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Lys'lastine™ Vby Beauty Creations

Lys'lastine™ V Reference A00077Description Aqueous Anethum Graveolens extract.

Regulatory data

IncI Water, Butylene glycol, Peucedanum Graveolens (Dill) Extract, Pentylene Glycol, Xanthan Gum.

Japanese cosmetic Denomination

Mizu, BG, Inondo ekisu, Penchiren Gurikoru, Kisantangamu.

cAS# 7732-18-5, 107-88-0, 90028-03-8, 5343-92-0, 11138-66-2.

EInEcS# 231-791-2, 203-529-7, 289-790-8, 226-285-3, 234-394-2.

Preservatives/Additives none.

Origin Lys'lastine™ V is an aqueous plant extract made from Anethum graveolens L.

Final specifications

Formulation data

cosmetic properties • LOXL synthesis stimulation,• Elastin functionality renewer.

Applications • Anti-aging face care,• Body care elasticity rebuilder.

concentration of use 1 to 2%.

Incorporation method Add at the end of the formula at temperature lower than 35°c.

Patent Cenizo V, Bouez C, Sommer P, Damour O, Gleyzal C, Andre V, Reymermier C, Perrier E.Stimulation of the synthesis and of the activity of an isoform of lysyl oxidase-like LOXL for stimulating the formation of elastic fibers.(Germany: 102004028302), (Korea: 100637934), (canada: pending), (France: pending), (UK: pending), (Japan: pending), (USA: pending).

Summary file

Beauty Care Solutions

ANALYSIS NAME SPECIFICATIONS METHOD(ref. COP#)

FINAL SPECIFICATIONS SHEET

Product

Ref.

Cosmetic Operational Procedure (COP) # 14-11

Version dated September 17, 2012

Max. shelf-life: 12 months - TBC

LYS'LASTINE V

A00077

CHEMICAL ANALYSES (Results are given in g for 100 g of finished product.)

- 0.2 - 1.0 57-25

Total Inorganic Matter Content (15 hours, 600°C) - 0.0 - 0.5 57-25

PHYSICAL ANALYSES

Appearance - clear liquid 57-75

Color - Brown 57-75

Odor - Characteristic 57-75

pH (Direct) - 5.0 - 7.0 57-31

Viscosity (Brookfield RVDV-II +) -(2; 6.0) (600 - 6667) cps 54-32

Refractive Index (20°C) - 1.35 - 1.37 54-94

BACTERIOLOGICAL ANALYSES

total aerobic bacteria (30 °C) - < or = 100/g BASF/08/2009

Pathogens : (S. Aureus; E. Coli; P.Aeruginosa; C.Albicans; A.Niger) - None BASF/08/2009

VISA Anne BENISTI Quality Control Manager

The Specifications Sheet indicates the range of values in wich the analytical characteristics of the product take place, and if necessary, the compliance of the product with some non-analytical analytical characteristics. These data apply exclusively to the sample or the Product indicated in reference.

The Specifications Sheets are progressively set, according to the number of analyzed batches, in accordance with the following process:

Preliminary Specifications Sheet The indicated values are determined after analysis of one batch only. They may be adjusted after analysis performed on several pilot or industrial batches.

Temporary Specification Sheet The indicated values are determined after analysis of at least 3 pilot or industrial batches.

Final Specifications Sheet The indicated values are determined after analysis of at least 10 pilot or industrial batches.

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Although all statements and information in this publication are believed to be accurate and reliable, they are presented gratis and for guidance only, and risks and liability for results obtained by use of the products or application of the suggestions described are assumed by the user. THERE ARE NO WARRANTIES OF ANY KIND. TO THE EXTENT PERMISSIBLE BY LAW, ALL EXPRESS AND IMPLIED WARRANTIES ARE DISCLAIMED. Statements or suggestions concerning possible use of the products are made without representation or warranty that any such use is free of patent infringement and are not recommendations to infringe any patent. The user should not assume that toxicity data and safety measures are indicated or that other measures may not be required.

Page 1/1

S.A.S. au capital de 5 325 177 euros - TVA FR 04 333 802 767 – SIRET 333 802 767 00025 - APE 731Z - R.C.S. LYON B 333 802 767

50 Health Sciences Drive,New York, NY 11790 Phone +1- 631-380-2300 Fax +1- 631-689-2904 [email protected]

Roppongi Hills Mori Tower 21F 6-10-1 Roppongi, Minato-ku Tokyo 106-6121 Phone +81-3-3796-9214 Fax +81-3-3796-9299 [email protected]

49, Av. Georges Pompidou 92593 Levallois Cedex Tél. +33 1 49 64 50 00 Fax +33 1 49 64 50 50 [email protected]

32, rue Saint Jean de Dieu 69366 Lyon Cedex 07 Tél. +33 4 72 76 60 00 Fax +33 4 78 58 09 71 [email protected]

Head office: In France

Sales office: France Japan USA

BASF Beauty Care Solutions France SAS

Although all statements and information in this publication are believed to be accu-rate and rel iable, they are presented gratis and for guidance only, and risks and l iabil ity for results obtained by use of the products or application of the suggestions described are assumed by the user. SELLER MAKES NO WARRANTY OF ANY KIND, EITHER EXPRESS OR IMPLIED, BY FACT OR LAW, INCLUDING WARRANTIES OF MERCHANTABILITY OR FITNESS FOR A PARTICULAR PURPOSE. Statements or sug-gestions concerning possible use of the products are made without representation or warranty that any such use is free of patent infringement and are not recommenda-tions to infringe any patent. The user should not assume that toxicity data and safety measures are indicated or that other measures may not be required. The claims and supporting data provided in this publication have not been evaluated for compliance with any jurisdiction’s regulatory requirements and the results reported may not be generally true under other conditions or in other matrices. Users must evaluate what claims and information are appropriate and comply with a jurisdiction’s regulatory requirements. Recipient of this publication agrees to ( i ) indemnify and hold harmless each entity of the BASF organization for any and all regulatory action arising from recipient’s use of any claims or information in this publication, including, but not l im-ited to, use in advertising and finished product label claims, and (i i ) not present this publication as evidence of f inished product claim substantiation to any regulatory authority.

Table of contents

3January 23, 2013 Lys'lastine™ V

Introduction .................................................................................. 5Description ........................................................................................ 5

Dermis and Extracellular matrix (ECM) ................................................. 6

Collagen fibers ............................................................................... 7

Elastic fibers ................................................................................. 8

Elastic fibers and aging ...................................................................12

Screening ....................................................................................13

Lys'lastine™ V: a demonstrated efficacy ...............................................14Dose effect of Lys'lastine™ V on LOXL expression ........................................15

Effect of Lys'lastine™ V on Mimederm™ and Mimeskin™ ..................................16

Visualization of elastic fibers in Mimeskin™ ...............................................18

Elasticity study of Mimederm™ treated by Lys'lastine™ V ...............................20

Evaluation in vivo of elastic properties of the skin .....................................21

Conclusion ...................................................................................27

Bibliography .................................................................................29

4 January 23, 2013 Lys'lastine™ V


IntroductionLys'lastine™ V is the new anti-age active substance developed and patented by BASF Beauty Care Solutions, that stimulates the enzyme LOXL (lysyl oxidase like).

LOXL is an enzyme in the extracellular matrix of the dermis and whose activity decreases with age(1-2). This enzyme is responsible for

the functionality of elastic fibers by ensuring assem-bly between microfibrils and tropoelastin.

Lys'lastine™ V thus boosts the synthesis of functional elastic fibers and is a new arm in the battle against the slack appearance of aging skin.

The loss of skin elasticity is one of the principal signs of aging, whether chronological or accelerated by exposure to sunlight.

Age and exposure to UV in solar radiation both lead to oxidative phenomena that desorganize the network of collagen fibers to become denser and that degrade elastic fibers.

The dermis becomes thinner, elastic fibers frag-ment; these fibers are resynthesized but are not functional, causing a loss of skin elasticity. collagen fibers replace the degraded elastic fibers, causing the appearance of lines and wrinkles …

Rather than targeting collagen or activating the syn-thesis of tropoelastin whose quantity varies little with age, BASF Beauty Care Solutions chose to in-vestigate a new anti-age approach to combat the loss of elasticity. This strategy has led to the de-velopment of an anti-age active substance that re-induces on the synthesis of LOXL, enzyme res-ponsible of the elastic fibers functionality …

(1) Ashcroft GS et al.Cell Tissue Res 1997 ; 290 : 581-591.

(2) Cenizo V et al.Exp Dermatol 2006 ; 12 : 1-10.

