A synthetic hexapeptide (Argireline) with antiwrinkle activity

Abstract

Synopsis
Botulinum neurotoxins (BoNTs) represent a revolution in cosmetic science because of their remarkable and long‐lasting antiwrinkle activity. However, their high neurotoxicity seriously limits their use. Thus, there is a need to design and validate non‐toxic molecules that mimic the action of BoNTs. The hexapeptide Ac‐EEMQRR‐NH 2 (coined Argireline) was identified as a result of a rational design programme. Noteworthy, skin topography analysis of an oil/water (O/W) emulsion containing 10% of the hexapeptide on healthy women volunteers reduced wrinkle depth up to 30% upon 30 days treatment. Analysis of the mechanism of action showed that Argireline significantly inhibited neurotransmitter release with a potency similar to that of BoNT A, although as expected, it displayed much lower efficacy than the neurotoxin. Inhibition of neurotransmitter release was due to the interference of the hexapeptide with the formation and/or stability of the protein complex that is required to drive Ca ²⁺ ‐dependent exocytosis, namely the vesicular fusion (known as SNARE) complex. Notably, this peptide did not exhibit in vivo oral toxicity nor primary irritation at high doses. Taken together, these findings demonstrate that Argireline is a non‐toxic, antiwrinkle peptide that emulates the action of currently used BoNTs. Therefore, this hexapetide represents a biosafe alternative to BoNTs in cosmetics.

Full text

A synthetic hexapeptide (Argireline) with
antiwrinkle activity
C. Blanes -Mira

, J. Clementey,G.Jodasy,A.Gil
3
,G.Ferna
¤ndez-Ballester

,B.Ponsatiy,L.Gutierrezz,
E. Pe
¤rez-Paya
¤‰and A. Ferrer-Montiel


Centro Biolog|¤a Molecular y Celular, Universitas Miguel Herna
¤ndez, 03202 Alicante, yLipotec, S.A., Santa Eulalia 240,
08902 L’Hospitalet de Llobregat, Barcelona, zInstituto de Neu rociencias-CSIC, Universitas Miguel Herna
¤ndez,03550
Alicante, and ‰De partamento de Bioqu|¤m ica y Biolog|¤a Molecular, Universidad deValencia,46100 Burjassot,Valencia,
Spain
Received 24 June 2002, Acc epted 3 September 2002
Keywords: ageing, botulinum, cosmetics, exocytosis, glabella, rejuvenation
Synopsis
Botulinum neurotoxins (BoNTs) represent a revolu-
tion in cosmetic science because of their remarkable
and long-lasting antiwrinkle activity. However, their
high neurotoxicity seriously limits their use. Thus,
there is a need to design and validate non-toxic mole-
cules that mimic t heac tiono f BoNTs.The hexapeptide
Ac-EEMQRR-NH
2
(coined Argireline) was identi¢ed
asaresult of a rationaldesignprogramme.Noteworthy,
skin topography analysis of an oil/water (O/W) emul-
sion containing 10% of the hexapeptide on healthy
women volunteers reduced wrinkle depth up to 30%
