Field of the Invention

The present invention relates to a method of treating skin which provides enhanced delivery of oil phase and oil phase components to the skin.

Background of the Invention

US2009136433 (BASF) discloses what appear to be prophetic compositions comprising cationic surfactant and hydrophobin. The hydrophobins are Class I fusion proteins and are said to be introduced into the compositions in order to deposit onto keratin or skin.

Skin care compositions are used to deliver a variety of actives. During everyday procedures such as bathing and exercise the skin actives are removed. There remains a need for skin care compositions which deliver skin care actives that are not easily removed.

The present inventors have found a way of mitigating the above problem. In addition, agents associated with the oil may be delivered more effectively. This effect is particularly enhanced when product is applied and then the skin is rinsed.

Summary of the Invention

The present invention provides a method of treating skin comprising the step of applying to the skin a composition obtainable by: (i) preparing an oil-in-water emulsion by dispersing an oil phase into an aqueous continuous phase, the aqueous continuous phase comprising an oil-in-water emulsifier which is selected from one or more class II hydrophobins, so that emulsified droplets of oil phase are formed; and ii) combining the emulsion so obtained with a skin base composition.

Description of the Invention

In the context of the present invention the term skin relates to all skin on the human body including scalp and underarm. Oil-in-water emulsion.

Dispersed Oil Phase

The oil phase may generally be formed from any physiologically acceptable lipophilic material having a liquid or semi-solid consistency at 25°C.

Lipophilic materials suitable for use as oil phase components in the invention include both natural and synthetically produced oils. Particularly preferred oils include fatty acid triglycerides, fatty acid monoglycerides or mixtures thereof.

Specific examples of suitable oil phase components include naturally or synthetically derived liquid hydrocarbons such as liquid paraffin, squalane, squalene and mineral oil; fatty esters having 6 to 50 carbon atoms in a molecule such as glyceryl monooleate, glyceryl monolinoleate, glyceryl monoisostearate, cetyl isooctanoate, octyldodecyl myristate, isopropyl myristate, isopropyl palmitate, isocetyl stearate, octyldodecyl oleate, sorbitan monooleate, sorbitan monopalmitate, sucrose mono-, di-or tri-palmitate, glyceryl trioctanoate and glyceryl triisostearate; higher fatty acids having 6 to 50 carbon atoms in a molecule such as isostearic acid, oleic acid, hexanoic acid and heptanoic acid; aliphatic higher alcohols having 6 to 50 carbon atoms in a molecule, such as isostearyl alcohol and oleyl alcohol; oils; triglyceride oils are particularly preferred especially those derived from plant sources such as castor oil, sunflower oil, olive oil, jojoba oil, rapeseed oil, soybean oil, palm kernel oil, babassu kernel oil and coconut oil.

The level of oil within the total composition is from 0.5 to 40 wt% of the total composition more preferably from 1 to 30 wt%, most preferably from 5-15 wt%.

Mixtures of any of the above described materials may also be used, and may be preferred in some cases. For example liquid materials may be used as diluents or carriers for semi-solid materials in order to improve processability.

The oil phase may also include further skin care benefit agents dissolved, dispersed or entrapped therein. Preferably the skin benefit agent is associated with the oil. Preferably skin care benefit agents are oil soluble, more preferably the skin benefit agents have a log P greater than 0.8, more preferably of 2 or greater, most preferably of 3 or greater.

In the context of the present invention Log P is the logarithm of the ratio (at 20°C) of the concentrations of a product between two solvents.

Preferably the skin benefit agent is soluble in soya bean oil at 20°C.

The term "skin care agent" in the context of the present invention generally means any material capable of providing a cosmetic or therapeutic benefit to the skin.

Preferably the skin benefit agent is selected from the group consisting of antimicrobial, antifungal and anti-aging agents, sun protection actives,

moisturisers, anti-inflammatory agents, antidandruff agents, skin conditioning agents, skin lightening and skin tanning actives.