Description

Lys'lastine™ V is a plant extract obtained from dill seeds harvested in India or Hungary. The Latin name of this plant is Anethum graveo-

lens L., family of the apiaceae (ombelliferae), ge-nus anethum. This annual plant reaches a height of about 1 meter and is native to the Mediterranean ba-sin (Anethum graveolens) or central Asia (Anethum sowa). It was used by Egyptians more than 5000 years ago as an anti-inflammatory, or soothing me-dicinal plant.

In addition to these virtues, the Greeks and Romans also used it for its aromatic properties. Today, dill is grown primarily as a condiment for its highly aroma-tic leaves and seeds.Lys'lastine™ V presents itself as a clear brown liquid. This vegetal extract is characterized by its content in dillapiole (1-Allyl-2,3-dimethoxy-4,5-(methylen-dioxy) benzen) an organic chemical compound (che-mical formula c12H14O4).

commercial sample of Lys'lastine™ V


Dermis and Extracellular matrix (ECM)

The dermis is located under the dermo-epider-mal junction (DEJ) and rests on the hypoder-mis. It is a dense connective tissue composed

primarily of extracellular matrix in which fibroblasts bathe.

There are two principal regions: the papillar or su-perficial dermis located just under the DEJ, and the reticular or deep dermis (figure 1).

The first is rich in cells (fibroblasts, dermal dendro-cytes …), blood vessels and nerve endings. It is for-med predominantly from collagen fibers and loose, fine elastic fibers arranged vertically to the skin sur-face and that bathe in the ground substance.The second is a denser connective tissue whose col-lagen and elastic fibers are arranged horizontally to the skin surface.

One of the essential functions of the dermis is to give the skin its consistency, but also its resistance and elasticity. This support function involves a large number of macromolecules belonging to four large families:

• collagens,

• elastic fibers,

• substances such as glycoproteins, and

• glycosaminoglycans.

The leading actor in the dermis is the fibroblast. It ensures the synthesis of the macromolecules that form the EcM. The EcM is composed of collagen fi-bers and elastic fibers that bathe in the ground subs-tance (water, inorganic salts, glycoproteins and gly-cosaminoglycans, principally hyaluronic acid).

Figure 1: Schematic representation of the skin

DEJ: Dermo-epidermal junction

Epidermis

DEJPapillary

dermis

Reticular dermis

Hypodermis


collagens are the physical support of all con-nective tissues and account for about 40% of all the proteins of the body.

Up to the present time, more than 27 forms of col-lagens have been described in two large families (fi-brillar collagens and non-fibrillar collagens) and 9 sub-classes according to their structures(3).

In the dermis, the main representatives are type I fibrillar collagens (collagen fibers) and type III (re-ticulin fibers).

The assembly of triple helices of collagen occurs via cross-linking, most often between the central pep-tide chains and the tips (telopeptides).

The fibrils generated aggregate in larger bundles, forming veritable fibers of several micrometers in diameter. The fibers in turn organize in a network and give connective tissues (bones, tendons, der-mis) their mechanical resistance by absorbing ten-sion forces (figure 2).

In contrast to the elastic fibers also present in con-nective tissues, collagen is inextensible and resists traction forces.

One shall keep in mind that, in addition to this prin-cipal mechanical role, collagens also are involved in cell development, growth and transfers of intercel-lular information.

Figure 2: Different stages of the formation of dermis collagenic fibers

Synt

hesi

s of

pep

tidi

c ch

ains

Gly

cosy

lati

on,

Hyd

roxy

lati

on

Trip

le h

elix

fo

rmat

ion

Sepa

rati

on o

f pr

opep

ides

(P

roco

llage

n-pe

ptid

ase)

Lysi

ne o

xyda

tion

(L

ysyl

oxyd

ase)

Fibe

r Fo

rmat

ion

and

cros

s-lin

king

FibroblastExtension Telopeptides peptides

Acido-soluble collagen

CHONH2

Collagen fibers

(3) Ricard-Blum S et al.Pathol Biol 2005 ; 53 : 430-442.

Schematic representation of the triple helix of collagen

Reminder: several definitions (source: Petit Larousse dictionary, 2001)

Plasticity = characteristic of a very malleable substance

Elasticity = property of certain bodies to return to their initial form or volume after a deformation.

Flexibility of the skin that is necessary for all movement is attributable to sinuous collagen fibers that in response to a stretching movement, become straight without stretching (they are not elastic); they determine the stretch limit of the tissue.


Although the EcM of the dermis is composed primarily of collagen fibers, the 3 or 4% of elastic fibers are just as important. In organs

such as the aorta or the lungs, they are even the major constituent …

These fibers are seen as a tight, three-dimensional interlacing of fibrils, intimately criss-crossed with collagen fibers (diagram 1 and photo 1). What is the composition of these fibers? Why are they so special and so important for the skin ?

a) Composition Elastic fibers result from a complex non-sponta-neous assembly that requires several coordinated steps(4). The two main players are tropoelastin and microfibrils:

• The elastin precursor, or tropoelastin (TE).The major constituent of elastic fibers (about 90%), TE gives them their elastic property. TE is a water-soluble protein of about 68 kDa, synthe-sized by the cells of elastic tissues (fibroblasts in the skin). The majority of its structure is an alter-nation between hydrophobic domains and other domains involved in the cross-linking process. This

step is directed by enzymes of the lysyl oxidase (LOX) family and leads to the transformation of soluble elastin into an insoluble elastin (figure 3).

(4) Jacob MP et al.Biomed Pharmcother 2001 ; 57 : 195-202.

Schematic representation of elastin

Elastic fibers

Elastin is synthesized as soluble tropoe-lastin that acquires its physicochemical properties (insolubility, elasticity) after its cross-linking by enzymes of the lysyl oxidase family and its deposit on micro-fibrils.

Diagram 1: network of interlaced fibers

Epidermis

Dermis

Elastin

Fibroblast

Fibrils

collagen

Elastic fibers straightened by the stretching forces use their property of elasticity to return the tissue to its normal position when the forces no longer act.

Photo 1: Section of human skin (young subject) observed by transmission electron microscopy. Magnifying x5700.Prof. Daniela Quaglino, Department of Biomedical Sciences, General Pathology, University of Modena and Reggio Emilia, Italy

Elastic fibers

Collagen fibers

Figure 3: components and formation of elastic fibers. They are modified during normal aging and that accelerates by hyper-cross-linking or calcification.

EBP = Elastin Binding Protein


• Microfibrils. These assemblies of glycoproteins are composed primarily of fibrillins 1 and 2, but also proteins such as MAGP-1 (Microfibril-Associated-GlycoProteins-1), MAGP-2 and 3 and other minor proteins synthetized in the fibroblasts. Once se-creted in the EcM, these glycoproteins form a complex multi-molecular structure in the shape of linear microfibrils (figure 3).

They are assembled in bundles or networks and are a sort of skeleton on which TE attaches. This structure plays a crucial role in the correct ali-gnment of TE molecules before the cross-linking process.

b) Formation Since 2000, molecular biology techniques and the analysis of genetic deficiencies have enabled giant steps to be taken in the understanding of the com-plex process of the physiological formation of elas-tic fibers. In particular, the analysis of the formation defects of elastic fibers(5) has led to the characteri-zation and structural study of the different simpli-fied proteins and glycoproteins.

The synthesis of elastic fibers starts around the 3rd or 4th month of fetal development and continues du-ring the growth phase of the person. Elastogenesis, the formation of elastic fibers, starts by the assem-bly of monomers of fibrillins 1 and 2 catalyzed by MAGPs and disulfide bonds formed by free cysteine residues. These assemblies form microfibrils. They are organized in the extracellular space to form the support on which newly synthesized TE mole-

cules will deposit. A caping protein, Elastin Binding Protein (EBP), binds to TE within the endoplasmic reticulum(6) to prevent its aggregation and prema-ture degradation in the cell and the ECM.

TE is escorted and secreted into the extracellular space. After contacting microfibrils, EBP undergoes a conformational change and releases TE that ali-gns and binds to fibrillins 1 and 2(7-8) (figure 4). Once aligned on the network of microfibrils, TE molecu-les undergo several maturation steps: they mutual-ly bind during the formation of cross-linking amino acids such as desmosine (cf figure 6, next page). This step is called cross-linking.

Recent studies have shown that this reaction is ini-tiated by two isoform enzymes of the family of lysyl oxidase, LOX and LOXL (LOX Like) and leads to the insolubilization of elastin(9-10).

The cross-linking of elastin gives the protein its pro-perties of elasticity, insolubility and resistance. This final step is characterized by the appearance of functional elastic fibers (figure 5).

(5) Milewicz DM et al.Matrix Biol 2000 ; 19 ; 471-480.

