WO2006053613A2 - Skin care composition comprising fusion polypeptide composition - Google Patents

Abstract

A composition is provided comprising (i) a fusion polypeptide comprising a first binding domain which comprises a complementarity determining region (CDR) which binds specifically to hair and a second binding domain which binds to a cosmetic or dermatologically active agent; and (ii) a cosmetic or dermatologically active agent bound to said second binding domain. The compositions can be used in methods of selectively delivering a cosmetic or dermatologically active agent to the skin.

Description

COMPOSITION

Field of the invention

The present invention relates to compositions for use in targeting skin benefit agents to skin by coupling the agents to fusion polypeptides that bind specifically to hair.

Summary of the invention

The present invention provides a composition comprising (i) a fusion polypeptide comprising a first binding domain which comprises a complementarity determining region (CDR) which binds specifically to hair and a second binding domain which binds to a cosmetic or dermatologically active agent; and (ii) a cosmetic or dermatologically active agent bound to said second binding domain. Preferably the cosmetic or dermatologically active agent is not a fragrance or perfume.

Preferably the first binding domain comprises a camelid antibody heavy chain fragment including said CDR.

In a preferred embodiment, the composition further comprises a third binding domain which comprises a CDR which binds specifically to hair. More preferably the third binding domain comprises a camelid antibody heavy chain fragment including said CDR.

Preferably the second binding domain is a binding domain that binds to an encapsulating material . More preferably the second binding domain is polysaccharide binding domain (such as a cellulose binding domain) or a melamine binding domain. In one embodiment the composition is a cosmetic composition comprising a cosmetically acceptable carrier or diluent.

In another embodiment the composition is a pharmaceutical composition comprising a pharmaceutically acceptable carrier or diluent.

The cosmetic or dermatologically active agent is preferably selected from surfactants, emollients, humectants, conditioners, sunscreens, anti-ageing actives, antimicrobials, skin lightening agents, moisturisers, appearance modifiers (e.g. opacifying or pearlescent actives)

The present invention also provides the use of a composition of the invention in a method of delivering a cosmetic or dermatologically active agent to the skin, which method comprises contacting hair associated with the skin with the composition. Preferably the eyelashes are excluded as hair associated with the skin.

In a preferred embodiment, the area of skin to which the agent is delivered is the scalp, face, upper lip or the underarm region.

In a related aspect, the present invention provides a method of selectively delivering a cosmetic or dermatologically active agent to the skin which method comprises contacting hair associated with the skin with a composition of the invention.

In a preferred embodiment, the area of skin to which the agent is delivered is the scalp, face, upper lip or the underarm region. Detailed description of the invention

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art (e.g. in cell culture, molecular genetics, nucleic acid chemistry, hybridisation techniques and biochemistry) . Standard techniques are used for molecular, genetic and biochemical methods (see generally, Sambrook et al., Molecular Cloning: A Laboratory Manual, 3^rd ed. (2001) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. and Ausubel et al . , Short Protocols in Molecular Biology (1999) 4^th Ed, John Wiley & Sons, Inc. - and the full version entitled Current Protocols in Molecular Biology, which are incorporated herein by reference) and chemical methods.

Fusion Proteins

Fusion proteins for use in the compositions of the present invention comprise at least two binding domains. The first binding domain binds specifically to hair.

The term "binds specifically to hair" means that, for example, the binding domain should not exhibit any significant binding to other external biological structures on an individual, such as skin. Typically, the relevant binding domain has a K_d for hair of lower than 10^"4 M, preferably lower than 10^"6 M, more preferably lower than 10^~8 M or 10^"10 M. Preferably the binding domain has a K_d for skin of greater than 10^"4 M, preferably greater than 10^"3 M, more preferably greater than 10^"2 M or 10^"1 M. It is also preferred that the ratio of the Ka for hair and the K_d for skin (K_d for skin / Ka for hair) is at least 100, more preferably at least 1000 or 10000. The first binding domain according to the present invention comprises an antibody complementarity determining region (CDR) . In a highly preferred embodiment the CDR is derived from a heavy chain variable domain derived from any immunoglobulin naturally devoid of light chains, such that the antigen-binding site is located exclusively in the heavy chain variable domain. Preferably, such CDRs are derived from camelid immunoglobulins as described in EP-A-0584421. The binding domains of these antibodies consist of a single polypeptide fragment. Procedures to obtain heavy chain immunoglobulins from

Camelidae, or (functionalized) fragments thereof, have been described in WO 94/04678 (Casterman and Hamers) and WO 94/25591 (Unilever and Free University of Brussels) . Alternatively, such binding domains can be obtained from the V_H fragments of classical antibodies by a procedure termed "camelization" .

Here, the classical V_H fragment is transformed, by substitution of a number of amino acids, into a V_HH-like fragment, whereby its binding properties are retained. This procedure has been described by Riechmann et al . in a number of publications (J. MoI. Biol. (1996) 259, 957-969; Protein. Eng. (1996) 9, 531- 537, Bio/Technology (1995) 13, 475-479) .

The second binding domain is capable of binding, directly or indirectly, a skin benefit agent. In a preferred embodiment, the second binding domain binds to polysaccharides or polysaccharide derivatives, to melamine-type polymers, or to polyurea. Preferably the second binding domain is a cellulose binding domain (CBD) , a melamine binding domain or a polyurea binding domain. Preferably the second binding domain is selected from CDRs, as described above, non-antibody derived peptides that bind specifically to polysaccharides or polysaccharide derivatives, such as a cellulose binding domain (CBD) and peptides that bind to melamine-type polymers. By indirect binding, we mean that the skin benefit agent is linked to another moiety which is bound by the second binding domain or is encapsulated by a material which is bound by the second binding domain e.g. a melamine or polysaccharide capsule. Thus, the second binding domain can be specific for an encapsulating material, i.e. a material that is used to encapsulate the skin benefit agent. It is preferred that indirect binding does not involve the use of antibodies i.e. that the second binding domain does not bind to an antibody to which the skin benefit agent is bound.

The cellulose binding domain is part of most cellulase enzymes and can be obtained therefrom. CBDs are also obtainable from xylanase and other hemicellulase degrading enzymes. Preferably, the cellulose binding domain is obtainable from a fungal enzyme origin such as Humicola, Trichoderma, Thermonospora, Phanerocfyaete, and Aspergillus, or from a bacterial origin such as Bacillus, Clostridium, Streptomyces, Cellulomonas and Pseudomonas.

Melamine binding domains are peptide/polypeptide binding domains that bind to melamine (a repeating unit of C₃N₆H₆ [1,3,5 triazine 2,4,6 triamine] ) or a melamine-like polymer. Melamine polymers are commonly used as encapsulation materials.

Other binding domains that can be used include peptides that are not derived from immunoglobulins. For example, a suitable peptide could be analogous to the active center of a protein analogous to a non-catalytic binding domain of a protein, e.g. a receptor. Suitable peptide binding domains can, for example, be designed de novo or selected using a variety of screening techniques such as yeast or phage display.

The fusion proteins of the present invention can comprise more than one binding domain comprising a CDR that binds specifically to hair. The different binding domains may bind specifically to the same target region, such as a hair epitope, or to different target regions.

