Salicyloyl-phytosphingosine: a novel agent for the repair ... · trans RA (0.025%) and vehicle were...

Salicyloyl-phytosphingosine: a novel agent for the

repair of photoaged skin

M. Farwick*, R. E. B. Watson�, A. V. Rawlings�, U. Wollenweber*, P. Lersch*, J. J. Bowden�,

J. Y. Bastrilles� and C. E. M. Griffiths�

*Degussa AG, Goldschmidt Personal Care, Goldschmidtstrasse 100, 45127 Essen, Germany, �Dermatopharmacology

Unit, Dermatology Centre, Hope Hospital, The University of Manchester, Manchester and �AVR Consulting Ltd,

Northwich, Cheshire, U.K.

Received 6 October 2006, Accepted 17 April 2007

Keywords: anti-ageing, ceramide, collagen, fibrillin, matrix metalloproteinase

Presented at the 24th IFSCC Congress, Osaka, Japan Oct 16–19, 2006.

Synopsis

In recent years the importance of sphingolipids

(cerebrosides, sphingomyelin, ceramides, sphingo-

sine-1-phospate, etc.) in skin biology is receiving

an increasing interest. Not only are ceramides

essential for the barrier function of the skin, espe-

cially through their phytosphingosine, sphingosine

and 6-hydroxysphingosine derivatives, they are

now also known to be cell-signalling mediators

which can improve epidermal differentiation. How-

ever, their effects on dermal anti-ageing markers

and reduction of wrinkles have not been estab-

lished. In this study, we were interested in the

effects of a sphingolipid derivative, salicyloyl-

phytosphingosine (SP), because of the known inde-

pendent beneficial effects of salicylic acid and

phytosphingosine on skin. Both of these agents are

known to reduce the activities of the activator pro-

tein-1 transcription factor, in a manner similar to

that observed with retinoic acid (RA) treatment.

Through this mechanism, RA was shown to

reduce the levels of matrix metalloproteases

(MMPs) and the increase levels of extracellular

matrix proteins. Therefore, we examined the effects

of SP on procollagen-I synthesis in fibroblasts

in vitro, its effects in vivo on the expression of der-

mal markers such as fibrillin-1, procollagen-I and

MMP-1 immunochemically in biopsies taken from

a short-term occluded patch test protocol and, its

effects on periorbital wrinkle reduction over

4 weeks using Fast Optical In Vivo Topometry of

Human Skin. In vitro we observed a significant

increase in the production of procollagen-I by adult

human fibroblasts (two fold increase, P < 0.01)

which encouraged us to test the effects of SP in vivo.

Initially, test products (SP at 0.05% and 0.2%, all-

trans RA (0.025%) and vehicle were applied under

occlusion for 8 days prior to biopsy and histological

assessment in photoaged volunteers (n ¼ 5).

Increased deposition of fibrillin-1 and procollagen-I,

together with reductions in the levels of MMP-1,

were observed for the SP treatments (P < 0.05).

Similar effects were observed for RA, except for the

increases in procollagen-I. With these beneficial

effects on the basement membrane and papillary

dermal markers, we evaluated the effects of SP in

an oil-in-water (O/W) cream for its effects in redu-

cing the appearance of periorbital wrinkles in a

4-week, half-face clinical study compared to

placebo cream (moderately photoaged female

subjects aged 41–69 years; n ¼ 30). Clear reduc-

tions in wrinkle depth and Rz (skin smoothness)

together with Ra (skin roughness) parameters were

observed (P < 0.05), indicating an anti-wrinkle

benefit. In conclusion, this series of studies demon-

strated for the first time that a ceramide derivative,

such as that SP, was a novel agent for the repair of

photoaged skin and highlight its effects at the

cellular, tissue and organ levels.