(6) Debelle L et al.The International Journal of Biochemistry & Cell Biology 1999 ; 31 : 261-272.

(7) Trask et al.The Journal of Biological Chemistry 2000 ; 275 (32) : 24400-24406.

(8) Jacob MP.Médecine et Sciences, 2003 ; N°4, vol. 19.

(9) Noblesse E et al.J Invest Dermatol 2004 ; 122 : 621-630.

(10) Liu X et al.Nat Genet 2004 : 36 (2) : 178–182.

The formation of cross-linking amino acids, a reaction catalyzed by LOXL or LOX, renders tropoelastin and collagen molecules insoluble and functional by conferring their respective properties of elasticity and resistance.

Fibulin-5Integrins (avb3, avb5, a9b1)

ca2+

ca2+

Microfibrils

Tropoelastin

Desmosine, cross-linking amino acids

Fibrillins synthesis

Lysyl oxydase

Elastic fibers organisation

Figure 4: Elastic fibers organisation(8)

Figure 5: Elastic proprieties of elastic fibers. The stretching of elastic fibers reduces entropy (a), enabling a return to the initial form with no supply of energy (b)

(a) Stretching (b)Relaxation

Tropoelastin monomer covalent bonds


c) Cross-linkingThe lysyl oxidase pathway is the principal mecha-nism for the cross-linking of collagens and elastin. This involves three extracellular events:

• the oxidative deamination of e-amines groups of lysine or hydroxylysine residues of tropoelastin molecules into the corresponding aldehyde deri-vatives (allysine or hydroxyallysine respectively),

• the spontaneous formation of reducible intra- and inter-molecular bonds, and

• the spontaneous reaction of the aldehyde with neighboring amine or aldehyde groups (figure 6).

The originality of the physical properties of TE is related to the particular presence of a cross-linking product, desmosine or isodesmosine (figures 4 and 6).

d) The family of lysyl oxidases (LOX)Lysyl oxidases are metalloenzymes belonging to the family of copper amine oxidases (cAO). At first, a single lysyl oxidase was considered to be responsible for the formation of covalent bonds between the lysine residues of tropoelastin.

In the 1990s, however, several other related genes were discovered. The family of lysyl oxidases cur-rently contains five members: LOX, LOXL, LOXL2, LOXL3 and LOXL4. The c-terminal regions have a high degree of homology(11).

LOXL2, LOXL3 and LOXL4, larger than LOX and LOXL, form a sub-group within this family. These proteins possess certain domains at their n-terminal end in-volving specific functions (interactions with secre-ted proteins or proteins bound to the cell membrane for example).

The LOXL protein was identified for the first time in 1998 as a secreted form expressed in the EcM in as-sociation with collagen fibers in cases of fibrosis(12).

A double immunolabeling ultrastructural study of human skin (photo 2) showed that LOX preferential-ly cross-links collagen and elastin, while LOXL cross-links almost exclusively elastin(9).

In human skin, as in our Mimeskin™ model of recons-tructed skin (BASF Beauty care Solutions, France), the expression of the LOXL gene (mRnA and protein) is preferentially coordinated with that of the elastin gene, while the expression of the LOX gene follows that of type I collagen(9).

In Mimeskin™ model, the first model of reconstruc-ted skin enabling the observation of elastic fibers, fibroblasts express LOXL and tropoelastin in a coor-dinated and relatively late way, while LOX and type I collagen are synthesized earlier in the course of cell culture.

(9) Noblesse E et al.J Invest Dermatol 2004 ; 122 : 621-630.

(11) Csiszar K.Research and Molecular Biology 2001 ; 70 : 2-28.

(12) Decitre M et al.Lab Invest 1998 ; 78 (2) : 143-151.

Figure 6: Formation of bonds by lysyloxidase on TE. LOX initiates the oxidative deamination of lysine residues in molecules of TE. Desmosine and isodesmosine are the specific bond agents of TE.

Photo 2: Section of human skin (young subject) observed by transmission electron microscopy. co-localization of LOXL and elastin. Visualization by labelling with gold conjugated anti-body. Magnifying x50000. Charbel BOUEZ, University Lyon IµF = microfibrils Eln = elastin Coll = collagen


In addition, a study in mice showed the fundamen-tal role of LOXL in the maintenance and renewal of elastic fibers in adults(10).In another study(13), it was shown that the deletion of the LOX gene, but not that of LOXL, was lethal.Mice not expressing the LOXL gene, on the other hand, had a typical Cutis laxa phenotype.This genetic disease is characterized by a loss of tro-poelastin and elastic fibers in the skin, vessels and other connective tissue compartments. In humans, certain factors responsible for an imper-fect elastogenesis can also cause considerable skin disorders.

e) Imperfect elastogenesisA copper deficiency for example in our diet, leads to a decrease in the activity of lysyl oxidase metal-loenzymes.Even more serious, lethal Merkes’ disease is due to the absence of copper-dependent enzymes inclu-

ding LOX. This disease is characterized by structural defects in connective tissue, retarded growth and skeletal anomalies.Another syndrome, that of Ehlers-Danlas type IX, is due to the mutation of procollagen or LOX genes. This disease results in a connective tissue malfor-mation. In the course of healing, there is generally a weak and non-functional neosynthesis. In this case, scar tissue lacks elasticity, causing esthetic and functio-nal disorders (solar elastosis or even more serious consequences in the case of burn victims).

(10) Liu X et al.Nat Genet 2004 : 36 (2) : 178–182.

(13) Hornstrat IK et al.J Biol Chem 2003 : 278 (16) : 14387-14393.

(14) Cenizo V.Thèse. Lyon : Université Claude Bernard Lyon I ; 2005.

Cross-linking amino acids formed during the cross-linking reaction catalyzed by LOXL stabilize the polymerized product that becomes insoluble: at this stage, elastic fibers become functional.LOXL is apparently the specific enzyme of the cross-linking of soluble elastin in micro-fibrils(14).


With aging, elastic fibers thicken instead of forming a homogeneous network; in this case, we speak of actinic elastosis.

In addition, as a result of their very long lifetime, elastic fibers and collagen fibers are very sensiti-ve to the phenomenon of glycation or non-enzyma-tic glycosylation, also called the Maillard reaction. This reaction resulting from the binding of reducing sugars to the free amine groups of elastic or col-

lagen fibers (covalent bonds), can generate AGEs (Advanced Glycosylation End products) .

This non-enzymatic pathway is associated with aging since this cross-linking causes tissues to thicken and become rigid, in contrast to the enzymatic pathway of lysyl oxidase that is associated with a maturation process(15).

During fetal development and the post-natal period, the expression of the elastin gene is considerable, decreases to a great extent in young children and then remains stable for the rest of the life (photos 3 opposite). This explains the notion of the elasticity capital at the end of adolescence(1).

The microfibrils of newborns are not completely co-vered with elastin, becoming so around puberty.

Starting at the age of about 40, inclusions appear on elastic fibers that fragment and then disappear under the dermo-epidermal junction (DEJ). This causes a loss of skin elasticity and the formation of lines, followed by deeper wrinkles.

It is to be noted that this phenomenon of appearan-ce of non-functional elastic fibers is accelerated in the case of photo-aging(16) (solar elastosis).

nevertheless, it has been shown that the princi-pal players in the final composition of elastic fibers (tropoelastin, microfibrils) are present throughout life(17). This study raised the hypothesis that these components are not limiting factors(2). This suggests that one or several factors responsible for the for-mation of functional elastic fibers during embryonic development up to adolescence become deficient in the course of aging.

One hypothesis could be a cross-linking fault of tro-poelastin with microfibrils, that would cause an im-mediate degradation of soluble elastin by enzymes such as metalloproteinases and serine proteases(18).

A deficit in lysyl oxidase could also explain this cross-linking deficiency. In order to substantiate this hypothesis, it was necessary to determine if the

(1) Ashcroft GS et al.Cell Tissue Res 1997 ; 290 : 581-591.


(15) Bouez C et al.Clin Cancer Res 2006 ; 12 (5) : 1463.

(16) Watson et al.J Invest Dermatol 1999 ; 112 : 782-787.

(17) Ashcroft G et al.J Pathol 1997 : 183 : 80-89.

Elastic fibers and agingAging of the skin and mucous membranes is accompanied by a modification of the fibril networks, especially that of elastic fibers, that degrade and no longer reform correctly.