Where the individual single binding domains which are joined together to form the fusion proteins used in the present invention have the same specificity, a binding protein which binds more than one molecule of the same type will be produced. Alternatively, multivalent and multispecific binding proteins which are able to bind different epitopes from each other may be obtained by assembling together single binding domains directed against different targets. The advantage of using fusion proteins with a plurality of binding domains that bind to hair specifically is an increase in avidity.

In a preferred embodiment, the fusion proteins used in the present invention comprise two or more binding domains that bind specifically to hair together with a binding domain that binds a cosmetic or dermatologically active agent.

The fusion proteins used in the invention may be formed by linking together the single binding domains in series, such that each binding domain unit is linked to at least one other binding domain. The individual binding domains may be linked sequentially by means of peptide linkers, conveniently flexible peptide linkers which allow the domains to flex in relation to each other such that simultaneous binding to multiple antigenic determinants may be achieved. It will be appreciated that the binding of the linker to the individual binding domains will be such that it does not affect the binding capacity of the binding domain. Such linkers include, e.g., peptides derived from known proteins, such as glucoamylase, cellobiohydrolase, or cell wall proteins (CWP) , or synthetic peptides which are rationally designed. The linker may suitably comprise from 1 to 400 or more amino acid residues; conveniently, the peptide linker comprises from 5 to 20 amino acid residues. This group of antigen binding proteins according to the invention with such a linker between the two binding domains is usually preferred because of its good production yields, functionality and stability in product forms.

In another preferred embodiment of the invention, the binding domains may be connected directly in series without any intervening linker. In this way, the binding sites in the multivalent binding proteins according to the invention are held in much closer proximity to each other than would be the case in an intact immunoglobulin from which binding domains can be derived. It might generally be expected that this would give rise to unfavourable steric interactions, but surprisingly, full binding activity is found to be retained. Furthermore, these fragments with directly linked binding domains appear to be more stable, e.g. towards proteolytic degradation.

Fusion polypeptides according to the invention may be prepared by transforming/transfecting a host cell with a nucleic acid encoding the polypeptide and cultivating said host cells under conditions which result in the expression of the polypeptide. Such techniques are well known in the art. Polypeptides may include fusion protein leaders which result in secretion of the polypeptide into the culture medium. The polypeptides may also optionally include an affinity tag to assist in purification of the polypeptide. Optionally, cleavage sites may be included such that these additional sequences can be cleaved from the polypeptide using, for example, proteases.

Suitably the host cell may be selected from prokaryotic bacteria, such as Gram-negative bacteria, for example E. coli, and Gram-positive bacteria, for example B. subtilis or lactic acid bacteria, lower eukaryotes such as yeasts, for example belonging to the genera Saccharomyces, Kluyveromyces, Hansenula or Pichia, or moulds such as those belonging to the genera Aspergillus or Trichoderma.

Preferred hosts for use in connection with the present invention are the lower eukaryotic moulds and yeasts.

Methods for producing antibody fragments or functionalised fragments thereof derived from the heavy chain immunoglobulin of Camelidae using a transformed lower eukaryotic host are described, for example in patent application WO 94/25591 and such techniques may suitably be applied to prepare constructs according to the present invention.

Proteins according to the invention may be recovered and purified using conventional techniques such as affinity chromatography, ion exchange chromatography or gel filtration chromatography. The activity of the fusion polypeptides may conveniently be measured by standard techniques known in the art such as enzyme- linked immunoadsorbant assay (ELISA) , radioimmune assay (RIA) or by using biosensors.

Compositions

Compositions of the invention comprise one or more fusion proteins described above together with at least one skin benefit agent, by which we mean a cosmetic or dermatologically active agent. In one embodiment, the cosmetic or dermatologically active agent is a skin irritant. Consequently, by targeting the agent to the skin via binding to hair, it is possible to reduce the irritant effect on the skin.

Skin and dermatological benefit agents include:

(a) silicone oils and modifications thereof such as linear and cyclic polydimethylsiloxanes; amino, alkyl, alkylaryl, and aryl silicone oils;

(b) fats and oils including natural fats and oils such as jojoba, soybean, sunflower, rice bran, avocado, almond, olive, sesame, persic, castor, coconut, mink oils; cacao fat; beef tallow, lard; hardened oils obtained by hydrogenating the aforementioned oils; and synthetic mono, di and triglycerides such as myristic acid glyceride and 2-ethylhexanoic acid glyceride;

(c) waxes such as carnauba, spermaceti, beeswax, lanolin, and derivatives thereof;

(d) hydrophobic and hydrophilic plant extracts; (e) hydrocarbons such as liquid paraffins, vaseline, microcrystalline wax, ceresin, squalene, pristan and mineral oil;

(f) higher fatty acids such as lauric, myristic, palmitic, stearic, behenic, oleic, linoleic, linolenic, lanolic, isostearic', arachidonic and poly unsaturated fatty acids (PUFA) ;

(g) higher alcohols such as lauryl, cetyl, stearyl, oleyl, behenyl, cholesterol and 2-hexydecanol alcohol;

(h) esters such as cetyl octanoate, myristyl lactate, cetyl lactate, isopropyl myristate, myristyl myristate, isopropyl palmitate, isopropyl adipate, butyl stearate, decyl oleate, cholesterol isostearate, glycerol monostearate, glycerol distearate, glycerol tristearate, alkyl lactate, alkyl citrate and alkyl tartrate;

(i) essential oils and extracts thereof such as mentha, jasmine, camphor, white cedar, bitter orange peel, ryu, turpentine, cinnamon, bergamot, citrus unshiu, calamus, pine, lavender, bay, clove, hiba, eucalyptus, lemon, starflower, thyme, peppermint, rose, sage, sesame, ginger, basil, juniper, lemon grass, rosemary, rosewood, avocado, grape, grapeseed, myrrh, cucumber, watercress, calendula, elder flower, geranium, linden blossom, amaranth, seaweed, ginko, ginseng, carrot, guarana, tea tree, jojoba, comfrey, oatmeal, cocoa, neroli, vanilla, green tea, penny royal, aloe vera, menthol, cineole, eugenol, citral, citronelle, borneol, linalool, geraniol, evening primrose, camphor, thymol, spirantol, penene, limonene and terpenoid oils; (j) lipids such as cholesterol, ceramides, sucrose esters and pseudo- ceramides as described in European Patent Specification No. 556,957;

(k) vitamins, minerals, and skin nutrients such as milk, vitamins A, E, and K; vitamin alkyl esters, including vitamin C alkyl esters; magnesium, calcium, copper, zinc and other metallic components;

(1) sunscreens such as octyl methoxyl cinnamate (Parsol MCX) and butyl methoxy benzoylmethane (Parsol 1789) ;

(m) phospholipids;

(n) antimicrobial agents such as the heavy metal salts of pyridinethione, climbazole, piroctone olamine, selenium sulphide and ketoconazole;

(o) antiaging compounds such as alpha hydroxy acids, beta hydroxy acids;

(p) pigment particles, such as solid dyes or colorants

(q) opacifying agents including higher fatty alcohols (e.g. cetyl, stearyl, arachidyl and behenyl) , solid esters (e.g. cetyl palmitate, glyceryl laurate, stearamide MEA-stearate) , high molecular weight fatty amides and alkanolamides and various fatty acid derivatives such as propylene glycol and polyethylene glycol esters. Inorganic materials include magnesium aluminium silicate, zinc oxide, and titanium dioxide. (r) Pearlescing agents such as C16-C22 fatty acids (e. g. stearic acid, myristic acid, oleic acid and behenic acid) , esters of C16-C22 fatty acid with alcohols and esters of C16- C22 fatty acid incorporating such elements as alkylene glycol units. Suitable alkylene glycol units may include ethylene glycol and propylene glycol . However, higher alkylene chain length glycols may be employed. Suitable higher alkylene chain length glycols include polyethylene glycol and polypropylene glycol. Further suitable pearlescing agents (^A) include inorganic materials such as nacreous pigments based on the natural mineral mica. An example is titanium dioxide coated mica. Particles of this material may vary in size from 2 to 150 microns in diameter. In general, smaller particles give rise to a pearly appearance, whereas particles having a larger average diameter will result in a glittery composition.

