WO2012080747A2

WO2012080747A2 - Anti-ageing agents

Info

Publication number: WO2012080747A2
Application number: PCT/GB2011/052498
Authority: WO
Inventors: Abigail Kate Langton; Christopher Ernest Maitland Griffiths; Michael John Sherratt; Rachel Elizabeth Beatrice Watson; Neil Kerslake Gibbs
Original assignee: The University Of Manchester
Priority date: 2010-12-17
Filing date: 2011-12-16
Publication date: 2012-06-21
Also published as: WO2012080747A3; GB2500549A; GB201312511D0; GB201021457D0

Abstract

This invention relates to a method for identifying an anti-ageing agent for use in skincare compositions, by determining the ability of a material or a combination of materials to modulate the expression of molecules associated with microfibril or elastic fibres. More specifically, the invention relates to a method for identifying an anti-ageing agent by determining the ability of a material or combination of materials to downregulate LOXL-1 expression. The invention also relates to methods for assessing the anti-ageing effect of a material or combination of materials on the skin, anti-ageing agents identified by such methods, and compositions containing them.

Description

Title - Anti-ageing agents Field of the invention The invention relates to a method for identifying an anti-ageing agent for use in skincare compositions, by determining the ability of a material or combination of materials to modulate the expression of molecules associated with microfibril or elastic fibres. More specifically, the invention relates to a method for identifying an anti-ageing agent by determining the ability of a material or combination of materials to modulate LOXL-1 expression. The invention also relates to methods for assessing the anti-ageing effect of a material or combination of materials on the skin, anti-ageing agents identified by such methods, and compositions containing them. Background of the invention

Cutaneous ageing is the result of two distinct, biological processes which may occur concurrently: i) the passage of time, termed intrinsic ageing, and ii) environmental influences, termed extrinsic ageing.

Anti-ageing agents are commonly used in skincare compositions to reduce or prevent the signs of extrinsic ageing. In particular, anti-ageing agents are included to protect the skin against the adverse effects of free radicals, eg effects mediated by UV-radiation, or other sources of oxidative stress. Such agents include sunscreening agents and antioxidant agents.

It would clearly be advantageous to provide an anti -ageing agent that targets intrinsic ageing to improve the condition and aesthetic appearance of skin, whether or not it has been exposed to environmental stress. It would also be advantageous to use anti-ageing agents that target intrinsic ageing in combination with current agents that reduce the signs of extrinsic ageing, to improve the overall anti-ageing benefit afforded by current skincare compositions. The clinical features of intrinsically aged skin are not usually evident until old age when, although smooth and unblemished, the skin surface appears pale and is characterised by fine wrinkles. Functionally, intrinsically aged skin is dry and less elastic than more youthful skin. The dermal elastic fibre network is responsible for providing the skin with its recoil capacity and loss of this property contributes to an aged appearance. Dermal elastic fibres have a complex biology due to their multiple components yet the effect of intrinsic ageing on all such molecules has not previously been explored. The present inventors have used an Affymetrix^® microarray-based approach to detect age-related changes in gene expression of molecules known to contribute to the elastic fibre network. Samples of photoprotected skin from young (18-30 years) and old (65-75 years) individuals were analysed. A comparison of the gene expression profiles from the young and old skin samples revealed that the majority of elastic fibre network components were unchanged with age. However, a gene was identified which showed significant differential expression. Given that intrinsic ageing of the skin is a slow and subtle process it is surprising that a significant alteration in expression level has been identified for this gene. This finding indicates that the identified gene and its expression product impact greatly on the organisation of elastic fibres in intrinsically aged skin. Thus, expression of this gene can be used to indicate the efficacy of a material or combination of materials as an anti-ageing agent.

The gene the inventors have identified is that which encodes LOXL-1 (Lysyl oxidase-like 1 ). The term "LOXL-1 " is used herein to denote, as the context requires, either the LOXL-1 enzyme or the gene that encodes it. Expression of LOXL-1 was found to be significantly increased in old skin compared to young skin. Thus it appears that the level of LOXL-1 in our skin increases as part of the intrinsic ageing process. Materials that are able to delay or prevent this natural increase in LOXL-1 necessarily hinder the intrinsic ageing process and therefore act as anti-ageing agents. Brief summary of the invention

According to a first aspect of the invention, there is provided a method for identifying an anti-ageing agent for use in skincare compositions, which method comprises determination of the ability of a material or combination of materials to decrease the expression of LOXL-1 .