Correspondence: Mike Farwick, Degussa AG, Goldschmidt

Personal Care, Goldschmidtstrasse 100, 45127 Essen,

Germany. Tel.: +49 20 117 323-51; fax: +49 20 117

319-20; e-mail: [email protected]

International Journal of Cosmetic Science, 2007, 29, 319–329

ª 2007 The Authors. Journal compilation

ª 2007 Society of Cosmetic Scientists and the Societe Francaise de Cosmetologie 319

Resume

Ces dernieres annees, on s’est interesse de plus en

plus au role des sphingolipids (cerebrosides, sphin-

gomyeline, ceramides, sphingosine-1-phosphate,

etc...) dans la biologie de la peau. Non seulement

les ceramides jouent un role essential dans la fonc-

tion barriere, particulierement les derives phyto-

sphingosine, sphingosine et 6-hydroxysphingosine,

mais on sait maintenant qu’ils sont aussi des med-

iateurs de signaux cellulaires qui peuvent ameli-

orer la differentiation epidermique. Cependant,

leurs effets sur les marqueurs d’anti-vieillissement

dermiques ainsi que sur la reduction des rides

n’ont pas ete etablis. Dans cette etude nous nous

sommes interesses aux effets d’un derive de sphin-

golipide, le salicylolphytosphingosine (SP), du fait

des effets benefiques connus de l’acide salicylique

et de la phytosphingosine sur la peau. Ces deux

agents sont connus pour reduire les activites du

facteur d’activation transcriptionnel de proteine-1,

de facon similaire a ce que l’on observe lors d’un

traitement a l’acide retinoıque (RA). A travers ce

mecanisme, il a ete montre que RA reduit les

teneurs en metalloproteases de la matrice (MMPs)

et augumente les teneurs en proteines de la

matrice extracellulaire. Nous avons donc examine

les effets de SP sur la synthese du procollagene-I

dans les fibroblasts in vitro. Nous avons egalement

etudie ses effets in vivo sur l’expression de mar-

queurs dermiques comme la fibrillin-1, le procolla-

gene 1 et la MMP-1 par immunochimie dans des

biopsies realisees dans le carde d’un protocole a

court terme de patch tests occlusifs. Finalement

nous avons etudie ses effets sur la reduction des

rides periorbitales sur plus de 4 semaines en utili-

sant la technique Fast Optical In Vivo Topometry

sur Peau Humaine. In vitro nous avons observe

une augmentation significative de la production de

procollagene 1 par les fibroblasts d’homme adulte

(augumentation double, P < 0.01) ce qui nous a

encourage a evaluer les effets de SP in vivo. Initia-

lement, les produits testes (SP a 0.05% et 0.2%,

acide retinoique all-trans, 0.025%; et vehicule) ont

ete appliques sous occlusion pendant 8 jours avant

biopsie et evaluation histologique chez des volon-

taires ayant une peau photo senescente (n ¼ 5).

Une augmentation simultanee du depot de fibrillin-

1 et de procollagene-I ainsi que des diminutions

des teneurs en MMP-1 ont ete observees pour les

traitements au SP (P < 0.05). Des effets semblables

ont ete observes pour RA a l’exception des

augmentations du procollagene 1. Ces effets benefi-

ques sur la membrane basale et les marqueurs der-

miques papillaries nous ont alors conduits a

evaluer les effets de SP dans une creme O/W sur

la reduction de l’apparition des rides periorbitales

dans le cadre d’une etude clinique de quatre

semaines, sur demie tete, comparativement a une

creme placebo (sujects ages de 41–69 ans;

n ¼ 30). Des diminutions nettes simultanees de la

profondeur de ride et des parametres Rz et Ra ont

ete observees (P < 0.05) indication d’un effet anti-

ride. En conclusion, cette serie d’etudes demontre

que SP est un nouvel agent pour la reparation de

peau photo endommagee et met en evidence ses

effets au niveau cellulaire, tissulaire et organique.

Introduction

Photoageing of skin is the combination of chronolo-

gical ageing and the effects of cumulative exposure

to ultraviolet radiation. Chronological skin ageing

produces characteristic fine lines, whereas exposure

to solar UV results in skin that is, in comparison,

coarse, roughened and is deeply wrinkled, as also

which is stiffer and less elastic [1–3]. Histologically,

photoaged skin exhibits numerous alterations to

the dermis. Destruction and loss of extracellular

matrix (ECM) constituents at the dermal epidermal

junction (DEJ) and in the dermis by matrix metallo-

proteinases (MMPs) are characteristic biochemical

features. Changes include the deposition of dys-

trophic elastic fibres in the papillary dermis, termed

solar elastosis [4], decreases in the major fibrillar

collagens types I and III [5, 6], reduction in the

numbers of anchoring fibrils together with a

reduced fibrillin-rich microfibrillar network (fibril-

lin-1 [7], fibulin-5 [8], etc.) proximal to the DEJ.