upon 30 days treatment. Analysis of the mechanism
of action showed that Argireline signi¢cantly inhib-
ited neurotransmitter release with a potency similar
to that of BoNT A, although as expected, it displayed
much lower e⁄cacy than the neurotoxin. Inhibition
of neurotransmitter release was due to the interfer-
ence of the hexapeptid e with the formationand/or sta-
bility of the protein complex that is required to drive
Ca
2þ
-dependent exocytosis, namely the vesicular
fusion (known as SNARE) complex. Notably, this pep-
tide did not exhibit inv ivo oraltoxicity norprimary irri-
tation at high doses. Taken together, these ¢ndings
demonstrate that Argireline is a non-toxic, antiwrin-
kle peptide that emulates the action of currently used
BoNTs.Therefore, this hexapet ide represents a biosafe
alternativeto B oNTsin cos metics.
Re
´sume
´
Les Botulinum neurotoxins (BoNTS) repre
¤sentent
l’e
¤volution de la science cosme
¤tique gra
“ce a
'leur
remarquables actions anti-rides longue du re
¤e. Cepe n-
dant, leur importante neurotoxicite
¤limite se
¤rieuse-
ment leur usage. Il est donc ne
¤cessaire d’e
¤tudier et
de valider des mole
¤cules non toxiques qui s’app-
arentent a
'l’action des BoNTS. L’Hexapeptide Ac-
EEMQRR-NH
2
(Argireline) a
'e
¤te
¤identi¢e
¤suite a
'un
re
¤sultat d’u n programme d’e
¤tude rationnel. Une ana-
lyse de la topographie de la peau des femmes volon-
taires montre que des applications d’une e
¤mulsion
(H/E) contenant 10% d’Argireline ont reduit leurs
rides jusqu’a
'30% sur un traitement de 30 jours.
L’a nal yse du me
¤canisme d’action montre que l’A rgire-
line, par i nhibition du relarguage du neurot ra nsmet-
teur, produit des e¡ets semblables a
'celle du BoNTA,
bien que, comme attendu, son e⁄cacite
¤soit beau-
coup moins importante que celle des neurotoxines.
L’in hibition des neurotra nsmissions es t due a
'l’inter-
fe
¤rence de l’Argireline avec une formation et/ou la
stabilite
¤du complexe de prote
¤ines ne
¤cessaire a
'l’exo-
cytose Caþþ de
¤pendante, appele
¤le complexe de fu-
sion vesiculaire (SNARE). Normalement, l’Argireline
ne montre pas de toxicite
¤in vivo. Mises ensemble, ces
de
¤couvertes de
¤montrent qu’il s’agit d’un peptide
non-toxique et a nti-rides qui im ite l’act ion des BoNT S
couramment utilise
¤s. L’Argireline repre
¤sente donc
une alte rnative‘‘bio-safe’’ aux BoNTS e n cosme
¤tique.
Introduction
One of the most striking signs of skin ageing is the
relative intensity of frown and wrinkle lines of
International Journal of Cosmetic Scien ce,2002,24,303^310
ß2002 Blackwell Sci ence Ltd 303
Correspondence: Dr Antonio Ferrer-Montiel, Centro de Bio-
log|¤a Molecular y Celular, Universitas Miguel Herna
¤ndez,
03202 Elche, Alicante, Spain. Tel.: þ34 966658727; fa x:
þ34 966658758; e-mail: aferrer@umh.es