According to a second aspect of the invention, there is provided a method for evaluating the anti-ageing effect of a material or combination of materials by assessing the ability of the material or combination of materials to downregulate the expression of LOXL-1 .

By a "material" is meant in the context of the present invention any single compound, or any mixture or extract. Preferred materials are commercially available cosmetic ingredients.

Due to the surprising identification in the differential expression of LOXL-1 in young compared to old skin, the inventors have discovered that measurement of LOXL-1 expression may be used to assess the potential usefulness of a material or combination of materials as an anti-ageing agent, and for screening large numbers of materials, both on their own and as combinations, for their anti -ageing effect.

It is relatively easy to determine the expression of a gene or gene product (ie protein) and methods for doing so are known in the art. Therefore, the present invention is advantageous because, due to the identification of LOXL-1 as a marker, the anti-ageing potential of a material may be determined quickly and easily. As a result, the invention provides useful methods for screening large numbers of materials, both on their own and as combinations, for their anti-ageing effect. Various methods are known for measuring gene or protein expression, including both in vitro and in vivo methods. The employment of both in vitro and in vivo testing enables the user to screen large numbers of materials for the desired effect in vitro before progressing to testing in vivo. This process means that unpromising agents can be eliminated from the search early, thereby saving time and resources.

The anti-ageing agents of the present invention are particularly advantageous because they target the intrinsic ageing process, and in particular the dermal elastic fibre network, which provides skin with its recoil capacity. In terms of visible signs, effects may be seen on the elasticity of the skin, its moisture balance and texture, and by the reduction or prevention of the appearance of fine lines and wrinkles.

Because they target the intrinsic ageing process, anti-ageing agents of the present invention may benefit those people who already protect their skin from

environmental factors, such as UV light, and who would consequently gain little benefit from the use of anti-ageing products which target extrinsic ageing. In addition, by targeting intrinsic ageing, the use of agents identified by the methods detailed here will benefit those areas of skin which are not normally exposed to environmental factors, for example areas of the body that are covered by clothes and therefore not exposed to sunlight. The intrinsic anti-ageing agents identified by this method can also be used in combination with anti-ageing agents that target extrinsic ageing affects, for example with sunscreening agents and antioxidants. As complementary processes, this would allow the consumer to derive the benefit from both the intrinsic and extrinsic anti-ageing agents.

The inventors have found that there is a smaller amount of LOXL-1 present in young skin compared to old skin, and hence that expression of LOXL-1 increases with age. Materials or combinations of materials that downregulate the expression of LOXL-1 may therefore be useful as anti-ageing agents.

Thus, according to a further aspect of the invention, there is provided a material or combination of materials which decreases the expression of LOXL-1 for use as an anti-ageing agent in a skincare composition. The expression of the LOXL-1 gene may be determined by measuring the amount of corresponding mRNA. However, biological relevance of the LOXL-1 gene expression should be additionally evaluated by measurement of the amount of the LOXL-1 protein, and the activity of the LOXL-1 enzyme.

Evaluation of protein expression may be carried out by any suitable method known in the art. Typically, in vitro methods are carried out on cultured cells

(keratinocytes). Protein expression can be determined by assaying the target proteins released from the cells into the growth media, for example using western blotting techniques.

Alternatively, the test material may be applied to the skin in vivo, in which case gene expression studies are most conveniently carried out on biopsied tissue samples. In preferred methods, biopsied tissue is frozen prior to

immunohistochemical analysis. Target proteins may be identified using

labeled-antibodies, in particular dye-labelled antibodies. Following incubation of the tissue sample with the appropriate unlabelled antibody, a dye-labelled secondary antibody is used to detect the presence of the first. Degree of antibody staining may be used to provide an indirect measure of protein expression.

The anti-ageing agents of the present invention are intended for use in skincare compositions. By "skincare composition" is meant in the context of the present invention a composition intended to benefit the skin. Skincare compositions may be oral compositions, eg food supplements. However, they are typically topical compositions, which are applied directly to the skin.

The anti-ageing agents of the present invention can be delivered to the skin via any of the conventional formulations known to those skilled in the art. The anti- ageing agents of the present invention are primarily intended for use in topical compositions, and in particular for use in topical compositions marketed as moisturisers, cleansers, toners, masks or scrubs etc, for the face or body.

The compositions may be "cosmetic" compositions that provide skincare benefits in addition to their principal cosmetic effect. Cosmetic skincare formulations may be marketed, for example, as foundations, powders, lipsticks, eyeliners,

eyeshadows, blushers, concealers etc.

