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A synthetic hexapeptide (Argireline) with antiwrinkle activity


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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.
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A synthetic hexapeptide (Argireline) with
antiwrinkle activity
C. Blanes -Mira
, J. Clementey,G.Jodasy,A.Gil
E. Pe
¤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
Alicante, and ‰De partamento de Bioqu|¤m ica y Biolog|¤a Molecular, Universidad deValencia,46100 Burjassot,Valencia,
Received 24 June 2002, Acc epted 3 September 2002
Keywords: ageing, botulinum, cosmetics, exocytosis, glabella, rejuvenation
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
(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
-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.
Les Botulinum neurotoxins (BoNTS) repre
¤volution de la science cosme
¤tique gra
ce a
remarquables actions anti-rides longue du re
¤e. Cepe n-
dant, leur importante neurotoxicite
¤limite se
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-
(Argireline) a
¤suite a
¤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
(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
¤rence de l’Argireline avec une formation et/ou la
¤du complexe de prote
¤ines ne
¤cessaire a
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
¤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
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
03202 Elche, Alicante, Spain. Tel.: þ34 966658727; fa x:
þ34 966658758; e-mail:
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
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
-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
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
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
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-
O (95/2.5/2.5, v/v/v,7 ml g
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
, eluent A 0.1% TFA, eluent B 0.07% TFA in
CN. Elution conditions: isocratic 11% and 0 ^40%
in 30-min g ra dient.
Recombinant SNARE proteins expression and
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
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 [
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
and were used
between t he third a nd sixth day after plati ng. A ll the
experiments were performed at 37 8C. Secreted
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
. 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
-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
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
. 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
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
) 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
.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 mm150 0 mm50 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
-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 [
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
-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
-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
. 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
(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
S]SNAP-25;(2)SNARE complex; (3) SNAREcomplex þ10 0 8C for 5 min;(4) SNAREcomplex þ1mMArgire-
line; (5) SNARE complex þ2m
Figure 2 Argireline inhibited Ca
-dependent exocytosis f rom permea-
bilized chroma⁄n cells. Digitonin
permeabilization lasted 5 min and
H]noradrenaline secretion was
evoked in the absence (5 mMEGTA)
or presence of 10 mMCa
. Shown is
the e¡ect on the basal and Ca
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
of 110 mMfor Argireline,
which is 5000higher than the characteristic of
BoNT A, and 400higher 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
of ESUP-E, the most
potent synthetic peptides, divided by the IC
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
Argireline attenuates wrinkle depth
in vivo
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
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
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
-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
) as compared with BoNT A
(20 ng k g
). 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
This work has bee n funded by Lip otec SA, and a grant
from the Ministry of Science and Technology
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A synthetic hexapeptide with antiwrinkle activity Blanes-Mira et al.
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... However, their high neurotoxicity presented a problem and created a need to find alternative options that produced similar results with lower toxicity. The first scientific publication on the usage of Argireline for aesthetic improvements of wrinkles occurred twenty years ago [1]. Argireline, an acetyl hexapeptide-8 formulation cited as having properties similar to botulinum, was provided to 10 healthy females in an oil/water emulsion that contained 10% Argireline. ...
... In comparison to the first study on Argireline by Blane in 2002, the current study involved a larger number of study participants [1]. Blane's study involved 10 participants, a sample size that is rather too small to draw any generalizable findings from. ...
... A lack of skin permeability to Argireline or its overall limited efficacy should also be considered. Another issue regarding previous research into the possible effects of Argireline is that such studies based their claims not only on a limited number of participants [1], [3] but also on subjective assessment methods [4]. A study by Wang used subjective assessments in the form of four-and five-point scales [5], [6]. ...
