WO2014095289A2 - Method of treating hair ageing - Google Patents

Method of treating hair ageing Download PDF

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Publication number
WO2014095289A2
WO2014095289A2 PCT/EP2013/074936 EP2013074936W WO2014095289A2 WO 2014095289 A2 WO2014095289 A2 WO 2014095289A2 EP 2013074936 W EP2013074936 W EP 2013074936W WO 2014095289 A2 WO2014095289 A2 WO 2014095289A2
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WIPO (PCT)
Prior art keywords
acid
hair
ethanol extract
composition
nrf2
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PCT/EP2013/074936
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French (fr)
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WO2014095289A3 (en
Inventor
Ranjit Kaur Bhogal
Iain Stuart HASLAM
Gail Jenkins
Ralf Ludwig Paus
Magdalena SAWICKA
Linda Jane Wainwright
Original Assignee
Unilever Plc
Unilever N.V.
Conopco, Inc., D/B/A Unilever
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Publication of WO2014095289A2 publication Critical patent/WO2014095289A2/en
Publication of WO2014095289A3 publication Critical patent/WO2014095289A3/en

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    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00Cosmetics or similar toiletry preparations
    • A61K8/18Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/30Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
    • A61K8/33Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds containing oxygen
    • A61K8/34Alcohols
    • A61K8/345Alcohols containing more than one hydroxy group
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00Cosmetics or similar toiletry preparations
    • A61K8/18Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/30Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
    • A61K8/33Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds containing oxygen
    • A61K8/34Alcohols
    • A61K8/347Phenols
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00Cosmetics or similar toiletry preparations
    • A61K8/18Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/30Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
    • A61K8/33Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds containing oxygen
    • A61K8/35Ketones, e.g. benzophenone
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00Cosmetics or similar toiletry preparations
    • A61K8/18Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/30Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
    • A61K8/33Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds containing oxygen
    • A61K8/35Ketones, e.g. benzophenone
    • A61K8/355Quinones
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00Cosmetics or similar toiletry preparations
    • A61K8/18Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/30Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
    • A61K8/33Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds containing oxygen
    • A61K8/36Carboxylic acids; Salts or anhydrides thereof
    • A61K8/361Carboxylic acids having more than seven carbon atoms in an unbroken chain; Salts or anhydrides thereof
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00Cosmetics or similar toiletry preparations
    • A61K8/18Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/30Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
    • A61K8/33Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds containing oxygen
    • A61K8/36Carboxylic acids; Salts or anhydrides thereof
    • A61K8/365Hydroxycarboxylic acids; Ketocarboxylic acids
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00Cosmetics or similar toiletry preparations
    • A61K8/18Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/30Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
    • A61K8/46Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds containing sulfur
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00Cosmetics or similar toiletry preparations
    • A61K8/18Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/30Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
    • A61K8/49Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds containing heterocyclic compounds
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00Cosmetics or similar toiletry preparations
    • A61K8/18Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/30Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
    • A61K8/49Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds containing heterocyclic compounds
    • A61K8/4973Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds containing heterocyclic compounds with oxygen as the only hetero atom
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00Cosmetics or similar toiletry preparations
    • A61K8/18Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/30Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
    • A61K8/49Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds containing heterocyclic compounds
    • A61K8/4973Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds containing heterocyclic compounds with oxygen as the only hetero atom
    • A61K8/498Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds containing heterocyclic compounds with oxygen as the only hetero atom having 6-membered rings or their condensed derivatives, e.g. coumarin
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00Cosmetics or similar toiletry preparations
    • A61K8/18Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/30Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
    • A61K8/63Steroids; Derivatives thereof
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61QSPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q7/00Preparations for affecting hair growth

Definitions

  • This invention relates to a method of treating hair ageing.
  • the method relates to treating hair ageing through application to hair fibres of a composition comprising a nuclear factor erythroid-2 related factor 2 (also known as NFE2L2 or Nrf2) agonist.
  • a nuclear factor erythroid-2 related factor 2 also known as NFE2L2 or Nrf2
  • the main function of the hair follicle is to produce a hair fibre.
  • the hair follicle develops from the embryonic epidermis as an epidermal finger which differentiates into the fibre, the outer root sheath (ORS) and the inner root sheath (IRS).
  • ORS outer root sheath
  • IFS inner root sheath
  • Figure 1 illustrates how mature follicles undergo follicular cycling through phases of organ growth and hair fibre production (anagen) for 3-7 years, cessation of fibre growth and organ involution (catagen) over about 2 weeks and a quiescent phase (telogen) which lasts about 3 months where the organ rests and the hair fibre remains anchored but no longer grows before the hair fibre falls (exogen) and is regenerated to start the cycle again (Dry, J Genet 16, 281 -340 (1926), Chase, Physiol Rev 34, 1 , 1 13-26 (1954) and Kligman, J Invest Dermatol 33, 307-16 (1959)).
  • Nrf2 is a transcription factor (protein) that in humans is encoded by the NFE2L2 gene. According to Lee et al (J. of Biochem & Mol Biol. 37, 139-143 (2004)) and Hybertson et al (Mol Aspects Med. 32, 234-46 (201 1 )), Nrf2 has been shown to be involved in the defence against oxidative injury in various tissues. Under basal conditions, Nrf-2 is inactive and bound in the cytoplasm by cytosolic regulatory protein Kelch-like ECH- associated protein 1 (Keapl ).
  • Nrf2 the protein Cullin 3 degrades Nrf2 by ubiquitination.
  • Kobayashi et al (Mol. Cell. Biol., 24, 16, 7130-9 (August 2004))
  • Keapl helps Cullin 3 ubiquitinate Nrf2.
  • Nrf2 When Nrf2 is ubiquitinated, it is transported to the proteasome where it is degraded and its components recycled such that under normal conditions, Nrf2 has a half-life of only 20 minutes. Yamamoto et al (Mol. Cell (Biol., 28, 8, 2758-70 (April 2008)) and Sekhar et al (Toxicol. Appl.
  • Nrf2 combines (forms a heterodimer) with a small Maf protein (a transcription factor) and binds to small regions of DNA known as Antioxidant Response Elements (ARE's) in the upstream promoter region of many anti-oxidative genes, and initiates their transcription.
  • ARE's Antioxidant Response Elements
  • the antioxidant genes include "phase II" enzymes such as NADP(H): quinone oxidoreductase (NQO-1 ) and hemoxygenase-1 (HO-1 ).
  • Phase II enzymes such as NADP(H): quinone oxidoreductase (NQO-1 ) and hemoxygenase-1 (HO-1 ).
  • Increased oxidative stress has been shown to have a detrimental effect on hair pigmentation (Lu et al, J Invest Dermatol, 129, 1790-804 (2009); Arck et al, FASEB J. 2, 1567-9 (2006)).
  • Hair ageing is a major age-related consumer issue (hair loss, thinning hair, loss of shine, increased number of grey hairs, etc).
  • Biological routes for hair growth or preventing hair greying provide effective opportunities to target consumer hair issues.
  • MinoxidilTM and FinasterideTM are the only clinically proven, mildly effective products available for hair growth and both are classified as medicines and therefore not suitable for cosmetic use.
  • the identification of cosmetic ingredients which are able to boost hair growth, maintain anagen and/or prevent catagen may prove to be effective anti ageing treatments to prevent or attenuate some of the symptoms associated with hair ageing.
  • US 2008/0187608 discloses use of certain novel compositions from clary sage (Salvia sclarea) which are inhibitors of nuclear factor kappa B (NF- ⁇ ), Nerf2 (an ETS (E- twenty six) transcription factor that is specifically expressed in endothelial cells (Dube et al, Circ Res, 84, 10, 1 177-85 (1999)) and the activity of the endothelin receptor.
  • NF- ⁇ nuclear factor kappa B
  • Nerf2 an ETS (E- twenty six) transcription factor that is specifically expressed in endothelial cells (Dube et al, Circ Res, 84, 10, 1 177-85 (1999)) and the activity of the endothelin receptor.
  • phase II detoxifying enzymes and antioxidant proteins Induction of phase II enzymes is predominantly mediated by a redox-sensitive transcription factor NF-E2 related factor-2 (Nrf2).
  • a variety of phytochemicals are able to activate Nrf2 thereby up- regulating a set of enzymes including nicotinamide adenine dinucleotide phosphate (NADP(H)): quinone oxidasereductase-1 (NQO-1 ), superoxide dismutase (SOD), glutathione S-transferase (GST), hemeoxygenase-1 (HO-1 ), and glutamyl cysteine ligase (GCL).
  • NADP(H) nicotinamide adenine dinucleotide phosphate
  • NQO-1 quinone oxidasereductase-1
  • SOD superoxide dismutase
  • GST glutathione S-transferase
  • HO-1 hemeoxygenase-1
  • GCL glutamyl cysteine ligase
  • a pharmaceutical, nutraceutical and cosmeceuticals composition suitable for the treatment and prevention of cancer, inflammation nrelated diseases, cardiovascular diseases, fungal and bacterial infections, as an anti-coagulant, enhancement of adenylate cyclise activity, anti-inflammatory, vasodilator, antimyocardiac, retardation of cholesterol biosynthesis, inhibition of lipoprotein oxidation, treatment of acne and eczema, prevent hair loss, whitening skin and protect myocardium from hypoxia-induced cardiac contractile failure.
  • Nerf2 expression is specific to the endothelial cell and not expressed in the hair follicle.
  • Nrf-2 agonists promote hair fibre growth in a hair growth model.
  • a method of treating hair ageing comprising the step of applying a composition, the composition comprising a nuclear factor erythroid-2 related factor 2 (Nrf2) agonist;
  • Nrf2 agonist is selected from the group consisting of andrographolide, apigenin, Asiatic acid, baicalein, tert-butyl hydroxyquinone, catechin, chrysin, chrysin dimethylether, curcumin, docosahexaenoic acid, epigallocatechin gallate, esculetin, fraxetin, galangin, genistein, guaiacol, hesperetin, 4-hydroxy-3-methoxyacetophenone, jasmonic acid, kaempferol, (S)-(+)-ketoprofen, kinetin, kinetin riboside, gamma linolenic acid, lipoic acid, lutein, luteolin, lycopene, nordihydroguaiaretic acid, oleanolic acid, parthenolide, peonidin chloride, phloroglucinol carboxaldehyde, pin
  • Nrf2 agonist is selected from the group consisting of andrographolide, apigenin, Asiatic acid, baicalein, tert-butyl hydroxyquinone, catechin, chrysin, chrysin dimethylether, curcumin, docosahexaenoic acid, epigallocatechin gallate, esculetin, fraxetin, galangin, genistein, guaiacol, hesperetin, 4-hydroxy-3-methoxyacetophenone, jasmonic acid, kaempferol, (S)-(+)-ketoprofen, kinetin, kinetin riboside, gamma linolenic acid, lipoic acid, lutein, luteolin, lycopene, nordihydroguaiaretic acid, oleanolic acid, parthenolide, peonidin chloride, phloroglucinol carboxaldehyde, pin
  • Figure 1 follicular cycling through phases of organ growth and hair fibre production (anagen), cessation of fibre growth and organ involution (catagen) and a quiescent phase (telogen); and Figure 2 human hair shaft length (mm) over time (hours) after treatment with 10 ⁇ sulforaphane versus a vehicle control.
  • a method of treating hair ageing comprising the step of applying a composition to hair fibres and/or scalp, the composition comprising a nuclear factor erythroid-2 related factor 2 (Nrf2) agonist;
  • Nrf2 agonist is selected from the group consisting of andrographolide, apigenin, Asiatic acid, baicalein, tert-butyl hydroxyquinone, catechin, chrysin, chrysin dimethylether, curcumin, docosahexaenoic acid, epigallocatechin gallate, esculetin, fraxetin, galangin, genistein, guaiacol, hesperetin, 4-hydroxy-3-methoxyacetophenone, jasmonic acid, kaempferol, (S)-(+)-ketoprofen, kinetin, kinetin riboside, gamma linolenic acid, lipoic acid, lutein, luteolin, lycopene, nordihydroguaiaretic acid, oleanolic acid, parthenolide, peonidin chloride, phloroglucinol carboxaldehyde, pin
  • the hair and/or scalp composition referred to in the first aspect of the invention may comprise 0.00001 -1 , preferably 0.001 -1 , most preferably 0.0001 -0.1 % w/w nuclear factor erythroid-2 related factor 2 (Nrf2) agonist.
  • composition will normally be in the form of a:
  • Hair colouring compositions will nearly always comprise a cleansing surfactant component in an aqueous base.
  • the cleansing surfactant may consist of a single surfactant, usually an anionic surfactant (to provide foam) such as sodium lauryl ether sulphate (SLES with one ethylene oxide), or more commonly a mixture of SLES with a co-surfactant to provide mildness.
  • an anionic surfactant such as sodium lauryl ether sulphate (SLES with one ethylene oxide), or more commonly a mixture of SLES with a co-surfactant to provide mildness.
  • the most preferred co-surfactant is cocoamidopropyl betaine (CAPB).
  • the total amount of surfactant (including any co-surfactant, and/or any emulsifier) in a shampoo composition is generally 1 -50 % w/w, preferably from 2-40 % w/w, more preferably from 10-25 % w/w. Compositions comprising more than 25 % w/w cleansing surfactant are commonly considered concentrated shampoos.
  • Anionic Surfactant to provide foam
  • anionic cleansing surfactants are the alkyi sulphates, alkyi ether sulphates, alkaryl sulphonates, alkanoyl isethionates, alkyi succinates, alkyi sulphosuccinates, alkyi ether sulphosuccinates, N-alkyl sarcosinates, alkyi phosphates, alkyi ether phosphates, and alkyi ether carboxylic acids and salts thereof, especially their sodium, magnesium, ammonium and mono-, di- and triethanolamine salts.
