m

Technical Report

Product
ARGIRELINE peptide

Date
December 2014

Revision
28
Contents

A MAIN CONCERN: THE EXPRESSION LINES 3

THE SNARE COMPLEX IN MUSCLE CONTRACTION 4
SNARE PROTEINS ARE SPECIAL TARGETS IN COSMETICS 5

ARGIRELINE peptide, THE FIRST PEPTIDE FOR EXPRESSION WRINKLES 6
IN VITRO EFFICACY
Modulation of SNARE complex formation 7
Inhibition of glutamate release 8
IN VIVO EFFICACY
Reduction of wrinkle depth 9
Roughness diminution in wrinkles 10
Effect on wrinkle volume and length 12
COSMETIC PROPERTIES 14
COSMETIC APPLICATIONS 14
TECHNICAL DATA
INCI name of the active ingredient 15
Presentation and Preservative 15
APPLICATION DATA
Processing 15
Incompatibilities 15
Solubility 15
Dosage 15
REFERENCES 16

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A main concern: the expression lines
One of the main concerns of a high percentage of population is avoiding the first and visible
signs of aging. Individuals wish to maintain a young appearance as long as possible.

The appearance of frown and wrinkle lines include the constant pull of gravity,
in the forehead, glabella, lateral periorbital frequent and constant positional pressure
area (of a relative intensity) as well as chin, of facial skin (during sleeping for instance)
upper lip wrinkling, nasolabial folds and or repeated facial movements caused by
platysma neck bands are among the most the contraction of the muscles of facial
striking signs that betray an aged skin [1]. expression causing characteristic folds,
These changes may arise naturally along furrows and creases [1, 2].
with the years being the result of certain
biochemical, histological and physiological The excessive stimulation of the muscle
changes which are usually enhanced by fibres in the face (which pulls the skin
environmental exposure. inwards) increases the well-known
expression lines, which can become evident
There are also other secondary factors that at the age of 30. Attenuating the muscle
may help in the wrinkles formation and contraction can be a useful strategy to
even originate them themselves. They remove or delay the appearance of these
undesirable lines as much as possible.

A cosmetic treatment targeting

the right mechanism implied in
muscle contraction can help to
minimise these undesirable lines.

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The SNARE complex in muscle contraction
Muscle contraction is a process where both the nerve and the muscle are involved in a synapse
known as the neuromuscular junction (NMJ). Muscles are contracted when they receive a
neurotransmitter released from inside a vesicle of the motor neuron. This neurotransmitter
release is a highly regulated cascade that proceeds through an orchestrated sequence of
protein-protein interactions that culminate in the fusion of the neurotransmitter-loaded vesicles
with the neuron membrane [3].

A localised change in membrane potential and synaptosomal associated protein 25

(action potential) is transmitted along the (SNAP-25) [3]. Such proteins directly
neuron to the terminal where it triggers the govern vesicle docking and fusion through
entry of calcium ions into the neuron [3]. the formation of a ternary structure known
When these ions enter the pre-synaptic as the SNARE complex, which is like a
terminal, the vesicles containing the cellular hook that captures vesicles and
acetylcholine (ACh) neurotransmitter are fuses them with the membrane [3, 4].
induced to join other elements of the
neuron in order to release ACh outwards. Once the fusion of these vesicles occurs,
ACh is liberated into the synapse between
This is a complicated process mediated by nerve and muscle cells. There, ACh binds to
the so-called SNAP Receptors (SNARE) acetylcholine receptors (AChR) located on
proteins, which include the vesicle- the surface of muscle cells, finally leading
associated membrane protein (VAMP) and to muscle contraction.
the membrane-associated protein syntaxin

Fig. 1. ACh release and muscle contraction.

The SNARE complex is essential for ACh release at the

neuromuscular synapse, which causes muscle contraction.