Skincare compositions provided in accordance with the present invention comprise an anti-ageing ingredient identified in accordance with the present invention and a pharmaceutically acceptable carrier therefor. Typically, said anti-ageing agent may be present in an amount of 0.0001 -10% (w/w). Optionally, the composition may include additional active ingredients. The carrier may form the remainder of the composition up to 100% w/w. Compositions according to the present invention may be formulated in numerous forms for topical application known to those skilled in the art. The composition may often take the form of an aqueous or oily solution or dispersion or emulsion or a gel, including creams, serums, pastes, lotions, milks, ointments, salves, sticks, spray, roll-on, powders, aqueous gels, suspension dispersions and emulsions. Preferably, any solvent system comprises water. An emulsion may be an oil-in-water emulsion, a water-in-oil emulsion, an

oil-in-water-in-oil emulsion or a water-in-oil-in-water emulsion. Preferred emulsions are oil-in-water emulsions.

Additional skincare actives known to have benefit on the skin may also be added. Examples include anti-acne actives, vitamins, anti-fungals, skin conditioners, organic or inorganic sunscreens, plant extracts and the active principle from these, skin identical lipids such as ceramides, moisturisers and other materials to promote the repair of the skin barrier such as hyaluronic acid and other suitable materials that are known to those skilled in the art. If desired, further anti-wrinkle or anti-ageing actives may be included, such as peptides and promoters of collagen and/or elastin production, hydroxy acids, anti -oxidants, anti-inflammatories and anti-microbials.

The composition may additionally comprise other conventional topical skincare ingredients known to those skilled in the art. These include, but are not limited to, one or more of the following: emollients, humectants or moisturizers, surfactants, thickeners/viscosity modifying agents, emulsifiers and/or emulsion stabilizers, pH adjusters, perfumes and/or colourings. Such topical ingredients will be

incorporated in conventional amounts known to those skilled in the art. The compositions according to the invention may be applied to and left on the skin.

Detailed description

1 Expression of elastic fibre components in intrinsically aged skin

The inventors of the present invention used an Affymetrix^® microarray-based approach to detect age-related changes in gene expression of molecules known to contribute to the elastic fibre network.

1 .1 Skin biopsy procurement

Skin biopsies (6mm diameter) were obtained from the photoprotected buttock of three young (18-30 years) and three aged (65-75 years) healthy volunteers. Each punch biopsy was obtained under 1 % lignocaine local anaesthesia and bisected.

Half of the biopsy was embedded in optimal cutting temperature compound (OCT,

Miles Laboratories, Elkhart, IN, USA), snap frozen in liquid nitrogen and stored at

-70°C for immunohistochemical analysis. The other half of the biopsy was immersed in RNA/ater solution (Ambion, Foster City, CA, USA), incubated at 4 °C overnight to allow the solution to permeate the tissue and stored at -70 °C for subsequent RNA extraction and Affymetrix® analysis.

An additional twelve volunteers were recruited to provide tissue for validation of the Affymetrix® results by quantitative real-time PCR (qPCR) and

immunohistochemical analysis. Skin biopsies (6mm diameter) were obtained from photoprotected buttock of six young ( age range 18-30 years) and six aged (age range 65-75 years) healthy volunteers. Each punch biopsy was embedded in OCT, snap frozen in liquid nitrogen and stored at -70 °C. Local ethical committee approval was obtained for all human studies and all subjects gave written, informed consent. 1 .2 Affymetrix® study

1 .2.1 RNA Extraction Reagents and protocols from the TRIzol^® Plus RNA Purification Kit (Invitrogen, Carlsbad, CA, USA) were used for total RNA extraction. Briefly, biopsies were removed from -70 °C storage and RNA/ater solution was discarded. Each biopsy was placed in a separate 2ml tube with 800μΙ TRIzol^®. Biopsies were