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Objective To analyze the effects of Argireline on skin surface wrinkles using the Visia® camera system developed by Canfield Scientific Inc., U.S.A., for facial image capture. Method Nineteen female participants were recruited from a plastic surgery clinic. Initial facial images captured the left, front, and right sides of the participants’ faces, which were documented as timepoint one. Following this, the participants immediately began to apply a facial skin serum containing triple hyaluronic acids produced by CNC cosmetic GmbH, Philippsburg, Germany. The serum was applied once in the morning and once in the evening. Participants received two identical containers labeled L for left and R for right, with each container to be used on the corresponding facial side, particularly around the eye area. One container contained Argireline, a synthetic hexapeptide, which previously was deemed to be a biosafe alternative to botulinum neurotoxin. The study was conducted as double-blind; neither the participants nor researchers knew which of the two containers contained Argireline. Participants were allowed to use their own cosmetic products throughout the study. After four weeks, the participants returned to have their faces recaptured using the Visia® camera, which was documented as timepoint two. The absolute scores of the wrinkles were noted, and results on both sides of the face were calculated and compared. The “TruSkinAge®” measurement provided by the Visia® camera was reviewed for each face side. Results between both time points and both sides of the face were compared. After the data analysis was complete, the company was contacted to determine which container contained Argireline. Results Nineteen participants returned for facial image capture. There were no significant adverse events, allergic reactions, or skin irritations. The investigation revealed that the wrinkle score slightly decreased for the right and left side of the face following four weeks of serum application. However, this decrease was not significant (p>0.05) based on the Wilcoxon matched pairs tests for the wrinkle scores (right side p=0.060 and left side p=0.176) and Truskin Ages® results (right side p=0.096 and left side p=0.489).Comparing the data from the right side with that from the left side of the face revealed that neither demonstrated a significant reduction in wrinkle score (p=0.829) or Truskin Ages® results (p=0.804). Argireline was included in the serum applied to the right side of the face. However, no statistical significance was seen in the results on this side of the face indicating any possible effects. Conclusion Wrinkle scores and Truskin Ages® results were observed to decrease non-significantly following the application of a skin serum involving hyaluronic acid. The Visia® imaging method was used to analyze the data objectively. Differences between both sides of the face that were treated with and without Argireline were not statistically significant. Therefore, the effect of Argireline was not proven. While Argireline presented with low toxicity, its efficacy was found not to be significant. Therefore, it is not deemed to be an alternative treatment to botulinum toxin.
... One of the best-known peptides with neurotransmitter-inhibiting properties is the synthetic hexapeptide, acetyl hexapeptide-3 or Argireline ® (Lipotec, Barcelona, Spain). It has shown promising clinical results in reducing wrinkle depth when applied topically at a 10% concentration, [84][85][86] and has been reported to induce collagen synthesis in mouse models. 87 Pentapeptide-18, or Leuphasyl ® (Lipotec), is another popular active peptide with neurotransmitter-inhibiting properties used in antiageing formulations. ...
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Skin ageing is a complex process involving the additive effects of skin’s interaction with its external environment, predominantly chronic sun exposure, upon a background of time-dependent intrinsic ageing. Skin health and beauty is considered one of the principal factors perceived to represent overall ‘health and wellbeing’; thus, the demand for skin rejuvenation strategies has rapidly increased, with a worldwide annual expenditure expected to grow from $US24.6 billion to around $US44.5 billion by 2030 ( Skin rejuvenation can be achieved in several ways, ranging from laser and device-based treatments to chemical peels and injectables; however, topical skin care regimes are a mainstay treatment for ageing skin and all patients seeking skin rejuvenation can benefit from this relatively low-risk intervention. While the most efficacious topical rejuvenation treatment is application of tretinoin (all-trans retinoic acid) – a prescription-only medicine considered to be the clinical ‘gold standard’ – a hybrid category of ‘cosmeceutical’ products at the midpoint of the spectrum of cosmetics and pharmaceutical has emerged. This article reviews the clinical manifestations of skin ageing and the available topical treatments for skin rejuvenation, including retinoids, peptides and antioxidants.
... Additionally, PO enhances the production of hyaluronic acid by dermal fibroblasts [97]. These findings suggest that the amounts of PO and OP in blood are important factors in showing the efficacy of collagen hydrolysates on skin health which leads to the improvement in facial skin conditions, including facial skin moisture, elasticity, wrinkles and roughness by topical application [97] [98] [99] [100] [101]. While uneven skin pigmentation is a significant cosmetic concern, the tetrapeptide PKEK was found to exert skin whitening effects based on one in vitro and four double-blinded vehicle-controlled in vivo studies [102]. ...