(s) antioxidants;

(t) natural hair root nutrients, such as amino acids and sugars. Examples of suitable amino acids include arginine, cysteine, glutamine, glutamic acid, isoleucine, leucine, methionine, serine and valine, and/or precursors and derivatives thereof. The amino acids may be added singly, in mixtures, or in the form of peptides, e.g. di- and tripeptides. The amino acids may also be added in the form of a protein hydrolysate, such as a keratin or collagen hydrolysate. Suitable sugars are glucose, dextrose and fructose. These may be added singly or in the form of, e.g. fruit extracts. A particularly preferred combination of natural hair root nutrients for inclusion in compositions of the invention is isoleucine and glucose. A particularly preferred amino acid nutrient is arginine; (u) fragrances and/or perfumes; and

(v) mixtures of any of the foregoing components.

The amount of skin benefit agent is preferably from 0.001 to 15 wt%, such as from 0.01 wt% to 10 wt%.

In one embodiment, the second binding domain binds to a material used to encapsulate the one or more skin benefit agents. In this embodiment, the skin benefit agent is encapsulated by the material and the skin benefit agent is not directly bound to the second binding domain. A variety of different capsule wall chemistries is possible, including but not limited to polysaccharides, melamine and polyurea.

In a related embodiment, the second binding domain binds to polysaccharides or polysaccharide derivatives e.g. the second binding domain is a cellulose binding domain. In this embodiment, the skin benefit agent is linked to or encapsulated by a polysaccharide, or mixture of polysaccharides.

In another related embodiment, the second binding domain binds to a melamine-type polymer. In this embodiment, the skin benefit agent is typically encapsulated in a melamine capsule.

The production of melamine capsules is well known in the art, see for instance WOOl/51197, WO01/49817, US6,248,703. They contain and are characterised by the repeating unit of C₃N₆H₆ [1,3,5 triazine 2,4,6 triamine] .

These melamine polymers are advantageously used in the manufacture of micro-capsules, preferably having a particle size of between 0.1 and 100 μm, more preferably of between 10 and 50 μm. Such micro-capsules are well known and have been described in US-A-2003078043 , JP-A-10139817, WO03/035245, US6,080,418. These melamine-polymer containing micro-capsules contain the skin benefit agents. Many processes for microencapsulation are known. These include methods for capsule formation such as described in US2, 730,456, US2, 800,457 and US2,800,458. Other useful methods for microcapsule manufacture are described in: US4, 001,140, US4, 081,376 and US4,089,802 describing a reaction between urea and formaldehyde; US4,100,103 describing reaction between melamine and formaldehyde; GB2, 062,570 describing a process for producing microcapsules having walls produced by polymerisation of melamine and formaldehyde in the presence of a styrenesulfonic acid. Micro-encapsulation is also taught in US-A-2 730 457 and US-A-4 197 346.

Pharmaceutical compositions of the invention comprising one or more fusion proteins described above together with a dermatologically active agent bound to the second binding domain, are intended for topical use and will therefore also comprise a pharmaceutically acceptable carrier or diluent suitable for topical application. Suitable carriers and diluents for topical compositions are well known in the art.

Cosmetic compositions of the invention comprising one or more fusion proteins described above together with a cosmetic or dermatologically active agent bound to the second binding domain, are intended for topical use and will therefore also comprise a cosmetically acceptable carrier or diluent suitable for topical application. Suitable carriers and diluents for topical compositions are well known in the art. The composition may be, for example, in the form of an ointment, a lotion, a foam, a cream, a gel, or a solution.

In one embodiment, compositions of the invention are "rinse- off" compositions to be applied to the hair / skin and then rinsed away. A particularly preferred product form is a shampoo composition or shower gel .

In one embodiment, cosmetic compositions of the invention comprise surfactant. The amount of surfactant generally ranges from 0.5 to 45%, preferably from 1.5 to 35%, more preferably from 5 to 20% by total weight surfactant based on the total weight of the composition.

Shampoo Compositions

Shampoo compositions of the invention are generally aqueous, i.e. they have water or an aqueous solution or a lyotropic liquid crystalline phase as their major component. Suitably, the composition will comprise from 50 to 98%, preferably from 60 to 90% water by weight based on the total weight of the composition.

Shampoo compositions according to the invention will generally comprise one or more anionic cleansing surfactants which are cosmetically acceptable and suitable for topical application to the hair. Examples of suitable anionic cleansing surfactants are the alkyl sulphates, alkyl ether sulphates, alkaryl sulphonates, alkanoyl isethionates, alkyl succinates, alkyl sulphosuccinates, alkyl ether sulphosuccinates, N-alkyl sarcosinates, alkyl phosphates, alkyl ether phosphates, and alkyl ether carboxylic acids and salts thereof, especially their sodium, magnesium, ammonium and mono-, di- and triethanolamine salts. The alkyl and acyl groups generally contain from 8 to 18, preferably from 10 to 16 carbon atoms and may be unsaturated. The alkyl ether sulphates, alkyl ether sulphosuccinates, alkyl ether phosphates and alkyl ether carboxylic acids and salts thereof may contain from 1 to 20 ethylene oxide or propylene oxide units per molecule.

Typical anionic cleansing surfactants for use in shampoo compositions of the invention include sodium oleyl succinate, ammonium lauryl sulphosuccinate, sodium lauryl sulphate, sodium lauryl ether sulphate, sodium lauryl ether sulphosuccinate, ammonium lauryl sulphate, ammonium lauryl ether sulphate, sodium dodecylbenzene sulphonate, triethanolamine dodecylbenzene sulphonate, sodium cocoyl isethionate, sodium lauryl isethionate, lauryl ether carboxylic acid and sodium N-lauryl sarcosinate.

The total amount of anionic cleansing surfactant in shampoo compositions of the invention generally ranges from 0.5 to 45%, preferably from 1.5 to 35%, more preferably from 5 to 20% by total weight anionic cleansing surfactant based on the total weight of the composition.

Optionally, a shampoo composition of the invention may contain further ingredients to enhance performance and/or consumer acceptability such as: co-surfactants, to help impart aesthetic, physical or cleansing properties to the composition; cationic polymers for enhancing conditioning performance; suspending agents .

A preferred example of a co-surfactant is an amphoteric or zwitterionic surfactant, which can be included in an amount ranging from 0.5 to about 8%, preferably from 1 to 4% by weight based on the total weight of the composition. The total amount of surfactant (including any co-surfactant, and/or any emulsifier) in a shampoo composition of the invention is generally from 1 to 50%, preferably from 2 to 40%, more preferably from 10 to 25% by total weight surfactant based on the total weight of the composition.