homogenised for 1 minute using an Ultra-Turrax T10 Basic homogeniser (IKA^® Werke GmbH & Co. KG, Staufen, Germany) with a dispersing element attached (S 10 N - 5 G; IKA^® Werke GmbH & Co. KG). The lysate was incubated at room temperature for 5 minutes to allow dissociation of nucleoprotein complexes. To each lysate, 160μΙ chloroform was added and the tube shaken vigorously by hand for 15 seconds. Samples were incubated at room temperature for 3 minutes and centrifuged at full speed for 15 mins at room temperature. The colourless, upper phase (approximately 300μΙ) was transferred to a fresh RNase-free tube and an equal volume of 70% ethanol was added. The sample was mixed by vortexing and transferred to a spin cartridge (with a collection tube). Cartridges were centrifuged at full speed for 15 seconds and the flow through was discarded. The spin cartridge was re-inserted into the same collection tube and 350μΙ Wash buffer I was added. The spin cartridges (with a collection tube) were centrifuged at full speed for 15 seconds and the flow through and collection tube were discarded. The spin cartridge was inserted into a new collection tube and 80μΙ DNase mix (8μΙ DNase reaction buffer, 10μΙ DNase, 62μΙ RNase-free water; Invitrogen) was added directly onto the surface of the spin cartridge membrane. Samples were incubated at room temperature for 15 minutes. At the end of the incubation period, 350μΙ Wash Buffer I was added to the spin cartridge and samples were centrifuged at full speed for 15 seconds. The flow through and collection tube were discarded and the spin cartridge was inserted into a new tube. Wash buffer II (500μΙ) was added to the spin cartridge and centrifuged at full speed for 15 seconds. The flow through was discarded and the spin cartridge was re-inserted into the same collection tube. The spin cartridge membrane was washed again in 500μΙ Wash buffer II and centrifuged at full speed for 15 seconds. The flow through was discarded and the spin cartridge was centrifuged at full speed for 1 minute to dry the membrane. The spin cartridge was inserted into a recovery tube and 30μΙ RNase-free water was added to the centre of the spin cartridge.

Samples were incubated at room temperature for 2 minutes and centrifuged at full speed for 1 minute to elute the RNA.

1 .2.2 Quantitative Polymerase Chain Reaction (QPCR) analysis

Following RNA extraction, cDNA was generated using cloned AMV (Avian

Myeloblastosis Virus) reverse transcriptase cDNA synthesis kit (Invitrogen, Ltd, Paisley, UK) to convert the RNA into cDNA, according to the manufacturers instructions, with 0.4 μg of cDNA being generated. QPCR analysis to quantify the gene expression was then performed using the ABI StepOne plus thermal cycler using ABI TaqMan fast universal mastermix with ABI probes for LOXL-1 and the loading control β-actin (Applied Biosystems, Carlsbad, California,. USA) according to the manufacturer. Gene expression of the LOXL-1 gene was assessed using cDNA from both age groups and normalised using the β-actin control, with values generated using ABI StepOne plus software. Relative expression of the two groups was used to indicate expression changes. 1 .2.3 Microarray analysis

Microarray experiments were performed using the human genome U133A oligonucleotide array (Affymetrix Inc, High Wycombe, UK) according to the manufacturer's instructions. Total RNA (100 ng) from three young and three aged volunteers was used with the Two-Cycle cDNA Synthesis Kit (P/N 900432

Affymetrix Inc; one sample hybridized per array). Technical quality control was performed with dChip. Background correction, quantile normalization, and gene expression analysis were performed using GCRMA. Differential expression between the young and old groups was tested statistically with CyberT on logarithmic scale data. False-discovery correction was performed with Q-value software. Significantly changed probe sets were selected on p-value (<0.05) and expression level (>50). 1 .3 I mmunohistochemical Analysis

1 .3.1 Cryosectionning Frozen tissue sections (5μηη) were cut on a cryostat (OTF 5000; Bright Instrument Co Ltd, Huntingdon, Cambridgeshire, UK) and mounted on poly-L-lysine coated slides (VWR, Leicestershire, UK). Slides were stored at -70°C prior to use.

1 .3.2 LOXL-1 immunostaining

Sections were defrosted at room temperature for 10 minutes and fixed in ice-cold acetone for 10 minutes. Sections were washed for 2 minutes in two changes of TBS. To prevent non-specific binding of the secondary antibody, 5% normal rabbit serum (Vector Laboratories Inc) in TBS was applied to each section for 30 minutes. Blocking serum was removed and mouse anti-human LOXL-1 primary antibody (Santa Cruz Biotechnology Inc., product # sc-166632, clone H-1 1 , dilution 1 :10 in 5% normal rabbit serum) was applied to each section for 1 hour at room temperature. Primary antibody was omitted from selected sections as a negative control. After washing twice in TBS for 2 minutes, sections were incubated for 30 minutes in rabbit anti-mouse Alexafluor 647 secondary antibody (diluted 1 :200 in TBS). After washing twice in TBS for 2 minutes, sections were incubated for 2 minutes in DAPI (1 g/ml in TBS). After washing twice in TBS for 2 minutes, sections were mounted and coverslipped in Fluoromount-G. 1 .3.3 Image Capture and analysis

