j. Cosmet.

Sci., 58, 157-171 (March/April 2007)

Preparationand stabilityof cosmeticformulationswith an

anti-agingpeptide
M. A. RUIZ, B. CLARES, M. E. MORALES, S. CAZALLA, and

V. GALLARDO, Departamento
deFarmaciay Tecnolog/a
Farmacgutica,Facultad de Farmacia, Universidadde Granada, 10871

Granada,Spain.
Accepted
for publication
November
21, 2006.

Synopsis

Wrinkling of the skinis the mostobvioussignof deterioration

of the humanbodywith age.This process
involvesa numberof genetic,constitutional,hormonal,nutritional, and environmentalfactors,in addition
to the influenceof frequentlyrepeatedfacialmovements
duringlaughing,smoking,etc.This articlereviews
the physiological
basisand mechanismof actionof the activecosmeticingredientacetylhexapeptide-8

(Argireline).
We prepared
twoformulations:
anemulsion
withanexternal
aqueous
phase
fornormalto dry
skin, and a gel for oily skin. Laboratoryanalyses,
theologytestsand in vitroreleaseassays
were usedto
evaluatethe stability of theseformulationsfor cosmetictreatment.

INTRODUCTION

The searchfor new compoundsto preventor attenuateskin aging and enhanceselfimage(1) is a priority of currentresearchon activecosmetics.
Given the socialimplicationssurroundingphysicalappearance,
we have undertakenwork to investigatethe
treatmentof facialexpression
wrinkles.Favorableresultswith botulin toxin infiltration
led to the developmentof a new activeprinciplewith effectssimilar to the botoxeffect,
namedArgireline, asan alternativeto botulinumtoxin.
Unlike othercreamsdevelopedto treat agingwrinkles,the formulastestedin this study
are intendedto treat expression
wrinkles.Substances
with a botox-likeeffectact upon
the samephenomenonas botulin toxin, but via a different mechanismof action. To
understandthe mechanismof action of the formulaswe tested, a brief review of how
expression
wrinklesare formedmay be helpful.
Expression
wrinkles(2) form asa resultof repeatedmusclecontractioncausedby dermal
atrophyand the appearance
of hypodermalfibrosis(3). Facialmovementscausecellsof
the dermisto contractand relax, and subjectfibroblastsanchoredby the networkof

Addressall correspondence
to M. A. Ruiz Martinez.
157

158

JOURNAL OF COSMETIC SCIENCE

collagenand elastinfibersto similar stresses.

As a result, the skin becomescontracted
into a permanentexpression
wrinkle, where the extracellularmatrix of collagenand
elastin has been found to break down (4).

Severalprocesses
arevulnerableto alterationfrom skin wrinkling in cosmeticterms(5):
Neuronalexocytosis
involvesneurotransmitter
releasefrom synapticvesiclesinto the
synapticspace.Synapticvesiclesbearingneurotransmitters
aretakenup by the soluble
N-ethylmaleimide-sensitive
factor attachmentprotein receptor(SNARE) complex
and fusedwith the cell membrane,releasingneurotransmitters
in the process.
The
receptorcomplexconsistsof three proteins:synaptobrevin(VAMP), syntaxin,and
synaptosomal-associated
protein(SNAP-25) (6).
Contractionand relaxationof fibroblasts,the cellsthat producecollagenand elastin
and are responsiblefor maintaining the extracellularmatrix, are transmittedto the
connectivetissue,wheretheseforcessuccessively
stretchand relax the skin.

Specifically,
type A botulintoxin producedby Clostridim
botlinmactsby irreversibly
destroyingSNAP-25 protein in the SNARE complex,thus preventingthe releaseof
acetylcholineand paralyzingthe involvedmuscle(7). Between 15 and 20 days after
infiltration,newnerveendingsareformed,andtheseendingsbecomeactivewithin two
or threemonths.After threeto six months,nervesignalsto the musclearecompletely
restored (8).