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SNARE proteins are special targets in cosmetics
Structurally, the SNARE complex is a parallel four-helix bundle formed by the coiled-coil
arrangement of two helices from SNAP-25 and one each from syntaxin and VAMP [3]. The
centre of the complex shows the presence of leucine-zipper layers interspersed with an ionic
layer and surrounded by a hydrophobic core.

The SNARE complex is the target of Although BoNTs and specially type A were
botulinum neurotoxins (BoNTs), a family of a revolution in cosmetics and have been
naturally occurring neurotoxins that extensively used to attenuate expression
potently block neurosecretion. These wrinkles, they have certain limitations, as
BoNTs cause a specific proteolysis of they need strict medical control (highly
SNAP-25 that avoids this complex assembly toxic) and require regular intervals of
and the fusion of ACh vesicles onto the application [5].
nerve membrane. Therefore, they impede
the exocytosis of the neurotransmitter Thus, topical cosmetics causing the same
and its release into the synaptic cleft, effects but safely are an interesting
making the affected nerve terminals alternative or even an additional and
incapable of stimulating muscle complementary treatment between BoNTs
contraction, which ends up in muscle sessions to diminish expression lines.
paralysis.

There is a need for topical

cosmetic alternatives to reduce
expression lines safely.

Fig. 2. SNARE complex inhibition by BoNT.

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ARGIRELINE peptide, the first peptide for expression wrinkles
ARGIRELINE peptide was designed to act in the pre-synaptic mechanism to reduce the
contraction that leads to the wrinkle formation.

ARGIRELINE peptide is a replica of the N- ARGIRELINE peptide targets the same

terminal end of SNAP-25, which competes protein complex as Botulinum Toxin A,
with this natural protein for a position in the modulating muscle contraction. Thus, this
SNARE complex, destabilizing its ingredient is a safer active with proved
formation, without breaking any of its efficacy in destabilizing the SNARE
components. If the SNARE complex is complex, reducing wrinkle formation and
slightly destabilized, the vesicle cannot minimising the depth, volume and length
release neurotransmitters efficiently. of existing wrinkles and expression lines in
Consequently, muscle is relaxed rather vivo, as well as skin roughness.
than paralysed, decreasing the formation
of lines and wrinkles.

ARGIRELINE peptide is an
excellent anti-wrinkle ingredient
that reduces muscle contraction
and its expression lines.

Fig. 3. SNARE complex modulation by ARGIRELINE peptide.

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In vitro efficacy
M ODULATION OF SNARE COMPLEX FORMATION

This test evaluates the antagonistic competitive efficacy of ARGIRELINE peptide with the wild
type SNAP-25 in assembling with syntaxin and synaptobrevin to form the SNARE complex.

SNARE complex was reconstituted using The SNARE complex assembly was stopped
recombinant VAMP and syntaxin proteins by addition of sodium dodecyl sulfate
and in vitro transcribed and translated [35S]- polyacrylamide gel electrophoresis (SDS-
SNAP-25. Equimolar amounts of VAMP and PAGE) buffer, and the samples were
syntaxin were incubated in the absence or analysed by SDS-PAGE on 12% gels,
presence of ARGIRELINE peptide for 2 h at followed by fluorographic detection on
4 C. Then, [35S]-SNAP-25 was added and Kodak X-Omat AR X-ray films. Heat
the mixture was further incubated at 4 C disassembles the SNARE complex, thus it
during 12 h. was used as the positive control.

SNARE complex

SNAP-25

SNARE Heat ARGIRELINE peptide

Fig. 4. Modulation of the SNARE complex formation by ARGIRELINE peptide.

The top band corresponds to the SNARE

complex and the bottom band shows
SNAP-25 presence. The first lane on the
left was a control with intact SNARE ARGIRELINE peptide destabilizes
complex while the second lane showed the SNARE complex formation.
SNARE complex disassembled by heat,
where only the SNAP-25 band was visible.
The other lanes showed how the number
of SNARE complexes was lower when
the hexapeptide was present.

