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A synthetic hexapeptide (Argireline) with antiwrinkle activity


Abstract and Figures

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.
310 ß2002 International Journal of Cosmetic Science,24,303^310
... However, many of the NCS peptides were reported to be more specific in their mechanism of action. For example, five of the NCS peptides (acetyl hexapeptide-3 [10,175], acetyl octapeptide-3 [176] and pentapeptide-3 [177]) acted as neuroinhibitors at the neuromuscular junction, causing muscle relaxation and subsequent reduction of wrinkles in a similar manner to botulinum toxin. The NCS acetyl tetrapeptide-2 mimicked the action of its original source protein, thrombopoietin, stimulating skin immune defences [178]. ...
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Extracellular matrix (ECM) proteins confer biomechanical properties, maintain cell phenotype and mediate tissue repair (via release of sequestered cytokines and proteases). In contrast to intracellular proteomes, where proteins are monitored and replaced over short time periods, many ECM proteins function for years (decades in humans) without replacement. The longevity of abundant ECM proteins, such as collagen I and elastin, leaves them vulnerable to damage accumulation and their host organs prone to chronic, age-related diseases. However, ECM protein fragmentation can potentially produce peptide cytokines (matrikines) which may exacerbate and/or ameliorate age- and disease-related ECM remodelling. In this review, we discuss ECM composition, function and degradation and highlight examples of endogenous matrikines. We then critically and comprehensively analyse published studies of matrix-derived peptides used as topical skin treatments, before considering the potential for improvements in the discovery and delivery of novel matrix-derived peptides to skin and internal organs. From this, we concluded that while the translational impact of matrix-derived peptide therapeutics is evident, the mechanisms of action of these peptides are poorly defined. Further, well-designed, multimodal studies are required.
... Acetyl hexapeptide-3 (Ac-Glu-Glu-Met-Gln-Arg-Arg-NH 2 ) is reported as an artificial peptide chemically modified from N-terminal, which can inhibit SNARE complex formation (Blanes-Mira et al. 2002). It is commercially available as Argireline V R (Dana and Rotsztejn 2017). ...
Skin ageing is a cumulative result of oxidative stress, predominantly caused by reactive oxygen species (ROS). Respiration, pollutants, toxins, or ultraviolet A (UVA) irradiation produce ROS with 80% of skin damage attributed to UVA irradiation. Anti-ageing peptides and proteins are considered valuable compounds for removing ROS to prevent skin ageing and maintenance of skin health. In this review, skin ageing theory has been illustrated with a focus on the mechanism and relationship with anti-ageing peptides and proteins. The effects, classification, and transport pathways of anti-ageing peptides and proteins across skin are summarized and discussed. Over the last decade, several novel formulations and advanced strategies have been developed to overcome the challenges in the dermal delivery of proteins and peptides for skin ageing. This article also provides an in-depth review of the latest advancements in the dermal delivery of anti-ageing proteins and peptides. Based on these studies, this review prospected several semi-solid dosage forms to achieve topical applicability for anti-ageing peptides and proteins.
... (Moh et al., 2011;Blanes-Mira et al., 2002;Hoppel et al., 2015;Lungu et al., 2013). 아세 틸 헥사펩타이드는 진통성 화학물질인 엔케팔린(enkephalins)이 표정 근에 보톡스처럼 작용하여 신경전달물질인 아세틸콜린(acetylcholine) 의 방출을 억제함으로써 근육을 긴장시키는 작용을 한다 (Giacomoni, 2008;Zhang & Falla, 2009 * present compared to only medium condition in each sample. ...
Purpose: The topical application of scalp-care cosmetics, infused with functional ingredients, can offer an optimal cosmetic approach to prevent problems associated with aging. In terms of the development of anti-aging cosmetics, we studied the use of acetyl hexapeptide-8 (AH-8) for improving elasticity of the scalp.Methods: Ampoules containing 3% and 5% concentrations of AH-8 as an active ingredient were prepared and their safety and efficacy were evaluated. HaCaT cells were used to evaluate the safety of AH-8 ampoules by measurement of in vitro cytotoxicity. In in vivo experiments, we tested the AH-8 ampoules by cumulative irritation test (CIT) on healthy adults, 20–50 years of age. Relative expressions of superoxide dismutase 2 (SOD2), forkhead box O1 (FOXO1), actinin alpha 1 (ACTN1), collagen type XVII alpha 1 chain (COL17A1), and integrin subunit beta 4 (ITGB4) were assessed using quantitative real-time PCR (Q-PCR).Results: AH-8 ampoules showed significant cytotoxicity to HaCaT cells in a dose-dependent manner. CIT assessment in 30 adults revealed no skin reactions to both 3% and 5% AH-8 ampoules. After treatment of HaCaT cells with the AH-8 ampoules, mRNA levels of SOD2 and FOXO1, which are directly implicated as antioxidant factors, were increased. Of the factors that improve elasticity, ACTN1 mRNA levels had the greatest increase when treated with the 5% AH-8 ampoules (1.75 µg/mL). Also, the AH-8 ampoules increased mRNA levels of COL17A1, and ITGB4 in HaCaT cells.Conclusions: In this study, we proved that 3% and 5% AH-8 ampoules are safe for use as a scalp-care cosmetic. AH-8 ampoule had efficacy in anti-aging and improving elasticity of the scalp. This comprehensive test showed that AH-8 can be developed as a cosmetic for anti-aging and improvement of scalp elasticity.