Suitable cationic polymers may be homopolymers which are cationically substituted or may be formed from two or more types of monomers. The weight average (M_w) molecular weight of the polymers will generally be between 100,000 and 2 million daltons. The polymers will have cationic nitrogen containing groups such as quaternary ammonium or protonated amino groups, or a mixture thereof. If the molecular weight of the polymer is too low, then the conditioning effect is poor. If too high, then there may be problems of high extensional viscosity leading to stringiness of the composition when it is poured.

Suitable cationic polymers include, for example, copolymers of vinyl monomers having cationic amine or quaternary ammonium functionalities with water soluble spacer monomers such as (meth) acrylamide, alkyl and dialkyl (meth) acrylamides, alkyl (meth) acrylate, vinyl caprolactone and vinyl pyrrolidine. The alkyl and dialkyl substituted monomers preferably have C1-C7 alkyl groups, more preferably Cl-3 alkyl groups. Other suitable spacers include vinyl esters, vinyl alcohol, maleic anhydride, propylene glycol and ethylene glycol.

Cationic polymer will generally be present in a shampoo composition of the invention at levels of from 0.01 to 5%, preferably from 0.05 to 1%, more preferably from 0.08 to 0.5% by total weight of cationic polymer based on the total weight of the composition. Preferably an aqueous shampoo 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 a 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.

Suspending agent will generally be present in a shampoo composition of the invention at levels of from 0.1 to 10%, preferably from 0.5 to 6%, more preferably from 0.9 to 4% by total weight of suspending agent based on the total weight of the composition.

Conditioner Compositions

Another preferred product form for compositions in accordance with the invention is a conditioner for the treatment of hair (typically after shampooing) and subsequent rinsing.

Such conditioner compositions will typically comprise one or more conditioning surfactants which are cosmetically acceptable and suitable for topical application to the hair.

Suitable conditioning surfactants include those selected from cationic surfactants, used singly or in a mixture. Preferably, the cationic surfactants have the formula N⁺R¹R²R³R⁴ wherein R¹, R², R³ and R⁴ are independently (C_x to C₃₀) alkyl or benzyl. Preferably, one, two or three of R¹, R², R³ and R⁴ are independently (C₄ to C₃₀) alkyl and the other R¹, R², R³ and R⁴ group or groups are (Ci-C₆) alkyl or benzyl. More preferably, one or two of R¹, R², R³ and R⁴ are independently (C₆ to C₃₀) alkyl and the other R¹, R², R³ and R⁴ groups are (Ci-C₆) alkyl or benzyl groups. Optionally, the alkyl groups may comprise one or more ester (-0CO- or -COO-) and/or ether (-0-) linkages within the alkyl chain. Alkyl groups may optionally be substituted with one or more hydroxy1 groups. Alkyl groups may be straight chain or branched and, for alkyl groups having 3 or more carbon atoms, cyclic. The alkyl groups may be saturated or may contain one or more carbon-carbon double bonds (eg, oleyl) . Alkyl groups are optionally ethoxylated on the alkyl chain with one or more ethyleneoxy groups.

Suitable cationic surfactants for use in conditioner compositions according to the invention include cetyltrimethylammonium chloride, behenyltrimethylammonium chloride, cetylpyridinium chloride, tetramethylammonium chloride, tetraethylammonium chloride, octyltrimethylammonium chloride, dodecyltrimethylammonium chloride, hexadecyltrimethylammonium chloride, octyldimethylbenzylammonium chloride, decyldimethylbenzylammonium chloride, stearyldimethylbenzylammonium chloride, didodecyldimethyl ammonium chloride, dioctadecyldimethylammonium chloride, tallowtrimethyl ammonium chloride, dihydrogenated tallow dimethyl ammonium chloride (eg, Arquad 2HT/75 from Akzo Nobel) , cocotrimethylammonium chloride, PEG-2-oleammonium chloride and the corresponding hydroxides thereof. Further suitable cationic surfactants include those materials having the CTFA designations Quaternium-5, Quaternium-31 and Quaternium-18. Mixtures of any of the foregoing materials may also be suitable.

In conditioners of the invention, the level of cationic surfactant will generally range from 0.01 to 10%, more preferably 0.05 to 7.5%, most preferably 0.1 to 5% by weight of the composition. Conditioners of the invention will typically also incorporate a fatty alcohol. The combined use of fatty alcohols and cationic surfactants in conditioning compositions is believed to be especially advantageous, because this leads to the formation of a lamellar phase, in which the cationic surfactant is dispersed.

Representative fatty alcohols comprise from 8 to 22 carbon atoms, more preferably 16 to 22. Fatty alcohols are typically compounds containing straight chain alkyl groups. Examples of suitable fatty alcohols include cetyl alcohol, stearyl alcohol and mixtures thereof. The use of these materials is also advantageous in that they contribute to the overall conditioning properties of compositions of the invention.

The level of fatty alcohol in conditioners of the invention will generally range from 0.01 to 10%, preferably from 0.1 to 8%, more preferably from 0.2 to 7%, most preferably from 0.3 to 6% by weight of the composition. The weight ratio of cationic surfactant to fatty alcohol is suitably from 1:1 to 1:10, preferably from 1:1.5 to 1:8, optimally from 1:2 to 1:5. If the weight ratio of cationic surfactant to fatty alcohol is too high, this can lead to eye irritancy from the composition. If it is too low, it can make the hair feel squeaky for some consumers.

Compositions of the invention may comprise further conditioning agents to optimise wet and dry conditioning benefits . Particularly preferred further conditioning agents are silicone emulsions.

Suitable silicone emulsions include those formed from silicones such as polydiorganosiloxanes , in particular polydimethylsiloxanes which have the CTFA designation dimethicone, polydimethyl siloxanes having hydroxyl end groups which have the CTFA designation dimethiconol, and amino- functional polydimethyl siloxanes which have the CTFA designation amodirnethicone.

Suitable silicone emulsions for use in compositions of the invention are available from suppliers of silicones such as Dow Corning and GE Silicones. The use of such pre-formed silicone emulsions is preferred for ease of processing and control of silicone particle size. Such pre-formed silicone emulsions will typically additionally comprise a suitable emulsifier such as an anionic or nonionic emulsifier, or mixture thereof, and may be prepared by a chemical emulsification process such as emulsion polymerisation, or by mechanical emulsification using a high shear mixer. Pre-formed silicone emulsions having a Sauter mean droplet diameter (D₃,₂) of less than 0.15 micrometers are generally termed microemulsions.

Silicone will generally be present in a composition of the invention at levels of from 0.05 to 10%, preferably 0.05 to 5%, more preferably from 0.5 to 2% by total weight of silicone based on the total weight of the composition.

A composition of the invention may contain other ingredients for enhancing performance and/or consumer acceptability. Such ingredients include fragrance, perfume, dyes and pigments, pH adjusting agents, pearlescers or opacifiers, viscosity modifiers, preservatives, and natural hair nutrients such as botanicals, fruit extracts, sugar derivatives and amino acids. Uses

The compositions of the invention can be used to deliver a cosmetic or dermatologically active agent to the skin by- contacting the associated hair with a composition of the invention. For example, the compositions of the invention can be used to deliver agents to the scalp, the underarm region, the face, including the beard area, i.e. the cheek, neck, upper lip, or chin, the legs, arms or torso. In a preferred embodiment, the area of skin to which the agent is delivered is the scalp, face, the underarm region or the upper lip.