A novelaspectof Argireline
is its ability to act via topicalapplication,
whichoffers
multiple advantages
in comparisonto formulationsbasedon botulin toxin. The main

advantage
of Argireline
liesin its lowertoxicityperunit weight.Onegramof botulin
toxinisenoughto kill onemillionpersons,
whereas
Argireline is about4000 timesless
potent, and thusconstitutesa saferalternativefor treatingwrinkles(9). With the latter,
injectionsto the face,whichare potentiallyuncomfortable
or painful,are unnecessary.

Moreover,Argireline can be usedas an interim treatmentbetweenbotulin toxin

injections,sinceit prolongsthe effectsof Botoxand reducesthe frequencyof microinjections.The syntheticpeptideis cheaper,and is indicatedfor personswho havedevelopedimmunity to botulin toxin after prolongeduse.

Argireline
(acetylhexapeptide-8),
theactiveprinciplein theformulations
studiedhere,
is a hexapeptideformedfrom a chainof six amino acidslinked via a syntheticprocess.
This peptidehastwo main actions.One is musclerelaxationby inhibiting the SNARE

complex,but unlikebotulintoxin,Argireline
doesnot irreversibly
destroythe SNAP25 protein,but modifiesits conformation
and competeswith it for sitesin the SNARE
complex.The hexapeptide,an analogof the N-terminal end of the SNAP-25 protein,
doesnot completelydestroythe SNARE complexbut only destabilizesit slightly, such
that the synapticvesiclescannotbind and releaseacetylcholineefficiently(10). As a
result,a degreeof neurotransmission
is preserved
in equilibriumwith musclerelaxation,

musclecontraction
is attenuated,
andwrinkleformationis prevented
(11). Argireline

is alsoable to relax fibroblaststhat relax the collagenand elastinmatrix, througha

mechanisminvolving calciumion uptake.

The main objectiveof this studywas to developa formulationthat ensuredtransformation of the activeprinciple(acetylhexapeptide-8)into a cosmeticallyactiveproduct.
We thereforestudiedstability,definedasthe ability of the formulationto maintainits
initial characteristics.
The parameterswe measuredasrelevantto structuralchangesthat
canoccurin cosmeticformulationswerechangesin organolepticcharacteristics
(a fun-

COSMETIC FORMULATIONS

WITH

ARGIRELINE

159

damentalconsideration
for useracceptability),heat stability, and rheologicalcharacteristicsover time and at different temperatures.

MATERIAL

AND

METHODS

MATERIALS

The productsusedas componentsin our formulationswere:

Argireline (acetylhexapeptide-8),
BatchF1460/04, suppliedby Lipotec(Barcelona,
Spain).

Neo PCL o/w Autoemulsionable

(ceraalba, stearylheptanoate,cetearyloctanoate,
cetyl palmirate,stearylalcohol,steareth-7,steareth-10,stearylcaprylate,isopropyl
myristate,myristyl alcohol,dimethicone,paraffinurnliquidurn), Batch 0512651,
suppliedby Roig Farma-Fagron(Terrasa,Spain).
Tefose 2561 (PEG-6 stearate,Ceteth-20, glyceryl stearate,Steareth-20), Batch
0503697, suppliedby Roig Farma-Fagron(Terrasa,Spain).
Cyclomethicone
pentamer(cyclopentasiloxane),
purity 99.26%, Batch0509565, supplied by Roig Farma-Fagron(Terrasa,Spain).
Sorbitol70%, Ph. Eur., purity 70.1%, Batch 0405020, suppliedby Roig FarmaFagron(Terrasa,Spain).
Glycerol,Ph. Eur., purity 99.8%, Batch05F0204, suppliedby Roig Farma-Fagron
(Terrasa, Spain).

Hispagel200, Batch0409242, suppliedby Roig Farma-Fagron

(Terrasa,Spain).
Propyleneglycol,Ph. Eur., watercontent<0.1%, Batch04K23FP, suppliedby Roig
Farma-Fagron(Terrasa,Spain).