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INHIBITION OF GLUTAMATE RELEASE

Inhibition of glutamate release by depolarised neuron cells is a validated cell assay for
measuring the potential activity of compounds on the inhibition of neuronal exocytosis. The
K+-induced depolarisation of hippocampal cultures in the presence of extracellular calcium ions
results in the release of glutamate, which is the most abundant excitatory neurotransmitter in
the nervous system.

Primary neuron cells were incubated with L- The culture media was collected and the
[3H]-glutamine to charge them with L-[3H]- quantity of L-[3H]-glutamate was
glutamate. Afterwards, the excess of L-[3H]- determined by a scintillation counter. The
glutamine was rinsed off and they were results were normalized regarding the
incubated with ARGIRELINE peptide. The release of L-[3H]-glutamate in absence of
release of L-[3H]-glutamate was carried out the test item (control) and corrected from
by depolarization in a physiologic buffer. the basal release in absence of calcium.

Inhibition glutamate release (%)

Fig. 5. Inhibition of glutamate release.

The active peptide decreased glutamate ARGIRELINE peptide effectively

release in a dose-dependent manner, up modulates glutamate release,
to -32.0% at 2 mM, implying a reduction indicative of anti-expression
of neuronal exocytosis. wrinkle activity.

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 In vivo efficacy
REDUCTION OF WRINKLE DEPTH

The anti-wrinkle power of a cream with ARGIRELINE peptide solution was evaluated in vivo.

A panel of 10 female volunteers (average after 15 and 30 days. Then, they were
age: 44) applied a cream containing 10% analysed by confocal microscopy.
ARGIRELINE peptide solution around one
eye and a placebo cream around the other The depth-coded image generated from
twice a day for 30 days. section series is a real map of the surface
structure of the sample.
The silicone imprints of the treated areas
were obtained before the treatment and

0 days 15 days 30 days

Placebo

10% ARGIRELINE
peptide solution

Fig. 6. Images belonging to imprints of the treated areas at different times.

The active peptide decreased wrinkle ARGIRELINE peptide noticeably

depth by an average of -16.9% and -27.0% diminished wrinkle depth.
after 15 and 30 days respectively.

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ROUGHNESS DIMINUTION IN WRINKLES

To assess the effect of ARGIRELINE peptide solution C in reducing skin roughness, 18 female
volunteers between 35-55 years old presenting wrinkles around the eyes were chosen.

Volunteers applied a cream containing 5% of the face were also taken and examined,
ARGIRELINE peptide solution C on the before and after the treatment.
face, including the crows feet area, twice a
day for 28 consecutive days. Skin replicas The evolution of the arithmetic roughness
were taken of the eye crows feet area and average (Ra) evolution, understood as the
analysed with the PRIMOS technique before average of all heights and depths to the
and after the 28 days. Macrophotographies reference plane, was represented.

Fig. 7. Mean results of the roughness average before and after the treatment (*p<0.05).

The active peptide reduced Ra by Skin roughness was clearly

-20.4% after 28 days (statistically
decreased by ARGIRELINE peptide.
significant value).

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 0 days 28 days

Fig. 8. Images of a volunteer before and after the treatment.

0 days 28 days

Fig. 9. Images of another volunteer before and after the treatment.

ARGIRELINE peptide improves the wrinkles of the crows feet area.

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E FFECT ON WRINKLE VOLUME AND LENGTH

Another study was carried out to evaluate the efficacy of a cream containing ARGIRELINE
peptide solution C in decreasing wrinkles, using the Fast Optical In vivo Topometry of human
Skin (FOITS) technique.

A panel of 24 female volunteers (between Pictures were taken and skin topography
35-45 years old) was selected to apply a was quantitatively evaluated, after FOITS
cream containing 2% ARGIRELINE peptide measurements (wrinkle volume and length)
solution C on half of the face, focusing on were recorded at the beginning and end of
the crows feet area, and a placebo cream the treatment.
on the other half twice a day for 7 days.