... 16,17,25 Thus, they prevent the signs of premature skin aging. 19,39 Signal peptides. Signal peptides can moderate the turnover of skin proteins. ...
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Cosmeceuticals, i.e., cosmetic products with active ingredients possessing the scientifically proven and thoroughly evaluated biological activity, are becoming progressively more common on the market. Consumer awareness has prompted manufacturers to create formulas with a valuable composition, good permeability through the skin, and prolonged stability. Peptides, short chains of amino acids, are good candidates for active ingredients. Due to the ease of their modification, uncomplicated synthesis, and the possibility of giving them the desired properties, they are becoming frequent ingredients in cosmeceuticals. This doctoral dissertation discusses two enzymes contributing to the appearance of signs of skin aging – elastase, responsible for the breakdown of collagen fibers, and tyrosinase – directly affecting the synthesis of melanin and skin discoloration. Design, synthesis, biological investigation, and molecular modeling of peptides and their conjugates with small organic molecules were discussed. In vitro studies of these compounds indicated inhibitors of the abovementioned enzymes, some of them with micromolar activity. The correlation between the structure of obtained compounds and their activity was discussed, and special attention was paid to the role of peptide conjugates in the design of biologically active compounds.
... 10,11 Like classic retinol, peptides are also one of the most attention-worthy actives, especially soothing peptides for expression lines that have been reported and popularized 10 years ago. 12,13 In addition to the reported anti-wrinkle effects of retinol and neurotransmitter suppressive peptides in Chinese populations, our clinical study examined improvements in skin textures, elasticity, firmness, pores, gloss and stratum corneum hydration. Our study validated the feasibility of using retinol twice daily and supplemented the data and records of retinol-related efficacy dimensions in the Chinese population. ...
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Background The anti‐ageing gold standard, retinol, has been widely recognized for its anti‐wrinkle benefits in the Chinese population. Studies have shown that Asians are more sensitive to retinol compared to their Caucasian counterparts, and it is generally recommended to use retinol once a day in the evening. However, there are few reports on the most appropriate concentration and frequency of retinol use in the general Chinese population. Objectives In this study, supramolecular retinol was prepared using cyclodextrin encapsulation technology, and the most appropriate concentration for the general Chinese population was investigated. Then, a cosmetic essence was developed by combining the classic supramolecular retinol, which promotes collagen regeneration, with acetyl hexapeptide‐1, a popular ingredient known for reducing expression lines. The safety and efficacy of this cosmetic essence were studied through clinical tests. Methods First, a patch test was conducted on 32 healthy Chinese subjects to compare the tolerance of supramolecular retinol to non‐encapsulated retinol and to select the optimal concentration of retinol. Then, an 8‐week clinical study was conducted using a twice‐daily cosmetic essence containing 0.1% supramolecular retinol and 0.02% acetyl hexapeptide‐1 to treat mild photoaging in 32 middle‐aged Chinese women. Dermatological evaluations and instrument measurements were taken at baseline, 4 weeks, and 8 weeks. Efficacy was assessed using facial skin wrinkles, textures, elasticity, firmness, pores, gloss and stratum corneum hydration. Tolerability was assessed throughout the study. Results Our patch test results showed that supramolecular retinol was better tolerated than non‐encapsulated retinol, and our findings suggest that 0.1% was the approximate optimal retinol concentration for the general Chinese population. The cosmetic essence studied was effective in improving the appearance of photoaged skin in the Chinese population in all aspects studied and was well tolerated. Conclusions 0.1% retinol is suitable for twice daily use in the general Chinese population. Data and records on efficacy dimensions of skin textures, elasticity, firmness, pores, gloss and stratum corneum hydration for retinol in the Chinese population are supplemented with our study. Cosmeceutical approaches targeting both static and dynamic wrinkles are of value for treating the photoaged Chinese population.