Preferably, the eyelashes are specifically excluded as the associated hair.

In one embodiment, the agents are skin irritants. Consequently by targeting the agents to the hair proximal to the skin area of interest, the irritant effect can be lessened whilst still providing a beneficial effect.

The present invention will now be described with reference to the following examples which are illustrative only and non- limiting. The examples refer to figures:

Figure 1 is a photograph showing the ability of the VHH26 anti- hair antibody fragment to bind to both terminal and vellus (facial) hair.

Figure 2 is a photograph showing the binding ability of a fusion protein comprised of both the VHH26 anti hair antibody fragment and a cellulose binding domain (CBD) . Figure 3A is a graph showing the retention of perfume notes by hair after incubating hair switches with capsules containing perfume A in the presence of conditioner formulations. Note the effect of adding the anti-hair / anti-capsule fusion protein.

Figure 3B is a graph showing the retention of perfume notes by hair after incubating hair switches with capsules containing perfume B in the presence of conditioner formulations. Note the effect of adding the anti-hair / anti-capsule fusion protein.

Figure 4 shows photographs illustrating the retention of capsules on hair after incubating hair fibres with capsules with or without the presence of the anti-hair/ anti-capsule fusion protein. Note the effect of adding the anti-hair / anti-capsule fusion protein.

Figure 5 is a graph showing the retention of capsules on hair after incubating hair fibres with capsules in the presence of PBST buffer or conditioner formulation. Note the effect of adding the anti-hair / anti-capsule fusion protein.

Figure 6 is a graph showing retention of capsules on hair after incubating hair fibres with anti-hair / anti-capsule fusion protein that had been pre-incubated with buffer or conditioner and capsules. Note that the activity of the fusion protein is maintained after 3 days incubation with formulation.

Figure 7 is a graph showing retention of perfume notes by hair after incubating hair switches with perfume-containing capsules and anti-hair / anti-capsule fusion protein that had been pre- incubated with conditioner formulation. Note that the activity of the fusion protein is maintained after 7 days storage in formulation.

Figure 8 is a graph showing retention of perfume notes by hair after incubating hair switches with perfume-containing capsules in the presence of conditioner formulations. Note that the addition of an unrelated fusion protein (one that cannot bind hair or capsules) does not increase perfume deposition.

EXAMPLES

1. Library construction and antigen preparation

A naϊve camelid expression library comprising a repertoire of llama heavy chain variable domain fragments derived from immunoglobulins naturally devoid of light chains as described previously in WO99/37681 was used to pan for antibody fragments binding to hair. Panning was carried out by two methods: in the first method, the antigens used were a protein extract of hair: in the second method, panning was against whole hair fibres.

1.1 Protein extract of undamaged hair

Virgin Spanish hair (5 g) was placed in potassium hydroxide solution in methanol (100 mM KOH in MeOH, 250 mL) . The container was sealed, agitated occasionally and left overnight at room temperature. The hair, with the cuticle now dissolved away, was removed. The remaining solution (described as MeOH/KOH extract) was stored at 4⁰C and used to isolate antibody fragments against undamaged hair.

2. Immobilisation of antigen and biopanning

Immunotubes (Nunc) were sensitised with MeOH/KOH supernatant (2 mL of 100 μg/mL) or PBS (negative control) overnight at 4°C. Biopanning was essentially as described in WO 99/37681 with minor modifications. After the initial round of panning, the antigen concentration was reduced in an attempt to identify antibody fragments of higher affinity: panning was also repeated using the same antigen concentration.

2.1 Panning against whole hair

When panning against whole hair fibres, 3 mL Isolute^R columns (Kinesis Ltd.) were used to immobilise the hair. The columns were first blocked by incubating with 2% Marvel, 1% BSA in PBST (3.5 mL) for 2 hours at room temperature on a rotary mixer. Meanwhile, 1 mL of 4% Marvel, 2% BSA in PBST was added to the precipitated phage library (1 mL) and this mixture was incubated for 30 minutes at room temperature on a rotary mixer. The blocking solution was removed from each column and 0.005 g (+/-0.0005 g) of hair fibres was added. Pre-blocked phage precipitated library (with blocking solution, 2 mL total) was added to the column containing the hair fibres and the columns were incubated at room temperature for 2 hours with rotation, and for 1 hour standing. After incubation, the precipitated phage library/blocking solution was removed and the columns were washed by incubating with 6 x 2 mL PBST for 5 minutes on a rotary mixer, then with 6 x 2 mL PBS for 5 minutes on a rotary mixer. To elute the bound phage, triethylamine (1 mL of 100 mM, Fluka) was added to the column and the column was incubated at room temperature .with rotation for 30 minutes. This solution was subsequently removed from the column, retained in a fresh tube and neutralised with Tris-HCl (500 mL of 1 M, pH 7.4) . The columns were neutralised by adding Tris-HCl (200 μL of 1 M, pH 7.4) .

The eluted phage were added to log-phase E.coli TGl (9 mL) and 3 mL log-phase E.coli TGl were added to the column. Cultures were incubated at 37°C without shaking for 1 hour to allow for phage infection of the bacteria. Serial dilutions of the infected E. coli were subsequently plated out on to SOBAG plates and incubated overnight at 37°C. The remainder of the 3 mL and 9 mL cultures were then combined as appropriate and were centrifuged at 4500 rpra for 10 minutes. The retained pellets were resuspended in 50 mL 2TY medium containing 100 μg/mL ampicillin and 1 % glucose and incubated overnight at 37°C, 180 rpm for phage rescue as described in WO 99/37681.

3. High throughput ELISA screening

Approximately 100 bacterial colonies from each round of panning were picked using sterile cocktail sticks into individual wells of a 96-well microtitre plate (Sterilin) containing 2TY medium, (300 μL, containing 1% glucose and 100 μg/mL ampicillin) for production of phage displayed and soluble antibody fragments as described in WO 99/37681. These VHH containing supernatants were tested for binding to the antigen used in the panning procedure by ELISA essentially as described in WO 99/37681 and summarised below.

a) Sensitise a 96-well flat-bottomed ELISA plates (Greiner High binding) with antigen (25 μg/mL, 100 μL per well) in carbonate buffer or PBS at 4⁰C overnight and also a blank plate without antigen. b) Wash the plates with PBST (4 x 200 μL) then block them with 2 % Marvel/PBST at 37⁰C for 1 h. c) Empty the blocking buffer from the wells and tap the plates dry. d) To each well, add supernatant (50 μL) from phage or soluble produced VHH and 2' % Marvel/PBST (50 μL) . Incubate at 37°C or RT for 1-2 h. e) Wash the plates with PBST (4 x 200 μL) . f) To each well, add mouse anti-myc monoclonal antibody (100 μL, Invitrogen, diluted according to the manufacturer's instructions) in 2 % Marvel/PBST and incubate at 37°C or RT for 1 h. g) Wash the plates with PBST (4 x 200 μL) . h) To each well, add rabbit anti-mouse alkaline phosphatase conjugate (Zymed,100 μL of a 1:1000 dilution in 2 % Marvel/PBST) and incubate at 37⁰C or RT for 1 h. i) Wash the plates with PBST (4 x 200 μL) . j) Finally, to develop a signal, add pNPP substrate (Sigma,

1 mg/mL in IM diethanolamine/lmM MgCl₂, pH 9.8, 100 μL per well) and read the optical density at 405 nm once the colour has developed.