Preferred skin lightening composition are resorcinol (log P 0.81 ) and derivates thereof especially derivatives including 4-substituted resorcinols. 12-hydroxy stearic acid is an alternative preferred skin lightening agent. Specific examples of sunscreens are ethylhexyl p-methoxycinnamate (log P 5.8) (available as Parsol MCX ^® ), avobenzene (available as Parsol 1789 ^® ),

octylsalicylate (available as dermablock OS ^® ), tetraphthalylidene dicamphor sulfonic acid (available as Mexoryl SX®), benzophenone-4 and benzophenone-3 (Oxybenzone).

Preferred skin rejuvenation agents include conjugated linoleic acid and/or retinoid. As used herein, "retinoid" includes all natural and/or synthetic analogs of Vitamin A or retinol-like compounds which possess the biological activity of Vitamin A in the skin as well as the geometric isomers and stereoisomers of these compounds. The retinoid is preferably retinol, retinol esters (e.g., C2 -C22 alkyl esters of retinol, including retinyl palmitate, retinyl acetate, retinyl propionate), retinal, and/or retinoic acid (including all-trans retinoic acid and/or 13-cis-retinoic acid), more preferably retinoids other than retinoic acid. Other suitable retinoids are

tocopheryl-retinoate [tocopherol ester of retinoic acid (trans- or cis-), adapalene {6-[3-(1 -adamantyl)-4-methoxyphenyl]-2-naphthoic acid}, and tazarotene (ethyl 6- [2-(4,4-dimethylthiochroman-6-yl)-ethynyl]nicotinate). Preferred retinoids are retinol, retinyl palmitate, retinyl acetate, retinyl propionate, retinal and

combinations thereof. Vitamin E is suitable for inclusion as a skin benefit agent.

Curcumin is suitable for inclusion as a skin benefit agent.

Further embodiments of suitable skin benefit agents include resveratrol, alpha- lipoic acid, ellagic acid, kinetin, retinoxytrimethylsilane (available from Clariant Corp. under the Silcare 1 M-75 trademark), dehydroepiandrosterone (DHEA) and combinations thereof. Ceramides (including Ceramide 1 , Ceramide 3, Ceramide 3B, Ceramide 6 and Ceramide 7) as well as pseudoceramides. A preferred antimicrobial agents is climbazole

Preferably skin benefit agents are present from 0.01 to 10%, more preferably from 0.5 to 2% by weight of the total composition. Aqueous Continuous Phase

The aqueous continuous phase (into which the oil phase is dispersed) generally comprises at least 10%, preferably at least 20% by weight water based on the total weight of the aqueous continuous phase.

Hydrophobin

The aqueous continuous phase comprises an oil-in-water emulsifier which is selected from one or more hydrophobins. Hydrophobins are a well-defined class of proteins (Wessels, 1997, Adv. Microb. Physio. 38: 1 -45; Wosten, 2001 , Annu Rev. Microbiol. 55: 625-646) capable of self-assembly at a hydrophobic/hydrophilic interface, and having a conserved sequence:

Xn-C-Xs-g-C-C-X -sg-C-Xs^S-C-Xs-g-C-C-Xe-ie-C-Xm

(SEQ ID No. 1 ) where X represents any amino acid, and n and m independently represent an integer. Typically, a hydrophobin has a length of up to 125 amino acids. The cysteine residues (C) in the conserved sequence are part of disulphide bridges. In the context of this invention, the term hydrophobin has a wider meaning to include functionally equivalent proteins still displaying the characteristic of self- assembly at a hydrophobic-hydrophilic interface resulting in a protein film, such as proteins comprising the sequence:

X _n -C-Xi _50-C-Xo-5-C-Xi -100-C-X1 -100-C-X1 _50-C-Xo-5-C-Xi -50-C-Xm (SEQ ID No. 2) or parts thereof still displaying the characteristic of self-assembly at a

hydrophobic-hydrophilic interface resulting in a protein film. In accordance with the definition of this invention, self-assembly can be detected by adsorbing the protein to Teflon and using Circular Dichroism to establish the presence of a secondary structure (in general, a-helix) (De Vocht et al., 1998, Biophys. J. 74: 2059-68).

The formation of a film can be established by incubating a Teflon sheet in the protein solution followed by at least three washes with water or buffer (Wosten et al., 1994, Embo. J. 13: 5848-54). The protein film can be visualised by any suitable method, such as labelling with a fluorescent marker or by the use of fluorescent antibodies, as is well established in the art. m and n typically have values ranging from 0 to 2000, but more usually m and n in total are less than 100 or 200. The definition of hydrophobin in the context of this invention includes fusion proteins of a hydrophobin and another polypeptide as well as conjugates of hydrophobin and other molecules such as polysaccharides.