the forehead, glabella, lateral periorbital area as well
as the intensity of chin, upper lip wrinkling, nasola-
bial folds, nasal £are and platysma ne ckba nds among
others [1]. This can occur naturally over time and is
identi¢ed by certain biochemical, histological and
physiological changes t hat a re enhanced by e nv iron-
mental exposure. There are, however, other second-
ary factors that can cause characteristic folds,
furrows and creases of the face, such as the constant
pull of gravity, frequent and c onstant positional pres -
sure of the skin of the face (e.g. during sleep) or
repeated fac ial move ments caused by the c ontraction
of the muscles of facial expression [1, 2].
Physiologically, the formation of wrinkles appears
to be due, at least partly, to the excessive stimulation
of the muscle ¢bres in the face, which pull inwards
the skin giving rise to the well-known wrinkle [1^3].
Thus, a useful strategy to reduce the intensity of
wrinkle lines is to down-regulate muscle action
either directly or by attenuating the activity of the
inner vati ng ne urone [1,3^5]. I n support of t his tenet,
treatment with botulinum neurotoxin (BoNT) A sig-
ni¢ca ntly reduces t he intensit y of frown a nd wrink le
lines. BoNTs strongly inhibits the Ca
2þ
-dependent
neurotransmitter release in neurones [6, 7]. These
proteins a re metalloprotea ses that s peci¢ca lly cleave
synaptic proteins essential for regulated neuronal
exocytosis [6, 7], speci¢cally the vesicular protein
VAMP, and the membrane proteins syntaxin and
SNAP-25 [7]. As a result, the critical protein fusion
complex assembled by these proteins, known as
SNARE complex, is destabilized preventing vesicle
fusion with plasma membrane, and consequently
abrogating Ca
2þ
-triggered exocytosis [7].
Although botulinum neurotoxins, specially BoNT
A (BOTOX), has been extensively used to attenuate
facial ageing signs, its use is limited because of its
high toxicity (human LD
50
2500 biologic mouse
units) [1,6]. Paradoxically, BoNTA is the most potent
toxin known to humankind and therefore BOTOX
treatment has to be under strict medical control [1,
3^5]. To circumvent this limitation, small molecules
that mimic the action of BoNTs is being pursued [8^
10]. Int his regard, synthetic pepti des of 20 me r that
emulate the a mino acid se quence of t he synaptic pro-
tein SNAP-25 were shown to be speci¢c inhibitors of
neurose cretion at micromolar c oncentrations [8^10].
The lengt h of these pept ides, however, a long with a
poor membrane permeability signi¢cantly limited
their cosmetic utility. Accordingly, there is a need to
identify new sequences that are shorter while pre-
serving a biological activity. We have used rational
design to address this issue.We report the identi¢ca-
tion of a 6 -mer peptide (Ac-EEMQRR-NH
2
),patterned
after the N-end of the N-terminal domain of SNAP-
25 (a a 12^17), t hat interfe r es with the assembly of
the SNARE ternary complex, and inhibits Ca
2þ
-
dependent catecholamine release from chroma⁄n
cells. The hexapeptide was coined with the name
Argireline. This peptide exhibited a signi¢cant skin
permeation. Noteworthy, topical use of O/W emul-
sions containing 10% of the peptide reduced the
intensity of wrinkles in the lateral preorbital area
healthy human volunteers. Toxicological and irrita-
tion primary data ind icate that Argireline is well tol-
erated. Collectively, these ¢ndings demonstrate the
feasibility of the rational strategy to ¢nd peptide-
based mimetics of BoNTs action, and indicate that
these peptides are a biosafe cosmetic alternative to
attenuate facial wr inkles.
Materials and methods
Peptide synthesis and puri¢cation
Peptides were synthesized by Fmoc chemistries by
solid-phas e synthesis as described [8]. Brie£y,Argi re-
line was synthesized by solid phase on a pMBHA-
resin (p-methylbenzydrilamine-resin) with AM-han-
dle, which allows the cleavage of the peptide amide
in acid condit ions with the concom itant deprotection
of the side chains protection. The elongation of the
peptide chain was carried out using the Fmoc/tBu
strategy. The resulting peptidyl resin was treated at
room temperature with a mixture of TFA/thioani-
sol/H
2
O (95/2.5/2.5, v/v/v,7 ml g
1
resin) for 2 h. The
crude peptides were precipitated by ¢ltration into
cold diethyl ether and vacuum-dried. The crude
product was d issolved i n 10 % acet ic acid for de-tert-
butylation at 60 8C and treated with DIAION for
puri¢cation. Characterization was done by ESI/MS
and analytical HPLC using Kromasil C8 column
(4.6 mm 250 mm, 5 mm, 100 —) £ow rate 1 ml
min
1
, eluent A 0.1% TFA, eluent B 0.07% TFA in
CH
3
CN. Elution conditions: isocratic 11% and 0 ^40%
in 30-min g ra dient.
Recombinant SNARE proteins expression and
puri¢cation
Recombinant VAMP (lacking the transmembrane
segment) and cytosolic domain of syntaxin were
expressed as GST fusion proteins in the E. coli strains
BL21DE3 and C43, respectively. Protein expression
A synthetic hexapeptide with antiwrinkle activity Blanes-Mira et al.
304 ß2002 International Journal of Cosmetic Science,24,303^310