  • the alkyi and acyl groups generally contain from 8 to 18, preferably from 10 to 16 carbon atoms and may be unsaturated.
  • alkyi ether sulphates, alkyi ether sulphosuccinates, alkyi ether phosphates and alkyi 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 dodecyl benzene sulphonate, triethanolamine dodecylbenzene sulphonate, sodium cocoyl isethionate, sodium lauryl isethionate, lauryl ether carboxylic acid and sodium N-lauryl sarcosinate.
  • Preferred anionic surfactants are the alkyl sulfates and alkyl ether sulfates. These materials have the respective formulae ROS0 3 M and RO(C 2 H 4 0) x S0 3 M, wherein R 2 is alkyl or alkenyl of from 8 to 18 carbon atoms, x is an integer having a value of from about 1 to about 10, and M is a cation such as ammonium, alkanolamines, such as triethanolamine, monovalent metals, such as sodium and potassium, and polyvalent metal cations, such as magnesium, and calcium. Most preferably R has 12 to 14 carbon atoms, in a linear rather than branched chain.
  • the level of alkyl ether sulphate is from 0.5-25 % w/w, more preferably from 3-18 % w/w, most preferably from 6-15 % w/w of the total shampoo composition.
  • the total amount of anionic cleansing surfactant in shampoo compositions of the invention generally ranges from 0.5-45 % w/w, more preferably from 1.5-20 % w/w.
  • Shampoo compositions of the invention may contain non-ionic surfactant. Most preferably non-ionic surfactants are present in the range 0-5 % w/w of the total shampoo composition.
  • Nonionic surfactants that can be included in shampoo compositions of the invention include condensation products of aliphatic (C8 - C18) primary or secondary linear or branched chain alcohols or phenols with alkylene oxides, usually ethylene oxide and generally having from 6 to 30 ethylene oxide groups.
  • Alkyl ethoxylates are particularly preferred. Most preferred are alkyl ethoxylates having the formula R(OCH 2 CH 2 ) n OH, where R is an alkyl chain of C12 to C15, and n is 5 to 9.
  • Other suitable nonionic surfactants include mono- or di-alkyl alkanolamides. Examples include coco mono- or di-ethanolamide and coco mono-isopropanolamide.
  • APG alkyl polyglycosides
  • APG is one which comprises an alkyl group connected (optionally via a bridging group) to a block of one or more glycosyl groups.
  • Preferred APGs are defined by the following formula:
  • R is a branched or straight chain alkyl group which may be saturated or unsaturated and G is a saccharide group.
  • R may represent a mean alkyl chain length of from about C5 to about C20.
  • R represents a mean alkyl chain length of from about C8 to about C12.
  • G may be selected from C5 or C6 monosaccharide residues, and is preferably a glucoside.
  • G may be selected from the group comprising glucose, xylose, lactose, fructose, mannose and derivatives thereof.
  • G is glucose.
  • the degree of polymerisation, n may have a value of from about 1 to about 10 or more.
  • the value of n lies from about 1.1 to about 2.
  • Most preferably the value of n lies from about 1.3 to about 1.5.
  • Suitable alkyl polyglycosides for use in the invention are commercially available and include for example those materials identified as: Oramix NS10 ex Seppic; Plantaren 1200 and Plantaren 2000 ex Henkel.
  • compositions of the invention include the C10-C18 N-alkyl (CI-C6) polyhydroxy fatty acid amides, such as the C12-C18 N-methyl glucamides, as described for example in WO 92/06154 and US 5 194 639, and the N-alkoxy polyhydroxy fatty acid amides, such as C10-C18 N-(3- methoxypropyl) glucamide.
  • C10-C18 N-alkyl (CI-C6) polyhydroxy fatty acid amides such as the C12-C18 N-methyl glucamides, as described for example in WO 92/06154 and US 5 194 639
  • N-alkoxy polyhydroxy fatty acid amides such as C10-C18 N-(3- methoxypropyl) glucamide.
  • Amphoteric or zwitterionic surfactant can be included in an amount ranging from 0.5-8 % w/w, preferably from 1 -4 % w/w of the total shampoo composition.
  • amphoteric or zwitterionic surfactants include alkyl amine oxides, alkyl betaines, alkyl amidopropyl betaines, alkyl sulphobetaines (sultaines), alkyl glycinates, alkyl carboxyglycinates, alkyl amphoacetates, alkyl amphopropionates, alkylamphoglycinates, alkyl amidopropyl hydroxysultaines, acyl taurates and acyl glutamates, wherein the alkyl and acyl groups have from 8 to 19 carbon atoms.
  • Typical amphoteric and zwitterionic surfactants for use in shampoos of the invention include lauryl amine oxide, cocodimethyl sulphopropyl betaine, lauryl betaine, cocamidopropyl betaine and sodium cocoamphoacetate.
  • a particularly preferred amphoteric or zwitterionic surfactant is cocamidopropyl betaine.
  • amphoteric or zwitterionic surfactants may also be suitable.
  • Preferred mixtures are those of cocamidopropyl betaine with further amphoteric or zwitterionic surfactants as described above.
  • a preferred further amphoteric or zwitterionic surfactant is sodium cocoamphoacetate.
  • Carbopol 980 is the commonly used suspending agent though there is a growing desire to find an alternative. Typically such suspending agents are present at around 2 % w/w of the total shampoo composition.
  • 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.
  • 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 and Carbopol 980.
  • 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 Goodrich.
  • 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.
  • suspending agents may be used.
  • Preferred is a mixture of cross-linked polymer of acrylic acid and crystalline long chain acyl derivative.
  • Suspending agent will generally be present in a shampoo composition of the invention at levels of 0.1 -10 %, preferably 0.5-6 %, more preferably 0.9-4 % w/w of the total weight of the composition.
  • 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.
  • the composition will comprise 50-98 %, preferably 60-90 % of the total weight of the composition.
  • shampoo compositions typically have a pH of around 5.5.
  • Optional ingredients Typically, shampoo compositions have a pH of around 5.5.
  • Conditioning actives might also contain the following optional ingredients. Conditioning actives
  • Conditioning actives are often included in shampoo compositions. These are sometimes called '2-in-1 ' formulations.
  • Conditioning actives fall into three classes:
  • the composition is likely to also contain a cationic deposition polymer for enhancing deposition of the silicone.
  • silicone-containing composition is likely to be lamellar as opposed to isotropic. Isotropic compositions do not deposit silicone effectively. Silicone conditioning actives
  • compositions of the invention can contain, emulsified droplets of a silicone conditioning agent, for enhancing conditioning performance.
  • Suitable silicones include polydiorganosiloxanes, in particular polydimethylsiloxanes which have the CTFA designation dimethicone. Also suitable for use compositions of the invention (particularly shampoos and conditioners) are polydimethyl siloxanes having hydroxyl end groups, which have the CTFA designation dimethiconol. Also suitable for use in compositions of the invention are silicone gums having a slight degree of cross-linking, as are described for example in WO 96/31 188.
  • the viscosity of the emulsified silicone itself (not the emulsion or the final hair conditioning composition) is typically at least 10,000 est at 25 °C the viscosity of the silicone itself is preferably at least 60,000 est, most preferably at least 500,000 est, ideally at least 1 ,000,000 est. Preferably the viscosity does not exceed 109 est for ease of formulation.
  • Emulsified silicones for use in the shampoo compositions of the invention will typically have an average silicone droplet size in the composition of less than 30, preferably less than 20, more preferably less than 10 micron, ideally from 0.01 to 1 micron. Silicone emulsions having an average silicone droplet size of 0.15 micron are generally termed microemulsions.
  • Emulsified silicones for use in the conditioner compositions of the invention will typically have an average silicone droplet size in the composition of less than 30, preferably less than 20, more preferably less than 15.
  • the average silicone droplet is greater than 0.5 micron, more preferably greater than 1 micron, ideally from 2 to 8 micron.
  • Silicone particle size may be measured by means of a laser light scattering technique, for example using a 2600D Particle Sizer from Malvern Instruments.
  • Suitable pre-formed emulsions include Xiameter MEM 1785 and microemulsion DC2-1865 available from Dow Corning. These are emulsions /microemulsions of dimethiconol. Cross-linked silicone gums are also available in a pre- emulsified form, which is advantageous for ease of formulation.
  • a further preferred class of silicones for inclusion in shampoos and conditioners of the invention are amino functional silicones.
  • amino functional silicone is meant a silicone containing at least one primary, secondary or tertiary amine group, or a quaternary ammonium group.
  • suitable amino functional silicones include: polysiloxanes having the CTFA designation "amodimethicone”.
  • Specific examples of amino functional silicones suitable for use in the invention are the aminosilicone oils DC2-8220, DC2-8166 and DC2-8566 (all ex Dow Corning).
  • Suitable quaternary silicone polymers are described in EP-A-0 530 974.
  • a preferred quaternary silicone polymer is K3474, ex Goldschmidt.
  • emulsions of amino functional silicone oils with non ionic and/or cationic surfactant are also suitable from suppliers of silicone oils such as Dow Corning and General Electric. Specific examples include DC939 Cationic Emulsion and the non-ionic emulsions DC2-7224, DC2-8467, DC2- 8177 and DC2-8154 (all ex Dow Corning).
  • the total amount of silicone is preferably 0.01 -10 %, more preferably 0.1 -5 %, most preferably 0.5-3 % w/w of the total composition.
  • Cationic deposition polymers are used to deposit the silicone droplets to the hair surface and are thus preferred ingredients in a shampoo composition of the invention for enhancing performance.
  • Suitable cationic polymers may be homopolymers which are cationically substituted or may be formed from two or more types of monomers.
  • the weight average (Mw) 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.
  • the cationic nitrogen-containing group will generally be present as a substituent on a fraction of the total monomer units of the cationic polymer.
  • the polymer when it is not a homopolymer it can contain spacer non-cationic monomer units.
  • spacer non-cationic monomer units Such polymers are described in the CTFA Cosmetic Ingredient Directory, 3rd edition.
  • the ratio of the cationic to non-cationic monomer units is selected to give polymers having a cationic charge density in the required range, which is generally from 0.2 to 3.0 meq/gm.
  • the cationic charge density of the polymer is suitably determined via the Kjeldahl method as described in the US Pharmacopoeia under chemical tests for nitrogen determination.
  • 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 C1 -3 alkyl groups.
  • Other suitable spacers include vinyl esters, vinyl alcohol, maleic anhydride, propylene glycol and ethylene glycol.
  • the cationic amines can be primary, secondary or tertiary amines, depending upon the particular species and the pH of the composition. In general secondary and tertiary amines, especially tertiary, are preferred.
  • Amine substituted vinyl monomers and amines can be polymerized in the amine form and then converted to ammonium by quaternization.
  • the cationic polymers can comprise mixtures of monomer units derived from amine- and/or quaternary ammonium-substituted monomer and/or compatible spacer monomers.
  • Suitable cationic polymers include, for example: - cationic diallyl quaternary ammonium-containing polymers including, for example, dimethyldiallylammonium chloride homopolymer and copolymers of acrylamide and dimethyldiallylammonium chloride, referred to in the industry (CTFA) as Polyquaternium 6 and Polyquaternium 7, respectively;
  • cationic polymers that can be used include cationic polysaccharide polymers, such as cationic cellulose derivatives, cationic starch derivatives, and cationic guar gum derivatives.
  • Cationic polysaccharide polymers suitable for use in compositions of the invention include monomers of the formula:
  • A is an anhydroglucose residual group, such as a starch or cellulose anhydroglucose residual
  • R is an alkylene, oxyalkylene, polyoxy
  • cationic cellulose includes the polymeric quaternary ammonium salts of hydroxyethyl cellulose reacted with lauryl dimethyl ammonium-substituted epoxide, referred to in the industry (CTFA) as Polyquaternium 24. These materials are available from the Amerchol Corporation, for instance under the tradename Polymer LM-200.
  • CTFA lauryl dimethyl ammonium-substituted epoxide
  • Suitable cationic polysaccharide polymers include quaternary nitrogen-containing cellulose ethers (e.g. as described in US 3 962 418), and copolymers of etherified cellulose and starch (e.g. as described in US 3 958 581 ).
  • a particularly suitable type of cationic polysaccharide polymer that can be used is a cationic guar gum derivative, such as guar hydroxypropyltrimethylammonium chloride (commercially available from Rhodia in their JAGUAR trademark series).
  • a cationic guar gum derivative such as guar hydroxypropyltrimethylammonium chloride (commercially available from Rhodia in their JAGUAR trademark series).
  • examples of such materials are JAGUAR C13S, JAGUAR C14, JAGUAR C15, JAGUAR C17 and JAGUAR C16 Jaguar CHT and JAGUAR C162.
  • Mixtures of any of the above cationic polymers may be used.
  • Cationic polymer will generally be present in a shampoo composition of the invention at levels of 0.01 -5 %, preferably 0.05-1 %, more preferably 0.08-0.5 % by total weight of cationic polymer based on the total weight of the composition.
  • Cationic surfactants may be used in 2-in-1 shampoos to provide a conditioning benefit.
  • a shampoo composition is likely to also comprise anionic cleansing surfactants the use of cationic surfactants is limited to compositions where the cationic surfactant is separated from the anionic phase by way of a stable conditioning gel phase made separately from the rest of the formulation and then incorporated afterwards.
  • the term used in the art for this phase is 'conditioning gel network'.
  • Fibre actives are provided to repair or coat the hair fibres.
  • Antidandruff actives are provided to repair or coat the hair fibres.
  • the azoles include ketoconazole and climbazole. These are fat soluble actives.
  • the pyrithiones include zinc pyrithione (ZPT) which is insoluble and delivered as a particle to the scalp.