The expression of MMP is implicated in the proteo-

lysis of these key proteins in both chronological but

especially photoaging [9–11].

It is well known that all-trans retinoic acid (RA)

has potent anti-ageing activity and induces partial

dermal repair of photoaged skin [12]. Effacement

of wrinkles following topical treatment with RA

arises through new collagen deposition and syn-

thesis in the skin [5, 13], together with increases

in the number of anchoring fibrils and improves

the fibrillin-rich microfibrillar network. Recent

data has also identified its potential for modulating

MMP-1 expression [14, 15].

Sphingolipids, especially ceramides, are the well

known stratum corneum barrier repair agents


ª 2007 Society of Cosmetic Scientists and the Societe Francaise de Cosmetologie

International Journal of Cosmetic Science, 29, 319–329320

Salicyloyl-phytosphingosine M. Farwick et al.

[16]. They have been reported to also improve epi-

dermal differentiation [17], especially the shorter

chain variants [18], and, as biological modifiers,

they also influence gene-transcription [19]. Phy-

tosphingosine has also been shown to possess

anti-inflammatory effects and be a peroxisomal

proliferator activator (PPAR) ligand [20]. Like RA,

PPAR ligands are known to function in skin via

their inhibition of the transcription factor activator

protein-1 (AP-1; 21) which leads to an increase in

procollagen-I production by fibroblasts [22]. Sali-

cylic acid and its variants have been reported to

improve the skin condition and reduce the signs of

ageing [23]. Aspirin has also been shown to inhi-

bit ultraviolet B-induced AP-1 activity [24].

Because of these biological actions, we recently

synthesized a novel sphingolipid, a combination of

salicylic acid N-acylated to phytosphingosine...

namely salicyloyl-phytosphingosine (SP, Fig. 1)

with the intent that it may possess biological activ-

ity that may mimic some of the effects of salicylic

acid and phytosphingosine.

In this respect, we were interested to ascertain if

SP could result in the increase in procollagen-I by

adult human fibroblasts in vitro, whether SP could

increase procollagen-I and fibrillin-1, while, at the

same time, reducing the MMP-1 expression in vivo

and whether SP had an anti-wrinkle benefit in vivo.

Materials and methods

In vitro procollagen-I synthesis in adult human

fibroblasts

Human adult dermal fibroblasts (adult cryopre-

served; Cambrex BioScience, Walkersville, MD,

USA,) were grown to sub-confluency in 96-well

plates in the FGM medium. These were collected

from breast biopsies of a 37-year-old female

subject. Five separate wells containing cells were

treated with solutions containing ethanol (50 lM)

or ethanol containing 10 lM SP. After 48-h incu-

bation, supernatants were removed and assayed

for procollagen-I C-Peptide EIA kit (Takara JP,

obtained by Cambrex Bio Science). Total superna-

tant protein levels were determined by Bradford

analysis. Procollagen-I levels were expressed as

percentage of the total supernatant protein. Statis-

tical significance was taken at the 95% confidence

level (Student’s t-test; graphpad prism 5.00 for

Windows, GraphPad Software, San Diego, CA,

USA, http://www.graphpad.com’.).

In vivo patch test assay and immunohistochemis-

try

The Covance Local Research Ethics Committee

(Leeds, U.K.) approved the study and all subjects

gave written informed consent. The methodology

of Watson et al. [25] was followed in this study.