Вступ. Відомо, що гідролізати (пептиди) колагену, отримані з побічних продуктів харчової промисловості, відіграють різноманітну біологічну роль як біологічно активні амінокислоти. Пептиди постійно синтезуються у всіх живих організмах для регулювання фізіологічних процесів. Насамперед, це органічні сполуки, які утворюються внаслідок поліконденсації амінокислот (взаємодії їх α-карбоксильної та α-аміногруп), створюють захисну гідроліпідну оболонку епідермісу та відіграють надважливу роль в процесі росту клітин шкіри, активуючи їх для виробництва колагену. Ці аспекти з успіхом застосовуються в галузі дерматології та косметології. Мета дослідження. Вивчити особливості технологічного процесу виготовлення емульсійного ЛКЗ із пластично-пружно-вʼязким дисперсійним середовищем (крему) на основі пептидів природного походження. Для досягнення означеної мети вирішувалися наступні задачі: 1) провести аналіз сучасного арсеналу поліпептидів; 2) розробити рецептуру емульсійного косметичного крему на основі пептидів рослинно-мікробного походження, охарактеризувати його основні складові; 3) вивчити особливості технології цієї лікарської форми, розробити відповідну блок-схему. Матеріали та методи. При проведенні експериментальних досліджень матеріалами слугували активні фармацевтичні інгредієнти – пептиди рослинно-мікробного походження – гексапептид-10 (Hexapeptide-10, Serilesine) та ацетил-гексапептид-8. При проведенні досліджень використовували загальноприйняті стандартні, описані в літературі методи проведення технологічних досліджень м’яких ЛФ і відповідні прилади, а також нові методики дослідження, які модифіковані для вивчення розробленого лікувально-косметичного засобу. Для проведення контролю якості зразків запропонованої лікарської форми дотримувалися рекомендацій і методик, наведених у ДФУ І вид., розділ «М’які лікарські засоби для місцевого застосування». Регламентацію показників якості та характеристик проводили згідно ДФУ, Настанов та іншим нормативним документам. Результати дослідження. Розроблена типова рецептура означеної мʼякої лікарської форми на основі пептидів з використанням емульсійної основи. До складу олійної фази (25 %) входять лише природні жири, воски та олії, які мають не лише формоутворюючу здатність, а й забезпечують живлючий ефект шкіри. Проведення лабораторних досліджень з обґрунтування технології емульсійного косметичного крему надало змогу побудувати послідовність технологічного процесу, що може бути покладено в основу технології промислового виробництва, яка гарантує одержання кінцевого продукту, що відповідає вимогам технологічного регламенту. Технологічний процес виробництва включає плавлення в окремому реакторі компонентів олійної фази, виготовлення в окремому реакторі водної фази – розчинів водорозчинних компонентів, емульгування суміші двох фаз при температурі 70-80 °С, охолодження до температури 40-45 °С, введення ароматичних речовин чи інших термолабільних компонентів, фасування та пакування продукції. Пакування емульсійного косметичного крему відповідає вимогам ГОСТ 22715:2019 (для крему густої консистенції) [ДСТУ 22715:2019], [ДСТУ 22715:2019], [ДСТУ 22715:2019]. Спожиткову тару заповнюють косметичним кремом відповідно до маси або об’єму, встановлених у технічних вимогах на крем конкретної назви із врахуванням допустимих відхилень від номінальної маси (об’єму). Висновки. Проведено аналіз сучасного арсеналу поліпептидів рослинно-мікробного походження, доведена їх унікальність. Запропоновано рецептуру емульсійного косметичного крему для доглядання за шкірою обличчя на основі пептидів рослинно-мікробного походження – гексапептиду-10 (Hexapeptide-10, Serilesine) та ацетил-гексапептиду-8. Визначено особливості технології виготовлення емульсійного лікувально-косметичного крему та розроблено блок-схему й описано технологічну схему його отримання з використанням біологічно-активних речовин у вигляді низькомолекулярних пептидів і певних видів технологічного обладнання.
The term cosmeceutical refers to a category of skincare products that are purported to have active ingredients whose physiological or pharmacological actions are capable of inducing cosmetic enhancements to the skin. Given a demand for brighter, healthier, younger‐appearing skin, and relatively limited regulatory control, thousands of different cosmeceutical formulations have found their way onto store shelves and onto the skin of hopeful consumers, despite a lack of scientific evidence. This chapter summarizes new and old, common and rare cosmeceutical ingredients; their uses and side effects; and the science, where available, behind the claims.