... Acetyl hexapeptide-8, also known as acetylhexapeptide-3, is a neurotransmitter inhibitor peptide designed from the N-terminal end of the synaptosomal-associated protein (SNAP25) [4,5]. It competitive inhibits the SNAP25 component of the said vesicle docking and fusion protein complex (SNARE) [6] which triggers the calcium-dependent neurotransmitter release into the synapse, a process necessary for muscle contraction [7][8][9][10]. Acetyl hexapeptide-8 is marketed as Argireline ® [11], and it has been efficiently used in cosmetics for smoothening the under-eye wrinkles and the forehead furrows [12][13][14]. ...
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Bioactive peptides are gaining more and more popularity in the research and development of cosmetic products with anti-aging effect. Acetyl hexapeptide-8 is a hydrophilic peptide incorporated in cosmetics to reduce the under-eye wrinkles and the forehead furrows. Hydrophilic interaction liquid chromatography (HILIC) is the separation technique of choice for analyzing peptides. In this work, a rapid HILIC method coupled to photodiode array detection operated at 214 nm was developed, validated and used to determine acetyl-hexapeptide-8 in cosmetics. Chromatography was performed on a Xbridge® HILIC BEH analytical column using as mobile phase a 40 mM ammonium formate water solution (pH 6.5)-acetonitrile mixture 30:70, v/v at flow rate 0.25 mL min−1. The assay was linear over the concentration range 20 to 30 μg mL−1 for the cosmetic formulations and 0.004 to 0.007% (w/w) for the cosmetic cream. The limits of quantitation for acetyl hexapeptide-8 were 1.5 μg mL−1 and 0.002% (w/w) for the assay of cosmetic formulations and cosmetic creams, respectively. The method was applied to the analysis of cosmetic formulations and anti-wrinkle cosmetic creams.
... As a consequence, DD04107 (1) produced dose-dependent, in vivo analgesic effects in chronic models of inflammatory and neuropathic pain, including inflammation and mechanical hyperalgesia induced by carrageenan, Freund's adjuvant (CFA) and osteosarcoma, and some peripheral neuropathies (chemotherapy, diabetes) [17]. The N-acetyl analogue of DD04107 (Ac-EEMQRR-NH 2 , 2, Ac-DD04107) is also an inhibitor of neuronal exocytosis, although with a lower potency than BoNTA and DD04107 (1) [15,18]. Peculiarly, this peptide is being used as a safe BoNT mimetic by the cosmetic industry for the reduction of expression wrinkles [19], and DD04107 (1) is currently active in phase II clinical trials for neuropathic pain [20]. ...
The analgesic peptide DD04107 (Pal-EEMQRR-NH2) and its acetylated analogue inhibit α-calcitonin gene-related peptide (α-CGRP) exocytotic release from primary sensory neurons. Examining the crystal structure of the SNARE-Synaptotagmin-1(Syt1) complex, we hypothesized that these peptides could inhibit neuronal exocytosis by binding to Syt1, hampering at least partially its interaction with the SNARE complex. To address this hypothesis, we first interrogate the role of individual side-chains on the inhibition of α-CGRP release, finding that E1, M3, Q4 and R6 residues were crucial for activity. CD and NMR conformational analysis showed that linear peptides have tendency to adopt α-helical conformations, but the results with cyclic analogues indicated that this secondary structure is not needed for activity. Isothermal titration calorimetry (ITC) measurements demonstrate a direct interaction of some of these peptides with Syt1-C2B domain, but not with Syt7-C2B region, indicating selectivity. As expected for a compound able to inhibit α-CGRP release, cyclic peptide derivative Pal-E-cyclo[EMQK]R-NH2 showed potent in vivo analgesic activity, in a model of inflammatory pain. Molecular dynamics simulations provided a model consistent with KD values for the interaction of peptides with Syt1-C2B domain, and with their biological activity. Altogether, these results identify Syt1 as a potential new analgesic target.