  • Antidandruff agents are compounds that are active against dandruff and are typically antimicrobial agents and preferably antifungal agents. Antifungal agents typically display a minimum inhibitory concentration of about 50 mg/ml or less against Malassezia spp.
  • Suitable antidandruff agents include compounds selected from ketoconazole, climbazole, octopirox, metal pyrithione salts, and mixtures thereof.
  • the preferred azole based antifungal agents are ketoconazole and climbazole.
  • Preferred metal pyrithione salts are zinc, copper, silver and zirconium pyrithione. The most preferred is zinc pyrithione.
  • the antidandruff active is present at 0.01 -5 %, more preferably 0.1 -2.5 % w/w of the composition.
  • Hair conditioning compositions are usually opaque lamellar phase compositions.
  • a conditioner which is to be used after a shampoo is known as a 'system conditioner' whereas one which is included in a shampoo composition is known as a '2-in-1 '.
  • the main ingredients in a system conditioner are the conditioning actives described above, the main actives being a cationic surfacant (e.g. behenyltrimmonium chloride (BTAC)), a conditioning silicone (e.g. aminosilicone (DC7134)) and a non-silicone oil, usually a fatty alcohol (e.g. cetearyl alcohol).
  • a cationic surfacant e.g. behenyltrimmonium chloride (BTAC)
  • BTAC behenyltrimmonium chloride
  • conditioning silicone e.g. aminosilicone (DC7134)
  • DC7134 non-silicone oil
  • non-silicone oil usually a fatty alcohol (e.g. cetearyl alcohol).
  • conditioners exist known as 'masks' or Overnight treatments' where the level of conditioning actives is merely increased over a standard system conditioning composition.
  • conditioning compositions may also be left on the head, i.e. not rinsed off after application. These are known as Leave-on-Treatments (LOTs) as opposed to Rinse-off- Treatments (ROTs).
  • LOTs Leave-on-Treatments
  • ROTs Rinse-off- Treatments
  • the desired ingredients are mixed in no particular order and usually at temperatures from 70 to 80 °C and under atmospheric pressure.
  • the packaging for the composition can be a bottle, tube, roll-ball applicator, propellant driven aerosol device, squeeze container or lidded jar.
  • a method of treating air ageing comprising the step of imbibing a composition, the composition comprising a nuclear factor erythroid-2 related factor 2 (Nrf2) agonist;
  • Nrf2 agonist is selected from the group consisting of andrographolide, apigenin, Asiatic acid, baicalein, tert-butyl hydroxyquinone, catechin, chrysin, chrysin dimethylether, curcumin, docosahexaenoic acid, epigallocatechin gallate, esculetin, fraxetin, galangin, genistein, guaiacol, hesperetin, 4-hydroxy-3-methoxyacetophenone, jasmonic acid, kaempferol, (S)-(+)-ketoprofen, kinetin, kinetin riboside, gamma linolenic acid, lipoic acid, lutein, luteolin, lycopene, nordihydroguaiaretic acid, oleanolic acid, parthenolide, peonidin chloride, phloroglucinol carboxaldehyde, pin
  • the oral composition referred to in the second aspect of the invention may comprise at least one unit dose comprising a daily dose of 1-1000, preferably 1 -500, most preferably 10-300 mg nuclear factor erythroid-2 related factor 2 (Nrf2) agonist.
  • Nrf2 nuclear factor erythroid-2 related factor 2
  • composition of the second aspect of the invention may be a foodstuff selected from the group consisting of a beverage, a supplement, a soup, margarine, a ready-to-eat meal, a dressing, a mayonnaise, mustard, a tomato-based condiment, a sauce, a seasoning, yoghurt and a frozen confection.
  • the composition may be in the form of a solid, a slurry, a solution, a suspension, a gel or an emulsion.
  • composition may be in the form of a beverage, in particular a fruit or tea based beverage.
  • composition may be in the form of a supplement of one or more unit dosages such as capsules, cachets, lozenges, pills, tablets, caplets.
  • the composition may be a soup in dry, paste or liquid form.
  • the composition may be a seasoning sold as unit doses in the form of a powder, a compressed powder in the form of, for example, a cube, a liquid or suspension, or a gel.
  • frozen confection means a sweet-tasting fabricated foodstuff intended for consumption in the frozen state (i.e. under conditions wherein the temperature of the foodstuff is less than 0°C, and preferably under conditions wherein the foodstuff comprises significant amounts of ice).
  • Frozen confections include ice cream, sorbet, sherbet, frozen yoghurt, water ice, milk ice and the like.
  • the frozen confection has a total solids content (i.e. the sum of the weights of all the ingredients other than water, expressed as a percentage of the total weight) of at least 20 %, more preferably at least 25 %.
  • Frozen confections may be aerated or unaerated.
  • the frozen confection is aerated.
  • the frozen confection may be manufactured by any suitable process, typically by preparing a mix of ingredients; then pasteurising and optionally homogenising the mix; and then freezing and optionally aerating the mix to produce the frozen confection.
  • EXAMPLE 1 NADP(H):QUINONE OXIDOREDUCTASE (NQO-1) AND HEMOXYGENASE-1 (HO-1) ASSAYS OF SKIN KERATINOCYTES
  • Cells were plated in 12 well plates, at a seeding density of 20,000 cells per ml of growth medium and incubated at 37 °C, in 5 % CO 2 until they were almost confluent, after approximately 48 hours incubation. Any change in cell morphology was noted after addition of test solutions.
  • Test solutions were prepared in ethanol or dimethyl sulfoxide (DMSO) and added directly to the cells and treated for 24 hours. The cells were then pelleted.
  • DMSO dimethyl sulfoxide
  • RNA samples were lysed using QIAzol lysis reagent and total RNA extracted using Qiagen RNeasy Mini or Micro Spin Columns. Total RNA concentration and purity were analysed using a Nanodrop 1000 (Thermo Scientific). The RNeasy MinElute Cleanup kit (Qiagen) was subsequently used to further concentrate the RNA samples if required.
  • RNA was reverse transcribed into cDNA using a High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems). Briefly, 0.1 ⁇ g of total RNA was prepared in a total of 15 ⁇ of ribonuclease (RNase) free water and added to an equal volume of 2X RT Master Mix (each reaction consisting of 3 ⁇ 10X RT Buffer, 1.2 ⁇ 25X deoxyribonucleotide triphosphate (dNTP) mix, 3 ⁇ 10X RT Random Primers, 1.5 ⁇ MultiScribe Reverse Transcriptase, 1.5 ⁇ RNase Inhibitor, 4.8 ⁇ RNase-free water).
  • RNase ribonuclease
  • qPCR human reference total RNA (Stratagene) was reverse transcribed at the same time to provide standard curves for gene expression analysis.
  • NQO-1 , HO-1 and 18S (housekeeper gene) were measured on an Eppendorf RealPlex qPCR machine using iQTMSYBR Green Supermix (BioRad) and QuantiTect Primers (Qiagen- NQO-1 and 18S). Briefly, cDNA was diluted 1 in 10 and 2 ⁇ added to 23 ⁇ of reaction mix (consisting of 12.5 ⁇ IQ SYBR Green Supermix, 5 ⁇ QuantiTect Primers and 5.5 ⁇ RNase-free water) on ice. Samples were run in triplicate for each of the genes.
  • the reverse transcribed qPCR human reference total RNA was used to generate Standard curve samples (20, 2, 0.2, 0.02 and 0.002 ng/ml) which were run in duplicate on every plate to allow multiple-plate analysis.
  • the polymerase chain reactions were monitored in real time on an Eppendorf RealPlex qPCR machine under the following conditions: 95 °C for 5 minutes; 45 cycles of 95 °C for 10 seconds and 60 °C for 30 seconds; 95 °C for 1 minute.
  • Unknown gene expression values were extrapolated from the standard curves, subsequently the NQO-1 values were normalised to the 18S housekeeper gene values.
  • Results are presented in table 1 which shows HO-1 and NQO-1 gene expression (fold change compared to 18S) in primary epidermal keratinocytes treated with certain test compounds.
  • Upregulation of gene expression of one or both of the detoxification enzymes (HO-1 and/or NQO-1 ) in the assay is indicative of Nrf2 activation and thus sulforaphane, resveratrol, curcumin, pterostilbene, lycopene, docosahexaenoic acid, gamma linolenic acid, allyl isothiocyanates and oleanolic acid are Nrf2 agonists.
  • EXAMPLE 2 NADP(H):QUININE OXIDOREDUCTASE (NQ01) AND HEMOXYGENASE-1 (HO-1) ASSAYS OF DERMAL FIBROBLASTS
  • DMEM Dulbecco's Modified Eagle Serum
  • FBS foetal bovine serum
  • Test solutions were prepared in ethanol or DMSO and added directly to the cells and treated for 24 hours.
  • the test solutions were prepared with selected concentrations of test material as set forth in Table 2. The cells were then pelleted. Preparation of cell lysate
  • the lysis buffer contained 1 % NP-40, 0.1 % sodium deoxycholate, 0.1 % SDS, 6 mM sodium chloride and 0.05 M Tris at pH 7.6.
  • Protease inhibitor cocktail 1000X; Sigma P8340 was added prior to use at a level of 10 ⁇ per ml of lysis buffer.
  • the clarified cell lysate was frozen at -80 °C until needed.
  • the total protein concentration of each cell lysate was measured using the Pierce BCA protein assay kit.
  • a set of eight standard solutions ranging from 0 to 1200 ⁇ g/ml protein was prepared from the supplied 2 mg/ml BSA stock solution. 10 ⁇ of standard or cell lysate was added to duplicate wells of a flat-bottomed, 96-well microtitre plate.
  • the reagent solution was prepared according to the kit instructions from 50 parts reagent A and 1 part reagent B. 200 ⁇ of the final reagent was added to each well of the microtitre plate. The plate was mixed, covered and incubated at 37 °C for 30 minutes and absorbance read at 562 nm. A protein standard curve was constructed and used to determine the protein concentration of each cell lysate.
  • NAD(P)H quinone oxidoreductase-1 (NQO-1 ) protein concentration of each cell lysate was assayed using the AdipoGenTM NQO-1 , human, intracellular, twin plex ELISA kit (AdipogenTM) according to the manufacturer's instructions.
  • NQO-1 standards were prepared in reagent diluent (0.5% Trition-X100 and 1 mM EDTA in phosphate buffered saline at pH 7.2) at concentrations ranging from 0.313 to 20 ng/ml, including a negative control.
  • 100 ⁇ of cell lysate, diluted 1/2, 1/5 or 1/10 or standard was added to duplicate wells of the assay plate which was pre-coated with the specific NQO-1 capture antibody by the manufacturer. The plate was incubated at before being washed three times with wash buffer. 100 ⁇ of NQO-1 ready to use detection antibody was added to each well and the plate incubated at 37 °C for 1 hour. The plate was washed as before.
  • TMB 3,3',5,5'-tetramethylbenzidine
  • hemeoxygenase-1 (HO-1 ) protein concentration of each cell lysate was assayed using the DuoSet Human Total HO-1/HMOX1 assay (R&D Systems DYC3776) according to the manufacturer's instructions.
  • Eight HO-1 standards were prepared in reagent diluent (0.5 % Trition-X100 and 1 mM EDTA in PBS at pH 7.2) at concentrations ranging from 0.15625 to 10 ng/ml, including a negative control.
  • the HO-1 capture antibody was diluted to a concentration of 8 ⁇ g/ml in PBS and was bound to the microtitre plate (Greiner Bio-One) overnight at room temperature.
  • the unbound antibody was then removed by washing 3 times with wash buffer (0.05 % Tween 20 in PBS) on an automatic plate washer.
  • the plate was blocked with 300 ⁇ per well of 1 % bovine serum albumin (BSA) in PBS for 1 hour and washed 3 times in wash buffer.
  • BSA bovine serum albumin
  • 100 ⁇ of cell lysate, diluted 1/5, or standard was added to duplicate wells.
  • the plate was incubated at room temperature for 2 hours before being washed three times with wash buffer.
  • HRP horseradish peroxidase
  • results were normalised using the total protein data and expressed as ng HO-1 per ⁇ g protein and are presented as percentage change in HO-1 compared to the vehicle control value.
  • Results are presented in table 2 which summarises levels of HO-1 and NQO-1 protein synthesis (ng HO-1 or NQO-1 per ⁇ g protein as a % of vehicle control) in dermal fibroblasts treated with certain test compounds.
  • HO-1 and/or NQO-1 synthesis is significantly up-regulated in dermal fibroblasts by andrographolide, apigenin, Asiatic acid, baicalein, catechin, chrysin, chrysin dimethylether, curcumin, docosahexaenoic acid, epigallocatechin gallate, esculetin, fraxetin, galangin, genistein, guaiacol, hesperetin, 4-hydroxy-3-methoxyacetophenone, jasmonic acid, kaempferol, (S)-(+)-ketoprofen, kinetin, kinetin riboside, lipoic acid, lutein, luteolin, nordihydroguaiaretic acid, parthenolide, peonidin chloride, phloroglucinol carboxaldehyde, pinocembrin, pinostrobin, protocatechinic acid, quercetin, rauwolscine,
  • test groups received Nrf2 agonist daily for 6 days and control groups (n>7 hair follicles) received vehicle alone.
  • the culture medium was replenished every other day and the hair shaft length was measured using an inverted binocular microscope as described in Philpot et al.
  • the vehicle was ethanol.
  • Results are presented in table 3 which shows that there is a significant increase in hair growth in the human hair follicle model versus the vehicle control when the human hair follicles are treated with 10 ⁇ sulforaphane. This is illustrated for 10 ⁇ sulforaphane in figure 1.