Phenonip
(phenoxyethanol,
methylparaben,
butylparaben,ethylparaben,
propylparaben),Batch0510592, suppliedby Roig Farma-Fagron(Terrasa,Spain).
KathonCG (methylchloroisothiazolinone
1.5%, methylisothiazolinone
0.37%), purity 75.2%, Batch0504885, pH 2.6.
Deionizeddistilled water, suppliedby Interapothek(Murcia, Spain).
METHODS

Preparation
ofjbrmalations.
We preparedanoil/wateremulsionfor normal-to-dryskinand
a creamforoilyskin.Argireline
is soldunderthebrandnameLipotec
asa transparent
aqueoussolutionthat contains0.5 g/1 acetylhexapeptide-8,0.5% Phenonip
, and
99.45% water.It wasrefrigerated
uponarrivalat the laboratoryand kept at 4C, and
addedcold to all formulations
at a concentration
of 5% Argireline
solution.The
compositions
of the creamand gel formulationsare listed in Table I.
The creamwaspreparedby weighingthe ingredientsof the oil andwaterphases,heating
the oil phaseuntil all componentshad melted, heating the water phaseto the same
temperatureundergentleshakingto ensurehomogeneity,
and obtainingthe emulsion
by addingthe waterphaseto the oil phase.The systemwasstabilizedby gentleshaking
while the formulation cooledto room temperature.
The gel waspreparedby weighingall ingredients,slowly addingwater, and shaking
gently(to avoidthe formationof bubbles)until a gel hadformed.The formulations
were

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JOURNAL OF COSMETIC SCIENCE

Table

Formulasfor the Gel andCreamFormulations

with Argireline

Formula
Gel

Composition
Hispagel 200 25%

Propylene
glycol3% + Argireline
5%
Phenonip0.3%
Water
Cream

to a volume of 100 ml

Oil phase
Neo PCL O/W
Tefose 1.5 %

23%

Cyclomethicone
pentamere2%
Water phase
Sorbitol 4%

Glycerine4%
Kathon

0.1%

Water to a volumeof 100 ml + Argireline 5%

storedat 4C and room temperature(25C). To preparecreamand gel we useda

propeller Heidolph RZR 1.

Organoleptic
characteristics.
Organolepticcharacteristics
were classifiedwith descriptive
terms(12) asthick, hard,creamy,smooth,soft,dry, thin, spreadable,
cool,or warm. The
creamandgel werescoredfor color,odor,texture,consistency,
andappearance
(exudates)
24 h afterpreparationandafterstorageat both temperatures
for 30, 60 and 90 days,six
months, and 12 months.

pH. ChemicalstabilitywasevaluatedaspH during storagefor threemonthsto predict

the behaviorof the formulationsin contactwith humanskin. To measurepH we used
a Crison 501 digital pH/mV-meter with the electrodefor viscoussamples.
Rheological
characteristics.
The rheologicalpropertiesof the formulationswerestudiedas
viscosity,a parametercloselyrelatedwith stability (13). Assayswere run at increasing
shearratesin a BrookfieldDV II+ viscosimeter(BrookfieldEngineeringLaboratories,
Stoughton,MA) connected
to a PC with the appropriatesoftware.Rheologicaldatawere
recordedperiodicallyduring a maximum period of 30 days.

Stability.The activityof Argireline peptidewasstudiedasthe effectof temperature

on
the stabilityof the activeprinciple.Samples
of the commercially
availableArgireline
solutionwerestoredat 25C,40C,and60Cin an incubatorfor 24 h, andactivitywas
then determinedwith high-performance
liquid chromatography.
In vitro release

Assayswith no membrane.To avoidmanipulationsand vehiclesthat might interfere

with the cutaneous
releaseof Argireline, we studiedreleasefrom the gel and cream
formulationsin vitro.In this studywe testeddiffusionin a systemwith no membrane,
in which the excipientand the receptorphasewere in direct contact(14,15). Both
formulationswere alsostudiedin an in vitroreleasesystemthat simulatedthe physiologicalconditionsof drug desorption(16). To simulatetheseconditions,the formulationswereplacedin a 32C bath at 60 rpm in the releasemediaphosphate-buffered
solution(PBS)at pH 5.6. Releasewasmeasuredby spectrophotometry
overtime and at