Background: Effective anti-aging treatments are an unmet consumer need. Aim: Ex vivoand clinical tests have evaluated the efficacy of a topical facial serum containing a proprietary blend of neuropeptides, proteins, amino acids, and marine extracts on aged skin. Methods: In the ex vivo study the facial serum was compared to a commercially marketed face serum and to an untreated control on skin explants using microrelief, smoothness, and epidermal thickness endpoints. The 12 weeks monadic clinical study was designed for the test product to be used on the whole face. Subjects functioned as their own control; evaluating change from baseline. Skin was evaluated clinically by a Dermatologist for tolerability and for efficacy. Also part of the product assessment was skin hydration measurements, imaging, and a subject questionnaire. Results: The facial serum improved skin condition by significant reductions in skin surface area occupied by microfolds and in skin roughness. Additionally, it increased epidermal thickness as compared to the untreated control as well as the commercially marketed face serum. The facial serum provided a statistically increased skin moisturization compared to pretreatment values. Dermatological evaluation of the skin concluded that there were statistically and clinically significant improvements in skin smoothness, wrinkles severity, fine lines visibility and lifting, and tightening effects at crow's feet area, forehead, and upper lip. Conclusion: A facial serum, containing a proprietary blend of neuropeptides, proteins, amino acids, and marine extracts, has been shown to improve the overall quality of aged skin in a series of ex vivo and clinical tests.
Peptides are biologically active communication tools that direct skin functioning. Peptides, proteins, and amino acids are often mislabeled and the terms applied as if they were interchangeable, yet they are different in their characteristics, uses, biological activities, and cosmetic potential. Peptides perform many important biologic signal functions. The incorporation of peptides into various galenic forms of skin care products can be challenging. The term matrikines is used to describe naturally occurring fragments of matrix macromolecules endowed with stimulatory, tissue repair activity. Concepts derived from wound healing research are but one approach to peptide‐based anti‐aging treatments. Neurotensin, vasointestinal peptide, neuropeptide Y, substance P, and calcitonin gene‐related peptide, although endowed with potent biological activity, are not candidates for cosmetic applications because of their size and irritation potential. Structural proteins are building blocks for the organs and tissues of the human body. Superoxide dismutase, an antioxidant enzyme, is present at the surface of the skin.
Introducción: Actualmente los péptidos sintéticos se han constituido en una novedosa alternativa para el tratamiento de la piel envejecida. Acetilhexapéptido-3 (Ac-EEMQRR-NH2) ha sido utilizado para inducir reducción de líneas de expresión de manera análoga a la toxina botulínica, pero sin efectos tóxicos. Objetivo: Sintetizar el acetilhexapéptido-3 y desarrollar un sistema liposomal para su encapsulación y favorecer su paso a través de una membrana modelo. Metodología: El péptido fue obtenido mediante síntesis en fase sólida (SFS) empleando la estrategia Fmoc/tBu, fue purificado y plenamente caracterizado. El sistema liposomal con acetilhexapéptido-3 encapsulado fue desarrollado mediante la formación de una emulsión con posterior inversión de fase por evaporación del solvente orgánico. Los sistemas fueron caracterizados en su tamaño, potencial zeta, eficiencia de encapsulación del neuropéptido y de manera preliminar se realizó un estudio de permeabilidad ex vivo. Resultados: Es posible sintetizar péptidos cortos con alto grado de pureza y buen rendimiento, utilizando la metodología de SFS Fmoc/tBu. De acuerdo con la caracterización, el sistema liposomal adelantado sugiere una buena estrategia para la encapsulación del acetilhexapéptido-3 y su potencial aplicación en el desarrollo un novedoso producto cosmecéutico.
With the emergence of social aging problem, people's demand for active ingredients for skin health protection and therapeutic efficacy increases. Bioactive peptides are the optimal substances for skin anti-aging with its great diversity of biological activities and high security, such as antioxidation, anti-aging, anti-diabetes, anti-hypertension, and antibacterial. In recent years, natural and synthetic anti-aging peptides have been extensively studied in vitro, in vivo and clinically. Anti-aging peptides, such as collagen peptides, can affect various physiological pathways of skin, and have significant skin protection effect when applied locally and eaten. These characteristics show that bioactive peptides can improve skin health by providing specific physiological functions. In this review, we summarized the research of anti-aging peptides and the finishing of anti-aging peptides on improving skin health which is mainly based on the collagen peptides and the related synthetic peptides.
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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.