  • EXAMPLE 4 NADP(H): QUINONE OXI DO REDUCTASE (NQO-1) AND HEMOXYGENASE-1 (HO-1) GENE EXPRESSION ASSAYS OF HAIR FOLLICLES CELLS
  • Nrf2 agonist sulforaphane or tert-butyl hydroxyquinone (tBHQ)
  • RNA 100 ng RNA was reverse transcribed using the Invitrogen Cloned AMV First Strand Synthesis kit, according to manufacturers' instructions.
  • TaqMan primer sets for HO-1 (Hs01 1 10250_m1 ), NQO-1 (Hs00168547_m1 ) and Nrf2 (Hs00232352_m1 ) were obtained from the Invitrogen inventoried assays (Life technologies Ltd.).
  • cDNA was diluted 1 : 10 in RNase free water. Each reaction consisted of 1 ⁇ _ 20x TaqMan assay, 10 ⁇ _ Taqman Fast Advance Mastermix, 5 ⁇ _ RNase free water and 2 ng of cDNA. Plates were run on an Applied Biosystems StepOnePlusTM Real Time PCR machine. Fast cycling conditions were used, utilising a 2 minute polymerase activation step at 95°C, followed by 45 cycles of denaturing at 95°C for 1 second, and annealing/extension at 60°C for 20 seconds. All PCR reactions were normalised to the housekeeping control PPIA, which showed no variation between treatments. Data was analysed by the AACt methodology
  • Results are presented in table 4 which shows NQO-1 and HO-1 gene expression (fold change compared to 18S) in hair follicles treated with sulforaphane or tBHQ.
  • EXAMPLE 5 IMMUNOHISTOCHEMICAL ANALYSIS OF NADP(H): QUINONE OXIDOREDUCTASE (NQO-1) AND HEMOXYGENASE-1 (HO-1) IN HAIR FOLLICLES Human hair follicle isolation
  • Anagen VI hair follicles were isolated and maintained according to the method described in Philpott et al (J. Cell Sci 97, 463-471 (1990)). Dhurat et al (Int J Trichology, 2(2), 96-100 (Jul-Dec 2010)) describe that anagen is conventionally divided into six stages, Anagen l-V (proanagen), when the hair shaft grows within the hair follicle, and Anagen VI (metanagen), when the tip of the hair shaft emerges from the hair follicle. Hair follicles were stabilised in RNAIater (for PCR). Five individual Anagen VI hair follicles were used for each culture condition/patient.
  • DMSO dimethyl sulfoxide
  • cryosections were stained for HO-1 and NQO-1 using mouse monoclonal antibodies (Abeam ab13248 and ab28947 (Clone A180) respectively). Indirect detection was performed using a secondary goat anti-mouse alexa fluor 488 by strepavidin- conjugated fluorescein (FITC).
  • FITC strepavidin- conjugated fluorescein
  • Immunolocalisation and intensity analyses were performed with the Biozero-8000 microscope (Keyence) and analysed using Image J software (National Institute of Health). Immunofluorescent images were taken using the same exposure time when using intensity of fluorescent stain for quantitative analyses. To compare staining intensity and positive cell numbers, the mean values from multiple cryosections from each patient were taken to allow for variation between the sections. Reference areas of a standard size were selected, encompassing the epithelial cells or mesenchymal connective tissue sheath, within which fluorescence intensity was determined using Image J software. These reference areas were the same for each cryosection/hair follicle/patient sample analysed.
  • HO-1 immunoreactivity was not observed in vehicle control (dimethyl sulfoxide) treated follicles in either the epithelial hair follicle or the mesenchymal connective tissue sheath (CTS).
  • CTS mesenchymal connective tissue sheath
  • NQO-1 immunoreactivity was detected in both the inner and outer root sheath in the vehicle control (dimethyl sulfoxide) treated follicles.
  • vehicle control dimethyl sulfoxide
  • a significant increase in NQO-1 immunofluorescence was seen in the epithelial cells of the isolated hair follicles.
  • Table 5 NQO-1 fluorescence levels (arbitrary units) of hair follicles (epithelial cells) after 24 hours of treatment with 2 ⁇ , 5 ⁇ , and 20 ⁇ sulforaphane with standard deviation and P-values (95 % confidence limits).
  • NQO-1 epidermal 8.4 ⁇ 3. 15.4 ⁇ 1 .1 18.8 ⁇ 1 .4 cells
  • Table 6 HO-1 fluorescence levels (arbitary units) of hair follicles after 24 hours of treatment with 2 ⁇ , 5 ⁇ , and 20 ⁇ sulforaphane with standard deviation and P- values (95 % confidence limits).
  • Treatment of hair follicles with sulforaphane leads to up-regulation of HO-1 and NQO-1.

Abstract

Hair ageing is a major age-related consumer issue (hair loss, thinning hair, loss of shine, increased number of grey hairs, etc). Biological routes for hair growth or preventing hair greying provide effective opportunities to target consumer hair issues. Currently, Minoxidil ™ and Finasteride™ are the only clinically proven, mildly effective products available for hair growth and both are classified as medicines and therefore not suitable for cosmetic use. The identification of cosmetic ingredients which are able to boost hair growth, maintain anagen and/or prevent catagen may prove to be effective anti ageing treatments to prevent or attenuate some of the symptoms associated with hair ageing. This invention relates to a method of treating hair ageing. In particular the method relates to treating hair ageing through application to hair fibres of a composition comprising a nuclear factor erythroid-2 related factor 2 (also known as NFE2L2or Nrf2) agonist.

Description

METHOD OF TREATING HAIR AGEING
This invention relates to a method of treating hair ageing. In particular the method relates to treating hair ageing through application to hair fibres of a composition comprising a nuclear factor erythroid-2 related factor 2 (also known as NFE2L2 or Nrf2) agonist.
The main function of the hair follicle is to produce a hair fibre. The hair follicle develops from the embryonic epidermis as an epidermal finger which differentiates into the fibre, the outer root sheath (ORS) and the inner root sheath (IRS). Figure 1 illustrates how mature follicles undergo follicular cycling through phases of organ growth and hair fibre production (anagen) for 3-7 years, cessation of fibre growth and organ involution (catagen) over about 2 weeks and a quiescent phase (telogen) which lasts about 3 months where the organ rests and the hair fibre remains anchored but no longer grows before the hair fibre falls (exogen) and is regenerated to start the cycle again (Dry, J Genet 16, 281 -340 (1926), Chase, Physiol Rev 34, 1 , 1 13-26 (1954) and Kligman, J Invest Dermatol 33, 307-16 (1959)).
Moi et al (Proc. Natl. Acad. Sci. U.S.A,. 91 , 21 , 9926-30 (October 1994)) discloses that Nrf2 is a transcription factor (protein) that in humans is encoded by the NFE2L2 gene. According to Lee et al (J. of Biochem & Mol Biol. 37, 139-143 (2004)) and Hybertson et al (Mol Aspects Med. 32, 234-46 (201 1 )), Nrf2 has been shown to be involved in the defence against oxidative injury in various tissues. Under basal conditions, Nrf-2 is inactive and bound in the cytoplasm by cytosolic regulatory protein Kelch-like ECH- associated protein 1 (Keapl ). According to Itoh et al (Genes Dev., 13, 1 , 76-86 (January 1999)) the protein Cullin 3 degrades Nrf2 by ubiquitination. According to Kobayashi et al (Mol. Cell. Biol., 24, 16, 7130-9 (August 2004)) Keapl helps Cullin 3 ubiquitinate Nrf2. When Nrf2 is ubiquitinated, it is transported to the proteasome where it is degraded and its components recycled such that under normal conditions, Nrf2 has a half-life of only 20 minutes. Yamamoto et al (Mol. Cell (Biol., 28, 8, 2758-70 (April 2008)) and Sekhar et al (Toxicol. Appl. Pharmacol., 244, 1 , 21-6 (June 2009)) disclose that oxidative stress or electrophilic stress disrupts critical cysteine residues in Keapl , disrupting the Keapl - Cullin 3 ubiquitination system. When Nrf2 is not ubiquitinated, it builds up in the cytoplasm and translocates into the nucleus. Itoh et al (Biochem. Biophys. Res. Commun., 236, 2, 313-22 (July 1997)) disclose that in the nucleus, Nrf2 combines (forms a heterodimer) with a small Maf protein (a transcription factor) and binds to small regions of DNA known as Antioxidant Response Elements (ARE's) in the upstream promoter region of many anti-oxidative genes, and initiates their transcription.
According to Lee et al and Hybertson et al, the antioxidant genes include "phase II" enzymes such as NADP(H): quinone oxidoreductase (NQO-1 ) and hemoxygenase-1 (HO-1 ). Increased oxidative stress has been shown to have a detrimental effect on hair pigmentation (Lu et al, J Invest Dermatol, 129, 1790-804 (2009); Arck et al, FASEB J. 2, 1567-9 (2006)).
Hair ageing is a major age-related consumer issue (hair loss, thinning hair, loss of shine, increased number of grey hairs, etc). Biological routes for hair growth or preventing hair greying provide effective opportunities to target consumer hair issues. Currently, Minoxidil™ and Finasteride™ are the only clinically proven, mildly effective products available for hair growth and both are classified as medicines and therefore not suitable for cosmetic use. The identification of cosmetic ingredients which are able to boost hair growth, maintain anagen and/or prevent catagen may prove to be effective anti ageing treatments to prevent or attenuate some of the symptoms associated with hair ageing.
US 2008/0187608 discloses use of certain novel compositions from clary sage (Salvia sclarea) which are inhibitors of nuclear factor kappa B (NF-κΒ), Nerf2 (an ETS (E- twenty six) transcription factor that is specifically expressed in endothelial cells (Dube et al, Circ Res, 84, 10, 1 177-85 (1999)) and the activity of the endothelin receptor. To counteract oxidative stress, higher animals have developed elaborate mechanisms, including phase II detoxifying enzymes and antioxidant proteins. Induction of phase II enzymes is predominantly mediated by a redox-sensitive transcription factor NF-E2 related factor-2 (Nrf2). A variety of phytochemicals are able to activate Nrf2 thereby up- regulating a set of enzymes including nicotinamide adenine dinucleotide phosphate (NADP(H)): quinone oxidasereductase-1 (NQO-1 ), superoxide dismutase (SOD), glutathione S-transferase (GST), hemeoxygenase-1 (HO-1 ), and glutamyl cysteine ligase (GCL). US 2008/0187608 further discloses that it would be highly desirable to enrich the fractions from clary sage roots that have activity against the NF-κΒ, Nerf2 and the endothelin receptors. In particular it would be desirable to provide standardised root extract from clary sage for use in a pharmaceutical, nutraceutical and cosmeceuticals composition suitable for the treatment and prevention of cancer, inflammation nrelated diseases, cardiovascular diseases, fungal and bacterial infections, as an anti-coagulant, enhancement of adenylate cyclise activity, anti-inflammatory, vasodilator, antimyocardiac, retardation of cholesterol biosynthesis, inhibition of lipoprotein oxidation, treatment of acne and eczema, prevent hair loss, whitening skin and protect myocardium from hypoxia-induced cardiac contractile failure. Nerf2 expression is specific to the endothelial cell and not expressed in the hair follicle.
This invention is based on the observation by the inventors that Nrf-2 agonists promote hair fibre growth in a hair growth model. SUMMARY OF THE INVENTION
In a first aspect of the invention, a method of treating hair ageing is provided, the method comprising the step of applying a composition, the composition comprising a nuclear factor erythroid-2 related factor 2 (Nrf2) agonist;
wherein the Nrf2 agonist is selected from the group consisting of andrographolide, apigenin, Asiatic acid, baicalein, tert-butyl hydroxyquinone, catechin, chrysin, chrysin dimethylether, curcumin, docosahexaenoic acid, epigallocatechin gallate, esculetin, fraxetin, galangin, genistein, guaiacol, hesperetin, 4-hydroxy-3-methoxyacetophenone, jasmonic acid, kaempferol, (S)-(+)-ketoprofen, kinetin, kinetin riboside, gamma linolenic acid, lipoic acid, lutein, luteolin, lycopene, nordihydroguaiaretic acid, oleanolic acid, parthenolide, peonidin chloride, phloroglucinol carboxaldehyde, pinocembrin, pinostrobin, protocatechinic acid, pterostilbene, quercetin, rauwolscine, resveratrol, silibinin, silichristin, taxifolin, theaflavin, vanillyl acetone, trans-zeatin riboside, chamomile oil, parsley peel ethanol extract, sage peel ethanol extract, sour orange peel ethanol extract, tangerine peel ethanol extract and thyme peel ethanol extract. In a second aspect of the invention, a method of treating hair ageing is provided, the method comprising the step of imbibing a composition, the composition comprising a nuclear factor erythroid-2 related factor 2 (Nrf2) agonist;
wherein the Nrf2 agonist is selected from the group consisting of andrographolide, apigenin, Asiatic acid, baicalein, tert-butyl hydroxyquinone, catechin, chrysin, chrysin dimethylether, curcumin, docosahexaenoic acid, epigallocatechin gallate, esculetin, fraxetin, galangin, genistein, guaiacol, hesperetin, 4-hydroxy-3-methoxyacetophenone, jasmonic acid, kaempferol, (S)-(+)-ketoprofen, kinetin, kinetin riboside, gamma linolenic acid, lipoic acid, lutein, luteolin, lycopene, nordihydroguaiaretic acid, oleanolic acid, parthenolide, peonidin chloride, phloroglucinol carboxaldehyde, pinocembrin, pinostrobin, protocatechinic acid, pterostilbene, quercetin, rauwolscine, resveratrol, silibinin, silichristin, taxifolin, theaflavin, vanillyl acetone, trans-zeatin riboside, chamomile oil, parsley peel ethanol extract, sage peel ethanol extract, sour orange peel ethanol extract, tangerine peel ethanol extract and thyme peel ethanol extract. BRIEF DESCRIPTION OF THE FIGURES
The invention is exemplified with reference to the figures which show in:
Figure 1 follicular cycling through phases of organ growth and hair fibre production (anagen), cessation of fibre growth and organ involution (catagen) and a quiescent phase (telogen); and Figure 2 human hair shaft length (mm) over time (hours) after treatment with 10 μΜ sulforaphane versus a vehicle control.