COSMETIC

FORMULATIONS

WITH

ARGIRELINE

161

a wavelengthof 260 nm, at which absorptionof the activeprincipleis maximal.The

sameformulationwith no activeprinciple wasassayedas a control.
Diffusion acrossa membrane.Most publishedstudiesinvolveFranz-typecells(17-19).
The FDC-400 cell (Vidra-Foc,Barcelona,Spain)consists
of two compartments
with a
membraneclampedbetweenthe donorandreceiverchambers.As the receptorphasewe
useda phosphate-buffered
solutionat pH 5.6 (normalskin pH). Three typesof membrane(all 47 mm in diameterwith 0.45-m pore size)were tested:methylcellulose,
nylon, and polysulfone(suppliedby Millipore, Madrid, Spain).

The concentrationof Argireline in the receptor cell was measuredby UVspectrophotometry

at 260 nm (kmax).The methodwaspreviously
validatedandverified
for accuracy,precision,and linearity (20). A Perkin Elmer UV/Vis Lambda40 UVspectrophotometer
was usedfor all measurements.

RESULTS

AND

DISCUSSION

The data aregiven asthe meanand standarddeviationof six determinationsmadewith

samplesof eachformulationat eachtemperatureandaftereachstorageperiod.All results
werecomparedby analysisof variance(ANOVA) for a 95% confidence
levelto identify
significantdifferences.
ORGANOLEPTIC

CHARACTERISTICS

TablesII and III showthe changesin organolepticpropertieswith time in the gel and
creamformulations,respectively.The temperatureor duration of storagedid not significantlyaffectthe externalappearance
or textureof eitherformulationafter12 months.
After 30 days,refrigeratedsamplesshowedbetter consistency
than samplesstoredat
room temperature.Consistencytended to decreasein the gel formulation after 12
months,with no differencesbetweensamplesstoredunder refrigerationor at room
Table

II

Changesin OrganolepticCharacteristics
of the Gel FormulationDuring Storage
Storageconditions

Time

Temp. (C)

0 days

30 days
60 days
90 days
6 months
12 months

Organolepticcharacteristics

Color

Texture

Transparent Smooth,thin, cool

Odor

Consistency Exudate

Noticeable

Viscous,easy

No
No

No
No
No
No
No
No
No
No
Yes
Yes

25

Transparent Smooth,thin

Noticeable

to spread
Viscous,easy
to spread

4
25
4
25
4
25
4
25
4
25

Unchanged
Unchanged
Unchanged
Unchanged
Unchanged
Unchanged
Unchanged
Unchanged
Unchanged
Unchanged

Unchanged
Unchanged
Unchanged
Unchanged
Unchanged
Unchanged
Unchanged
Unchanged
Change
Change

Unchanged
Thinner
Unchanged
Unchanged
Unchanged
Unchanged
Unchanged
Unchanged
Decrease
Decrease

Unchanged
Unchanged
Unchanged
Unchanged
Unchanged
Unchanged
Unchanged
Unchanged
Unchanged
Unchanged

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JOURNAL OF COSMETIC SCIENCE

Table

III

Changesin OrganolepticCharacteristics
of the CreamFormulationDuring Storage
Storageconditions
Time

Temp. (C)

0 days

60 days
90 days
6 months
12 months

Color

White

25

30 days

Organolepticcharacteristics

4
25

Texture

Odor

Consistency

Smooth,creamy Noticeable Viscous,easy

Exudate

No

to spread
White
Smooth,creamy Noticeable Viscous,easy
to spread
Unchanged Unchanged
Unchanged Viscous,harder
Unchanged Unchanged
Unchanged Viscous,softer

No
No

No

Unchanged Unchanged

Unchanged Unchanged

No

25
4
25
4
25
4

Unchanged
Unchanged
Unchanged
White
White
White

Unchanged
Unchanged
Unchanged
Smooth,creamy
Smooth,creamy
Creamy,hard

Unchanged
Unchanged
Unchanged
Unchanged
Unchanged
Unchanged

No
No
No
No
No
Yes

25

White

Smooth,creamy Unchanged Viscous,harder

Unchanged
Unchanged
Unchanged
Viscous,harder
Viscous,harder
Viscous,harder,
crust formation