Clones were ranked according to the strength of the developed signal. The number of VHHs giving signals greater than 0.2 increased with the later pans and lower antigen concentrations.

4. Dilution ELISAs Dilutions of VHHs in crude E. coli supernatants were tested for binding to the antigen they were raised against. VHH-antigen binding was determined by ELISA using either monoclonal mouse anti-myc as detection antibody or polyclonal rabbit anti-llama antibody. Twenty-one VHHs of special interest were selected for further analysis.

5. Specificity ELISAs

The binding specificity of selected VHHs was determined by testing their binding to a number of irrelevant antigens in an ELISA format. Again, two different antibodies were used to detect the VHHs: mouse anti-myc and rabbit anti-llama. 6. Construction of a fusion protein molecule

An application of the anti-hair VHHs is in the construction of a fusion protein with a second VHH (eg. to a capsule using a melamine binding domain, see below) and optionally a third binding domain, for example a cellulose binding domain (see WO01/46356) to generate a vehicle for the delivery of encapsulated actives to hair or regions of hair. An example of how this is achieved is detailed below.

Bihead protein sequences:

(i) Anti-melamine capsule, anti-hair, CBD_tr

NH₂- QVQLQESGGGLVQAGGSLRLSCAASGRTGSSYAMGWFRQAPGKEREFVASISRSGSRTYY

ADSVKGRFTISRDNAKNTVYLQMSSLKPEDTAVYYCMYGRITRDYSNYWGQGTQVTVSSQVQL QESGGGLVQAGESLRLSCAASGSIFSFNHMAWYRQAPGKQRESVASIRSGGYTSYADSVKGRF TISRDNAKNTVYLQMNNLKPEDTAVYYCTAGRVWWWYWGQGTLVTVSSGSHHHHHHGSTSIEG RTQSHYGQCGGIGYSGPTVCASGTTCQVLNPYYSQCL -COOH [SEQ ID No.l]

Amino acids 1-120 = anti-melamine Vhh Amino acids 121-135 = anti-hair Vhh

(ii) Anti-hair, anti-melamine capsule, CBD_tr

NH₂-

QVQLQESGGGLVQAGESLRLSCAASGSIFSFNHMAWYRQAPGKQRESVASIRSGGYTSYADSV KGRFTISRDNAKNTVYLQMNNLKPEDTAVYYCTAGRVWWWYWGQGTLVTVSSQVQLQESGGGL VQAGGSLRLSCAASGRTGSSYAMGWFRQAPGKEREFVASISRSGSRTYYADSVKGRFTISRDN AKNTVYLQMSSLKPEDTAVYYCNYGRITRDYSNYWGQGTQVTVSSGSHHHHHHGSTSIEGRTQ SHYGQCGGIGYSGPTVCASGTTCQVLNPYYSQCL -COOH [SEQ ID No.2] Amino acids 2-116 = anti-hair Vhh Amino acids 117-136 = anti-melamine Vhh

6.1. Linearisation and dephosphorylation of plasmid DNA Depending upon the desired orientation of the VHHs (i.e. anti- hair anti-capsule or anti-capsule anti-hair) , DNA of either or both plasmids (10 μg) containing the anti-capsule CBD fusion protein and the anti-hair CBD fusion protein was linearised by incubating with BstEII (10 Units, Roche) at 60⁰C for 1 hour. Vector DNA was purified away from salt and enzymes using the QIAquick PCR purification kit (Qiagen) according to the manufacturer' s instructions.

To prevent recircularization, the ends of each plasmid were dephosphorylated by incubating DNA (5 μg) with calf intestinal alkaline phosphatase (CIAP, 2 Units, Roche) for 60 min at 37°C. The CIAP was inactivated by adding EDTA (4 μL of 500 mM) and incubating at 6°C for a further 10 minutes. Salt and enzymes were subsequently removed by purifying the DNA on a QIAquick PCR purification column as before.

6.2 PCR of plasmid DNA to introduce a BstEII site at 5¹ end of the Vhh

Two oligonucleotide primers were designed, one containing a sequence of DNA analogous to the 3 ' end of the Vhh sequence (3 ' primer) , the other containing a BstEII restriction enzyme site and a sequence of DNA analogous to the 5 ' end of the Vhh sequence (5 ' primer) . 5 ' Vhh bihead primer sequence:

5 ' AGG GGA CCC AGG TCA CCG TCT CCT CAC AGG TGC AGC TGC AGC AGT CTG G 3' [SEQ ID No.3]

3 ' Vhh bihead primer sequence:

5' GGG AAT TCA AGC TTA AGC AAT TG 3¹ [SEQ ID No.4]

For the PCR reaction, Plasmid DNA (0.5 μL) containing either the anti-hair or anti-capsule VHH-CBD fusions, dNTPs (1 μL of 20 mM, Roche), 10x Taq buffer with MgCl₂ (10 μL, Roche), Taq Polymerase (2.5U, Roche), Pfu polymerase (0.1875U, Stratagene) , 5' and 3' primers (1 μM of each) and water were added together in a 0.5 mL Eppendorf tube to give a total volume of 100 μL. Each PCR reaction was overlaid with 50 μL mineral oil and was incubated as follows:

94°C 4 minutes

94°C 1 minute ]

55°C 1 minute ] x 33 cycles

72°C 1 minute ] ^' 72°C 10 minutes

After incubation, the PCR products were examined by electrophoresis on a 1.5% agarose gel containing ethidium bromide (0.5 μg/mL) . Salt and enzymes were removed from the remainder of each product by purifying .on a QIAquick PCR purification column as before.

Each PCR product was incubated with BstEII at 60⁰C for 1 hour 30 minutes. The digested DNA was visualised by electrophoresis on a 1.5% agarose gel containing ethidium bromide (0.5 μg/mL) and the higher molecular weight fragment containing the Vhh sequence was excised from the gel. This DNA fragment was purified using the QIAquick gel extraction kit (Qiagen) according to the manufacturer's instructions. This procedure yielded a Vhh fragment with a BstEII compatible end at both the 5 ' and 3 ' ends of the fragment.

6.3 Ligation of PCR products and dephosphorylated plasmid DNA and transformation into E.coli XL-I Blue cells Digested PCR product (5 μL) , dephosphorylated plasmid DNA (5μL) , 5x Ligation Buffer (4 μL, Invitrogen) , water (5 μL) and DNA Ligase (lμL, Invitrogen) were added together and mixed gently by stirring with a pipette tip. The reactions were incubated at room temperature for approximately 5 hours. After incubation, 10 μL of each ligation mix was added to 200μL XLl Blue competent cells (recAl endAl gyrA96 thi-1 hsdR17 supE44 relAl lac [F¹ proAB lac^qZΔM15Tnl0 Tet^r) ] , Stratagene) . The contents of the tubes were gently mixed and were incubated on ice for 30 minutes. The tubes were then placed at 42°C for exactly 2 minutes to heat-shock, ImL 2xTY medium was added to each tube and the tubes were incubated at 37°C for 1 hour. Each tube was then briefly microfuged and the pellets were resuspended in 100 μL 2xTY medium. 5 μL, 20 μL and 25 μL aliquots were then plated out on to SOBAG plates and incubated overnight at 37°C.