was induc ed with 1 mMIPTG for 5 h at 30 8C, and
puri¢ ed from bacterial extract sby a⁄nity chromato-
graphy on glutathione agarose as described [11].
Resin-bound fusion proteins were released by diges-
tion with thrombin protease (Pharmacia) for 2 h at
23 8C [11]. Proteins concentration was assayed with
the BCA kit (Pierce), andpurity veri¢ed bygel analysis.
In vitro
expression of SNAP-25
In vitro translation of the cDNA clone coding for
SNAP-25 from rat brain in the presence of
[
35
S]methionine involved a transcription^transla-
tion-coupled reticulocyte lysate system (Promega) as
described [12].
In vitro
reconstitution of SNARE complex and
modulation by Argireline
SNARE complex was reconstituted using the recom-
binant VAMP and syntaxin proteins and in vitro
translated [
35
S]SNAP-25. Brie£y, equimolar amounts
of VAMPa nd syntaxi n were incubated in t he absence
or presence of Argireline for 2 h at 4 8C. Thereafter,
SNAP-25 was added to the mixture and the reaction
proceeded for 3 h at 4 8C. Complex assembly was
stopped by addition of SD S-PAGE sa mple bu¡er. Sam-
ples were analysed by SDS-PAGE on 12% gels, fol-
lowed by £uorographic detection on Kodak X-Omat
AR X-ray ¢lms.
Chroma⁄n cell cultures and secretion assays
Chroma⁄n cell cultures were prepared from bovine
adrenal glands by collagenase digestion and further
separated from debris and erythrocytes by centrifu-
gation on Percoll gradients as described [8^10].
Brie£y, cells were maintained in monolayer cultures
at a density of 625 000 cells cm
2
and were used
between t he third a nd sixth day after plati ng. A ll the
experiments were performed at 37 8C. Secreted
[
3
H]noradrenaline was determined in digitonin-per-
meabilized cells as described [8^10]. The CPM
released from control cells under basal conditions
were 3000, and they were increased to 11000,
when st imulated w ith 10 mMCa
2þ
. The tota l number
of counts obtained from detergent-permeabilized
cells was 110 000. Thus, the normalized basal
release represents the 3.5% of the total secretion,
and the Ca
2þ
-evoked 10%. Statistical signi¢cance
was calculated using Student’s t-test with data from
four or more i ndependent expe riments.
Stratum corneum assay
Human ski n wa s obtained from d i¡erent donors who
underwent cosmetic surgery. All fat was removed
from fresh or frozen skin pieces with a scalpel. The
skin was then submitted to ammonia vapours in a
closed recipient at room temperature for 30 min.
The skin was placed, stratum corneum side upper-
most, on a glass surface and the epidermiswas teased
gently away from the underlying dermis using the
tip of a glowed ¢nger. To prepare stratum coreum
samples, the epidermal membranes were £oated
overnight stratum corneum side up, on a aqueous
solution of trypsin solution (0.01%, pH ¼8^8.6). To
remove digested matter, membranes were squeezed
between two ¢lter papers, placed on the ¢lter paper
with nucleated tissue side uppermost, and any
remaining digested material was removed by wash-
ing with water and gentle swabbing. The SC pieces
were £oated on 0.001% aqueous NaN
3
solution for
10 min before drying inserted between ¢lter paper
sheets in a desiccator. Immediately before use, mem-
branes were £oated with the stratum corneum side
up, on 0.002% aqueous s odium azi de for1 h.
Permeation experiments were carried out using a
static cell manually sampled as described [13]. The
cell was fabricated of glass and had two chambers,
an upper chamber (donor chamber), and a lower one
(receptor chamber); the average di¡usion area was
1.3 c m
2
. The receptor chamber volume was 4 ml in-
cluded that of the out£ow tubing. Receptor reservoir
was continuously stirred and thermostated through
its connection to a circulating bath maintained at
37 8C. A disk of ¢lter paper (to act as rigid support)
was located between both chambers and skin disks
of about 2 cm
2
were mounted on them. Isotonic phos-
phate bu¡er, pH ¼7.4, with 0.01% sodium azide as
preservative, was used as the receptor £uid. Samples
(0.5 ml of an aqueous solution of Argireline) were
poured softly in the donor chamber and 100 mlali-
quots of receptor £uid were periodically withdrawn
for analysis and replaced with an equal volume of
fresh receptor £uid. Argireline concentration in the
receptor £uid was quanti¢ed by HPLC on C-18 col-
umns, workingi n isocraticway11% ACN (0.05% T FA).
Antiwrinkle test on healthy humans
Skin topography analysis for measur ing the e¡ective-
ness of an O/Wemul sionc ontaining10% o f Argireline
(solution presentation) was performed obtaining
silicon imprints from the lateral preorbital region of
A synthetic hexapeptide with antiwrinkle activity Blanes-Mira et al.
ß2002 Internati onal Journ al of Cosmetic Science,24,303^310 305

