE 1

Exemplary Liposomal Compounds

[0129] Embodiments of the liposomal compounds according to the present invention are detailed in Table 1 below. The exemplary embodiments list the core ingredients such that other components can be included in the composition (with water making up the balance of the ingredients to 100% w/w). TABLE 1

Formulation Component Amount (w/w percent)

1 Polyglycerol oleic acid ester 1 .0%

Plant Extract ¹ 0.5%

Phospholipid 0.05%

Polyglycerol oleic acid ester 1 .0%

Plant Extract ¹ 0.5%

Phospholipid 0.1 %

Polyglycerol oleic acid ester 1 .0%

Plant Extract ¹ 1.0%

Phospholipid 0.05%

Polyglycerol oleic acid ester 2.0%

Plant Extract ¹ 0.5%

Phospholipid 0.05%

Polyglycerol oleic acid ester 2.0%

Plant Extract ¹ 1.0%

Phospholipid 0.05%

Polyglycerol oleic acid ester 2.0%

Plant Extract ¹ 1.0%

Phospholipid 0.1 %

Polyglycerol oleic acid ester 2.5%

Plant Extract ¹ 0.5%

Phospholipid 0.05%

Polyglycerol oleic acid ester 2.5%

Plant Extract ¹ 0.5%

Phospholipid 0.1 %

Polyglycerol oleic acid ester 2.5%

Plant Extract ¹ 1.0%

Phospholipid 0.1 % Polyglycerol oleic acid ester 2.0%

Plant Extract ¹ 1.5%

Phospholipid 0.05%

Polyglycerol oleic acid ester 2.0%

Plant Extract ¹ 1.5%

Phospholipid 0.1 %

Polyglycerol oleic acid ester 2.0%

Plant Extract ¹ 2.0%

Phospholipid 0.05%

Polyglycerol oleic acid ester 2.0%

Plant Extract ¹ 2.0%

Phospholipid 0.1 %

Polyglycerol oleic acid ester 2.0%

Plant Extract ¹ 3.0%

Phospholipid 0.05%

Polyglycerol oleic acid ester 2.0%

Plant Extract ¹ 3.0%

Phospholipid 0.1 %

Polyglycerol oleic acid ester 2.0%

Plant Extract ¹ 4.0%

Phospholipid 0.1 %

Polyglycerol oleic acid ester 5.0%

Plant Extract ¹ 5.0%

Phospholipid 1.0%

Polyglycerol oleic acid ester 2.5%

Plant Extract ¹ 2.5%

Phospholipid 0.5%

Polyglycerol oleic acid ester 1 .9%

Plant Extract ¹ 1.0%

Phospholipid 0.1 % 20 Polyglycerol-2-oleate (diglycerol monooleate ester) 1 .9%

Horseradish extract ¹ 1 .0%

Hydrogenated Lecithin 0.1 %

NOTE: ¹ The extract comprises a plant protein with peroxidase activi ty-

EXAMPLE 2

Preparation of the Liposomal Compound

[0130] An exemplary liposomal compound is prepared by the method below. The components listed in Table 2, part A are heated to 90°C and evenly dissolved and the components listed in part B are added gradually to part A while stirring to yield a liposomal compound according to the invention.

TABLE 2

EXAMPLE 3

Plant protein with peroxidase activity promotes production of type I and type IV collagen by normal human skin fibroblasts

Cell Culture

[0131 ] Normal human skin fibroblasts (NB1 RGB) were cultured in Dulbecco's minimum essential media (DMEM) containing 10% FBS at 37°C and 5% C0 ₂ , and then harvested by trypsin treatment. The harvested cells were suspended at 1 .6 * 10 ⁵ cells/mL in the same culture medium. A 100 μί aliquot was added to each well of a 96-well microplate, and the cells were cultured overnight. The culture medium was discarded and 150 μί of a solution of horse radish extract (in 50% glycerin) diluted in Dulbecco's DMEM containing 0.25% FBS was added to each well, and the cells were cultured for 3 days. After culture, the culture media was collected and the amount of collagen I and IV in the culture medium in each well was measured using the ELISA method. 25 μg/mL magnesium ascorbyl phosphate was used as a positive control.

[0132] Type I Collagen Results: The rate of promotion of type I collagen production was calculated as follows:

Rate of promotion of type I collagen production (%) = A / B ^χ 100

A: Amount of type I collagen when sample is added

B: Amount of type I collagen when sample is not added

Cell survival rate (%) = C / D * 100

C: Absorbance in cells when sample is added

D: Absorbance in cells when sample is not added

[0133] It was found that treatment of normal human skin fibroblasts with horse radish extract promoted type I collagen production at all doses (Table 3).