DETAILED DESCRIPTION OF THE INVENTION
In a first aspect of the invention, a method of treating hair ageing is provided, the method comprising the step of applying a composition to hair fibres and/or scalp, the composition comprising a nuclear factor erythroid-2 related factor 2 (Nrf2) agonist;
wherein the Nrf2 agonist is selected from the group consisting of andrographolide, apigenin, Asiatic acid, baicalein, tert-butyl hydroxyquinone, catechin, chrysin, chrysin dimethylether, curcumin, docosahexaenoic acid, epigallocatechin gallate, esculetin, fraxetin, galangin, genistein, guaiacol, hesperetin, 4-hydroxy-3-methoxyacetophenone, jasmonic acid, kaempferol, (S)-(+)-ketoprofen, kinetin, kinetin riboside, gamma linolenic acid, lipoic acid, lutein, luteolin, lycopene, nordihydroguaiaretic acid, oleanolic acid, parthenolide, peonidin chloride, phloroglucinol carboxaldehyde, pinocembrin, pinostrobin, protocatechinic acid, pterostilbene, quercetin, rauwolscine, resveratrol, silibinin, silichristin, taxifolin, theaflavin, vanillyl acetone, trans-zeatin riboside, chamomile oil, parsley peel ethanol extract, sage peel ethanol extract, sour orange peel ethanol extract, tangerine peel ethanol extract and thyme peel ethanol extract. Treating hair ageing is through promoting hair fibre growth or reducing hair fibre loss.
The hair and/or scalp composition referred to in the first aspect of the invention may comprise 0.00001 -1 , preferably 0.001 -1 , most preferably 0.0001 -0.1 % w/w nuclear factor erythroid-2 related factor 2 (Nrf2) agonist.
Such a composition will normally be in the form of a:
1. Shampoo
2. Hair conditioner
3. Hair styling composition
4. Hair colouring composition Shampoo compositions will nearly always comprise a cleansing surfactant component in an aqueous base.
Cleansing surfactant
The cleansing surfactant may consist of a single surfactant, usually an anionic surfactant (to provide foam) such as sodium lauryl ether sulphate (SLES with one ethylene oxide), or more commonly a mixture of SLES with a co-surfactant to provide mildness. The most preferred co-surfactant is cocoamidopropyl betaine (CAPB). The total amount of surfactant (including any co-surfactant, and/or any emulsifier) in a shampoo composition is generally 1 -50 % w/w, preferably from 2-40 % w/w, more preferably from 10-25 % w/w. Compositions comprising more than 25 % w/w cleansing surfactant are commonly considered concentrated shampoos. Anionic Surfactant
Examples of suitable anionic cleansing surfactants are the alkyi sulphates, alkyi ether sulphates, alkaryl sulphonates, alkanoyl isethionates, alkyi succinates, alkyi sulphosuccinates, alkyi ether sulphosuccinates, N-alkyl sarcosinates, alkyi phosphates, alkyi ether phosphates, and alkyi ether carboxylic acids and salts thereof, especially their sodium, magnesium, ammonium and mono-, di- and triethanolamine salts. The alkyi and acyl groups generally contain from 8 to 18, preferably from 10 to 16 carbon atoms and may be unsaturated. The alkyi ether sulphates, alkyi ether sulphosuccinates, alkyi ether phosphates and alkyi 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 dodecyl benzene sulphonate, triethanolamine dodecylbenzene sulphonate, sodium cocoyl isethionate, sodium lauryl isethionate, lauryl ether carboxylic acid and sodium N-lauryl sarcosinate. Preferred anionic surfactants are the alkyl sulfates and alkyl ether sulfates. These materials have the respective formulae ROS03M and RO(C2H40)xS03M, wherein R2 is alkyl or alkenyl of from 8 to 18 carbon atoms, x is an integer having a value of from about 1 to about 10, and M is a cation such as ammonium, alkanolamines, such as triethanolamine, monovalent metals, such as sodium and potassium, and polyvalent metal cations, such as magnesium, and calcium. Most preferably R has 12 to 14 carbon atoms, in a linear rather than branched chain.
Preferred anionic cleansing surfactants are selected from sodium lauryl sulphate and sodium lauryl ether sulphate(n)EO, (where n is from 1 to 3); more preferably sodium lauryl ether sulphate(n)EO, (where n is from 1 to 3); most preferably sodium lauryl ether sulphate(n)EO where n=1.
Preferably the level of alkyl ether sulphate is from 0.5-25 % w/w, more preferably from 3-18 % w/w, most preferably from 6-15 % w/w of the total shampoo composition.
The total amount of anionic cleansing surfactant in shampoo compositions of the invention generally ranges from 0.5-45 % w/w, more preferably from 1.5-20 % w/w. Nonionic surfactant
Shampoo compositions of the invention may contain non-ionic surfactant. Most preferably non-ionic surfactants are present in the range 0-5 % w/w of the total shampoo composition. Nonionic surfactants that can be included in shampoo compositions of the invention include condensation products of aliphatic (C8 - C18) primary or secondary linear or branched chain alcohols or phenols with alkylene oxides, usually ethylene oxide and generally having from 6 to 30 ethylene oxide groups. Alkyl ethoxylates are particularly preferred. Most preferred are alkyl ethoxylates having the formula R(OCH2CH2)nOH, where R is an alkyl chain of C12 to C15, and n is 5 to 9. Other suitable nonionic surfactants include mono- or di-alkyl alkanolamides. Examples include coco mono- or di-ethanolamide and coco mono-isopropanolamide.
Further nonionic surfactants which can be included in shampoo]compositions of the invention are the alkyl polyglycosides (APGs). Typically, APG is one which comprises an alkyl group connected (optionally via a bridging group) to a block of one or more glycosyl groups. Preferred APGs are defined by the following formula:
RO - (G)n wherein R is a branched or straight chain alkyl group which may be saturated or unsaturated and G is a saccharide group.
R may represent a mean alkyl chain length of from about C5 to about C20. Preferably R represents a mean alkyl chain length of from about C8 to about C12. Most preferably the value of the mean alkyl chain length of R lies between about 9.5 and about 10.5. G may be selected from C5 or C6 monosaccharide residues, and is preferably a glucoside. G may be selected from the group comprising glucose, xylose, lactose, fructose, mannose and derivatives thereof. Preferably G is glucose.
The degree of polymerisation, n, may have a value of from about 1 to about 10 or more. Preferably, the value of n lies from about 1.1 to about 2. Most preferably the value of n lies from about 1.3 to about 1.5. Suitable alkyl polyglycosides for use in the invention are commercially available and include for example those materials identified as: Oramix NS10 ex Seppic; Plantaren 1200 and Plantaren 2000 ex Henkel.
Other sugar-derived nonionic surfactants which can be included in compositions of the invention include the C10-C18 N-alkyl (CI-C6) polyhydroxy fatty acid amides, such as the C12-C18 N-methyl glucamides, as described for example in WO 92/06154 and US 5 194 639, and the N-alkoxy polyhydroxy fatty acid amides, such as C10-C18 N-(3- methoxypropyl) glucamide.
Amphoteric/zwitterionic surfactant
Amphoteric or zwitterionic surfactant can be included in an amount ranging from 0.5-8 % w/w, preferably from 1 -4 % w/w of the total shampoo composition.
Examples of amphoteric or zwitterionic surfactants include alkyl amine oxides, alkyl betaines, alkyl amidopropyl betaines, alkyl sulphobetaines (sultaines), alkyl glycinates, alkyl carboxyglycinates, alkyl amphoacetates, alkyl amphopropionates, alkylamphoglycinates, alkyl amidopropyl hydroxysultaines, acyl taurates and acyl glutamates, wherein the alkyl and acyl groups have from 8 to 19 carbon atoms. Typical amphoteric and zwitterionic surfactants for use in shampoos of the invention include lauryl amine oxide, cocodimethyl sulphopropyl betaine, lauryl betaine, cocamidopropyl betaine and sodium cocoamphoacetate.
A particularly preferred amphoteric or zwitterionic surfactant is cocamidopropyl betaine.
Mixtures of any of the foregoing amphoteric or zwitterionic surfactants may also be suitable. Preferred mixtures are those of cocamidopropyl betaine with further amphoteric or zwitterionic surfactants as described above. A preferred further amphoteric or zwitterionic surfactant is sodium cocoamphoacetate.
Suspending agents
Carbopol 980 is the commonly used suspending agent though there is a growing desire to find an alternative. Typically such suspending agents are present at around 2 % w/w of the total shampoo 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. 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 and Carbopol 980. 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 Goodrich.
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.
Mixtures of any of the above suspending agents may be used. Preferred is a mixture of cross-linked polymer of acrylic acid and crystalline long chain acyl derivative.
Suspending agent will generally be present in a shampoo composition of the invention at levels of 0.1 -10 %, preferably 0.5-6 %, more preferably 0.9-4 % w/w of the total weight of the composition. Water
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 50-98 %, preferably 60-90 % of the total weight of the composition.
Typically, shampoo compositions have a pH of around 5.5. Optional ingredients
Shampoo compositions might also contain the following optional ingredients. Conditioning actives
Conditioning actives are often included in shampoo compositions. These are sometimes called '2-in-1 ' formulations.
Conditioning actives fall into three classes:
-silicones (and cationic deposition polymers to assist in silicone deposition)
-cationic surfactants
-non-silicone oils
Where silicones are included, the composition is likely to also contain a cationic deposition polymer for enhancing deposition of the silicone.
Further, a silicone-containing composition is likely to be lamellar as opposed to isotropic. Isotropic compositions do not deposit silicone effectively. Silicone conditioning actives
The compositions of the invention can contain, emulsified droplets of a silicone conditioning agent, for enhancing conditioning performance.
Suitable silicones include polydiorganosiloxanes, in particular polydimethylsiloxanes which have the CTFA designation dimethicone. Also suitable for use compositions of the invention (particularly shampoos and conditioners) are polydimethyl siloxanes having hydroxyl end groups, which have the CTFA designation dimethiconol. Also suitable for use in compositions of the invention are silicone gums having a slight degree of cross-linking, as are described for example in WO 96/31 188. The viscosity of the emulsified silicone itself (not the emulsion or the final hair conditioning composition) is typically at least 10,000 est at 25 °C the viscosity of the silicone itself is preferably at least 60,000 est, most preferably at least 500,000 est, ideally at least 1 ,000,000 est. Preferably the viscosity does not exceed 109 est for ease of formulation.
Emulsified silicones for use in the shampoo compositions of the invention will typically have an average silicone droplet size in the composition of less than 30, preferably less than 20, more preferably less than 10 micron, ideally from 0.01 to 1 micron. Silicone emulsions having an average silicone droplet size of 0.15 micron are generally termed microemulsions.
Emulsified silicones for use in the conditioner compositions of the invention will typically have an average silicone droplet size in the composition of less than 30, preferably less than 20, more preferably less than 15. Preferably the average silicone droplet is greater than 0.5 micron, more preferably greater than 1 micron, ideally from 2 to 8 micron.
Silicone particle size may be measured by means of a laser light scattering technique, for example using a 2600D Particle Sizer from Malvern Instruments.
Examples of suitable pre-formed emulsions include Xiameter MEM 1785 and microemulsion DC2-1865 available from Dow Corning. These are emulsions /microemulsions of dimethiconol. Cross-linked silicone gums are also available in a pre- emulsified form, which is advantageous for ease of formulation.
A further preferred class of silicones for inclusion in shampoos and conditioners of the invention are amino functional silicones. By "amino functional silicone" is meant a silicone containing at least one primary, secondary or tertiary amine group, or a quaternary ammonium group. Examples of suitable amino functional silicones include: polysiloxanes having the CTFA designation "amodimethicone". Specific examples of amino functional silicones suitable for use in the invention are the aminosilicone oils DC2-8220, DC2-8166 and DC2-8566 (all ex Dow Corning).
Suitable quaternary silicone polymers are described in EP-A-0 530 974. A preferred quaternary silicone polymer is K3474, ex Goldschmidt.
Also suitable are emulsions of amino functional silicone oils with non ionic and/or cationic surfactant. Pre-formed emulsions of amino functional silicone are also available from suppliers of silicone oils such as Dow Corning and General Electric. Specific examples include DC939 Cationic Emulsion and the non-ionic emulsions DC2-7224, DC2-8467, DC2- 8177 and DC2-8154 (all ex Dow Corning). The total amount of silicone is preferably 0.01 -10 %, more preferably 0.1 -5 %, most preferably 0.5-3 % w/w of the total composition.
Cationic deposition polymers are used to deposit the silicone droplets to the hair surface and are thus preferred ingredients in a shampoo composition of the invention for enhancing performance.
Suitable cationic polymers may be homopolymers which are cationically substituted or may be formed from two or more types of monomers. The weight average (Mw) 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. The cationic nitrogen-containing group will generally be present as a substituent on a fraction of the total monomer units of the cationic polymer. Thus when the polymer is not a homopolymer it can contain spacer non-cationic monomer units. Such polymers are described in the CTFA Cosmetic Ingredient Directory, 3rd edition. The ratio of the cationic to non-cationic monomer units is selected to give polymers having a cationic charge density in the required range, which is generally from 0.2 to 3.0 meq/gm. The cationic charge density of the polymer is suitably determined via the Kjeldahl method as described in the US Pharmacopoeia under chemical tests for nitrogen determination. 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 C1 -3 alkyl groups. Other suitable spacers include vinyl esters, vinyl alcohol, maleic anhydride, propylene glycol and ethylene glycol.