Hydrophobins identified to date are generally classed as either class I or class II. Both types have been identified in fungi as secreted proteins that self-assemble at hydrophobic-hydrophilic interfaces into amphipathic films.

Hydrophobin-like proteins have also been identified in filamentous bacteria, such as Actinomycete and Streptomyces sp. (WO01/74864; Talbot, 2003, Curr. Biol, 13: R696-R698). These bacterial proteins by contrast to fungal hydrophobins, may form only up to one disulphide bridge since they may have only two cysteine residues. Such proteins are an example of functional equivalents to hydrophobins having the consensus sequences shown in SEQ ID Nos. 1 and 2, and are within the scope of this invention.

The hydrophobins can be obtained by extraction from native sources, such as filamentous fungi, by any suitable process. For example, hydrophobins can be obtained by culturing filamentous fungi that secrete the hydrophobin into the growth medium or by extraction from fungal mycelia with 60% ethanol. It is particularly preferred to isolate hydrophobins from host organisms that naturally secrete hydrophobins. Preferred hosts are hyphomycetes (e.g. Trichoderma), basidiomycetes and ascomycetes. Particularly preferred hosts are food grade organisms, such as Cryphonectria parasitica which secretes a hydrophobin termed cryparin (MacCabe and Van Alfen, 1999, App. Environ. Microbiol 65:

5431 -5435). Alternatively, hydrophobins can be obtained by the use of recombinant

technology. For example host cells, typically micro-organisms, may be modified to express hydrophobins and the hydrophobins can then be isolated and used in accordance with the present invention. Techniques for introducing nucleic acid constructs encoding hydrophobins into host cells are well known in the art. More than 34 genes coding for hydrophobins have been cloned, from over 16 fungal species (see for example WO96/41882 which gives the sequence of hydrophobins identified in Agaricus bisporus; and Wosten, 2001 , Annu. Rev. Microbiol. 55: 625- 646). Recombinant technology can also be used to modify hydrophobin sequences or synthesise novel hydrophobins having desired/improved properties.

Typically, an appropriate host cell or organism is transformed by a nucleic acid construct that encodes the desired hydrophobin. The nucleotide sequence coding for the polypeptide can be inserted into a suitable expression vector encoding the necessary elements for transcription and translation and in such a manner that they will be expressed under appropriate conditions (e.g. in proper orientation and correct reading frame and with appropriate targeting and expression sequences). The methods required to construct these expression vectors are well known to those skilled in the art.

A number of expression systems may be used to express the polypeptide coding sequence. These include, but are not limited to, bacteria, fungi (including yeast), insect cell systems, plant cell culture systems and plants all transformed with the appropriate expression vectors. Preferred hosts are those that are considered food grade - 'generally regarded as safe' (GRAS).

Suitable fungal species, include yeasts such as (but not limited to) those of the genera Saccharomyces, Kluyveromyces, Pichia, Hansenula, Candida, Schizo saccharomyces and the like, and filamentous species such as (but not limited to) those of the genera Aspergillus, Trichoderma, Mucor, Neurospora, Fusarium and the like.

The sequences encoding the hydrophobins are preferably at least 80% identical at the amino acid level to a hydrophobin identified in nature, more preferably at least 95% or 100% identical. However, persons skilled in the art may make

conservative substitutions or other amino acid changes that do not reduce the biological activity of the hydrophobin. For the purpose of the invention these hydrophobins possessing this high level of identity to a hydrophobin that naturally occurs are also embraced within the term "hydrophobins".

Hydrophobins can be purified from culture media or cellular extracts by, for example, the procedure described in WO01/57076 which involves adsorbing the hydrophobin present in a hydrophobin-containing solution to surface and then contacting the surface with a surfactant, such as Tween 20, to elute the

hydrophobin from the surface. See also Collen et al., 2002, Biochim Biophys Acta. 1569: 139-50; Calonje et al., 2002, Can. J. Microbiol. 48: 1030-4; Askolin et al., 2001 , Appl Microbiol Biotechnol. 57: 124-30; and De Vries et al., 1999, Eur J Biochem. 262: 377-85.