Introduction We propose a new facial lifting protocol using polydioxanone (PDO) threads embedded in acetyl hexapeptide-8 (Argireline [Arg]). We assume that Arg reinforces the effects of PDO threads, as it is a mimetic of botulinum toxin. Because the PDO suture is hydrolyzable, this assumption is analyzed by instrumental analysis. Objective To demonstrate the capacity of the PDO suture as a system for the controlled release of acetyl hexapeptide-8 to apply in deep wrinkles of the upper third. Materials and Methods Three segments of 1-cm long 21G PDO threads immersed in 1 mL of Arg. PDO threads were observed under an optical, electron microscope at 24, 48, and 72 h later. They were also weighed before and after being soaked in Arg, and employing ultraviolet (UV)-visible spectroscopy, the release rate of Arg from the PDO suture was measured. Finally, was insert the thread PDO-Arg following a protocol designed especially for deep static wrinkles in the upper third. Results The electronic weighing revealed that the PDO thread enjoys capillarity by the peptide, doubling its weight every 24 h. UV spectra revealed that PDO thread is a well-controlled release system for Arg, allowing its sustained release for 1 h. Optical and electronic photomicrographs confirm the swelling of the PDO thread by absorbing Arg by its capillarity, but this hydrophilicity does not lead to its premature physical degradation. Conclusions The PDO thread system with Arg is an intelligent bioactive system useful in facial harmonization. It recommend conduct clinical trial to verify his superior lifting effect.
Background: Skin aging is a complex multifactorial progressive process. With age, intrinsic and extrinsic factors cause the loss of skin elasticity, with the formation of wrinkles, resulting in skin sagging through various pathways. A combination of multiple bioactive peptides could be used as a treatment for skin wrinkles and sagging. Objectives: This study aimed to evaluate the cosmetic efficacy of a multi-peptide eye serum as a daily skin-care product for improving the periocular skin of women within the ages of 20-45 years. Methods: The stratum corneum skin hydration and skin elasticity were assessed using a Corneometer CM825 and Skin Elastometer MPA580, respectively. The PRIMOS CR technique based on digital strip projection technology was used for skin image and wrinkle analysis around the "crow's feet" area. Self-assessment questionnaires were filled on Day 14 and 28 of product use. Results: This study included 32 subjects with an average age of 28.5 years. On Day 28, there was a significant decrease in the number, depth, and volume of wrinkles. Skin hydration, elasticity, and firmness increased continuously during the study period, consistent with typical anti-aging claims. A majority of the participants (75.00%) expressed overall satisfaction with their skin appearance after using the product. Most participants noted a visible skin improvement, with an increase in skin elasticity and smoothness, and confirmed the extensibility, applicability, and temperance of the product. No adverse reactions related to product use were observed. Conclusions: The multi-peptide eye serum uses a multi-targeted mechanism against skin aging to improve the skin appearance, making it an ideal choice for daily skincare.
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Clostridial neurotoxins' metalloprotease domain selectively cleaves proteins implicated in the process of synaptic vesicle fusion with the plasma membrane and, accordingly, blocks neurotransmitter release into the synaptic cleft. Here we investigate the potential modulation of these neurotoxins by intracellular cascades triggered by environmental signals, which in turn may alter its activity on target substrates. We report that the nonreceptor tyrosine kinase Src phosphorylates botulinum neurotoxins A, B, and E and tetanus neurotoxin. Protein tyrosine phosphorylation of serotypes A and E dramatically increases both their catalytic activity and thermal stability, while dephosphorylation reverses the effect. This suggests that the biologically significant form of the neurotoxins inside neurons is phosphorylated. Indeed, in PC12 cells in which tyrosine kinases such as Src and PYK2 are highly abundant, stimulation by membrane depolarization in presence of extracellular calcium induces rapid and selective tyrosine phosphorylation of internalized light chain, the metalloprotease domain, of botulinum toxin A. These findings provide a conceptual framework to connect intracellular signaling pathways involving tyrosine kinases, G-proteins, phosphoinositides, and calcium with the action of botulinum neurotoxins in abrogating vesicle fusion and neurosecretion.