Fluorescent images were captured using All-in-one Type Fluorescence

Microscope Biozero-8000 (Keyence, Osaka, Japan). LOXL-1 was also tested using plot profiles, area under the curve analysis and t-tests. 1 .4 Assessment of in situ LOX/LOXL-1 enzyme activity

Tissue cryosections were air-dried to room temperature before use. Amine substrate solution was prepared by adding 1 , 2-diaminobutane to a final concentration of 40mg/ml in phosphate buffer solution (5 mM di -sodium hydrogen phosphate, 5 mM sodium di-hydrogen phosphate monohydrate; pH 7.2).

Horseradish peroxidise was added to a final concentration of 4μΙ/ιηΙ. Immediately prior to use, an equal volume of lumino (ECL chemiluminescence detection reagent 2; GE Healthcare, Buckinghamshire, UK) was added. Substrate solution was applied to each tissue section and incubated at 37 °C for 5 minutes. Following incubation, excess substrate solution was carefully blotted from the tissue section and a glass coverslip was applied. The slides were placed in a developer cassette with an ECL film and incubated at 4 °C for 1 hour. The film was fixed and developed using standard procedures. Analysis of enzyme distribution was performed by scanning the autoradiography films and quantifying the intensity of signal using ImageJ software. Line profiles extending from a midpoint at the dermal-epidermal junction (DEJ) to the outer surface of the skin and into the dermis were generated an analysed across the different age groups. 1 .5 Results

1 .5.1 Microarray data

Microarray data was examined for expression levels of reported microfibril and elastic fibre associated molecules (Kielty et al. Journal of Cell Science, 2002).

Molecules that were tested included Fibrillin-1 , Fibrillin-2, Fibrillin-3, Latent TGFB Binding Protein-1 , Latent TGFB Binding Protein-2, Latent TGFB Binding Protein-3, Latent TGFB Binding Protein-4, Decorin, Biglycan, Versican, MFAP-1 , MFAP-3, MFAP-4 (MAGP-36), Elastin, Lysyl Oxidase, LOXL (LOXL-1 ), LOXL-2, LOXL-3, Fibulin-2, Fibulin-5, Emilin-1 , Emilin-2, Vitronectin, Endostatin, Collagen VIII, Collagen XVI and Collagen VI. Significantly changed probe sets were selected on p-value (<0.05) and expression level (>50).

1 .5.2 Immunohistochemical Analysis

Using immunofluroescence, it was found that LOXL-1 protein was distributed predominantly in the epidermal basal cell layers in young skin. In intrinsically aged skin, the protein had a wider distribution, extending further away from the dermal epidermal junction into the suprabasal layers of the epidermis.

The distribution of epidermal LOXL-1 was analysed by plotting line profiles of fluorescence from the dermal-epidermal junction towards the outer surface of the skin for both young and aged skin. The fluorescence activity of LOXL-1 activity was calculated using area under curve.

Assessment of in situ LOX/LOXL-1 enzyme activity

Table 1 : Results of assessment of in situ LOX/LOXL-1 enzyme activity in young and old skin.

An increase in LOX/LOXL-1 activity was detected in both the epidermis and dermis of aged skin (Table 1 ).

LOX activity was shown to be localized within the epidermal basal layer, hair follicles, blood vessels and glandular tissue in young skin. This distribution is in accordance with the immunohistochemical localisation of the LOX family member, LOXL-1 . In intrinsically aged skin, LOXL-1 distribution is significantly increased in the epidermal basal layer and a similar increase in LOX activity is observed. 2 Identifying materials that may reverse effects of intrinsic ageing

A two phase approach may be used to screen potential active ingredients for activity at the target binding sites. In vitro testing on cultured skin cells

(keratinocytes) allows for the screening of large numbers of materials both on their own and as combinations. Ingredients with the desired effect in vitro can then be incorporated into simple o/w emulsion vehicles before being tested in a 12 day in vivo patch test. In both cases, the aim is to identify whether the selected

ingredients have changed the expression of LOXL-1 protein, either from the cultured keratinocytes or in the patch test biopsies.