Typically, the hydrophobin is in an isolated form, typically at least partially purified, such as at least 10% pure, based on weight of solids. By "isolated form", we mean that the hydrophobin is not added as part of a naturally-occurring organism, such as a mushroom, which naturally expresses hydrophobins. Instead, the hydrophobin will typically either have been extracted from a naturally-occurring source or obtained by recombinant expression in a host organism.

Hydrophobin proteins can be divided into two classes: Class I, which are largely insoluble in water, and Class II, which are readily soluble in water. Hydrophobins for use with the present invention are Class II hydrophobins.

Preferably the hydrophobins used are Class II hydrophobins such as HFBI, HFBII, HFBIII, or Cerato ulmin. The hydrophobin can be from a single source or a plurality of sources e.g. a mixture of two or more different hydrophobins.

The amount of hydrophobin in the total composition is preferably at least 0.001 wt%, more preferably at least 0.005 wt% most preferably at least 0.01 wt%, and preferably no greater than 2 wt%, more preferably 1wt% or less.

Base Formulations

Suspending Agent/ Thickening Agent

Preferably the aqueous composition of the invention further comprises a suspending agent. Suitable suspending agents are selected from polyacrylic acids, cross-linked polymers of acrylic acid, copolymers of acrylic acid with Hydrophobic monomer, copolymers of carboxylic acid-containing monomers and acrylic esters, cross-linked copolymers of acrylic acid and acrylate esters, heteropolysaccharide gums and crystalline long chain acyl derivatives. The long chain acyl derivative is desirably selected from ethylene glycol stearate, alkanolamides of fatty acids having from 16 to 22 carbon atoms and mixtures thereof. Ethylene glycol distearate and polyethylene glycol 3 distearate are preferred long chain acyl derivatives, since these impart pearlescence to the composition. Polyacrylic acid is available commercially as Carbopol 420,

Carbopol 488 or Carbopol 493. Polymers of acrylic acid cross-linked with a polyfunctional agent may also be used; they are available commercially as

Carbopol 910, Carbopol 934, Carbopol 941 , Carbopol 980, Carbopol ETD2020, Carbopol Ultrez 10 and Carbopol ETD2050. An example of a suitable copolymer of a carboxylic acid containing monomer and acrylic acid esters is Carbopol 1342. All Carbopol (trademark) materials are available from Lubrizol Corp.

Suitable cross-linked polymers of acrylic acid and acrylate esters are Pemulen TR1 or Pemulen TR2. A suitable heteropolysaccharide gum is xanthan gum, for example that available as Kelzan mu.

The aqueous continuous phase may if necessary include a thickener in order to reduce creaming or coalescence of the particles of the dispersed oil phase.

Examples of suitable thickeners include organic polyols having 3 or more hydroxyl groups in the molecule (hereinafter termed "organic polyols"). Examples of such materials include glycerol, sorbitol, xylitol, mannitol, lactitol, maltitol, erythritol, and hydrogenated partially hydrolyzed polysaccharides. The most preferred organic polyol is glycerol. Mixtures of any of the above described materials may also be used.

Mixtures of any of the above suspending agents/thickeners may be used.

Preferred is a mixture of cross-linked polymer of acrylic acid and crystalline long chain acyl derivative.

Suspending agents and/or thickening agents if included, will generally be present in the composition of the invention at levels of from 0.1 to 10%, preferably from 0.2 to 4%, more preferably from 0.3 to 1 .0% by total weight of suspending agent based on the total weight of the composition.