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The interaction of the presynaptic membrane proteins SNAP-25 and syntaxin with the synaptic vesicle protein synaptobrevin (VAMP) plays a key role in the regulated exocytosis of neurotransmitters. Clostridial neurotoxins, which proteolyze these polypeptides, are potent inhibitors of neurotransmission. The cytoplasmic domains of the three membrane proteins join into a tight SDS-resistant complex (Hayashi et al., 1994). Here, we show that this reconstituted complex, as well as heterodimers composed of syntaxin and SNAP-25, can be disassembled by the concerted action of the N-ethylmaleimide-sensitive factor, NSF, and the soluble NSF attachment protein, alpha-SNAP. alpha-SNAP binds to predicted alpha-helical coiled-coil regions of syntaxin and SNAP-25, shown previously to be engaged in their direct interaction. Synaptobrevin, although incapable of binding alpha-SNAP individually, induced a third alpha-SNAP binding site when associated with syntaxin and SNAP-25 into heterotrimers. NSF released prebound alpha-SNAP from full-length syntaxin but not from a syntaxin derivative truncated at the N-terminus. Disassembly of complexes containing this syntaxin mutant was impaired, indicating a critical role for the N-terminal domain in the alpha-SNAP/NSF-mediated dissociation process. Complexes containing C-terminally deleted SNAP-25 derivatives, as generated by botulinal toxins type A and E, were dissociated more efficiently. In contrast, the N-terminal fragment generated from synaptobrevin by botulinal toxin type F produced an SDS-sensitive complex that was poorly dissociated.
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Excitation-secretion uncoupling peptides (ESUPs) are inhibitors of Ca2+-dependent exocytosis in neural and endocrine cells. Their mechanism of action, however, remains elusive. We report that ESUP-A, a 20-mer peptide patterned after the C terminus of SNAP-25 (synaptosomal associated protein of 25 kDa) and containing the cleavage sequence for botulinum neurotoxin A (BoNT A), abrogates the slow, ATP-dependent component of the exocytotic pathway, without affecting the fast, ATP-independent, Ca2+-mediated fusion event. Ultrastructural analysis indicates that ESUP-A induces a drastic accumulation of dense-core vesicles near the plasma membrane, mimicking the effect of BoNT A. Together, these findings argue in favor of the notion that ESUP-A inhibits ATP-primed exocytosis by blocking vesicle docking. Identification of blocking peptides which mimic sequences that bind to complementary partner domains on interacting proteins of the exocytotic machinery provides new pharmacological tools to dissect the molecular and mechanistic details of neurosecretion. Our findings may assist in developing ESUPs as substitute drugs to BoNTs for the treatment of spasmodic disorders.
BACKGROUND: Décolleté wrinkles develop with age. In women more than 35 years of age these wrinkles are still transient, but become permanent at about age 50. In a substantial number of women décolleté wrinkles, seem to be associated with an insertion of platysma exceeding the second intercostal space. We report botulinum A toxin therapy of these wrinkles in five patients. OBJECTIVE: To show the effect of botulinum A toxin on décolleté wrinkles. METHODS: All five women with décolleté lines treated with botulinum A had different varieties of the platysma muscle inserting deep down beneath the fourth intercostal space. During platysma contraction injection points were marked and a certain dosage of toxin was applied. RESULTS: Two weeks after therapy a significant improvement of the treated décolleté region was observed. CONCLUSION: These observations indicated that botulinum A toxin can be an effective and safe method in the temporary management of décolleté wrinkles. It should therefore be considered as a new adjuvant treatment in cosmetic décolleté rejuvenation.
This article deals with the office treatment of several common cosmetic problems. The topic of benign lesions is discussed, which includes the management of lentigines, milia, spider nevi, telangiectasis, xanthelasma, and keloids. The use of several modalities for the treatment of acne scarring is advocated; these include dermabrasion, punch-transplant replacement techniques, and collagen implants. Chemical face peels for actinic damage and premature wrinkling are described, and the use of hair transplants, scalp-reduction techniques, and scale flaps to surgically correct male-pattern alopecia are discussed.