10 hea lthy womenvoluntee r who apply the emul sion
twice a day. Volunteers apply the emuls ion c ontain-
ing 10% of Argireline in one lateral preorbital area,
and the emulsion alone in the contralateral side. Sili-
con impri nt s were obta ined a fter 0, 15 and 30 days,
and analysed by confocal laser scanning microscopy
to assess the evolution of the skin surface before and
after treatment. Confocal Microscopy in re£ection
mode and three-dimensional analysis to assess the
di¡erent parameters of roughness was used.
The same skin areas were selected before the pro-
cessing (Day 0) and after the processing (Days 15 and
30), by means of observation under magnifying glass
(Leica M LZ III, augment 10) with outer white light.
Three sample areas (2.25 mm
2
) were measured for
each replica (n¼30, for each t ime point). The selected
regions were clean, free of any strange particle, with-
out zones of ¢ssure or pores in silicone. Later, a frag-
ment of the selec ted wrinkles was cut to and mounted
on a microscope slide. The observation was made by
means of a con focal microscope TCS SPII adapted to a
motorized microscope Leitz DMIRB (lens Leitz 10,
NA 0,3 s, n¼1).Thepickupwasmadeinmodeofre£ec-
tion with an only photomultiplicator. The con¢gura-
tion of ¢lters was: re£ection:excitation:488-nm, Beam
Splitter RT30/70. The exploration surface was of
2.25 mm
2
.Inrouteinzwas of 500 mm. Each series of
images consists of 201 optical sections separated
among them 2.5 mm. Final measures of obser vation of
each ¢eld (x,y,z):1500 mm150 0 mm50 0 mm.
Roughness parameters were calculated according
to the UNE EN ISO 4287 normative and roughness
di¡erences for each volunteer were assessed by cal-
culating the decrease percentage of the Pa (arith-
metic roughness, equivalent to the Ra, DIN 4768)
between days 0 and 15 or 30. Roughness di¡erences
between groups are expressed as a mean of the Pa
decrease percentages. Comparisons between control
and treated areas were evaluated with the F-Fisher’s
test with statistical signi¢cance set at P<0.075.
Three-dimensional reconstructions
For each sample the following three observations of
macro-relief were made:
Topographic image (depth-coded image)
The topographic image obtained from the series of
sections is a real map of the structure of super¢cie of
the sample. It examines the points of the sample
(voxel) that are superposed throughout z-axis of all
the ser ies of optical sections.
Three-dimensional graph
It was obtained from the topographic image. It pro-
vides a three-dimensional image of the map of sur-
face of the sample. They have been made graphical
three-dimensional from the topographic image of
the retort and the real topographic image of the skin
(inverted process) in di¡erent angles.
Measures of roughness parameters
The measurement of the parameters of roughness
(Ra) was p erformed with Leica TCS SPII sof tware, fol-
lowing the UNE EN ISO 4287 norm (geometric pro-
duct Speci¢cation).
Results and discussion
Rational design of Argireline
Sequence a nd structure a na lysis of the N-ter minal of
SNAP-25 revealed t he s equence E EMQRR (aa12^17)
that display a high propens ity to acquire an a-helical
structure along with a pronounced coiled-coil prob-
ability (Fig. 1A). AGADIR, a programme that estimates
helical propensity of peptides, predicted a remarkable
12%probabilityfor this smallpeptide.These properties
suggest that a peptide patterned after this sequence
may modulate Ca
2þ
-dependent exocytosis, similar to
those peptides derived from the C-terminal of SNAP-
25 [8^10]. The acetylated and amidated peptide was
coined with the term A rgireline.
Argireline interferes with the formation of the
SNARE complex
To evaluate the potential antiwrinkle activity of
the hexapeptide, we ¢rst determined if the peptide
prevents or destabilizes the formation of the SNARE
complex in vitro. For this task, we used recombinant
synaptic proteins VAMP, syntaxin and in vitro tran-
scribed and translated [
35
S] S N A P-25. As d e p i c ted
in Fig. 1B, i ncubation of the thre e sy naptic proteins
led to the formation of a protein complex of 75 kDa
that was resistant to the chaotropic detergent SDS
(lane 2), but sensitive to heat (lane 3), two well-
known properties of the SNARE complex [14].When
the proteins were incubated with Argireline, the
formation of the SNARE complex was prevented in a
dose-dependent manner (lanes 4 and 5). Note that
at 2 mMthe 75 kDa band was undetectable, suggest-
ing complete abrogation of complex formation by
the small peptide. These results demonstrate that
Argireline can prevent the assembly of the protein
complex that drives Ca
2þ
-dependent exocytosis in
A synthetic hexapeptide with antiwrinkle activity Blanes-Mira et al.
306 ß2002 International Journal of Cosmetic Science,24,303^310