Fig. 10. Evolution of the wrinkle parameters after 7 days (*p<0.05).

The peptide diminished the

average volume and length of the ARGIRELINE peptide reduced wrinkle
wrinkles by -20.6% and -15.9% volume and length after 7 days.
respectively, after 7 days. Both
values were statistically significant.

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0 days 7 days

Fig. 11. Images of a volunteer before and after the active treatment.

ARGIRELINE peptide visibly ameliorated

wrinkle appearance after just 1 week.

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Cosmetic properties
ARGIRELINE peptide:

is an anti-wrinkle hexapeptide that destabilizes the SNARE complex arrangement,

reducing the formation of expression lines (induced by muscle contraction) on the skin
surface.

decreased glutamate release (up to -32.0% at 2 mM) in a dose-dependent manner,

implying a noticeable reduction of neuronal exocytosis.

is an excellent and effective active ingredient against wrinkles, which offered an

average wrinkle depth decrease of -16.9% and -27.0% after 15 and 30 days of in vivo
treatment respectively (10% peptide solution).

reduced the average roughness (Ra) of skin in volunteers, by -20.4% after 28 days
(5% peptide solution C).

ameliorates wrinkle appearance, proven by the decrease of wrinkle volume

by -20.6% and wrinkle length by -15.9% after just 7 days (2% peptide solution C).

Cosmetic applications
ARGIRELINE peptide can be incorporated into formulations where the removal of deep lines
or wrinkles on the forehead or around the eyes area is desired. It especially targets the
appearance of the expression lines and delays their formation.

Thus, it is perfect to add into facial creams, sera and treatments intended to fight the first
expression wrinkles and to incorporate into anti-aging products.

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Technical data
INCI NAME OF THE ACTIVE INGREDIENT

Active ingredient INCI name

ARGIRELINE peptide Acetyl Hexapeptide-8

P RESENTATION AND PRESERVATIVE

Solution containing 0.05% of active ingredient.

Code Product presentation Preservative

P06-PD010 ARGIRELINE peptide solution C Preservative free

Other versions available. Please contact your sales representative for further information.

Application data
P ROCESSING
ARGIRELINE peptide solution C can be formulated in the aqueous phase in the final step of the
manufacturing process. In case of preparing an emulsion, it should be added once the emulsion
is formed. In both cases, it should always be provided that the temperature is below 40 C
although it can exceptionally remain stable for 2 h at 80 C.

Recommended pH range between 3.0 and 8.0 for ARGIRELINE peptide.

INCOMPATIBILITIES
Oxidants, electrophiles and tannins.

SOLUBILITY
Soluble in water.

DOSAGE
A dosage of 2-10% of ARGIRELINE peptide solution C is recommended in final cosmetic
formulations.

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 References

1. Benedetto AV. Environmental and skin aging. Clin Derm. [Link], 1998.

2. Stegman SJ, Tromovitch TA, Clogau RG. The skin of the aging face in cosmetic
dermatologic surgery. 2nd edn. Mosby year book. St Louis. MO, pp:5-15, 1990.

3. Blanes-Mira C, Merino JM, Valera E, et al. Small peptides patterned after the N-terminus
domain of SNAP25 inhibit SNARE complex assembly and regulated exocytosis. J
Neurochem. 88:124-135, 2004.

4. Chen YA, Scheller RH. SNARE-mediated membrane fusion. Nature Reviews Molecular Cell
Biology 2: 98-106, 2001.

5. Blanes-Mira C, Clemente J, Jodas G, et al. A synthetic hexapeptide (Argireline) with

antiwrinkle activity. J Cosm Sci. 24:303-310, 2002.

AIMTEC and ARGIRELINE are owned by The Lubrizol Corporation.

The other tradenames and trademarks used herein belong to their respective and lawful owners.

Note: Graphs and photographs of efficacy tests are available for customer use provided that the final product contains the same
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The specific situation of the trademark in each country may vary and we recommend that you contact us for updated
information.

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