No

temperature.In the creamformulation,consistency

increasedafter six months,and a
crusthad formedafter storagefor 12 monthsat 4C.

pH

The data in Table IV showthat pH wasacidicin both of the freshlymadeup formulations,but washigherin the gel than in the cream.No significantchangesovertime
wereseenin eitherformulationregardless
of storagetemperature,a finding that makes
theseformulationssuitablefor topicalapplication(only in regardto pH value).

RHEOLOGICAL

CHARACTERISTICS

Rheologicalassays
to measureviscosityunderdifferentstorageconditionsand at different timesindicatedthat bothformulationsshowedpseudoplastic
behavior.Figures1 and
2 plot the meanvaluesfor the creamformulationafter24 h, sevendays,and 30 daysof
storageat 4C and 25C, respectively.
Storagetemperaturehadno significanteffecton
Table

IV

Changesin pH During Storage

Time (days)

pH
Gel
Cream

30

60

90

4C
25C
4C

6.18
6.36
4.1

6.1
6
4

6.18
6.25
4.2

6.12
6
4.1

25C

3.92

3.95

4.05

COSMETIC

FORMULATIONS

WITH

ARGIRELINE

163

80000

---25

C 24 h

25C7days

25C 30 days

60000

- 40000
o

20000

0.5

2.5

10

100

Speed (rpm)
Figure 1. Viscosityvs speedof Argireline
creamsmaintainedat 25Casa functionof storagetime.

viscosity,and shearrateswere the samein both samplesat all assaytimes, a result that
suggests
that the creamformulationcan be safelystoredat room temperature.

Figures3 and4 showthe findingsfor the gel formulationafterstoragefor up to 30 days

at the two temperatures.
Viscositywas slightly lower in refrigeratedsamplesthan in
sampleskept at roomtemperature,asa resultof thermalgelling (seenat low shearrate)
(21). In samplestestedafter 30 daysof storage,viscositywas the sameat both temperatures.

At both storagetemperatures,
viscositywaslower in the gel than in the creamformulation. However, in general,temperaturedid not affecteither formulationunder our
study conditions.No significant changesin rheologicalcharacteristicswere seen in
either formulationduring the 30-day period in which viscositywas studied.
STABILITY

The chromatographic
dataare shownin Table V. Figures5 to 7 are chromatograms
of
acetylhexapeptide-8at roomtemperature(25C)and after beingheatedto 40C and
60C for 24 h. The presenceof the activeprinciple decreased
to 58.8% and 41%,
respectively,
making extremetemperatures
a factorto take into consideration
in efforts
to improvethe stabilityof the activeingredientduring storageand during heating,if
this is requiredin the processof formulation.

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80000

--I

4 C 24 h

4 C 7 days

4C30days

60000

40000

20000

0.5

2.5

1o

1oo

Speed (rpm)
Figure 2. Viscosityvs speedof Argireline
creamsmaintained
at 4Casa functionof storagetime.
IN

VITRO

RELEASE

Release
assays
with nomembrane.
Figure 8 showsthe percentageof acetylhexapeptide-8
released
from the creamand gel excipientinto the mediumwith time in samplesstored
at 4C and 25C.In the creamformulation,releasewasgreaterfrom samplesstoredat
roomtemperaturethan from refrigeratedsamples.The viscosityof the creamformulation at 25C waslower than at 4C; hencethe fasterreleaseof the activeprinciple.
However,in the gel formulation,percentagereleasewaslower from samplesstoredat
25Cthanfromrefrigerated
samples,
because
of gellingasnotedabovein the rheological
assays(22).

The data showedan increasein releasefrom both excipientswith time at both temperatures,
with maximalreleaseafter90 min. The rateof releaseof the activeprinciple
was consideredsuitablefor use in topicalpreparationssinceit did not interferewith
other processes
that take placewhen the activeprincipleis placedin contactwith the
skin.