Newborn: microfibrils and elastin deposit in the course of formation

Young subject: microfibrils well formed and functional elastic fibers

Photos 3: Section of human skin observed by transmission electron microscopy. Magnifying x18000.Prof. Daniela Quaglino, Department of Biomedical Sciences, General Pathology, University of Modena and Reggio Emilia, ItalyEln = elastin (TE)Coll = collagen

Adult: microfibrils well formed but onset of degradation, especially at the edges of the fibril network

Elderly subject: network of elastic fibers has lost its structure (desorganization and darker areas)

A

C

B

D

Elastic fibers

CollColl

Eln

Coll

Coll

Elastic fibers


expression of the LOX and LOXL genes decreases with age, suggesting that one of them could be a limiting factor in the synthesis of functional elas-tic fibers.

Fibroblasts from young subjects (prepuce, n=5, age less than 10) and elderly donors (abdominal skin af-ter plastic surgery, n=6, age = 43±24) were grown to confluence in defined medium at 37°c in an atmo-sphere containing 5% cO2.

After extraction of total RnA from the cells in cul-ture, the levels of expression of mRnA of the elastin, LOX and LOXL genes were determined using quan-titative RT-PcR (Reverse Transcriptase Polymerase chain Reaction).

The results showed that TE mRnA was expressed to a considerable extent until the age of 60 and that its expression did not differ between the two age classes(2).

On the other hand, the levels of expression of LOX and LOXL were significantly lower in elderly donors compared to the young (–41% and –66%, respectively p < 0.001). This reduced expression of LOX and prin-cipally LOXL, is thus a limiting factor of the func-tionality of elastic fibers during aging(2). Therefore, we developed a specific screening model allowing to select the most effective active substance on the stimulation of LOXL synthesis.


(18) Ashworth et al.Biochem J 1999 ; 340 : 171–181.

Based on the previous results, the goal was to use an in vitro model of fibroblasts from an elderly donor to test more than 1000 molecu-

les potentially capable of stimulating the expression of LOXL.

Following this screening step, the active substan-ce most effective for re-inducing the formation of functional elastic fibers was selected.

A simplified monolayer model was developed and validated by positive controls to pre-select poten-tial active substances before their final evaluation in our model of Mimederm™ reconstructed dermis (BASF Beauty care Solutions, France).

Screening methodology was the following(2) :

1 Development of the in vitro model by analysis of the mRnA of elastin, LOX and LOXL in monolayers of fibroblasts from a 63 year-old donor.

2 Validation of the model by quantitative RT-PcR measurements of the effect of positive controls on the stimulation of transcription of elastin mRnA (TGF b1, retinoic acid and IL1 b). Regarding LOXL, a slightly inducible gene, IL1 b was showed as a good positive control.

3 Screening for substances capable of stimulating the transcription of under-expressed mRnA by qualitative RT-PcR: about 1000 compounds tes-ted at 1% (algae, plants, peptides, characterized molecules or biotechnology compounds).

4 Selection of the 3 best active substances capa-ble of stimulating the transcription of under-ex-pressed mRnA by quantitative RT-PcR and study of their dose effect.

5 Selection of Lys'lastine™ V (plant extract from dill), the best active substance among the 3 previously tested.

Screening


Lys'lastine™ V: a demonstrated efficacy

The strategy of BASF Beauty care Solutions during the development of this new patented active subs-tance involved 6 complementary orientations:

• quantitative RT-PcR to determine the dose effect of Lys'lastine™ V used in the previous model of fibroblast monolayers, on the expression of LOXL mRnA,

• systemic application of Lys'lastine™ V in the model of Mimederm™ reconstructed dermis (BASF Beauty care Solutions, France) and quantitative RT-PcR assay of increased LOXL expression,

• validation of the co-localization of tropoelastin and LOXL by immuno-histochemistry on Mimeskin™ (BASF Beauty care Solutions, France), followed by quantification of LOXL,

• transmission electron microscopy visualization of newly synthesized functional elastic fibers in the Mimeskin™ model (BASF Beauty care Solutions, France),

• elasticity study of Mimederm™ treated with Lys'lastine™ V,

• validation of the efficacy of a cosmetic formula containing 1% of Lys'lastine™ V on the biomechani-cal properties of skin versus a placebo formula by an in vivo clinical study on human volunteers.


MATERIALS & METHODS

Test systemAs with screening, the dose effect was assessed in the same monolayer culture model of normal human fi-broblasts from a 59 year-old donor.

The cells were grown in defined medium until post-confluence and were then treated or not (control) with Lys'lastine™ V at 0.01, 0.1, 0.5, 1, 2, 3 and 5% during IL1b 50 pg/ml (positive control) 24 hours.

MethodTotal RnAs from fibroblasts were extracted with the «SV 96 total RnA Isolation System» (Promega, Madison, WI, USA). RT-PcR analyses were con-ducted with the «QuantiTect SYBR Green RT-PcR kit» (Qia-gen, Courtaboeuf, France) in a fluorescence thermocycler (Opticon, MJ Research, USA). The reaction mix contained SYBR Green 1X buffer, specific primers of the LOXL gene used at 0.5 μM, 0.5 μl of enzymatic mix, 50 ng of total RnA, qs 50 μl with RnAse/DnAse free water.

Each condition was tested in triplicate (n=3).

During amplification, the in-corporation of a fluorophore (SYBR Green, Qiagen) ena-bled quantification by fluo-rescence and the real time monitoring of the quantity of newly formed amplification product. It was then possible to compare the difference in mRnA levels before and after treatment (Opticon™, MJ Re-search, USA).

Standardizing the results with respect to the quantity of reference mRnA (actin gene) enabled the results between treated and untreated condi-tions to be compared.

Dose effect of Lys'lastine™ V on LOXL expression

OBJECTIVEThe aim of this study was to use quantitative RT-PCR in the in vitro model ofold fibroblasts to measure the effect of Lys'lastine™ V at different concentrationson the expression of mRNA of LOXL.

RESULTS

The quantity of mRnA was standardized with respect to the reference actin gene and was expressed as stimulation factor in comparison to the untreated control.

The results of quantitative RT-PcR in comparison to the control showed that the positive control IL1b induced on average the LOXL gene stimulation by 1.8+0.5 fold; this validated the experiment. Lys'lastine™ V tested between 0.1 and 5% increased mRnA expression of LOXL up to 3.7-fold (figure 7).Lys'lastine™ V 1% already stimulated the expression byf LOXL 1.6-fold.

CONCLUSIONThis experiment confirmed the results obtained in the previous screening step: Lys'lastine™ V tested on fi-broblasts of an old donor significantly boosted the expression of LOXL mRnA (up to 3.7-fold the untreated control). This increase was observed in the concentration range of 0.1 to 5%.Moreover, with a stimulation of 1.6-fold, Lys'lastine™ V 1% appeared to be a relevant concentration for the next tests.

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Figure 7: Quantification of LOXL mRnA by quantitative RT-PcR with treatment by Lys'lastine™ V at different concentrations on normal human fibroblasts from a 59 year-old donor.

*: statistically different from control p<0.05***: statistically different from control p<0.001

x1.6 *

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OBJECTIVEThe aim of these experiments was to verify that Lys'lastine™ V stimulates the synthesis of the LOXL protein. Lys'lastine™ V was systemically applied in a culture of Mimederm™ reconstructed dermis in order to quantify the synthesis of LOXL mRNA and the resulting protein deposited.

RESULTSa) Quantitative RT-PCR

The application of Lys'lastine™ V at 1% in the conditions of this study resul-ted in a significant increase (+64%,

p<0.001) of the expression of LOXL mRnA by Mimederm™ after 28 days of culture (figure 8). In parallel, Lys'lastine™ V did not lead to an increase of the quantity of tropoe-lastin (TE) mRnA, while treatment with TGF-b led to a significant increase of +28% (p<0.05).

b) HistologyLys'lastine™ V applied at 1% in the Mimeskin™ culture medium exhibited efficacy towards the formation of the ECM (Extra Cellular Matrix) that was denser and present to a greater extent (photo 4b) than in the section of the untreated control (photo 4a).Even if this technique is not quantitative, the detection of tropoelastin (TE), maroon here, increased in recons-tructed skin grown in the presence of Lys'lastine™ V (photo 4b) compared to the untreated control (photo 4a).

The same was true for the detection of LOXL: the maroon color was more intense, especially under the dermo-epidermal junction, but also in the EcM, in the case of reconstructed skin treated with Lys'lastine™ V (photo 4d) compared to untreated control reconstructed skin (photo 4c).

MATERIALS & METHODSTest systemMimederm™ were obtained from normal human fibro-blasts deposited on a Mime-disc™ porous sponge (BASF Beauty Care Solutions, Fran-ce). These fibroblasts from adults of varying ages were grown in defined medium and incubated at 37°c in an at-mosphere of cO2/air (5%/95%, v/v) for 4 weeks.