Ten healthy, but clinically photoaged, volunteers

were recruited (age range: 54–71 years) and

15 lL of test substances: vehicle, SP (0.05% and

0.2%) and all-trans RA (0.025%; Retin-A� cream,

Janssen-Cilag Ltd., High Wycombe, UK) were

applied separately under the occlusive patch (6-

mm Finn chambers) to the extensor aspect of the

forearm. The formulations were aqueous solutions

of propylene glycol (74%), ethanol (25%) and pre-

servative. All treatments were compared to a vehi-

cle-control site. Formulations were applied to clean

skin on days 1 and 4 of the assay except for all-

trans RA which was applied to an untreated site

on the fourth day only. On the eighth day, Finn

chambers were removed and 3-mm punch biopsies

were obtained under 1% lignocaine anaesthesia

from each test-site. Biopsies were embedded in

OCT compound (Tissue-Tek�, Miles, IN, USA) and

snap frozen in liquid nitrogen. Frozen sections

were fixed in paraformaldehyde (4%) and hydrated

in Tris-buffered saline (TBS; 100 nM Tris, 150 nM

NaCl; pH 7.4) and mounted onto gelatin-coated

slides prior to histological analysis for the DEJ and

dermal ECM-marker proteins. Following hydration

in TBS (100 mM Tris, 150 mM NaCl), sections

were solubilized by the addition of 0.5% Triton-

X100 (10 min). Following washing, the endog-

enous peroxidase activity was abolished by

incubation with an excess of hydrogen peroxide in

methanol (30 min). Sections were blocked prior to

the application of primary antibodies (overnight

incubation; 4�C). These were: rat anti-human

OH

OH

O

HO

HONH

Figure 1 Structure of salicyloyl-phytosphingosine.



International Journal of Cosmetic Science, 29, 319–329 321


procollagen-I (clone M-58, Chemicon Inc., Teme-

cula, CA, USA, diluted 1 : 1000); mouse anti-

human fibrillin-1 (clone 11C1.3, NeoMarkers,

Fremont, CA, USA, diluted 1 : 100); and mouse

anti-human MMP-1 (Oncogene Research Products,

Cambridge, MA, USA, diluted 1 : 100) Negative

controls were concurrently incubated with either

block alone or control serum. Following incubation,

sections were thoroughly washed with TBS prior to

the application of an appropriate biotinylated secon-

dary antibody. Antibodies were localized and visual-

ized, using a well-characterized immunoperoxidase

reaction (VectaStain Elite ABC system, Vector

Laboratories, Burlingame, CA, USA) using Vector

SG� as chromogen. Sections were counterstained,

using nuclear fast red and, finally, dehydrated

through serial alcohols, cleared and mounted.

Sections were randomized, blinded and exam-

ined on a Nikon OPTIPHOT microscope (Tokyo,

Japan). For assessment, the degree of immuno-

staining was assessed on a 5-point semi-quantita-

tive scale where 0 ¼ no staining and

4 ¼ maximal staining. The numbers of keratino-

cytes positive for MMP-1 were quantified per high

power field. Four sections (including control) were

examined per subject per site. The degree of immu-

nostaining for each marker was scored for three

high power fields per section, and the average

score calculated for each site/test area. Differences

in the distribution between the test sites, and after

application of test substances for varying periods

of time were assessed for significance, using the

repeated measures ANOVA test. To assess whether

age or baseline values affected outcome measures,

data were tested using paired Student’s t-tests.

Both models were tested using spss+ software

(v. 11.5, SPSS Inc., Chicago, IL, USA) with signifi-

cance taken at the 95% confidence level.

Periorbital anti-wrinkle study using fast optical

in vivo topometry of human skin

Thirty female volunteers with moderate photodam-

age (aged 41–69 years) applied twice daily an

oil-in-water (O/W) cream without (placebo) and

with SP (0.2%, treatment) during a period of four

weeks to their periorbital areas of the face in a

half-face study design. Before and after the product

treatment, the subjects were acclimated to an envi-

ronment of 22�C and 50% relative humidity for

45 min before the non-contact FOITS was per-

formed to measure the 3-dimensional profile of the

periorbital skin areas [26]. Changes in the skin

profile can be quantified with the Rz (skin smooth-

ing) and Ra (skin roughness) parameters. The

distribution of the wrinkle depth can be measured

via the frequency distribution of depth (FDD). Start-

ing close to the eye, 50 singular lines in a distance

of 250 lm were analysed. The distribution of depth

is represented by the differences related to the

initial values, whereas the depth is distinguished in

micro structure (0–50 lm, should be approx. 5%

of total), fine structure (55–170 lm, should be

approx. 65% of total) and macro structure

(>170 lm, approx. 30% of total). An improvement

of the skin’s macro structure is given when the

values related to the macro structure are

reduced and, in parallel, the micro and fine

structure values are increased. Comparison to

untreated skin at baseline was made using the

ANOVA (graphpad prism 5.00 for Windows,

GraphPad Software, San Diego, CA, USA, http://

www.graphpad.com).