The resulting colonies were screened for the correct insert in the correct orientation by either sequencing or PCR of the miniprep DNA. One or more suitable clones were then taken forward for expression of the fusion protein, purification and analysis. 7. Antibody binding to various hair types

Antibody binding was investigated using virgin hair of Spanish origin and terminal and vellus hair from facial biopsies.

Hair samples were cut to approximately 0.5cm lengths and were placed into wells of a 2 μm filter plate. The hairs were then blocked with 5% goat sera in 1% TTBSA for 1 hour at room temperature (TBS = 0.5M Tris + 1.5M NaCl, pH7.6, TTBSA = 3% BSA in TBS) . The block was removed, Vhh.26 antibody fragment was added to the samples (1 in 2 dilution in 1% TTBSA) and the plate was incubated overnight at 4⁰C. The samples were then washed in TBS 4 times. Mouse anti-myc antibody was diluted 1 in 1000 in 1% TTBSA and added to the samples. The plate was then incubated at room temperature with shaking for 1 hour. After 4 washes with TBS, detection antibody was applied (goat anti- mouse Alexa Fluor 594, diluted 1 in 100 in 1% TTBSA/TBS) , and the plate was incubated for 1 hour at room temperature. After 4 more washes in TBS, the hair samples were analysed using scanning laser confocal microscopy.

Binding to hair is shown in Figure 1, which illustrates the ability of the VHH26 fragment to bind to both terminal and Vellus (facial) hair. This targeting allows delivery of skin benefit actives to both fine hair structures on the face or the more structural human body hair. Benefits agents include visual enhancing compounds to active ingredients such as vitamins, growth factors etc.

8. Binding of anti-hair CBD fusion to hair Hair samples were cut to approximately 0.5cm lengths and were placed into wells of a 2 μm filter plate. The hairs were then blocked with 5% goat sera in 1% TTBSA for 1 hour at room temperature. The block was removed, Vhh26CBD in 1% TTBSA (1 in 2 dilution) was added to the samples and the plate was incubated overnight at 4⁰C. The samples were then washed in TBS 4 times. Anti-His antibody was then diluted 1 in 1000 in 1% TTBSA, added to the samples and the plate was incubated at room temperature with shaking for 1 hour. After 4 washes with TBS, goat anti-mouse Alexa 594, diluted 1 in 100 in 1% TTBSA, was added. After 4 more washes in TBS, the hair samples were analysed using scanning laser confocal microscopy.

Fusion protein binding to hair is shown in Figure 2, which illustrates the binding ability of a fusion protein comprised of both the VHH26 anti hair antibody fragment and a cellulose binding domain (CBD) . The presence of the CBD adds functionality of delivering polysaccharide coated benefit agents (e.g. encapsulated) or any benefit agents associated with the polysaccharide by some other means.

9. Demonstration of activity of an anti-hair / anti-capsule fusion protein An application of the anti-hair VHHs is in the construction of a fusion protein with a second VHH (e.g. to a capsule using a melamine binding domain) to generate a vehicle for the delivery of encapsulated actives to hair.

The following examples illustrate the ability of such an anti- hair / anti-capsule fusion protein to deposit capsules on to hair fibres and to deliver encapsulated active to hair.

9.1 Demonstration of activity of the anti-hair / anti-capsule fusion protein in formulation To demonstrate that activity of the anti-hair / anti-capsule fusion protein is maintained in hair-care formulations, fusion protein, virgin Spanish hair and capsules (containing one of two different types of perfume) were incubated together in the presence of hair conditioners. Diluted conditioner formulations (diluted 1:5 with water) were added to hair switches with or without the anti-hair / anti-capsule fusion protein and the mixes were incubated on a roller mixer for 30 minutes at room temperature. Capsules containing either perfume A or perfume B were then added to the hair/conditioner mixes and the incubation was continued for a further 30 minutes. The hair switches were then placed into fresh vials and washed with water for a further 30 minutes. After air drying, the ends of the hair switches were cut off using scissors and any perfume remaining on the hair ends was solvent extracted. The amount of perfume retained by the hair ends was determined by GCMS analysis.

Figures 3a and 3b show the retention of perfume notes by hair as analysed by GCMS, after incubation of hair switches and capsules (perfume A or perfume B) , in the presence of conditioner, with and without the addition of the anti-hair / anti-capsule fusion protein. For the three conditioners tested, more perfume notes were retained by those hair switches incubated with the anti-hair / anti-capsule fusion protein, which indicates that activity of the fusion protein is maintained in formulation.

9.2 Demonstration that presence of the anti-hair / anti- capsule fusion protein can increase deposition of capsules on to hair fibres

To demonstrate 'that the presence of the anti-hair / anti- capsule fusion protein can increase deposition of capsules on to hair fibres, hair fibres and capsules were incubated with or without the anti-hair / anti-capsule fusion protein. Virgin Spanish hair fibres were incubated on a roller mixer for 30 minutes at room temperature with or without the anti-hair / anti-capsule fusion protein. Capsules were then added to the mixes and the incubation was continued for a further 30 minutes. The hair fibres were then placed into fresh vials and washed for a further 30 minutes. After air drying, retention of capsules on the surface of the hair fibres was examined by- microscopy.

Figure 4 shows the retention of capsules on hair after incubating hair fibres with capsules with or without the presence of the anti-hair/ anti-capsule fusion protein. More capsules were retained on the surface of hair fibres incubated with anti-hair / anti-capsule fusion protein than on the surface of hair fibres incubated without anti-hair / anti- capsule fusion protein. This indicates that presence of the anti-hair / anti-capsule fusion protein can increase deposition of capsules on to hair fibres.

9.3 Demonstration that presence of the anti-hair / anti- capsule fusion protein can increase deposition of capsules on to hair fibres in formulation

To demonstrate that the presence of the anti-hair / anti- capsule fusion protein can increase deposition of capsules on to hair fibres in formulation, fusion protein, virgin Spanish hair fibres, capsules and hair conditioner were incubated together. Hair fibres with or without the anti-hair / anti- capsule fusion protein were incubated on a roller mixer in the presence of PBST buffer or conditioner formulation (diluted 1:5 with water) for 30 minutes at room temperature. Capsules were then added to the mixes and the incubation was continued for a further 30 minutes. The hair fibres were then placed into fresh vials and washed for a further 30 minutes. After air drying, the hair fibres were examined by microscopy. For each treatment, capsules bound to the surface of 4 hair fibres (each 2cm in length) were counted.

Figure 5 is a graph showing the number of capsules retained on the surface of the hair fibres. More capsules were retained on the surface of hair fibres incubated with anti-hair / anti- capsule fusion protein than on the surface of hair fibres incubated without anti-hair / anti-capsule fusion protein, in the presence of buffer and conditioner. This indicates that activity of the fusion protein is maintained in formulation.

9.4 Demonstration that activity of 9the anti-hair / anti- capsule fusion protein is maintained after pre-incubation of fusion protein with formulation To demonstrate that activity of the anti-hair / anti-capsule fusion protein is maintained after pre-incubation of fusion protein with hair-care formulation, fusion protein and capsules were incubated together in the presence of PBST buffer or hair conditioner. Anti-hair / anti-capsule fusion protein and capsules were added to PBST buffer or conditioner formulation (diluted 1:5 with water) and incubated at 4°C for 3 days.