Mimeskin™, a 3-dimensional model of reconstructed skin (see insert), was prepared by keratinocytes from adult donors of variable ages, on the Mimederm™ previously obtained. This keratinocy-tes/Mimederm™ co-culture was maintained immersed in proliferation medium for one week. The reconstructed skin was then raised to the air-li-quid interface to enable the epidermis to develop correct-ly. These culture conditions were continued for 3 weeks in differentiation medium changed every two days(19).

Materials and test condition• 3 Mimederm™ and 3 Mimes-

kin™ untreated controls• 3 Mimederm™ and 3 Mimes-

kin™ treated with a positive control, TGF-b at 1 ng/ml in the culture medium

• 3 Mimederm™ and 3 Mimeskin™ treated with Lys'lastine™ V at 1% (v/v) in the culture me-dium.

There were 6 donors of dif-ferent ages for each of these three conditions (from 19 to 54 years old) for a total of 18 dermis samples and 18 re-constructed skin preparations for each condition.

The positive control TGF-b and Lys'lastine™ V were added daily to the culture medium of reconstructed dermis between days 3 and 28 of culture (D28).

The products were also ap-plied daily to the Mimeskin™ between D3 and D28 and then from D31 to D49 only 3 times a week during the immersion period.

Effect of Lys'lastine™ V on Mimederm™ and Mimeskin™

(15) Bouez C et al.Clin Cancer Res 2006 ; 12 (5) : 1463.

(19) Black et al.Tissue Eng 2005 ; 11 (5–6) : 723–733.

(20) Duplan-Perrat F et al.J Invest Dermatol 2000 ; 114 (2) : 365-370.

Mimeskin™ is the first model available whose extracellular matrix exhibits an ultrastructural organization similar to that of the skin, with elastic fibers and collagen fibers organized in bundles. In this Mimeskin™ model, keratinocytes induce the synthesis of tropoelastin and its deposit on microfibrils(20).

Bouez et al.(15) investigated these properties by testing an inhibitor of lysyl oxidase (beta-amino proprionitrile) on Mimeskin™. In these conditions, they observed a disorganization of collagen and elastin fibers and a deviation from the program of kerati-nocyte differentiation, with a reduction of the expression of terminal differentiation markers such as filaggrin.

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Figure 8: Quantitative RT-PcR analysis of the regulation of quantities of mRnA of tropoelastin (TE) and LOXL from Mimederm™ on D28 (n=18). The results were calculated as the ratio over actin mRnA and expressed as stimulation factor compared to the control.

*: statistically different from control p<0.05***: statistically different from control p<0.001

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c) QuantificationIn the case of the untreated control, the quantity of LOXL detected was normalized at 100%.

Treatement with Lys'lastine™ V significantly increased(+12.5%, p<0.001) the quantity of labeled LOXL pro-tein in Mimeskin™ compared to the untreated con-trol (figure 9).

CONCLUSIONThis study validated that Lys'lastine™ V increases the synthesis of LOXL mRnA in a model of Mimederm™ reconstructed dermis without stimulating tropoe-lastin mRnA expression. We used an immuno-histo-chemical technique to confirm the co-localization of tropoelastin (TE) and LOXL in the dermis. Finally, the quantification of LOXL labeling in Mimeskin™ re-constructed skin treated with Lys'lastine™ V showed a +12.5% increase in LOXL synthesis. This increase was accompanied by a better detection of tropoelastin (TE) in the dermis, suggesting its improved cross-linking with microfibrils.

In order to extend and illustrate these results, we examined sections of reconstructed skin treated with Lys'lastine™ V in transmission electron microscopy (TEM).

Methodologya) Quantitative RT-PCRAfter a 24 hours of treatment on D28, the Mimederm™ pre-parations were frozen in li-quid nitrogen and ground in a mortar. After dispersion in a buffer, the samples were cen-trifuged. The supernatants were loaded on purification columns and mRnAs were eluted with ultra-pure water for use in quantitative RT-PcR with the technique described before (see page 15).

b) HistologyThe Mimeskin™ preparations were fixed in 10% formalde-hyde and embedded in paraf-fin. Sections about 6 µm thick were cut with a microtome and were subjected to an un-masking step for the detec-tion of tropoelastin (TE). The primary antibody used was either polyclonal anti-human elastin (Novotec, Lyon, Fran-ce) or a polyclonal anti-LOXL (anti-human protein, not commercialized). The secon-dary antibody was rabbit anti-IgG (DAKO, Trappes, France) coupled to peroxidase to de-tect immunocomplexes. The labeling obtained is maroon and counterstaining with he-matoxylin is blue.

The primary antibody was not added to the controls.

c) QuantificationFor the quantification step, two reconstructed skin pre-parations from each condi-tion were sectioned and de-posited on slides. Sectioning and staining described for Mimeskin™ were used but wi-thout counterstaining. Digital photos of each sample were taken with a nikon Dxm1200F digital camera and an Axios-kop 2 Plus microscope (Zeiss, Germany). The images taken with the nikon Dxm1200 5.0 were recorded with LUcIA G software for quantification of the labeling.

On each image (n=8 treated conditions and n=8 untreated conditions), 6 fields were se-lected for signal quantifica-tion. The average pixel den-sity of each field was noted and the mean of the 6 fields was calculated.

a) non-treated control b) Mimeskin® treated by Lys'lastine™ V 1%

c) non-treated control d) Mimeskin® treated by Lys'lastine™ V 1%

Tropoelastin labelling

LOXL labelling

+12.5%

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Photos 4: Mimeskin™ histological sections treated by Lys'lastine™ V 1% or not (control). Intensity difference of tropoelastin and LOXL labelling. Observation with Axiostop 2 plus microscope (Zeiss, Germany).

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Figure 9: Quantification of labeled LOXL protein in Mimeskin™ treated with Lys'lastine™ V 1% or untreated.

***: significantly different from non treated control p<0.001

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Photos 5: Sections of reconstructed skin Mimeskin™ treated by Lys'lastine™ V 1%, observed by transmission electron microscopy. Magnifyings from x50,000 to x3,00000.

Visualization of elastic fibers in Mimeskin™

OBJECTIVEThe aim of this study was to use transmission electron microscopy to visualize functional elastic fibers in a model of Mimeskin™ reconstructed skin treated with Lys'lastine™ V 1%.

RESULTS

This work enabled us to visualize the ultrastructure of our model of Mimeskin™ reconstructed skin, in particular keratinocytes, the dermo-epidermal junction, intercellular junctions, fibroblasts and the extracellular matrix (ECM).

In these conditions, certain elements of the newly synthesized matrix could be observed. Thanks to the ex-pertise of Simone Peyrol (research engineer at the common Imaging center of Laënnec in Lyon, France), we observed and identified collagen fibers (type I and VII) and Microfibrils Associated with Elastin (MAE).

Photo AThis photo is a fibroblast bathing in the EcM. By paying close attention to the fibroblast membrane, it can be seen that this cell is fully active in the production and deposit of the principal constituents of the EcM.

Photo BAfter magnifying a part of photo A, it can be clearly seen that collagen fibers pass through the fibroblast membrane to enter the EcM. Microfibrils, much thiner than collagen, are also present.

MATERIALS & METHODSSampleMimeskin™ (63 year-old do-nor) treated with Lys'lastine™ V 1% (n=3).

Preparation The samples were fixed in a mixture of 2.5% glutaralde-hyde – 0.1M cacodylate buffer (Sigma, St Quentin Fallavier, France). The samples were then post-fixed with a mix-ture of 1% osmium tetroxide (Electron Microscopy Scien-ces, Fort Washington, USA)

- 0.1 M cacodylate buffer. The samples were then dehydra-ted in a graded ethanol series (30 to 100% absolute ethanol (Prolabo, Brioure le Canal, France)). The dehydration step terminated with a subs-titution bath in propylene oxide (Prolabo). The samples were then impregnated with epoxy resin (Prolabo) that was polymerized for 3 days at 56°c.

Sections70 nm ultrathin sections were cut from the inclusion blocks with an Ultracut S ultrami-crotome (Leica, Germany). The sections were then suc-cessively contrasted with a solution of 7% uranyl acetate (Prolabo) solution in methyl alcohol and then in lead ci-trate (Prolabo).

VisualizationImages were taken at the common Imaging center of Laënnec (Lyon, France) using a Jeol transmission electron microscope at an accelera-tion voltage of 80 kV. Photo A Photo B

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Synthesis and deposits of EcM constituents MAE = Microfibrils Associated with Elastincoll = collagen

collagen fibers diameter ≈ 50 nmMicrofibril fibers diameter ≈ 10-12 nm


Photo CAt even higher magnification, longitudinal microfibrils are very clearly seen. At regular intervals on these microfibrils, it is also possible to distinguish denser and darker zones that are the deposit of elastin on microfibrils.