The cationic amines can be primary, secondary or tertiary amines, depending upon the particular species and the pH of the composition. In general secondary and tertiary amines, especially tertiary, are preferred.
Amine substituted vinyl monomers and amines can be polymerized in the amine form and then converted to ammonium by quaternization. The cationic polymers can comprise mixtures of monomer units derived from amine- and/or quaternary ammonium-substituted monomer and/or compatible spacer monomers.
Suitable cationic polymers include, for example: - cationic diallyl quaternary ammonium-containing polymers including, for example, dimethyldiallylammonium chloride homopolymer and copolymers of acrylamide and dimethyldiallylammonium chloride, referred to in the industry (CTFA) as Polyquaternium 6 and Polyquaternium 7, respectively;
- mineral acid salts of amino-alkyl esters of homo-and co-polymers of unsaturated carboxylic acids having from 3 to 5 carbon atoms, (as described in US 4 009 256);
- cationic polyacrylamides(as described in WO 95/2231 1 ).
Other cationic polymers that can be used include cationic polysaccharide polymers, such as cationic cellulose derivatives, cationic starch derivatives, and cationic guar gum derivatives.
Cationic polysaccharide polymers suitable for use in compositions of the invention include monomers of the formula:
A-O-[R-N+(R1)(R2)(R3)X-], wherein: A is an anhydroglucose residual group, such as a starch or cellulose anhydroglucose residual; R is an alkylene, oxyalkylene, polyoxyalkylene, or hydroxyalkylene group, or combination thereof; Ri , R2 and R3 independently represent alkyl, aryl, alkylaryl, arylalkyl, alkoxyalkyl, or alkoxyaryl groups, each group containing up to about 18 carbon atoms (the total number of carbon atoms for each cationic moiety (i.e., the sum of carbon atoms in Ri , R2 and R3) is preferably about 20 or less); and X is an anionic counterion.
Another type of cationic cellulose includes the polymeric quaternary ammonium salts of hydroxyethyl cellulose reacted with lauryl dimethyl ammonium-substituted epoxide, referred to in the industry (CTFA) as Polyquaternium 24. These materials are available from the Amerchol Corporation, for instance under the tradename Polymer LM-200. Other suitable cationic polysaccharide polymers include quaternary nitrogen-containing cellulose ethers (e.g. as described in US 3 962 418), and copolymers of etherified cellulose and starch (e.g. as described in US 3 958 581 ). A particularly suitable type of cationic polysaccharide polymer that can be used is a cationic guar gum derivative, such as guar hydroxypropyltrimethylammonium chloride (commercially available from Rhodia in their JAGUAR trademark series). Examples of such materials are JAGUAR C13S, JAGUAR C14, JAGUAR C15, JAGUAR C17 and JAGUAR C16 Jaguar CHT and JAGUAR C162.
Mixtures of any of the above cationic polymers may be used.
Cationic polymer will generally be present in a shampoo composition of the invention at levels of 0.01 -5 %, preferably 0.05-1 %, more preferably 0.08-0.5 % by total weight of cationic polymer based on the total weight of the composition.
Cationic surfactants
Cationic surfactants may be used in 2-in-1 shampoos to provide a conditioning benefit. However, since a shampoo composition is likely to also comprise anionic cleansing surfactants the use of cationic surfactants is limited to compositions where the cationic surfactant is separated from the anionic phase by way of a stable conditioning gel phase made separately from the rest of the formulation and then incorporated afterwards. The term used in the art for this phase is 'conditioning gel network'. Non-silicone Oily Conditioning Components
These are typically hydrocarbon oils or fatty alcohols. A fatty alcohol is nearly always included in a conditioning composition and often included in 2-in-1 shampoos. Cetearyl alcohol is one of the preferred examples. Fibre actives
Fibre actives are provided to repair or coat the hair fibres. Antidandruff actives
There are two classes of antidandruff active: the azoles and the pyrithiones, both are active against the target fungi malasezzia spp. The azoles include ketoconazole and climbazole. These are fat soluble actives. The pyrithiones include zinc pyrithione (ZPT) which is insoluble and delivered as a particle to the scalp.
Antidandruff agents are compounds that are active against dandruff and are typically antimicrobial agents and preferably antifungal agents. Antifungal agents typically display a minimum inhibitory concentration of about 50 mg/ml or less against Malassezia spp.
Suitable antidandruff agents include compounds selected from ketoconazole, climbazole, octopirox, metal pyrithione salts, and mixtures thereof.
The preferred azole based antifungal agents are ketoconazole and climbazole. Preferred metal pyrithione salts are zinc, copper, silver and zirconium pyrithione. The most preferred is zinc pyrithione. Preferably, the antidandruff active is present at 0.01 -5 %, more preferably 0.1 -2.5 % w/w of the composition.
Hair conditioner
Hair conditioning compositions are usually opaque lamellar phase compositions.
A conditioner which is to be used after a shampoo is known as a 'system conditioner' whereas one which is included in a shampoo composition is known as a '2-in-1 '.
The main ingredients in a system conditioner are the conditioning actives described above, the main actives being a cationic surfacant (e.g. behenyltrimmonium chloride (BTAC)), a conditioning silicone (e.g. aminosilicone (DC7134)) and a non-silicone oil, usually a fatty alcohol (e.g. cetearyl alcohol).
A subset of conditioners exists known as 'masks' or Overnight treatments' where the level of conditioning actives is merely increased over a standard system conditioning composition.
Further, conditioning compositions may also be left on the head, i.e. not rinsed off after application. These are known as Leave-on-Treatments (LOTs) as opposed to Rinse-off- Treatments (ROTs).
When making the hair and/or scalp composition referred to in the first aspect of the invention, the desired ingredients are mixed in no particular order and usually at temperatures from 70 to 80 °C and under atmospheric pressure. The packaging for the composition can be a bottle, tube, roll-ball applicator, propellant driven aerosol device, squeeze container or lidded jar.
In a second aspect of the invention, a method of treating air ageing is provided, the method comprising the step of imbibing a composition, the composition comprising a nuclear factor erythroid-2 related factor 2 (Nrf2) agonist;
wherein the Nrf2 agonist is selected from the group consisting of andrographolide, apigenin, Asiatic acid, baicalein, tert-butyl hydroxyquinone, catechin, chrysin, chrysin dimethylether, curcumin, docosahexaenoic acid, epigallocatechin gallate, esculetin, fraxetin, galangin, genistein, guaiacol, hesperetin, 4-hydroxy-3-methoxyacetophenone, jasmonic acid, kaempferol, (S)-(+)-ketoprofen, kinetin, kinetin riboside, gamma linolenic acid, lipoic acid, lutein, luteolin, lycopene, nordihydroguaiaretic acid, oleanolic acid, parthenolide, peonidin chloride, phloroglucinol carboxaldehyde, pinocembrin, pinostrobin, protocatechinic acid, pterostilbene, quercetin, rauwolscine, resveratrol, silibinin, silichristin, taxifolin, theaflavin, vanillyl acetone, trans-zeatin riboside, chamomile oil, parsley peel ethanol extract, sage peel ethanol extract, sour orange peel ethanol extract, tangerine peel ethanol extract and thyme peel ethanol extract. Treating hair ageing is through promoting hair fibre growth or reducing hair fibre loss.
The oral composition referred to in the second aspect of the invention may comprise at least one unit dose comprising a daily dose of 1-1000, preferably 1 -500, most preferably 10-300 mg nuclear factor erythroid-2 related factor 2 (Nrf2) agonist.
The composition of the second aspect of the invention may be a foodstuff selected from the group consisting of a beverage, a supplement, a soup, margarine, a ready-to-eat meal, a dressing, a mayonnaise, mustard, a tomato-based condiment, a sauce, a seasoning, yoghurt and a frozen confection.
In general terms, the composition may be in the form of a solid, a slurry, a solution, a suspension, a gel or an emulsion.
More specifically, the composition may be in the form of a beverage, in particular a fruit or tea based beverage.
The composition may be in the form of a supplement of one or more unit dosages such as capsules, cachets, lozenges, pills, tablets, caplets.
The composition may be a soup in dry, paste or liquid form.
The composition may be a seasoning sold as unit doses in the form of a powder, a compressed powder in the form of, for example, a cube, a liquid or suspension, or a gel.
The term "frozen confection" means a sweet-tasting fabricated foodstuff intended for consumption in the frozen state (i.e. under conditions wherein the temperature of the foodstuff is less than 0°C, and preferably under conditions wherein the foodstuff comprises significant amounts of ice). Frozen confections include ice cream, sorbet, sherbet, frozen yoghurt, water ice, milk ice and the like. Preferably the frozen confection has a total solids content (i.e. the sum of the weights of all the ingredients other than water, expressed as a percentage of the total weight) of at least 20 %, more preferably at least 25 %. Frozen confections may be aerated or unaerated. Preferably the frozen confection is aerated. The frozen confection may be manufactured by any suitable process, typically by preparing a mix of ingredients; then pasteurising and optionally homogenising the mix; and then freezing and optionally aerating the mix to produce the frozen confection.
EXAMPLE 1 : NADP(H):QUINONE OXIDOREDUCTASE (NQO-1) AND HEMOXYGENASE-1 (HO-1) ASSAYS OF SKIN KERATINOCYTES
Culture of Keratinocytes
Primary human epidermal keratinocytes, obtained from Clonetics, were routinely cultured and passaged in Clonetics KGM-2 calcium free bullet kit (CC3108) with 70 μΜ calcium chloride added. Cells were passaged by washing the cells gently with 4-(2- hydroxyethyl)-1 -piperazineethanesulfonic acid (HEPES) Buffered Saline Solution and trypsinised with 0.05 % trypsin ethylenediaminetetraacetic acid (EDTA) solution. When the cells detached they were neutralised with Trypsin Neutralising Solution to produce a cell suspension. Cells were plated in 12 well plates, at a seeding density of 20,000 cells per ml of growth medium and incubated at 37 °C, in 5 % CO2 until they were almost confluent, after approximately 48 hours incubation. Any change in cell morphology was noted after addition of test solutions.
Addition of test solutions
Test solutions were prepared in ethanol or dimethyl sulfoxide (DMSO) and added directly to the cells and treated for 24 hours. The cells were then pelleted.
Preparation of Total RNA from Epidermal keratinocytes
Epidermal keratinocytes were lysed using QIAzol lysis reagent and total RNA extracted using Qiagen RNeasy Mini or Micro Spin Columns. Total RNA concentration and purity were analysed using a Nanodrop 1000 (Thermo Scientific). The RNeasy MinElute Cleanup kit (Qiagen) was subsequently used to further concentrate the RNA samples if required. cDN A Synthesis
RNA was reverse transcribed into cDNA using a High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems). Briefly, 0.1 μg of total RNA was prepared in a total of 15 μΙ of ribonuclease (RNase) free water and added to an equal volume of 2X RT Master Mix (each reaction consisting of 3 μΙ 10X RT Buffer, 1.2 μΙ 25X deoxyribonucleotide triphosphate (dNTP) mix, 3 μΙ 10X RT Random Primers, 1.5 μΙ MultiScribe Reverse Transcriptase, 1.5 μΙ RNase Inhibitor, 4.8 μΙ RNase-free water). Samples were kept on ice until the reaction was initialized on a thermal cycler (MiniOpticon, BioRad) under the following conditions: 25 °C for 10 minutes, 37 °C for 2 hours and 85 °C for 5 minutes. Freshly transcribed cDNA was cooled on ice and stored at -20 °C until needed.
In addition, 1 μg of qPCR human reference total RNA (Stratagene) was reverse transcribed at the same time to provide standard curves for gene expression analysis.
Gene Expression
Gene expression of NQO-1 , HO-1 and 18S (housekeeper gene) were measured on an Eppendorf RealPlex qPCR machine using iQTMSYBR Green Supermix (BioRad) and QuantiTect Primers (Qiagen- NQO-1 and 18S). Briefly, cDNA was diluted 1 in 10 and 2 μΙ added to 23 μΙ of reaction mix (consisting of 12.5 μΙ IQ SYBR Green Supermix, 5 μΙ QuantiTect Primers and 5.5 μΙ RNase-free water) on ice. Samples were run in triplicate for each of the genes. The reverse transcribed qPCR human reference total RNA was used to generate Standard curve samples (20, 2, 0.2, 0.02 and 0.002 ng/ml) which were run in duplicate on every plate to allow multiple-plate analysis. The polymerase chain reactions were monitored in real time on an Eppendorf RealPlex qPCR machine under the following conditions: 95 °C for 5 minutes; 45 cycles of 95 °C for 10 seconds and 60 °C for 30 seconds; 95 °C for 1 minute. Unknown gene expression values were extrapolated from the standard curves, subsequently the NQO-1 values were normalised to the 18S housekeeper gene values.
Results
Results are presented in table 1 which shows HO-1 and NQO-1 gene expression (fold change compared to 18S) in primary epidermal keratinocytes treated with certain test compounds.
Table 1 : HO-1 and NQO-1 gene expression (fold change compared to 18S) of keratinocytes treated with test compounds (n = 3) versus a vehicle control (n = 3). All P- values relate to significance to vehicle control. P < 0.05 at 95 % confidence limits unless otherwise stated (NS = not significant).