Further Ingredients

Adjunct humectants may be employed in the end use compositions of the present invention. These are generally polyhydric alcohol-type materials. Typical polyhydric alcohols include glycerol, propylene glycol, dipropylene glycol, polypropylene glycol, polyethylene glycol, sorbitol, hydroxypropyl sorbitol, hexylene glycol, 1 ,3-butylene glycol, isoprene glycol, 1 ,2,6-hexanetriol, ethoxylated glycerol, propoxylated glycerol and mixtures thereof. If used, the amount of adjunct humectant may range anywhere from 0.5 to 40%, preferably between 1 and 30 % by weight of the end use composition

A composition of the invention may contain other ingredients for enhancing performance and/or consumer acceptability. Such ingredients include fragrance, colouring agents, dyes and pigments, pH adjusting agents, pearlescers or opacifiers, surfactants in particular nonionic surfactants,viscosity modifiers, preservatives, and natural hair/skin nutrients such as botanicals, fruit extracts, sugar derivatives and amino acids, silicones, chelating agents such as EDTA, antioxidants such as vitamin E acetate, antimicrobials and sunscreens. Each of these ingredients will be present in an amount effective to accomplish its purpose. Generally these optional ingredients are included individually at a level of up to about 5% by weight of the total composition, more preferably at a level of up to 2%, most preferably up to 1 %, by weight of the total composition.

Method of preparation

A typical process used to form the oil-in-water emulsion described above comprises the following steps: mixing one or more hydrophobins with water and optionally a thickener such as glycerol to form an aqueous phase; mixing one or more oil phase components (as described above) in a separate vessel to form an oil phase; adding the oil phase to the aqueous phase, agitating to form a mixture and subjecting the resultant mixture to a mechanical emulsification treatment, thereby forming an oil-in-water emulsion in which the emulsified particles of oil phase are emulsified with the one or more hydrophobins.

The mechanical emulsification treatment may suitably be carried out using high shear mixing or homogenizing equipment known to those skilled in the art, such as a Silverson® mixer or a Microfluidizer®. Heating may be employed if necessary to aid processing during any or all of the process steps described above.

The oil-in-water emulsion is then added to a base composition followed by mixing in a conventional manner .

The invention is further illustrated with reference to the following, non-limiting Examples.

EXAMPLES

Skin Cream Compositions

Emulsions of Example 1 were prepared by mixing the Tween 20 (Sigma-Aldrich Code P1379) with the glycerol (from Pricene) and water. The Myritol 318 (from Cognis) was then added and a pre-emulsion formed using a Silverson LR4 mixer running at maximum speed for 30 seconds at room temperature. The pH was adjusted to 4.9 and the pre-emulsion was treated with a Branson® ultra sonic probe (Branson Sonifier 250) running at duty cycle of 50% for 30 seconds. The Carbopol® ETD 2050 (from Lubrizol) was hydrated in deionised water by gentle mixing at 150rpm on an IKA RET Basic magnetic stirrer overnight. The pH was adjusted to 4.9 and the hydrated Carbopol® was gently mixed with the emulsion to give the final skin care composition. Composition was stored at chill

temperature until required.

Emulsions of Example 2 were prepared by sonication of the hydrophobin stock solution in a VWR® USC200T ultrasonic bath for 10 minutes. The hydrophobin was then added to the water and glycerol. The Myritol® 318 was added and a pre-emulsion was formed using a Silverson LR4 mixer running at maximum speed for 30 seconds at room temperature. The pH was adjusted to 4.9 and the pre- emulsion was passed through a Microfluidics ® Microfluidiser M1 10S Materials Processor running at 250 bar pressure. The emulsion was passed through the Microfluidiser twice. The Carbopol® ETD 2050 (from Lubrizol) was hydrated in deionised water by gentle mixing at 150rpm on an IKA RET Basic magnetic stirrer overnight. The pH was adjusted to 4.9 and the hydrated Carbopol® was then carefully mixed into the emulsion to give the final skin care composition. The composition was stored at chill temperature until required.

Application and rinse-off protocol for skin cream formulations (Examples 1 and 2)

The porcine skin (waste product from meat processing) was pre-treated in the following way:

A large piece of clipped pig back skin was washed by wiping with 70% ethanol 10 times and rinsed under tap water for 1 minute. Skin was washed (by rubbing) with Salon C shampoo & water for 2 minutes and rinsed under tap water for a further 1 minute. The washed skin was patted dried.