2.1 In vitro screening

Keratinocyte cell lines (eg normal human epidermal keratinocytes (NHEK)

(Promocell) and the immortalized human cell lines HaCaT) are grown at 37°C in a humidified incubator with 5% C0₂, undergoing passage at regular intervals. Cells are cultured in growth medium with appropriate supplements (eg NHEK cells can be cultured in the Keratinocyte growth medium 2 kit with supplements (Promocell). Samples are then trypsinised using 1 xTrypsin-EDTA (0.5g/L trypsin, 0.2g/L EDTA) (Lonza).

2.1 .1 Western blotting

To identify LOXL-1 release into the media, western blotting of conditioned media can be performed. Briefly, 10μΙ of media is mixed with 2xSDS loading buffer (100mM Tris-HCI pH6.8, 200mM DTT, 4% SDS, 0.2% Bromophenol blue, 25% glycerol), heated to 90 °C for 3mins and run using 12% SDS polyacrylamide gels with a 4% stacking gel. Samples are run under I xTris Glycine SDS (TGS) buffer at 125V for 1 hour 30 minutes in an X-cell sure lock electrophoresis tank

(Invitrogen). Following separation, samples are transferred to Immobilion-P nitrocellulose membranes (Millipore) and then transferred by a semi wet method using an X-cell II blot module (Invitrogen) transfer chamber and transferred at 25V for 1 .5 hours as described by the manufacturer. Blots are then blocked in 5% milk. Once transferred, membranes are probed using a primary antibody to LOXL-1 . Following incubation with appropriate secondary antibody, membranes are then investigated using horse radishes peroxidise and the degree of staining assessed.

2.2 In vivo screening

10 healthy subjects are recruited in the age range 60-80. 20μΙ of test product is applied to the extensor photoaged aspect of the forearm under standard 6mm Finn chambers (Scanpore, Tuulsula, Finland). An area is left untreated but occluded to provide a baseline control. Test products are applied on days 1 , 4 and 8 of the assay. On day 12 of the assay, the Finn chambers are removed and 3mm punch biopsies taken under 1 % lignocaine local anaesthesia from the test sites. Biopsied tissue is embedded in optimal cutting temperature compound (Tissue Tek, Miles Laboratory, Elkhart, IN, U.S.A.), snap frozen in liquid nitrogen and stored at -70°C prior to immunohistochemical analysis. 10μιτι frozen sections are then prepared using a Cryostat and mounted onto gelatine coated slides prior to histological analysis. A primary antibody to LOXL is applied overnight at 4°C. A negative control is by incubation of the isotype sera at the appropriate concentration or by omission of the primary antibody. Sections are washed in TBS prior to incubation with the appropriate biotinylated secondary antibody for 30 minutes. Antibody staining is visualised using a well characterised immunoperoxidase reaction or more preferably using immunofluorescence. Sections are then lightly stained with nuclear fast red before being dehydrated and permanently mounted. A semiquantitative scale can be used for grading the degree of antibody staining.

Claims

15 Claims

1 . Method for identifying an anti-ageing agent for use in skincare

compositions, which method comprises determination of the ability of a material or combination of materials to decrease the expression of LOXL-1 .

2. Method for evaluating the anti-ageing effect of a material or combination of materials by assessing the ability of the material or combination of materials to downregulate the expression of LOXL-1 .

3. A method as claimed in any preceding claim, which involves determining the amount of LOXL-1 protein present in a sample.

4. A method as claimed in any preceding claim, which is an in vitro method.

5. A method as claimed in claim 4, which uses cultured cell lines.

6. A method as claimed in claim 4 or claim 5, wherein western blot analysis is used to identify and quantify the release of LOXL-1 .

7. A method as claimed in any one of clai ms 1 to 3, in which the material under test is applied to the skin in vivo.

8. A method as claimed in claim 7, which method uses biopsied tissue.

9. A method as claimed in claim 7, wherein the biopsied tissue is frozen prior to immunohistochemical analysis.

10. A method as claimed in any one of claims 7 to 9, wherein the protein is identified using a labeled antibody.

1 1 An anti-ageing agent identified by the method of any preceding claim. 16

12. A skincare composition comprising an anti-ageing agent identified by the method of any one of claims 1 to 10.

13. A material or combination of materials which decreases the expression of LOXL-1 , for use as an anti-ageing agent in a skincare composition.

14. Use of a material or combination of materials which decreases the expression of LOXL-1 as an anti-ageing agent in the manufacture of a skincare composition.

15. Any novel method for identifying an anti-ageing agent generally as herein described.