HO-1 NQO-1
Fold (x18S) Fold (x18S)
Vehicle 1.1 ±0.18 1.0±0.07
2 μΜ sulforaphane 5.6±0.83 3.8±0.52
5 μΜ resveratrol 4.0±0 1.8±0.07
10 μΜ resveratrol 6.9±1.4 2.4±0.25
2 μΜ curcumin 3.4±0.59 2.1 ±0.36
4 μΜ curcumin 7.5±0.87 2.8±0.1 1
Vehicle 0.96±0.13 0.84±0.14
2 μΜ sulforaphane 7.0±0.97 4.2±0.72
5 μΜ pterostilbene 141 .8±25.1 5.4±0.22
10 μΜ pterostilbene 104.1 ±7.2 5.5±0.22
5 μΜ lycopene 1.8±0.25 1.6±0.1 1
10 μΜ lycopene 5.3±0.73 2.5±0
Vehicle 0.98±0.03 1.2±0.29
5 μΜ docosahexaenoic acid 6.4±0.54 3.2±0.21
10 μΜ docosahexaenoic acid 42.7±6.64 4.3±0.49
5 μΜ gamma linolenic acid 1.6±0.09 2.4±0.15
10 μΜ gamma linolenic acid 12.1 ±0.69 4.8±0.41 Vehicle 1.0±0.04 0.84±0.14
2.5 μΜ allyl isothiocyanate 1 .63±0.44 (NS) 1.7 +0.36
5 μΜ allyl isothiocyanate 2.3±0.48 2±0.2
Vehicle 0.99±0.17 1.0±0.24
2 μΜ sulforaphane 3.2±0.13 4.2±0.17
5 μΜ apigenin 0.19±0.02 (NS) 0.62±0 (NS)
10 μΜ apigenin 0.12±0.02 (NS) 00.61 ±0.09 (NS)
5 μΜ kaempferol 0.97±0.23 (NS) 1 .4±0.29 (NS)
10 μΜ kaempferol 0.98±0.10 (NS) 1.3±0.2 (NS)
10 μΜ oleanolic acid 0.86±0.13 (NS) 0.82±0.11 (NS)
20 μΜ oleanolic acid 3.2± 0.13 0.68±0.05 (NS)
Conclusion
Upregulation of gene expression of one or both of the detoxification enzymes (HO-1 and/or NQO-1 ) in the assay is indicative of Nrf2 activation and thus sulforaphane, resveratrol, curcumin, pterostilbene, lycopene, docosahexaenoic acid, gamma linolenic acid, allyl isothiocyanates and oleanolic acid are Nrf2 agonists.
EXAMPLE 2: NADP(H):QUININE OXIDOREDUCTASE (NQ01) AND HEMOXYGENASE-1 (HO-1) ASSAYS OF DERMAL FIBROBLASTS
Culture of dermal fibroblast cells
Primary human dermal fibroblast cells were obtained from Cell Research Corporation and cultured and passaged in Dulbecco's Modified Eagle Serum (DMEM) (Gibco) supplemented with 10 % foetal bovine serum (FBS). Cells were routinely plated out in 6- well tissue culture dishes, at a seeding density of -5000 cells/cm2 in 2 ml complete medium/well for 24 hours, and incubated at 37 °C in 5 % CO2.
Addition of test solutions
Test solutions were prepared in ethanol or DMSO and added directly to the cells and treated for 24 hours. The test solutions were prepared with selected concentrations of test material as set forth in Table 2. The cells were then pelleted. Preparation of cell lysate
All cell pellets were lysed on ice for 30 minutes in 1 ml cell lysis buffer per 2.5 x 106 cells. The lysis buffer contained 1 % NP-40, 0.1 % sodium deoxycholate, 0.1 % SDS, 6 mM sodium chloride and 0.05 M Tris at pH 7.6. Protease inhibitor cocktail (1000X; Sigma P8340) was added prior to use at a level of 10 μΙ per ml of lysis buffer. The clarified cell lysate was frozen at -80 °C until needed.
Total protein assay (Pierce)
The total protein concentration of each cell lysate was measured using the Pierce BCA protein assay kit. A set of eight standard solutions ranging from 0 to 1200 μg/ml protein was prepared from the supplied 2 mg/ml BSA stock solution. 10 μΙ of standard or cell lysate was added to duplicate wells of a flat-bottomed, 96-well microtitre plate. The reagent solution was prepared according to the kit instructions from 50 parts reagent A and 1 part reagent B. 200 μΙ of the final reagent was added to each well of the microtitre plate. The plate was mixed, covered and incubated at 37 °C for 30 minutes and absorbance read at 562 nm. A protein standard curve was constructed and used to determine the protein concentration of each cell lysate.
NQO-1 mRNA assay
The NAD(P)H: quinone oxidoreductase-1 (NQO-1 ) protein concentration of each cell lysate was assayed using the AdipoGen™ NQO-1 , human, intracellular, twin plex ELISA kit (Adipogen™) according to the manufacturer's instructions.
Eight NQO-1 standards were prepared in reagent diluent (0.5% Trition-X100 and 1 mM EDTA in phosphate buffered saline at pH 7.2) at concentrations ranging from 0.313 to 20 ng/ml, including a negative control. 100 μΙ of cell lysate, diluted 1/2, 1/5 or 1/10 or standard was added to duplicate wells of the assay plate which was pre-coated with the specific NQO-1 capture antibody by the manufacturer. The plate was incubated at before being washed three times with wash buffer. 100 μΙ of NQO-1 ready to use detection antibody was added to each well and the plate incubated at 37 °C for 1 hour. The plate was washed as before. 100 μΙ of 1/100 diluted detector was added to each well and incubated for 1 hour at 37 °C. The plate was washed five times and then 100 μΙ of 3,3',5,5'-tetramethylbenzidine (TMB) substrate solution was added to each well and incubated, in the dark at room temperature for 20 minutes. 100 μΙ of stop solution was added to each well and the plate read on a microplate reader (Dynex MRX) at 450 nm with wavelength correction set at 540 nm within 30 minutes.
A standard curve was plotted of mean OD versus NQO-1 concentration and the line of best fit calculated by regression analysis. The unknown concentration of NQO-1 protein in all the samples was calculated from this.
The results were normalised using the total protein data and expressed as pg NQO-1 per μg protein and are presented as percentage change in NQO-1 compared to the vehicle control value. HO-1 ELISA (R & D Systems)
The hemeoxygenase-1 (HO-1 ) protein concentration of each cell lysate was assayed using the DuoSet Human Total HO-1/HMOX1 assay (R&D Systems DYC3776) according to the manufacturer's instructions. Eight HO-1 standards were prepared in reagent diluent (0.5 % Trition-X100 and 1 mM EDTA in PBS at pH 7.2) at concentrations ranging from 0.15625 to 10 ng/ml, including a negative control. The HO-1 capture antibody was diluted to a concentration of 8 μg/ml in PBS and was bound to the microtitre plate (Greiner Bio-One) overnight at room temperature. The unbound antibody was then removed by washing 3 times with wash buffer (0.05 % Tween 20 in PBS) on an automatic plate washer. The plate was blocked with 300 μΙ per well of 1 % bovine serum albumin (BSA) in PBS for 1 hour and washed 3 times in wash buffer. 100 μΙ of cell lysate, diluted 1/5, or standard was added to duplicate wells. The plate was incubated at room temperature for 2 hours before being washed three times with wash buffer. 100 μΙ of HO-1 detection antibody, diluted to a concentration of 200 ng/ml, was added to each well and the plate incubated at room temperature for 2 hours. The plate was washed as before. 100 μΙ of 1/200 diluted streptavidin horseradish peroxidase (HRP) was added to each well and incubated in the dark for 20 minutes at room temperature. The plate was washed as before and then 100 μΙ of substrate solution (R&D Systems DY999, 1 : 1 mixture of Colour Reagent A and Colour Reagent B) was added to each well and incubated, in the dark at room temperature, until colour developed (approx 20 minutes). 50 μΙ of stop solution (2M H2SO4) was applied to each well and the plate read on a microplate reader (Dynex MRX) at 450 nm with wavelength correction set at 540 nm.
A standard curve was plotted of mean OD versus HO-1 concentration and the line of best fit calculated by regression analysis. The unknown concentration of HO-1 protein in all the samples was calculated from this.
The results were normalised using the total protein data and expressed as ng HO-1 per μg protein and are presented as percentage change in HO-1 compared to the vehicle control value.
Results
Results are presented in table 2 which summarises levels of HO-1 and NQO-1 protein synthesis (ng HO-1 or NQO-1 per μg protein as a % of vehicle control) in dermal fibroblasts treated with certain test compounds.
Table 2: HO-1 and NQO-1 protein (ng HO-1 or NQO-1 per μg protein as a % of vehicle control) in dermal fibroblasts treated with certain test compounds (n = 3). All P-values relate to significance to vehicle control. P < 0.05 at 95 % confidence limits unless otherwise stated (NS = not significant). ng HO-1 per μg protein as a ng NQO-1 per μg protein as a
% of vehicle control % of vehicle control
10 μΜ andrographolide 162±5.6
10 μΜ apigenin 106±1 1.9(NS) 123±7.7
10 μΜ asiatic acid 122±5.1
10 μΜ baicalein 115±10.7 25 μΜ catechin - 103±6.1 (NS)
20 μΜ chrysin 103±10.8(NS) 115±10.3
10 μΜ chrysin dimethylether - 141 ±8
1 μΜ curcumin - 132±8.7
10 μΜ docosahexeanoic acid - 123±9.6
25 μΜ epigallocatechin gallate 104±7.3 106±5.5 (NS)
10 μΜ esculetin - 164±5.7
1 μΜ ferulic acid 13±4.4 (NS) 0±9.4 (NS)
10 μΜ fraxetin - 142±8.4
25 μΜ Galangin - 112±7.2 (NS)
10 μΜ genistein - 113±4.3
25 μΜ guaiacol 110±13.5 110±4.7
5 μΜ hesperetin - 106±7.2 (NS)
25 μΜ 4-hydroxy-3- 108±9.5 104±2.8 (NS) methoxyacetophenone
10 μΜ (+/-)-jasmonic acid - 113±5.2
10 μΜ kaempferol 101 ±17.2 114±8.4
10 μΜ (S)-(+)-ketoprofen - 113±4.9
10 μΜ kinetin - 109±5 (NS)
10 μΜ kinetin riboside - 119±8.6
50 μΜ lipoic acid 118±13.7
10 μΜ lutein - 141 ±23.8
5 μΜ luteolin 102±9.8 110±7.4 (NS)
10 μΜ nordihydroguaiaretic acid - 141 ±5.1 (NDGA)
10 μΜ parthenolide - 122±4.8
25 μΜ pelargonidin chloride 117±15.3 (NS)
20 μΜ peonidin chloride 1 1 ±10.3
10 μΜ phloroglucinol carboxaldehyde 108±4.8
20 μΜ pinocembrin - 106±16.1 (NS)
10 μΜ pinostrobin 129±10.3 106±4.6 (NS)
10 μΜ protocatechinic acid 107±23.6
10 μΜ quercetin 112±9.7
20 μΜ rauwolscine ± 131 ±19.7
1 μΜ resveratrol 143±22.9
1 μΜ silibinin - 111 ±9.5 (NS)
1 μΜ silichristin - 112±8.9 (NS) 1 μΜ sulforaphane 141 ±10
1 μΜ taxifolin 113±9.6 (NS)
25 μΜ theaflavin 111 ±8.6 (NS)
25 μΜ vanillyl acetone 113±10 (NS) 110±1.7
10 μΜ trans-zeatin riboside 125±5.3
100 μς/ηηΙ chamomile oil (Sigma 105±6.1 133±16.7
Aldrich)
100 μς/ιηΙ parsley ethanol extract - 113±8.4
50 g/ml sage ethanol extract - 106±6.4 (NS)
1 μg/g sour orange peel ethanol extract 126±14
(KGK Synergize Inc)
1 μg ml tangerine peel ethanol extract 153±7.8
(KGK Synergize Inc)
100 μg/ml thyme ethanol extract - 106±8.3
Conclusion
HO-1 and/or NQO-1 synthesis is significantly up-regulated in dermal fibroblasts by andrographolide, apigenin, Asiatic acid, baicalein, catechin, chrysin, chrysin dimethylether, curcumin, docosahexaenoic acid, epigallocatechin gallate, esculetin, fraxetin, galangin, genistein, guaiacol, hesperetin, 4-hydroxy-3-methoxyacetophenone, jasmonic acid, kaempferol, (S)-(+)-ketoprofen, kinetin, kinetin riboside, lipoic acid, lutein, luteolin, nordihydroguaiaretic acid, parthenolide, peonidin chloride, phloroglucinol carboxaldehyde, pinocembrin, pinostrobin, protocatechinic acid, quercetin, rauwolscine, resveratrol, silibinin, silichristin, sulforaphane, taxifolin, theaflavin, vanillyl acetone, trans-zeatin riboside, chamomile oil, parsley ethanol extract, sage ethanol extract, sour orange peel ethanol extract, tangerine peel ethanol extract and thyme ethanol extract, and thus all the foregoing compounds and extracts are Nrf2 agonists. EXAMPLE 3: HAIR GROWTH
Isolated human hair follicle culture model
Temporal or occipital human scalp skin was obtained from subjects undergoing face-lift or hair transplant surgery with informed consent and ethics approval. Anagen VI hair follicles were isolated and maintained according to the method described in Philpott et al (J. Cell Sci 97, 463-471 (1990)). Dhurat et al (Int J Trichology, 2(2), 96-100 (Jul-Dec 2010)) describe that anagen is conventionally divided into six stages, Anagen l-V (proanagen), when the hair shaft grows within the hair follicle, and Anagen VI (metanagen), when the tip of the hair shaft emerges from the hair follicle.
In order to evaluate the effect of Nrf-2 agonists on hair follicles, test groups (n>7 hair follicles) received Nrf2 agonist daily for 6 days and control groups (n>7 hair follicles) received vehicle alone. The culture medium was replenished every other day and the hair shaft length was measured using an inverted binocular microscope as described in Philpot et al. The vehicle was ethanol.