SNAP-25, a synaptosomal associated membrane protein of 25 kDa, participates in the presynaptic process of vesicle-plasma membrane fusion that results in neurotransmitter release at central nervous system synapses. SNAP-25 occurs in neuroendocrine cells and, in analogy to its role in neurons, has been implicated in catecholamine secretion, yet the nature of the underlying mechanism remains obscure. Here we use an anti-SNAP-25 monoclonal antibody to show that SNAP-25 is localized at the cytosolic surface of the plasma membrane of chromaffin cells. This antibody inhibited the Ca(2+)-evoked catecholamine release from digitonin-permeabilized chromaffin cells in a time- and dose-dependent manner. Remarkably, a 20-mer synthetic peptide representing the sequence of the C-terminal domain of SNAP-25 blocked Ca(2+)-dependent catecholamine release with an IC50 = 20 microM. The inhibitory activity of the peptide was sequence-specific as evidenced by the inertness of a control peptide with the same amino acid composition but random order. The C-terminal segment of SNAP-25, therefore, plays a key role in regulating Ca(2+)-dependent exocytosis, presumably mediated via interactions with other protein components of the fusion complex.
A new low-volume flow-through diffusion cell (LVFC) was designed to provide accurate determinations of penetrant flux across skin while minimizing the dilution of penetrant in receptor fluid and eliminating the need for magnetic stirring. The performance of the 0.3-mL LVFC was compared to a magnetically stirred, 4.3-mL high-volume flow cell (HVFC) and to a magnetically stirred, manually sampled 7.5-mL static cell (SC) with hydrophilic and lipophilic penetrants. The clearance of 14C-labeled benzoic acid from the LVFC and HVFC followed an exponential profile expected for complete mixing when the LVFC and HVFC were run at flow rates of 0.4-0.9 and 4.0-5.2 mL/h, respectively. The in vitro dispositions of 14C-labeled benzoic acid and estradiol were determined in the LVFC and HVFC by applying the compounds to split-thickness pig skin at a 4 micrograms/cm2 dose. Additionally, the effects of receptor fluid flow rate (1.2 vs 3.5 cell volumes/h) and method of skin attachment (O-ring vs compression) were determined on disposition in the HVFC. The percutaneous penetration of benzoic acid and the residue of estradiol within skin did not differ between the LVFC and HVFC. However, the percutaneous penetration of benzoic acid increased significantly (p < 0.05) using the O-ring attachment as compared to compression at a flow rate of 1.2 cell volumes/h. The in vitro permeation of benzoic acid-saturated water and 17 beta-estradiol-saturated propylene glycol monolaurate through human epidermis was compared between the LVFC, HVFC, and SC. The LVFC and HVFC had flow rates of 0.9-1.0 mL/h.(ABSTRACT TRUNCATED AT 250 WORDS)
Botulinum neurotoxin E (BoNT E) cleaves SNAP-25 at the C-terminal domain releasing a 26-mer peptide. This peptide product may act as an excitation-secretion uncoupling peptide (ESUP) to inhibit vesicle fusion and thus contribute to the efficacy of BoNT E in disabling neurosecretion. We have addressed this question using a synthetic 26-mer peptide which mimics the amino acid sequence of the naturally released peptide, and is hereafter denoted as ESUP E. This synthetic peptide is a potent inhibitor of Ca2+-evoked exocytosis in permeabilized chromaffin cells and reduces neurotransmitter release from identified cholinergic synapses in in vitro buccal ganglia of Aplysia californica. In chromaffin cells, both ESUP E and BoNT E abrogate the slow component of secretion without affecting the fast, Ca2+-mediated fusion event. Analysis of immunoprecipitates of the synaptic ternary complex involving SNAP-25, VAMP and syntaxin demonstrates that ESUP E interferes with the assembly of the docking complex. Thus, the efficacy of BoNTs as inhibitors of neurosecretion may arise from the synergistic action of cleaving the substrate and releasing peptide products that disable the fusion process by blocking specific steps of the exocytotic cascade.