Photo DThis photo also shows the deposit of elastin on microfibrils. Elastin forms dark zones, as nodes in the network of microfibrils.

CONCLUSIONUsing this method of observation of Mimeskin™ reconstructed skin treated with Lys'lastine™ V, we were able to visualize the deposit of elastin on microfibrils in the ECM. This enabled us to conclude on the presence of newly synthesized and functional elastic fibers in our model of reconstructed skin from a 63 year-old donor.

The following study allowed to demonstrate the effect of Lys'lastine™ V on the biomechanical properties of Mimederm™, our reconstructed dermis model.

MAE = Microfibrils Associated with Elastin

Photos 6: Sections of reconstructed skin Mimeskin™ treated by Lys'lastine™ V 1%, observed by transmission electron microscopy. Magnifying x30,0000.

Photo C Photo D

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MATERIALS & METHODSCulture of Mimederm™ and treatmentcf materials & methods of study page 16. n=6 Mimederm™ treated with Lys'lastine™ V 1%n=6 Mimederm™ non treated (control).

This study was performed at the “École centrale” of Lyon, in France, jointly with Pr. Zahouani and his team who developed a fully automated bio-tribometer to assess the mechanical properties of soft and complex materials like skin.In this study, we measured the elasticity of reconstructed dermis Mimederm™ treated with Lys’lastine™ V 1% compa-red with untreated ones.

Measurement of the elasticityThe tribometer is an instru-ment that allows measuring mechanical properties of a ma-terial either in compression or laterally. The tribometer com-prises a smooth spherical me-tallic probe called an “inden-ter” thanks to which one can create a strain at the surface of the studied material. The indenter is linked to sensors which measure the variations of forces and displacement all along the study (figure 11).

To explore the lateral elasticity of skin, the tribometer uses a specific configuration called the “friction test” described below.The friction test allows stret-ching the material laterally and measuring the related parame-ter of elasticity (calculation of the “shear modulus of elastici-ty”). The indenter is positioned at the surface of skin on which it presses slightly. By moving the indenter laterally at the surface of the skin, a friction force is created. This force first increases linearly until it rea-ches a threshold corresponding to the elastic limit of the skin. Since forces and displacement are measured throughout the study, a curve representing va-riations of forces in function of indenter displacement can be drawn (Figure 12). The slope of this curve at the starting point corresponds to a parameter named “lateral stiffness”, which is highly cor-related to the lateral elasticity of skin given by what physi-cians name the “shear* modu-lus of elasticity”. This latter is the parameter that we have measured in this study.

*in physical sciences, a “shear” is a lateral stretch.

Elasticity study of Mimederm™ treated by Lys'lastine™ V

OBJECTIVEAfter checking that Lys'lastine™ V was able to stimulate the synthesis of LOXL protein, the goal of this study was to obtain, thanks to a tribometer, some physical measurements of lateral elasticity on Mimederm™ reconstructed dermis from a 62 year-old donor, after systemic treatment or not by Lys'lastine™ V.

RESULTS

After getting friction curve, the shear modulus of elasticity was calculated for each assay.

The results (figure 13) showed that the reconstruc-ted dermis treated with Lys'lastine™ V reached to a significant increase of their elastic component by +29% compared to the non treated dermis (p<0.01).

CONCLUSIONThanks to the friction test, it was possible to de-termine and to assess the mechanical properties of reconstructed dermis models. After application of a friction force, the strained elastic fibers take the direction of the traction that permits a measurement of the lateral elasticity.

In theses conditions, the reconstructed dermis Mimederm™ treated with Lys'lastine™ V showed significant increase of their elasticity by +29% compared to the non treated dermis.

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Figure 13: Friction test approach: improvement of the lateral elasticity of Mimederm™ treated with Lyslastine™ V 1%, compared to a non treated control. n=6 Mimederm™ per condition.

**: significantly different from non-treated control p<0.01

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Figure 11: Tribometer device. Assessment of the elastic properties of the reconstructed dermis Mimederm™ by friction test approach.

Reconstructed dermis

Spherical indenter

Force measurement

Figure 12: classical curve representing friction forces and lateral displacement variations

Slope = lateral stiffness

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MATERIALS & METHODS50 women between 43 and 56 years of age were recruited and selected to participate in the clinical study by the use of specific criteria in terms of wrinkles/lines and elasticity of their skin.2 groups of 25 subjects were formed and each group tes-ted one product.Products tested:• O/W formulation +

Lys'lastine™ V 1%• O/W formulation without active

substance (placebo)It was a randomized double blind study.

The subjects in each group ap-plied Lys'lastine™ V or the pla-cebo to their entire face twice a day for 3 months in normal conditions of use.

Measuring skin elasticity by cutometry

Measurements were made on the temples with an SEM 575™ cutometer (Courage & Kha-zaka, Germany).

The principle involves crea-ting a suction force on the surface of the skin. Deforma-tion of the skin is recorded and the parameters Ur/Uf (rate of elastic return) and Uf (final deformation) are de-termined. An improvement in skin elasticity can be claimed if the parameters Ur/Uf and Uf increase.

Evaluation in vivo of the elastic properties of the skin

OBJECTIVEThe aim of this study was to evaluate and compare the efficacy of Lys'lastine™ V versus a placebo in healthy subjects with wrinkles and reduced elasticity of facial skin after 3 months of treatment.

RESULTS

The percentages of improvement were calculated with the following formula:

% improvement = (Tn - T0) x 100

Tn – (Tn – T0)

Measurements of the skin biomechanical properties by cutometry • After 2 and 3 months of application, the skin of the volunteers treated with the formula containing

Lys'lastine™ V at 1%, was significantly more elastic than the skin of the placebo group. The Ur/Uf parameter increased by +19% (p=0.06) and by +18% (p<0.05) compared to the baseline respec-

tively after D56 and D84 days (figure 14). The positive change of the parameter Ur/Uf could be explained by a significant improvement of the elastic return of the skin after stress by cutometer suction.

As far as the placebo is concerned, no significant effect was shown in comparison to the baseline neither at 54 or 86 days timepoint.

• After 2 months of application, the skin of the volunteers treated with the formula containing Lys'lastine™ V at 1%, was significantly firmer than the skin of the placebo group.

The Uf parameter increased by +17% and by +13% compared to the baseline respectively after D56 and D84 days (figure 15).

As far as the placebo is concerned, no significant effect was shown in comparison to the baseline neither at 54 or 86 days timepoint.

Tn: mean of scores of each subject at time TT0: mean of scores of each subject at T0

Figure 14: comparison of the elasticity recovery rate parameter (Ur/Uf) between placebo and Lys’lastine™ V 1% after 56 and 84 days of treatment (variations in % (Dn-D0), n=21 subjects per product).

*: significantly different from the baseline (Student t test, p<0.05)°: significantly different from the placebo (Student t test, p<0.05)

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Figure 15: comparison of the firmness parameter (Uf) between placebo and Lys’lastine™ V 1% after 56 and 84 days of treatment (variations in % (Dn-D0), n=21 subjects per product).

*: significantly different from the baseline (Student t test, p<0.05)**: significantly different from the baseline (Student t test, p<0.01)°: significantly different from the placebo (Student t test, p<0.05)

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Clinical evaluationThe visual and tactile clinical evaluation of each descriptor was conducted using an ana-logue scale defined by the ex-tremes «no intensity» (0) and «maximum intensity» (10).

The following descriptors were evaluated: elasticity and slackness of the jaw line. The results were given in the form of clinical scores (/10).

Clinical evaluation

In vivo touching evaluation of each descriptor by the clinician has given the following results (fi-gures 16 & 17):

• After 84 days of treatment with Lys'lastine™ V, sub-jects showed a significant improvement regarding with the elasticity of their skin face compared to the placebo group.

Moreover, the skin elasticity of the subjects trea-ted by Lys'lastine™ V was significantly better com-pared to the baseline (+5% and +11%, respecti-vely, after 2 and 3 months).

As far as the placebo is concerned, no significant change was shown in comparison to the baseline neither at 54 days or 86 days timepoints.

• After 84 days of treatment with Lys'lastine™ V, the improvement of the slackeness of the jaw line concerning the subjects treated by Lys'lastine™ V was significantly better compared to the baseline after 3 months (+10%, p<0.01).

As far as the placebo is concerned, no significant change was shown in comparison to the baseline neither at 54 days or 86 days timepoints.

Figure 16: comparison of the clinical evaluation of skin elasticity between the placebo group and the Lys’lastine™ V 1% group after 56 and 84 days of treatment (variations in % (Dn-D0), n=20 subjects per product).