Results

In vitro procollagen-I synthesis in adult human

fibroblasts

Following the incubation of fibroblasts for 48 h

with 10 lM SP, EIA quantification demonstrated

more than a two fold increase in the levels of pro-

collagen-I found in the supernatant culture med-

ium (P < 0.05, Fig. 2).

In vivo patch test assay and immunohistochemis-

try

All volunteers tolerated the patch test protocol

well. Erythema was not observed for the vehicle or

10

8

6

4

2

0Medium + EtOH

PIP

rel

ativ

e to

tota

lpr

otei

n (%

)

SP

Figure 2 Effect of salicyloyl-phytosphingosine (SP) on

procollagen-I synthesis by adult fibroblasts. Significant

increases in procollagen are induced by SP (P < 0.05).





at either of the SP test sites. However, the RA

treatment site produced marked erythema as in

previous studies, and, was the reason for only a 4-

day patch protocol as opposed to the 8-day proto-

col used for the novel formulations.

The application of RA had very little effect on

the deposition of procollagen-I proximal to the DEJ

following 4-day occluded application (in agree-

ment with previous short-term studies). However,

0.2% SP significantly increased the levels of pro-

collagen-I relative to control (Fig. 3A, P < 0.05).

Increased levels of pro-collagen-1 can be visually

observed, especially after treatment with SP

(Fig. 3B).

Application of the gold standard, all-trans RA,

produced deposition of fibrillin-1 - proximal to the

DEJ – in 5 of the 10 volunteers (Fig. 4A,B). Simi-

larly, 0.2% SP resulted in significantly increased

fibrillin deposition in this volunteer subset

(Fig. 4A,B; P < 0.05). The re-appearance of the

candelabra-type fibrillin-1 microfibrils extending

down from the DEJ can be seen following RA- and

SP-treatment (white arrows; Fig. 4). Volunteers

who did not respond to either treatment had signi-

ficantly higher baseline fibrillin-1 levels [i.e. lower

photodamage despite there being no significant

differences in their age groups (data not shown)].

The application of RA had no effect on the

mean number of epidermal keratinocytes expres-

sing MMP-1 following a 4-day occluded applica-

tion. However, both 0.05% and 0.2%

concentrations of SP decreased the epidermal cell

expression of the MMP-1 levels significantly in the

8-day protocol (Fig. 5A,B).

Periorbital anti-wrinkle study using fast optical

in vivo topometry of human skin

Treatment of periorbital skin with 0.2% SP resul-

ted in significant improvements in skin smooth-

ing (Rz) and skin roughness (Ra) relative to

initial assessment values and treatment with

vehicle (P < 0.05; Fig. 6A). FOITS analysis indi-

cates that the application of SP improves both

fine lines and the macro structure of the skin.

The improvement of the skin’s structure was also

confirmed by the FDD method (Fig. 6B): SP was

able to reduce the wrinkle depth in the macro

structures, and supports the restructuring of fine

structure. The results obtained by the FOITS

method were further substantiated by a photo-

graphical analysis of the skin structure before,

and immediately after, the application period

(Fig. 7).

Discussion

Here, we describe the ability of a synthetically-

derived sphingolipid derived from the combination

of N-acylated salicylic acid to phytosphingosine to

form SP. This novel sphingolipid increases the syn-

thesis of procollagen-I by human adult dermal

fibroblasts in vitro, increases the expression of

fibrillin-1 and procollagen-I – key dermal ECM

components – while, at the same time, reducing

MMP-1 levels in an extended in vivo patch test

assay, and reduces the appearance of periorbital

wrinkles in a clinical study.