secretory cells, thus implying that this peptide may
modulate neurotran smitter release f rom these cells.
Argireline inhibits catecholamine release from
chroma⁄ n cells
To test the cellular activity of the hexapeptide, we
measured the inhibitory activity of this peptide on
Ca
2þ
-evoked neurotransmitter release from digito-
nin-permeabiliz ed chroma⁄n cells. This is a reliable
assay that allows a rapid assess ment of the biological
activity of toxins and peptides [8^10]. As illustrated
in Fig. 2, detergent-permeabilized chroma⁄n cells
release both noradrenaline and adrenaline in
response to a raise in intracellular Ca
2þ
. Catechola-
mine relea se was inhibited up to 60% by 20 nMBoNT
Figure 1 Rational design (A) and in vitro activity (B) of a 6-mer peptide derived from the N-terminal domain of SNAP-25. (A)
Amino acid sequence of SNAP-25 was analysed forcoile d-coil propensity (ExPaSy) and a-helicalcontent (AGADIR).Thesequence
Ac-EEMQRR-NH
2
(aa12^17), c oined Argireline,showed a signi¢cant probability for both properties. (B) In vitro reconstitutionof
the SNARE complex. Assembly of the complex was performed using recombinant VAMP and syntaxin, and in vitro translated
[
35
S]SNAP-25.(1)[
35
S]SNAP-25;(2)SNARE complex; (3) SNAREcomplex þ10 0 8C for 5 min;(4) SNAREcomplex þ1mMArgire-
line; (5) SNARE complex þ2m
MArgireline.
Figure 2 Argireline inhibited Ca
2þ
-dependent exocytosis f rom permea-
bilized chroma⁄n cells. Digitonin
permeabilization lasted 5 min and
[
3
H]noradrenaline secretion was
evoked in the absence (5 mMEGTA)
or presence of 10 mMCa
2þ
. Shown is
the e¡ect on the basal and Ca
2þ
-sti-
mulatedrelease of100 mMofthe hexa-
peptide. Data are mean from three
di¡erent experiments. Data for
ESUPE were taken from [10] and for
BoNTA from[9].
A synthetic hexapeptide with antiwrinkle activity Blanes-Mira et al.
ß2002 Internati onal Journ al of Cosmetic Science,24,303^310 307