After 3 days incubation, virgin Spanish hair fibres were added to the mixes and the mixes were incubated on a roller mixer for 30 minutes at room temperature. The hair fibres were then placed into fresh vials and washed with PBST buffer or water for a further 30 minutes. Control hair fibres were incubated with buffer or conditioner (diluted 1:5 with water) on a roller mixer for 30 minutes at room temperature. Capsules were then added to the mixes and the incubation was continued for a further 30 minutes. The hair fibres were then placed into fresh vials and washed with PBST buffer or water for a further 30 minutes. After air drying, the control and treated hair fibres were examined by microscopy. For each treatment, capsules bound to the surface of 4 hair fibres (each 2cm in length) were counted .

Figure 6 is a graph showing the number of capsules retained on the surface of the hair fibres. More capsules were retained on the surface of hair fibres incubated with anti-hair / anti- capsule fusion protein than on the surface of hair fibres incubated without anti-hair / anti-capsule fusion protein in the presence of conditioner, indicating that activity of the fusion protein is maintained after 3 days incubation with formulation.

9.5 Demonstration that activity of the anti-hair / anti- capsule fusion protein is maintained after storage of fusion protein with formulation To demonstrate that activity of the anti-hair / anti-capsule fusion protein is maintained after storage of fusion protein in hair-care formulation, fusion protein was incubated in the presence of hair conditioner (diluted 1:5 with water) at room temperature for 7 days. After 7 days, virgin Spanish hair switches were added to the fusion protein / conditioner mix and the mix was incubated on a roller mixer for 30 minutes at room temperature. Capsules containing perfume were then added to the mix and the incubation was continued for a further 30 minutes. The hair switches were then placed into fresh vials and washed with water for a further 30 minutes.

Hair switches were also treated with anti-hair / anti-capsule fusion protein that had not been pre-incubated with conditioner formulation. Fusion protein and conditioner formulation (diluted 1:5 in water) were added to hair switches and the mix was incubated on a roller mixer for 30 minutes at room temperature. Capsules containing perfume were then added to the mix and the incubation was continued for a further 30 minutes. The hair switches were then placed into fresh vials and washed with water for a further 30 minutes.

Control hair switches were incubated with conditioner (diluted 1:5 with water) on a roller mixer for 30 minutes at room temperature. Capsules containing perfume were then added to the mix and the incubation was continued for a further 30 minutes. The hair switches were then placed into fresh vials and washed with water for a further 30 minutes.

After air drying, the ends of the treated and control hair switches were cut off using scissors and any perfume remaining on the hair ends was solvent extracted. The amount of perfume retained by the hair ends was determined by GCMS analysis.

Figure 7 is a graph showing the retention of perfume notes by hair as analysed by GCMS. The amount of perfume notes retained by the hair switches treated with fusion protein that had been pre-incubated with conditioner was very similar to the amount retained by the switches treated with fusion protein that had not been pre-incubated with conditioner. This indicates that activity of the fusion protein is maintained after 7 days storage in formulation.

9.6 Demonstration that an unrelated fusion protein cannot increase deposition of capsules on to hair

To demonstrate that presence of an unrelated fusion protein (one that cannot bind hair or capsules) cannot increase deposition of capsules on to hair, virgin Spanish hair and capsules were incubated with or without unrelated fusion protein. Diluted conditioner formulations (diluted 1:5 with water) were added to hair switches with or without the unrelated fusion protein and the mixes were incubated on a roller mixer for 30 minutes at room temperature. Capsules containing perfume were then added to the hair/conditioner mixes and the incubation was continued for a further 30 minutes. The hair switches were then placed into fresh vials and washed with water for a further 30 minutes. After air drying, the ends of the hair switches were cut off using scissors and any perfume remaining on the hair ends was solvent extracted. The amount of perfume retained by the hair ends was determined by GCMS analysis.

Figure 8 is a graph showing the retention of perfume notes by hair as analysed by GCMS. Addition of unrelated fusion protein did not increase retention of perfume notes by the hair switches. This indicates that the presence of an unrelated fusion protein cannot increase deposition of capsules on to hair.

The various features and embodiments of the present invention, referred to in individual sections above apply, as appropriate, to other sections, mutatis mutandis. Consequently features specified in one section may be combined with features specified in other sections, as appropriate.

All publications mentioned in the above specification are herein incorporated by reference. Various modifications and variations of the described methods and products of the invention will be apparent to those skilled in the art without departing from the scope of the invention. Although the invention has been described in connection with specific preferred 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 which are apparent to those skilled in the relevant fields are intended to be within the scope of the following claims.

Claims

Claims

1. A composition comprising (i) a fusion polypeptide comprising a first binding domain which comprises a complementarity determining region (CDR) which binds specifically to hair and a second binding domain which binds to a cosmetic or dermatologically active agent; and (ii) a cosmetic or dermatologically active agent bound to said second binding domain, with the proviso that the cosmetic or dermatologically active agent is other than a fragrance or perfume.

2. A composition according to claim 1 wherein the first binding domain comprises a camelid antibody heavy chain fragment including said CDR.

3. A composition according to claim 1 or claim 2 which further comprises a third binding domain which comprises a CDR which binds specifically to hair.

4. A composition according to claim 3 wherein the third binding domain comprises a camelid antibody heavy chain fragment including said CDR.

5. A composition according to any one of the preceding claims wherein the second binding domain is a polysaccharide binding domain.

6. A composition according to any one of the preceding claims wherein the composition is a cosmetic composition comprising a cosmetically acceptable carrier or diluent.

7. A composition according to any one of the preceding claims wherein the cosmetic or dermatologically active agent is selected from surfactants, emollients, humectants, conditioners, sunscreens, anti-ageing actives, antimicrobials, skin lightening agents, moisturisers and appearance modifiers.

8. A composition according to any one of claims 1 to 5 wherein the composition is a pharmaceutical composition comprising a pharmaceutically acceptable carrier or diluent.

9. Use of a composition comprising (i) a fusion polypeptide comprising a first binding domain which comprises a complementarity determining region (CDR) which binds specifically to hair and a second binding domain which binds to a cosmetic or dermatologically active agent; and (ii) a cosmetic or dermatologically active agent bound to said second binding domain, in a method of delivering a cosmetic or dermatologically active agent to the skin, which method comprises contacting hair associated with the skin with the composition.

10. Use according to claim 9 wherein the composition is as defined in any one of claims 1 to 8.

11. Use according to claim 9 or claim 10 wherein the area of skin to which the agent is delivered is the scalp, face, upper lip or the underarm region.

12. A method of selectively delivering a cosmetic or dermatologically active agent to the skin which method comprises contacting hair associated with the skin with a composition comprising (i) a fusion polypeptide comprising a first binding domain which comprises a complementarity- determining region (CDR) which binds specifically to hair and a second binding domain which binds to a cosmetic or dermatologically active agent; and (ii) a cosmetic or dermatologically active agent bound to said second binding domain.

13. A method according to claim 12 wherein the composition is as defined in any one of claims 1 to 8.

14. A method according to claim 12 or claim 13 wherein the area of skin to which the agent is delivered is the scalp, face, upper lip or the underarm region.