It is widely reported that in skin-ageing, numer-

ous alterations occur both in the structure and

function of the skin, especially in the dermis,

including reductions in the levels of fibrillar colla-

gens types I and III [5, 6], and severe truncation

of the fibrillin-rich microfibrillar apparatus at the

DEJ [7]. It is in this region where the exogenous

MMPs are thought to act, so remodelling these key

dermal proteins.

Partial dermal repair of the photoaged skin can

be induced by treatment with all-trans RA [12]

through the induction of new collagen and fibrillin

synthesis, while, at the same time, reducing the

levels of MMPs. Reduction in the activity of the

transcription factor AP-1 is believed to mediate

these RA-induced effects [27].

We were interested in the reported effects of sali-

cylic acid and its variants for their effects on skin

ageing [20]. It is also known that ligands for the

PPAR transcription factor are known to have

some skin anti-ageing effects, by increasing the

synthesis of both procollagen-I and decorin by

fibroblasts [22]. In this respect in vitro, both salicy-

lates [24] and PPAR ligands [21] are reported to

decrease the AP-1 activity. Recently, phytosphing-

osine was reported to be a PPAR-ligand which

may explain its anti-inflammatory activity [20].

As it is well established that skin collagen levels

decline in skin-ageing [1], we used an in vitro

procollagen-I fibroblast cell-based assay as the

primary screen to initially quantify the effects of

SP at the cellular level. This was the first time that

SP was shown to increase procollagen-I synthesis

in vitro, but it was crucial to confirm this action

in vivo and to ascertain whether other dermal-

marker proteins could be influenced.





A

BVehicle

Mea

n f

ibri

llin

imm

un

ost

ain

ing

4

3

2

1

0t-RA 0.05% SP 0.2% SP

**

Figure 3 (A) Effect of salicyloyl-phytosphingosine (SP) and all-trans retinoic acid (RA) application on dermal fibrilin-1

levels of photoaged skin. Formulations were applied under occlusion for 4 (all-trans RA) or 8 days (SP) and compared

to vehicle-treated skin. Significant increases in fibrillin-1 levels were observed for the all-trans RA (0.025%) and SP

(0.2%) formulations (P < 0.05). (B) Representative photomicrographs detail the positive effect of the occurrence of fibril-

lin, following treatment with all-trans RA (4-day treatment, d) or SP (8-day treatment, b and c). Vehicle (a). Magnifica-

tion, ·400.





A

BVehicle

Mea

n p

CI i

mm

un

ost

ain

ing

4

3

2

1

0

t-RA 0.05% SP 0.2% SP

*

Figure 4 (A) Effect of salicyloyl-phytosphingosine (SP) application on procollagen-I levels of photoaged skin. Formula-

tions were applied under occlusion for 4 (all-trans RA) or 8 days (SP) and compared to vehicle-treated skin. Significant

increases in procollagen-I levels were observed for the SP (0.2%) formulation (P < 0.05). (B) Representative photomicro-

graphs detail the positive effect of the occurrence of procollagen-I following treatment with SP (8-day treatment, b and

c). RA had no effect (d). Vehicle (a). Magnification, ·400.





A

B

Figure 5 (A) Effect of salicyloyl-phytosphingosine (SP) application on epidermal cell matrix metalloprotease-1 (MMP-

1) levels of photoaged skin. Formulations were applied under occlusion for 4 [all-trans retinoic acid (RA)] or 8 days

(SP) and compared to vehicle-treated skin. Significant decreases in MMP-1 levels were observed for the SP (0.05%

and 0.2%) formulation (P < 0.05). (B) Representative photomicrographs detail the diminution effect of the occurrence

of MMP-1, following treatment with SP (8-day treatment b and c). RA had no effect (d). Vehicle (a). Magnification,

·400.





We previously showed that topical application of

all-trans RA under occlusion for 4-days resulted in

the significant up-regulation of fibrillin-1 at both

mRNA and protein levels, similar to the partial

repair seen when used in the long-term (up to

4 years; 25). This identifies fibrillin-1 as a sensitive

biomarker for gauging dermal responses to topical

agents. More extensive treatment periods with all-

trans RA were shown to cause extensive erythema,

and, as a result, RA was only used for 4 days. As

SP had not previously been evaluated in vivo, we

utilized an 8-day protocol to maximize its activity.