*: significantly different from the baseline (Student t test, p<0.05)**: significantly different from the baseline (Student t test, p<0.01)°°: significantly different from the placebo (Student t test, p<0.01)

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Figure 17: comparison of the clinical evaluation of the slackeness of the jaw line between the placebo group and the Lys’lastine™ V 1% group after 56 and 84 days of treatment (variations in % (Dn-D0), n=20 subjects per product).

**: significantly different from the baseline (Student t test, p<0.01)

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Auto-evaluation

The satisfaction of subjects was evaluated with a questionnaire they filled out at D56 days and D84 days. The questionnaire contained items describing the qualities and efficacy of the products tested. Analysis of the responses showed that:

• after 56 days (figure 18), 90% of subjects treated with Lys'lastine™ V reported an improvement in the tex-ture and elasticity of their skin. 81% of them declared that the jaw line seemed remodeled and 76% found that their wrinkles and lines were less visible.

• after 84 days (figure 19), favorable opinions were reported for the following items:- Skin texture: 90%- Skin elasticity: 95%- Slackness of the jaw line: 76%- Wrinkles and lines smoother: 95%- Satisfaction with the Lys'lastine™ V treatment: 100%

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Figure 18: Auto-evaluation of product efficacy after 56 days. (n= 21 subjects per product).*: significant difference in favor of the product p<0.05

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moisturized

The texture of my skin

is improved

My skin seems smoother

The elasticity of my skin is better

The firmness of my skin is better

The jaw line is better

Wrinkles and lines

are smoother

Satisfaction with Lys'lastine™ V

treatment

70

90 *

Figure 19: Auto-evaluation of product efficacy after 84 days. (n= 21 subjects per product).*: significant difference in favor of the product p<0.05

70

90 *

75 *

95 *

95 *

80 *

86 *80

*80 *

7070

76 *

100 *95

*

Placebo

Lys'lastine™ V 1%

50

60

70

80

90

100

110

The skin ismore

moisturised





slackness ofthe jaw line is

improved

wrinkles & finelines are

smoothened

satisfaction withthe testedproduct ?

freq

uenc

y of

the

posi

tive

answ

ers

(%)


Auto-evaluationFor each product, the sub-jects filled out a question-naire to recover their opinion and evaluate their satisfac-tion for certain items at D56 and DT84 days. For each item, the frequency of «completely agree» and «agree» responses were pooled.


Measuring the anti-wrinkle effect by image analysisFor each subject, a silicone replica (Silflo™, Flexico De-velopments Ltd, UK) of one of their randomly selected crow’s feet was prepared at D0, D56 and D84 days.

The replicas were analyzed using the method of projec-ted shadows. The technique involves illuminating the re-plica with a light source at an angle of 35°. Digitized ima-ges were taken with a ccD-RGB camera (Sony) and the following parameters were determined with dedicated software:• average length (µm)• average surface (mm²)• number of wrinkles/lines• total length (µm)• total surface (mm²)

An anti-wrinkle effect could be shown if these parameters decreased.

Measurements of anti-wrinkle effect by digital image analysis

After 2 months of applica-tions, subjects who were treated with Lys'lastine™

V showed a decrease of all the parameters characterizing fi-ne lines and wrinkles. This de-crease was significantly better than the placebo for the wrinkle length parameter. This impro-vement of the cutaneous relief was evolved stronger in the time and after 3 months of applica-tions, surface and length of the wrinkles were decreased signifi-cantly compared to the placebo group (figures 20 & 21).

comparatively to the baseline, Lys'lastine™ V also showed a significant decrease concerning the parameters number of objects (-22%), surface (-20%), length (-13%), total length (-24%) and total surface (-27%) after 86 days of applications (figures 22, 23 & 24).

On the other hand, placebo did not show any significant effect in comparison to baseline either at 54 or 86 days timepoint.

T0

T+56

T+84

Number of wrinkles Total length Total surface

Empreintes Silflo

Longueur des ridesPlacebo Lys'lastine

D56 8,2 -8,3D84 11,5 -12,7

Surface des ridesPlacebo Lys'lastine

D56 5,1 -10,3D84 9,8 -19,6

Nombre des ridesPlacebo Lys'lastine

D56 -11,6 -7,3D84 -10,9 -22

Longueur total des ridesPlacebo Lys'lastine

D56 -1,2 -5,4D84 -0,4 -23,6

Total surfaces des ridesPlacebo Lys'lastine

D56 0 -6,1D84 6,7 -27,3

-28-24-20-16-12-8-4048

12

D56D84

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12

D56D84

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12

D56D84

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12

D56D84

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12

D56D84

Placebo Lys'lastine™ V D56 D84 D56 D84





Empreintes Silflo


D56 8,2 -8,3D84 11,5 -12,7


D56 5,1 -10,3D84 9,8 -19,6


D56 -11,6 -7,3D84 -10,9 -22


D56 -1,2 -5,4D84 -0,4 -23,6


D56 0 -6,1D84 6,7 -27,3

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12

D56D84

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12

D56D84

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12

D56D84

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12

D56D84

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12

D56D84






Empreintes Silflo


D56 8,2 -8,3D84 11,5 -12,7


D56 5,1 -10,3D84 9,8 -19,6


D56 -11,6 -7,3D84 -10,9 -22


D56 -1,2 -5,4D84 -0,4 -23,6


D56 0 -6,1D84 6,7 -27,3

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12

D56D84

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12

D56D84

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12

D56D84

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12

D56D84

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12

D56D84






-7%

**-22%

-11.5% -11%

-1% 0.5%

-5%

**-24%

-6%

+7%

0%

*-27%

Placebo Lys'lastine™ V 1%D56 D84 D56 D84



Figures 22, 23, 24: Evolution of the relief parameters "number of wrinkles", "total length" and "total surface" (variations in % (Dn-D0) n=20 or 21 subjects per product)

*: significantly different from T0 p<0.05**: significantly different from T0 p<0.01

Wrinkle length

Empreintes Silflo


D56 8,2 -8,3D84 11,5 -12,7


D56 5,1 -10,3D84 9,8 -19,6


D56 -11,6 -7,3D84 -10,9 -22


D56 -1,2 -5,4D84 -0,4 -23,6


D56 0 -6,1D84 6,7 -27,3

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12

D56D84

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12

D56D84

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12

D56D84

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12

D56D84

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12

D56D84






-10%* °

-20%

+5%

+10%

Empreintes Silflo


D56 8,2 -8,3D84 11,5 -12,7


D56 5,1 -10,3D84 9,8 -19,6


D56 -11,6 -7,3D84 -10,9 -22


D56 -1,2 -5,4D84 -0,4 -23,6


D56 0 -6,1D84 6,7 -27,3

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12

D56D84

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12

D56D84

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12

D56D84

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12

D56D84

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12

D56D84






+8%+11.5%

° -8% *

°° -13%



Wrinkle surface

Figures 20, 21: Evolution of the relief parameters “wrinkle length” and “wrinkle surface” (variations in % (Dn-D0), n=20 or 21 subjects per product)

*: significantly different from T0 p<0.05°: significantly different from placebo p<0.05°°: significantly different from placebo p<0.01


CONCLUSION

The aim of this study was to evaluate and compare the efficacy of Lys'lastine™ V versus a placebo after 3 months of treatment by healthy subjects showing a lack of skin elasticity and wrinkles.

The analysis of cutometry data showed that the skin elasticity of subjects treated with Lys'lastine™ V was improved after 56 days. This improvement was significantly higher than the placebo group one.

clinical evaluation indicated a significant improvement for the descriptors «skin elasticity» (starting at 56 days) and «slackness of the jaw line» in subjects treated with Lys'lastine™ V. At the end of the study, the facial skin of subjects in the Lys'lastine™ V group was more elastic than that of the placebo group.

The results showed that subjects treated with Lys'lastine™ V presented a significant reduction in the appea-rance of their wrinkles and lines at 56 days. This anti-wrinkle effect was significantly better than that for the placebo group, in which no efficacy could be shown.

Finally, all the improvements noted for the Lys'lastine™ V group and evaluated by either instrumental methods or by a clinician were confirmed by the subjects themselves. 100% of the volunteers declared to be very satisfied with Lys'lastine™ V and the large majority perceived a significant improvement of the general state of their skin, in particular a better elasticity, a reduction of slackness of the jaw line and the appearance of wrinkles and lines.

This auto-evaluation enabled us to show a correlation between the responses of the volunteers and the re-sults obtained with instrumental techniques and clinical evaluations.

Lys'lastine™ V fulfills the expectations of consumers by offering a visible anti-age efficacy and by opening new claims in cosmetology field such as "face re-architecture" and "skin elasticity renewer".