Results
Results are presented in table 3 which shows that there is a significant increase in hair growth in the human hair follicle model versus the vehicle control when the human hair follicles are treated with 10 μΜ sulforaphane. This is illustrated for 10 μΜ sulforaphane in figure 1.
Table 3: Human hair shaft length (mm) over time (hours) after treatment with either 5 or 10 μΜ sulforaphane versus a vehicle control (n = 7). P-values relates to significance of sulforaphane to vehicle control at 95 % confidence limits.
Follicle elongation (mm)
Time (hours) 0 24 48 72 96 120 144
Vehicle 0 - 0.99±0.14 1.22±0.14 1.50±0.16 1.74±0.16
5 μΜ sulforaphane 0 - 1.07±0.07 1.31 ±0.09 1.57±0.13 1.85±0.16
P-value 1.00 - 0.15 0.16 0.31 0.18
Vehicle 0 0.28±0.07 - 0.85±0.18 1.03±0.22 1.19±0.28
10 μΜ sulforaphane 0 0.33±0.06 - 1.22±0.14 1.44±0.14 1.60±0.17
P-value 1.00 0.20 - 0.00 0.00 0.01
Vehicle 0 - 0.55±0.20 0.79±0.30 1.00±0.38 1.23±0.46 1.42±0.50 10 μΜ sulforaphane 0 - 0.89±0.09 1.21 ±0.16 1.51 ±0.19 1.75±0.28 1.92±0.37
P-value 1.00 - 0.0014** 0.006** 0.006** 0.018* 0.044*
Conclusion
Sulforaphane at 10 μΜ significantly up-regulates human hair follicle growth compared to vehicle control.
EXAMPLE 4: NADP(H): QUINONE OXI DO REDUCTASE (NQO-1) AND HEMOXYGENASE-1 (HO-1) GENE EXPRESSION ASSAYS OF HAIR FOLLICLES CELLS
Human hair follicle isolation
Temporal or occipital human scalp skin was obtained from subjects undergoing face-lift or hair transplant surgery with informed consent and ethics approval. Anagen VI hair follicles were isolated and maintained according to the method described in Philpott et al (J. Cell Sci 97, 463-471 (1990)). Dhurat et al (Int J Trichology, 2(2), 96-100 (Jul-Dec 2010)) describe that anagen is conventionally divided into six stages, Anagen l-V (proanagen), when the hair shaft grows within the hair follicle, and Anagen VI (metanagen), when the tip of the hair shaft emerges from the hair follicle. Hair follicles were stabilised in RNAIater (for PCR). Five individual Anagen VI hair follicles were used for each culture condition/patient. In order to evaluate the effect of Nrf2 agonists on hair follicles, test groups (n=4 donor subjects) received Nrf2 agonist (sulforaphane or tert-butyl hydroxyquinone (tBHQ)) for 24 hours and control groups (n=4 donor subjects) received dimethyl sulfoxide vehicle alone. Preparation of total RNA from hair follicles
RNA was extracted using the Qiagen RNeasy Microkit (maximum 5 mg tissue per extraction). cDN A synthesis
100 ng RNA was reverse transcribed using the Invitrogen Cloned AMV First Strand Synthesis kit, according to manufacturers' instructions. TaqMan primer sets for HO-1 (Hs01 1 10250_m1 ), NQO-1 (Hs00168547_m1 ) and Nrf2 (Hs00232352_m1 ) were obtained from the Invitrogen inventoried assays (Life technologies Ltd.).
Gene expression analysis
cDNA was diluted 1 : 10 in RNase free water. Each reaction consisted of 1 μΙ_ 20x TaqMan assay, 10 μΙ_ Taqman Fast Advance Mastermix, 5 μΙ_ RNase free water and 2 ng of cDNA. Plates were run on an Applied Biosystems StepOnePlus™ Real Time PCR machine. Fast cycling conditions were used, utilising a 2 minute polymerase activation step at 95°C, followed by 45 cycles of denaturing at 95°C for 1 second, and annealing/extension at 60°C for 20 seconds. All PCR reactions were normalised to the housekeeping control PPIA, which showed no variation between treatments. Data was analysed by the AACt methodology
Results
Results are presented in table 4 which shows NQO-1 and HO-1 gene expression (fold change compared to 18S) in hair follicles treated with sulforaphane or tBHQ.
Table 4: NQO-1 and HO-1 gene expression (fold change compared to 18S) of hair follicle cells after 24 hour treatment with either 2, 5 or 20 μΜ sulforaphane or 20 μΜ tert- butyl hydroxyquinone (tBHQ) versus a vehicle control (n = 4). P-values relates to significance of sulforaphane or tBHQ to vehicle control at 95 % confidence limits (NS=not significant, i.e. P > 0.05).
NQO-1 HO-1
Fold change compared to 18S Fold change compared to 18S
Vehicle 1 ±0.11 1 ±0.07
2 μΜ sulforaphane 4.05±1 .83 0.98±0.34
P-value NS NS
5 μΜ sulforaphane 8.93±1 ,75 0.84±0.35 P-value 0.024* NS
20 μΜ sulforaphane 20.79±14.37 0.86±0.20
P-value 0.026* NS
Vehicle 1 ±0.11 1 ±0.07
20 μΜ tBHQ 39.6±32.58 4.93±0.35
P-value 0.04* 0.004:
Conclusion
Sulforaphane and tBHQ significantly up-regulate NQO-1 and HO-1 gene expression in hair follicles and thus both compounds are Nrf2 agonists.
EXAMPLE 5: IMMUNOHISTOCHEMICAL ANALYSIS OF NADP(H): QUINONE OXIDOREDUCTASE (NQO-1) AND HEMOXYGENASE-1 (HO-1) IN HAIR FOLLICLES Human hair follicle isolation
Temporal or occipital human scalp skin was obtained from subjects undergoing face-lift or hair transplant surgery with informed consent and ethics approval. Anagen VI hair follicles were isolated and maintained according to the method described in Philpott et al (J. Cell Sci 97, 463-471 (1990)). Dhurat et al (Int J Trichology, 2(2), 96-100 (Jul-Dec 2010)) describe that anagen is conventionally divided into six stages, Anagen l-V (proanagen), when the hair shaft grows within the hair follicle, and Anagen VI (metanagen), when the tip of the hair shaft emerges from the hair follicle. Hair follicles were stabilised in RNAIater (for PCR). Five individual Anagen VI hair follicles were used for each culture condition/patient.
In order to evaluate the effect of Nrf-2 agonists on hair follicles, test groups (n=4 hair follicles) received Nrf2 agonist for 24 hours and control groups (n=4 hair follicles) received dimethyl sulfoxide (DMSO) vehicle alone.
6 μηη cryosections were stained for HO-1 and NQO-1 using mouse monoclonal antibodies (Abeam ab13248 and ab28947 (Clone A180) respectively). Indirect detection was performed using a secondary goat anti-mouse alexa fluor 488 by strepavidin- conjugated fluorescein (FITC).
Immunohistochemical analysis
Immunolocalisation and intensity analyses were performed with the Biozero-8000 microscope (Keyence) and analysed using Image J software (National Institute of Health). Immunofluorescent images were taken using the same exposure time when using intensity of fluorescent stain for quantitative analyses. To compare staining intensity and positive cell numbers, the mean values from multiple cryosections from each patient were taken to allow for variation between the sections. Reference areas of a standard size were selected, encompassing the epithelial cells or mesenchymal connective tissue sheath, within which fluorescence intensity was determined using Image J software. These reference areas were the same for each cryosection/hair follicle/patient sample analysed.
Results
HO-1 immunoreactivity was not observed in vehicle control (dimethyl sulfoxide) treated follicles in either the epithelial hair follicle or the mesenchymal connective tissue sheath (CTS). Upon treatment with the highest concentration of sulforaphane (20 μΜ) a significant increase in HO-1 immunofluorescence was seen throughout the CTS, with no change in expression in the epithelial cells.
NQO-1 immunoreactivity was detected in both the inner and outer root sheath in the vehicle control (dimethyl sulfoxide) treated follicles. Upon treatment with 5 μΜ or 20 μΜ sulforaphane, a significant increase in NQO-1 immunofluorescence was seen in the epithelial cells of the isolated hair follicles.
The results are summarised in Tables 5 and 6. Table 5: NQO-1 fluorescence levels (arbitrary units) of hair follicles (epithelial cells) after 24 hours of treatment with 2 μΜ, 5 μΜ, and 20 μΜ sulforaphane with standard deviation and P-values (95 % confidence limits).
Fluorescence (arbitrary units)
2 μΜ
Ctrl sulforaphane 5 μΜ sulforaphane 20 μΜ sulforaphane
NQO-1 (epithelial 8.4±3. 15.4 ± 1 .1 18.8 ± 1 .4 cells) 1 9.0 ± 4.6 (*p=0.011 ) (**p=0.003)
Table 6: HO-1 fluorescence levels (arbitary units) of hair follicles after 24 hours of treatment with 2 μΜ, 5 μΜ, and 20 μΜ sulforaphane with standard deviation and P- values (95 % confidence limits).
Fluorescence (arbitrary units)
2μ M 5 μΜ
Control sulforaphane sulforaphane 20 μΜ sulforaphane
HO-1 (epithelial
cells) 2.9 ± 1 .4 2.8 ± 1 .2 3.2 ± 1.5 3.7 ± 1.6
HO-1 (CTS) 1.9 ± 0.8 1.9 ± 0.7 2.4 ± 0.2 8.5 ± 1.2 (**p=0.005) Conclusions
Treatment of hair follicles with sulforaphane leads to up-regulation of HO-1 and NQO-1.

Claims

1. A method of treating hair ageing comprising the step of applying a composition to hair fibres and/or scalp, the composition comprising a nuclear factor erythroid-2 related factor 2 (Nrf2) agonist;
wherein the Nrf2 agonist is selected from the group consisting of andrographolide, apigenin, Asiatic acid, baicalein, tert-butyl hydroxyquinone, catechin, chrysin, chrysin dimethylether, curcumin, docosahexaenoic acid, epigallocatechin gallate, esculetin, fraxetin, galangin, genistein, guaiacol, hesperetin, 4-hydroxy-3-methoxyacetophenone, jasmonic acid, kaempferol, (S)- (+)-ketoprofen, kinetin, kinetin riboside, gamma linolenic acid, lipoic acid, lutein, luteolin, lycopene, nordihydroguaiaretic acid, oleanolic acid, parthenolide, peonidin chloride, phloroglucinol carboxaldehyde, pinocembrin, pinostrobin, protocatechinic acid, pterostilbene, quercetin, rauwolscine, resveratrol, silibinin, silichristin, taxifolin, theaflavin, vanillyl acetone, trans-zeatin riboside, chamomile oil, parsley peel ethanol extract, sage peel ethanol extract, sour orange peel ethanol extract, tangerine peel ethanol extract and thyme peel ethanol extract.
2. A method of treating hair ageing comprising the step of imbibing a composition, the composition comprising a nuclear factor erythroid-2 related factor 2 (Nrf2) agonist;
wherein the Nrf2 agonist is selected from the group consisting of andrographolide, apigenin, Asiatic acid, baicalein, tert-butyl hydroxyquinone, catechin, chrysin, chrysin dimethylether, curcumin, docosahexaenoic acid, epigallocatechin gallate, esculetin, fraxetin, galangin, genistein, guaiacol, hesperetin, 4-hydroxy-3-methoxyacetophenone, jasmonic acid, kaempferol, (S)- (+)-ketoprofen, kinetin, kinetin riboside, gamma linolenic acid, lipoic acid, lutein, luteolin, lycopene, nordihydroguaiaretic acid, oleanolic acid, parthenolide, peonidin chloride, phloroglucinol carboxaldehyde, pinocembrin, pinostrobin, protocatechinic acid, pterostilbene, quercetin, rauwolscine, resveratrol, silibinin, silichristin, taxifolin, theaflavin, vanillyl acetone, trans-zeatin riboside, chamomile oil, parsley peel ethanol extract, sage peel ethanol extract, sour orange peel ethanol extract, tangerine peel ethanol extract and thyme peel ethanol extract.
A method according to claim 1 or claim 2 wherein the Nrf2 agonist is selected from the group consisting of apigenin, Asiatic acid, catechin, curcumin, docosahexaenoic acid, epigallocatechin gallate, esculetin, genistein, hesperetin, kaempferol, lipoic acid, lutein, luteolin, lycopene, pinocembrin, pinostrobin, quercetin, resveratrol, theaflavin, vanillyl acetone, chamomile oil, sour orange peel ethanol extract and tangerine peel ethanol extract.
A method according to claim 1 or claim 3 wherein the composition comprises 0.00001 -1 , preferably 0.001 -1 , most preferably 0.0001 -0.1 % w/w nuclear factor erythroid-2 related factor 2 (Nrf2) agonist.
A method according to any one of claims 1 , 3 and 4 wherein the composition is selected from the group consisting of a shampoo, a conditioner, a leave-on product, a wash-off product.
A method according to claim 2 or claim 3 wherein the composition comprises at least one unit dose comprising a daily dose of 1 -1000, preferably 1 -500, most preferably 10-300 mg nuclear factor erythroid-2 related factor 2 (Nrf2) agonist.
A method according to any one of claims 2, 3 and 6 wherein the composition is a foodstuff selected from the group consisting of a beverage, a supplement, a soup, margarine, a ready-to-eat meal, a dressing, a mayonnaise, mustard, a tomato-based condiment, a sauce, a seasoning, yoghurt and a frozen confection.
A method according to claim 7 wherein the composition is in the form of a supplement of one or more unit dosages such as capsules, cachets, lozenges, pills, tablets, caplets.
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