Using all-trans RA as our positive control, we

applied our panel of synthetic sphingolipids under

occlusion for 8 days, and assayed biopsy samples

for procollagen-I, fibrillin-1 and MMP-1. And, SP,

applied at a concentration of 0.5%, produced

increases in the deposition of fibrillin-1 proximal

to the DEJ comparable to that observed following

topical RA use. The response exactly mirrored that

of all-trans RA in five of our 10 volunteers. Lack

of response – even to the ‘gold’ standard positive

control, all-trans RA – is probably due to the

higher baseline fibrillin-1 levels in some of these

volunteers [i.e. although clinically photoaged, their

dermal components showed reduced dermal altera-

tions histologically (not shown)]. Fibrillin-1 expres-

sion by both keratinocytes and fibroblasts suggests

that both the cell types contribute to maintaining

the microfibrillar network that extends from the

DEJ into the papillary dermis [7]. Haynes et al.

[30] reported that keratinocytes actively co-ordin-

A

B

Figure 6 (A) Improvement of skin profile and periorbital

wrinkle reduction, using Fast Optical In Vivo Topometry

of Human Skin following treatment with an SP-contain-

ing cream over 4 weeks on different skin-sites related to

initial value and vehicle (all values P < 0.05). (B)

Improvement in the micro, fine and macro structure of

the skin following treatment with an SP-containing

cream.

Figure 7 Representative photographical analysis of the periorbital skin structure. (A) Before application of SP-contain-

ing cream; (B) After 4 weeks of application; (C) Profile width (black: baseline, red: after 4 weeks).





ate the secretion, deposition and assembly of these

elements. Duplan-Perrat et al. [31] demonstrated

the influence of keratinocytes on the maturation

and organization of the elastic network, while

Dzamba et al. [32] demonstrated that keratinocytes

deposit fibrillin into the ECM in a non-fibrillar

form. More recently, Marrionet et al. [33] reported

that in reconstructed skin models, only fibroblasts

are involved in fibrillinogenesis, but that keratino-

cytes clearly influence them.

Surprisingly, the application of SP also resulted

in increased procollagen-I deposition in the papil-

lary dermis. All-trans RA does not result in the

deposition of procollagen-I in this occluded in vivo

system. This may be due to the short application

time period for RA: occlusion for longer than

4 days often results in deleterious side-effects (i.e.

blistering or epidermal fragility; 28, 29). Identifica-

tion of increased amino-pro-peptide within the

papillary dermis implies increased procollagen-I

synthesis.

To assess whether SP has a positive effect on ECM

remodelling, we examined its effects on the expres-

sion and distribution of MMP-1 – the major MMP

acting on fibrillar collagens in the papillary dermis.

All-trans RA in vitro has been shown to down-regu-

late the MMP-1 synthesis, but had very little effect

in our in vivo system. However, the sphingolipid

formulations containing the SP significantly

reduced the numbers of epidermal keratinocytes

expressing MMP-1. These enzymes may directly

degrade ECM proteins after their diffusion into the

dermis, but, as AP-1 controls the expression of

MMP levels in both keratinocytes as well as fibro-

blasts (34), we anticipate similar reductions in the

levels of dermal MMPs following these treatments.

The ultimate effect of SP was proven in a perior-

bital anti-wrinkle study using fast optical in vivo

topometry of the human skin (FOITS, 26). Com-

pared with placebo treatments, reduction in the

wrinkle-depth was quantified, together with a

skin-smoothening effect. We believe that these

improvements in the skin condition are related to

the increase in ECM marker proteins and the dimi-

nution in the levels of MMP-1.

Clearly, SP cannot only improve the levels of der-

mal marker proteins in aged skin, but it can also

clinically improve the skin condition. Although we

do not yet know the mechanism of action we postu-

late that its effects are due to the combined reported

effects of salicylic acid and phytosphingosine as a

PPAR ligand [20] on reducing the AP-1 activity

[21, 24]. Nevertheless, this remains to be deter-

mined, and will be clarified in the next phase of our

studies.

Acknowledgements

This work was totally funded by Degussa.

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