ConclusionElastic fibers: a new anti-age target.BASF Beauty Care Solutions innovates and broadens the areas of anti-age claims.

In partnership with the cnRS (laboratories of Dr. P. Sommer and O. Damour), BASF Beauty care Solutions has made a major scientific discovery:

LOXL, an enzyme in the dermal extracellular ma-trix, is specifically responsible for the functionality of elastic fibers.

Until today, the classical scientific approach in cos-metics was based on stimulating the synthesis of so-luble elastin. It is known, however, that the quantity of this elastin changes very little with age, while elas-tic fibers thicken and degrade as the years go by.

With aging of the skin, soluble elastin or tropoelas-tin is no longer assembled normally on microfibrils and non-functional elastic fibers appear. In the case of photo-aging, this phenomenon accelerates and we speak of solar elastosis.

According to the hypothesis that imperfect elasto-genesis is not due a reduction of the principal com-ponents of elastic fibers, we discovered the missing link that can explain the appearance of non-func-tional fibers.

We have shown that expression of the LOXL gene decreases with age, making it a target of choice for re-establishing functional elastogenesis.

This is why we developed a model of screening in vitro to select the most effective active substan-ce capable of activating this new target.Among more than 1000 substances tested, Lys'lastine™ V was selected. Lys'lastine™ V was the most effective for stimulating expression of the ge-ne and the synthesis of LOXL protein.

We also observed functional elastic fibers in our mo-del of Mimeskin™ reconstructed skin from an old do-nor and treated with Lys'lastine™ V.

Finally, these results were validated in a clinical stu-dy of 50 human volunteers. In this study, instrumen-tal and clinical evaluations, as well as perception by the consumers confirmed the anti-age efficacy of Lys'lastine™ V. In particular, volunteers treated with Lys'lastine™ V presented skin that was more elastic, along with a reduction of the appearance of wrin-kles and of slackness of the jaw line.

Lys'lastine™ V, a patented anti-age active substan-ce, opens new perspectives in cosmetology: re-architecture of the face, skin elasticity, correc-tion of wrinkles…


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(2) Cenizo V, André V, Reymermier C, Sommer P, Damour O, Perrier E. LOXL as a target to increase the elastin content in adult skin: a dill extract induces the LOXL gene expression. Exp Dermatol 2006 ; 12 : 1-10.

(3) Ricard-Blum S, Ruggiero F.The collagen superfamily: from the extracellular matrix to the cell membrane. Pathol Biol 2005 ; 53 : 430-442.

(4) Jacob MP, Sauvage M, Osborne-Pellegrin M.Extracellular matrix remodeling and matrix metalloproteinases in the vascular wall during aging and in pathological conditions. Biomed Pharmcother 2001 ; 57 : 195-202.

(5) Milewicz DM, Urban Z and Boyd C.Genetic disorders of the elastic fiber system. Matrix Biol 2000 ; 19 ; 471-480.

(6) Debelle L, Tamburro AM.Elastin: molecular description and function. The International Journal of Biochemistry & Cell Biology 1999 ; 31 : 261-272.

(7) Trask et al.Interaction of tropoelastin with the amino-terminal domains of fibrillin-1 and fibrillin-2 suggests a role for the fibrillins in elastic fiber assembly. The Journal of Biological Chemistry 2000 ; 275 (32) : 24400-24406.

(8) Jacob MP.L’invalidation du gène de la fibuline-5 induit une élastinopathie. Médecine et Sciences, 2003 ; N°4, vol. 19.

(9) Noblesse E, Cenizo V, Bouez C, Borel A, Gleyzal C, Peyrol S, Jacob MP, Sommer P, Damour O. Lysyl oxidase-like and lysyl oxidase are present in the dermis and epidermis of a skin equivalent and in a human skin and are associated to elastic fibers. J Invest Dermatol 2004 ; 122 : 621-630.

(10) Liu X, Zhao Y, Gao J et al.Elastic fiber homeostasis requires lysyl oxidase-like 1 protein. Nat Genet 2004 : 36 (2) : 178–182.

(11) Csiszar K.Lysyl oxidases: A novel multifunctional amine oxidase family, nucleic Acid. Research and Molecular Biology 2001 ; 70 : 2-28.

(12) Decitre M, Gleyzal C, Raccurt M, Peyrol S, Aubert-Foucher E, Csiszar K, Sommer P. Lysyl oxidase-like protein localizes to sites of de novo fibrinogenesis in fibrosis and in the early stromal reaction of ductal breast carcinomas. Lab Invest 1998 ; 78 (2) : 143-151.

(13) Hornstrat IK, Birge S, Starcher B, Bailey AJ, Mecham RP, Shapiro SD. Lysyl oxidase is required for vascular and diaphragmatic development in mice. J Biol Chem 2003 : 278 (16) : 14387-14393.

(14) Cenizo V.Etude de la Lysyl oxidase-Like : Rôle dans la formation des fibres élastiques et régulation au cours du vieillissement cutané. Thèse de l’Université Claude Bernard – Lyon 1, 2005.

(15) Bouez C, Reynaud C, Noblesse E, Thepot A, Gleyzal C, Kanitakis J, Perrier E, Damour O, Sommer P. The Lysyl Oxidase LOX is absent in basal and squamous cell carcinomas and its knockdown induces an invading pheno-type in a skin equivalentmodel. Clin Cancer Res 2006 ; 12 (5) : 1463.

(16) Watson et al.Fibrillin-rich microfibrils are reduced in photoaged skin. Distribution at the dermal-epidermal junction. J Invest Dermatol 1999 ; 112 : 782-787.

(17) Ashcroft G, Kielty C, Horan M, Ferguson M.Age-related changes in the temporal and spatial distributions of fibrillin and elastin mRnAs and proteins in acute cutaneous wounds of healthy humans. J Pathol 1997 : 183 : 80-89.

(18) Ashworth JL, Murphy G, Rock MJ.Fibrillin degradation by matrix metalloproteinases: implications for connective tissue remodelling. Biochem J 1999 ; 340 : 171–181.

(19) Black AF, Bouez C, Perrier E, Schlotmann K, Chapuis F, Damour O. Optimization and characterization of an engineered human skin equivalent. Tissue Eng 2005 ; 11 (5–6) : 723–733.

(20) Duplan-Perrat F, Damour O, Montrocher C.Keratinocytes influence the maturation and organization of the elastin network in a skin equivalent. J Invest Dermatol 2000 ; 114 (2) : 365-370.

Cenizo V, Bouez C, Sommer P, Damour O, Gleyzal C, Andre V, Reymermier C, Perrier E. Stimulation of the synthesis and of the activity of an isoform of lysyl oxidase-like LOXL for stimulating the formation of elastic fibers. (Germany: 102004028302), (Korea: 100637934),

(Canada: pending), (France: pending), (UK: pending), (Japan: pending), (USA: pending).

For more contact please contact: EuropeBASF Beauty Creations49, avenue Georges Pompidou92593 Levallois-Perret CedexFranceTel: +33 (0) 1 49 64 53 97Fax: +33 (0) 1 49 64 53 85

Japan & Asia-PacificBASF Japan Ltd.21F Roppongi Hills Mori Tower, 6-10-1 Roppongi, Minato-ku, Tokyo, 106-6121 JapanTel: +81 (0) 3-3796-9214Fax: +81 (0) 3-3796-9299

AmericasBeauty Creations BASF Corporation50 Health Sciences DriveStony Brook, NY 11790USATel: +1 631-380-2300Fax: +1 631-689-2904

Although all statements and information in this publication are believed to be accurate and reliable, they are presented gratis and for guidance only, and risks and liability for results obtained by use of the products or application of the suggestions described are assumed by the user. SELLER MAKES NO WARRANTY OF ANY KIND, EITHER EXPRESS OR IMPLIED, BY FACT OR LAW, INCLUDING WARRANTIES OF MERCHANTABILITY OR FITNESS FOR A PARTICULAR PURPOSE. Statements or suggestions concerning possible use of the products are made without representation or warranty that any such use is free of patent infringement and are not recommendations to infringe any patent. The user should not assume that toxicity data and safety measures are indicated or that other measures may not be required. The claims and supporting data provided in this publication have not been evaluated for compliance with any jurisdiction’s regulatory requirements and the results reported may not be generally true under other conditions or in other matrices. Users must evaluate what claims and information are appropriate and comply with a jurisdiction’s regulatory requirements. Recipient of this publication agrees to (i) indemnify and hold harmless each entity of the BASF organization for any and all regulatory action arising from recipient’s use of any claims or information in this publication, including, but not limited to, use in advertising and finished product label claims, and (ii) not present this publication as evidence of finished product claim substantiation to any regulatory authority.

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