TABLE 3

Rate of promotion of type I collagen production

Mean+S.E., n = 5; ^*** : p<0.001.

[0134] Type IV Collagen results: The rate of promotion of type IV collagen production was calculated as follows:

Rate of promotion of type IV collagen production (%) = A / B ^χ 100 A: Amount of type IV collagen when sample is added

B: Amount of type IV collagen when sample is not added

Cell survival rate (%) = C / D * 100

C: Absorbance in cells when sample is added

D: Absorbance in cells when sample is not added

[0135] It was found that treatment of normal human skin fibroblasts with horse radish extract promoted type IV collagen production at all doses (Table 4).

TABLE 4

Rate of promotion of type IV collagen production

EXAMPLE 4

Promotion of profilaggrin/filaggrin production

[0136] This study evaluated the effect of horseradish peroxidase on profilaggrin/filaggrin production.

Cell Culture

[0137] Normal human epidermal keratinocytes (NHEK) were cultured in a 75 cm ² flask with keratinocyte growth medium (KGM) at 37°C under 5% C0 ₂ , and cells were harvested by treating with Trypsin/EDTA. The harvested cells were suspended at 1 .5 * 10 ⁵ cells/mL in KGM, a 2 mL aliquot was added to each well of a collagen-coated 6-well plate and the cells were cultured for 3 days at 37°C under 5% C0 ₂ . After culture, the culture medium was exchanged for 2 ml. of a solution of KGM containing the sample (horseradish extract in 50% gylcerin) dissolved in 0.2% dimethyl sulfoxide (DMSO) or with no sample (control), and the cells were cultured for 5 days at 37°C under 5% C0 ₂ . After culture, total protein was prepared according to the standard protocol.

Western blotting

[0138] The prepared sample was separated by SDS-PAGE at 10 μg/lane, and then transferred to PVDF. After blocking with 4% Block Ace solution, the separated sample was sequentially reacted with anti-human filaggrin monoclonal antibody (Santa Cruz Biotechnology, Inc.), biotin-labelled anti-mouse Ig (whole Ab) (Amersham Biosciences), and streptavidin-peroxidase conjugate (CALBIOCHEM), respectively diluted 5,000-, 10,000- and 10,000-fold with a solution of 0.1 % Tween 20 and 0.3% Block Ace. Profilaggrin and filaggrin were detected through the luminescence of ECL Plus Western Blotting Detection Reagents (GE Healthcare) using the ChemiDoc XRS+ imaging device (Bio-Rad Laboratories, Inc.). The detected bands were quantitatively analyzed by Image Lab Software version 2.0 (Bio-Rad Laboratories, Inc.).

[0139] Promotion of profilaggrin/filaggrin production by the sample was evaluated using the net intensity of the profilaggrin and filaggrin bands from the respective 10 μg protein preparations obtained from cell cultures incubated with or without the sample.

[0140] The rate of promotion of profilaggrin and filaggrin production was calculated as follows:

Rate of promotion of profilaggrin and filaggrin production (%) = A / B ^χ 100

A: Net intensity when sample is added

B: Net intensity when sample is not added (control)

[0141 ] It was found that treatment of normal human epidermal keratinocytes with horse radish extract has a weak effect on profilaggrin/filaggrin production at lower concentrations (Table 5). TABLE 5

Promotion of profilaggrin/filaggrin production

[0142] This study evaluated the transcutaneous absorption of a liposomal compound containing horseradish peroxidase according to an embodiment of the invention. Reconstructed epidermal skin (EPI-200; Martek, USA) was used in this study. The liposomal compound and a control vesicle were formulated in water or serum. Nile Red was incorporated into the liposomal compound and control vesicle for detection. Each sample was applied on reconstructed epidermal skin treated and incubated at 37° C for 1 hour. The reconstructed epidermal skin was then collected and frozen sections were prepared for observation using a confocal laser microscope LSM510 (Carl Zeiss, Germany). The diameter of the liposomal compound according to the invention and control vesicle was determined by photon correlation spectroscopy. The diameter of the liposomal compound was approximately 80 nm. The diameter of the control vesicle was approximately 650 nm.

[0143] As shown in Figure 1 , when compared to a control vesicle, the liposomal compound was observed to permeate deeper into the reconstructed epidermis, whether formulated in water or serum.

[0144] It will be appreciated that various modifications and variations of the methods and compositions of the invention described herein will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention that are apparent to those skilled in the art are intended to be within the scope of the present invention.