The washed porcine skin was cut into 1 .5 x 2cm piece and super-glued onto a glass slide. 12μΙ (4μΙ/αη ² ) of the emulsion described above stained with Nile blue. The stain comprised 0.1 % solution of Nile Blue (in water). 1 part Nile Blue solution was added to 9 parts emulsion (and mixing gently). The Nile Blue was added to the emulsion just before applying to the skin. The Nile Blue used was Nile Blue hydrogen sulphate from Gurr microscopy materials, BDH Chemicals Ltd. The stained emulsion was applied to the skin with a circular motion using a finger tip for 10 seconds, then incubated at room temperature for 5 minutes. Excess emulsion was rinsed off under running tap water for 5 seconds.

Deposition was assessed using a Leica DM IRBE confocal microscope with a Leica TCS SP blue laser and with a Leica Z16 APOA stereo macroscope

The porcine skin was washed under a continuous flow of tap water for 5 seconds for up to 5 times without rubbing and examined immediately and after washes 1 , 3 and 5.

The results showed that deposition of oil on the skin was enhanced in Example 2 compared with Example 1 .

Furthermore after 5 further washes with rubbing Example 1 most of the oil was removed, Example 2 still had a significant level of oil present.

Examples 3 to 4

Emulsion of Example 3 was prepared by dissolving Curcumin (supplied by Sigma Aldrich) in Grinsted MCT oil (supplied by Danisco) whilst stirring at 75°C for 10 minutes. The hydrophobin (HPC-MTA, supplied by Danisco) was sonicated for 10 minutes in a VWR USC200T ultrasonic water bath. An aqueous solution of hydrophobin was prepared with water and the pH was adjusted to pH 4.2. The curcumin/MCT mixture was added to the aqueous phase and mixed for 1 minute at full speed using the Silverson with the smallest mesh. The pre-emulsion was then processed on a Branson Ultrasonic probe (Branson Sonifer 250) for 2 minutes running at a duty cycle of 50%. The emulsions were wrapped up in foil to protect from photo bleaching and stored at chill temperature until required.

Emulsion for Example 4 was prepared by Curcumin (supplied by Sigma Aldrich) was dissolved in Grinsted MCT oil (supplied by Danisco) whilst stirring at 75°C for 10 minutes. An aqueous solution of Tween20 (supplied by Sigma Aldrich) was prepared with water and the pH was adjusted to pH 4.2 using citric acid and NaOH. The curcumin/MCT mixture was added to the aqueous phase and mixed for 1 minute at full speed using the Silverson with the smallest mesh. The pre- emulsion was then processed on a Branson Ultrasonic probe (Branson Sonifer 250) for 1 minute running at a duty cycle of 50%. The emulsion was wrapped up in foil to protect from photo bleaching and stored at chill temperature until required.

Application and rinse-off protocol for Examples 3 and 4 Porcine back skin was clipped and treated with the depilatory product Veet®, according to the manufacturer's instructions, to remove all hair fibres. The skin was then washed extensively under tap water to remove all traces of Veet®. The washed skin was cut into 1 .5 x 2cm pieces and super-glued on to glass slides. Before applying emulsion, the skin was allowed to equilibrate for 30 minutes by incubating in a water bath set at 32°C. 12ml_ of emulsion (4mL/cm ² ) was then applied to each skin piece for 10 seconds with a circular motion using a finger tip. The skin and emulsion were then incubated at room temperature for 5 minutes. The skin was incubated in a water bath set at 32°C for a further 1 hour. Assessment of Curcumin Delivery for Examples 3 and 4

After incubation the surface of the pig skin was tape stripped using pieces of Sellotape®. The Sellotape® was pressed firmly on to the surface of the skin and was then peeled from the surface to remove the upper skin layer. The skin was tape stripped 6 times. Each tape was placed into a vial containing 1 .5ml_ ethanol (0.5 ml_ per cm ² of tape) and sonicated for 15 minutes to remove any curcumin present from the tape. The tape was then removed from each vial and discarded.

Absorbance at 425nm of each ethanol sample was then recorded and the amount of curcumin present in each sample was calculated using standard curve data. The absorbance of defined amounts of each emulsion was also measured and the percentage of the dose applied in each ethanol sample was calculated. For each emulsion 4 skin pieces were treated.

Results

Table 1

^* significantly different to MCT Tween control p = 0.0326

* ^* significantly different to MCT Tween control p < 0.005

The results shown in Table 1 indicate that the curcumin was delivered more effectively from emulsion of the invention.