Figure 3 Argireline ex hibits invivo activity. Skin topographic imprints of t hepre orbital region of a healthyhea lthyvoluntee r(age 38) treated wit ha ndO/W e mulsion without (base) or with
10% the hexapeptide. Silicone imprints were taken before the onset of treatment, after 15 days treatment and after 30 days treatment. Imprints were processed by confocal microscopy.
Three-d imensional reconst ructions were obtained a s described in method s.
A synthetic hexapeptide with antiwrinkle activity Blanes-Mira et al.
308 ß2002 International Journal of Cosmetic Science,24,303^310

Aandupto55%by1mMof a 26-mer peptide derived
from the C-terminal end of SNAP-25 (ESUP-E).
Remarkably, 100 mMArgireline inhibited 30% of the
total catecholamine exocytosis. Dose^response
curves indicated an IC
50
of 110 mMfor Argireline,
which is 5000higher than the characteristic of
BoNT A, and 400higher than that of ESUP-E. To
homogeniz e the potential antiw rinkle act ivityo f syn-
thetic peptides that mimic the action of BoNT A, we
propose to de¢ne the antiwrinkle activity unit
(AAU) as the ratio of the IC
50
of ESUP-E, the most
potent synthetic peptides, divided by the IC
50
of the
desired peptide. Accordingly, Argireline would have
0.003 AAU. Because this parameter refers to the
activity blocking exocytosis from detergent-permea-
bilized cells, it may be modi¢ed depending on the
ability to permeate through cell membranes. None-
theless, the AAU provides an operational observable
to classify the family of synthetic peptides that emu-
late the activity of naturally occurring botulinum
neurotoxins.
Argireline attenuates wrinkle depth
in vivo
in
healthy volunteers
To assess the antiwrinkle activity of Argireline, we
¢rst evaluated the skin permeation ability of the
peptide. For this purpose, we investigated in vitro its
ability to permeate through stratum corneum
samples from human skin. The hexapeptide was
placed into the donor chamber, and the content
of peptide was determined in the receptor reser-
voir 2 h after placement. The total content of
peptide in the receptor reservoir was a signi¢cant
30% of the amount deposited onto the membrane in
the donor chamber. This result indicates that Ar-
gireline has the capability to permeate through the
skin and, therefore, may exhibit in vivo antiwrinkle
activity.
Accordingly, we next performed skin topography
analysis to determine the e¡ectiveness of an O/W
emulsion containi ng 10% o f A rgireline us ing sil icon
imprints from the lateral preorbital area in healthy
women volunteers. Subjects applied the O/W emul-
sion containing Argireline in one lateral preorbital
side, although admi nistered O/W emulsion alone in
the contralateral side. All subjects applie dt wice daily
the emuls ion for 30 days. Silicone imprints were ana-
lysed at days 0, 15 and 30 byconfocal microscopy. As
illustrated in Fig. 3, topic al application of t he O/W
emulsion containing the hexapeptide resulted in a
signi¢cant attenuation of the depth and roughness
of the wrinkles. Use of the O/Wemuls ion for the same
period of time did not result in signi¢cant changes in
the skin topography. Quantitative analysis and nor-
malization of the silicon replicas show that, whereas
the O/W base emulsion reduced by 10% the depth of
skin wr inkles, the O/W emulsion contain ing the pep -
tide decreased them by 30% (Fig. 4). These ¢ndings
demonstrate a signi¢cant antiwrinkle activity for
Argireline, in agreement with its in vitro and cellular
activities.
Conclusion
A 6-mer peptide patterned after the N-end of the
SNAP-25 protein that mimics the activity of BoNTs
in terms of inhibiting Ca
2þ
-dependent exocytosis,
display also remarkable antiwrinkle activity when
applied topically. Although much less potent than
BoNT A (12 vs. 0.003 AAUs), this small p eptide exhi-
bits the great advantage o f its ins igni¢cant acute t oxi-
city (2000 mg kg
1
) as compared with BoNT A
(20 ng k g
1
). Furthermore, the hexapeptide does not
exhibit primary skin irritation in an intracutaneous
test nor ge notoxicity as dete rmined by the A MES tes t
(data not shown), thus making its use highly safe
and physician-independent. Therefore, peptides that
mimic t he action of B oNTs, s uch as Argireline, repre-
sent the next generation of biosafe products with
antiwri nkle activit y which could be e xtensively used
in cosme tic preparations.
Figure 4 Argireline atte nuates w rinkle intens ity. The pe r-
centage of wrinkle formation as a function of day of treat-
ment with O/W emulsion without or with 10% the
hexapeptide.Wrinkle formation was obtained by meas uring
the depth of the wrinkle, and normalizing the values to
those obtained before initiating the treatment. Data are
mean of 10 healt hy women volunteers.
A synthetic hexapeptide with antiwrinkle activity Blanes-Mira et al.
ß2002 Internati onal Journ al of Cosmetic Science,24,303^310 309

Acknowledgements
This work has bee n funded by Lip otec SA, and a grant
from the Ministry of Science and Technology
(CICYT-PETRI) toA.F-M.
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