Diffusionacross
the membrane.
We selectedas the most suitablemembranethat which
offeredthe leastresistance
to diffusionof the activeprinciple,in orderto minimizethe

COSMETIC

FORMULATIONS

WITH

ARGIRELINE

165

60000

----

25 C 24 h

----

25 C 7 days

"

25 C 30 days

40000
o

20000

0.5

2.5

10

100

Speed (rpm)
Figure 3. Viscosityvs speedof Argireline
gel maintained
at 25Casa functionof storagetime.

influence
of the membrane
on the results.
Forthisstudy,a 5 mg m1-1solution
of
Argireline
wasusedasthe donorphase,andPBS(pH 5.6) wasusedasthe solventto
preparethe drugin the donorphase.This bufferwasalsousedasthe receptorphase.We
previouslyverifiedthat sink conditionswere maintained.Diffusionwasslightly more
rapidwith the nylonmembrane,and we thereforeusedthis membranefor subsequent
studies.Both preparations
weresubjectedto i, vitrodiffusionassays
in a Franzcell. All
assayswere performedunder the sameconditionsas describedabovein the sectionon
membrane

selection.

Releaseassays
to measurethe amountandpercentage
of activeprinciplein the receptor
cell(Figures9 and 10) showedthat release
wasgreaterfrom the creamformulation(50%
after five hours)than from the gel (20% after five hours).The differencemay reflect
thermalgelling of the latter formulationupon contactwith the dispersionmedium
i, vitro,an effectthat wouldbe expectedto increase
viscosity.Diffusionof the peptide
wasfirst detectedten minutesafter the essaywasstarted.

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60000

4 C 24 h

4 C 7 days
',','
,,,

4 oC 30 days

40000
o
u

20000

0.5

2.5

10

100

Speed (rpm)
Figure 4. Viscosityvs speedof Argireline
gel maintained
at 4Casa functionof storagetime.
Table

Chromatographic
Parameters
of Stabilityat DifferentTemperatures
Temperature(C)

RT

AUC

im/ml

25

18.5

1899098

500

40
60

18.5
18.5

1119000
781954

294
205

%
100

58.80
41

Althoughthe rateof absorption

below50% with bothformulations
mayappearlow, the
resultsin generalshowthat both the creamand the gel formulationssatisfiedthe
requirements
for cosmeticproductsintendedfor topicalapplication,sincethe cosmetically activesubstance,
8-acetylhexapeptide,
is targetedto treat the most superficial
layersof the skin (23).

COSMETIC

FORMULATIONS

WITH

ARGIRELINE

167

020

000'

oo

Figure 5. Chromatogram
of Argireline
at initial time.

-o,o ,',

, , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , ,
500

iO,00

15,00

20,00

25,00

30=00

15,00

Figure6. Chromatogram
of Argireline
24 hoursafterbeingsubjected
to temperature
of 40C.

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JOURNAL OF COSMETIC SCIENCE

Figure 7. Chromatogram
of Argireline
24 hoursafterbeingsubjected
to temperature
of 60C.

Its mechanismof action differsfrom that of botulinum toxin (24). It penetratesthe

stratum comeurnbut doesnot penetratethe dermis(5). Its sitesof actionare the
nociceptors,
thermoreceptors,
and mechanoreceptors
connectedto the nervoussystemvia
afferentfibers,which in turn areconnected
to the underlyingmusculature.
This enables
8-acetyl hexapeptideto act upon musclefiberswithout penetratingthe muscletissue
(25).

CONCLUSIONS

The formulationswe testedshowedgoodthixotropyand a slightly acidpH, and their

rheologicalbehaviorandorganoleptic
propertieswerestablefor the mostpart underthe
temperatureand storageconditionsreportedhere (4C and 25C). Interestingly,we
foundevidenceof activityof the activeprincipleunderextremetemperatureconditions
(40C and 60C).

The excipientsdid not impede the releaseof 8-acetylhexapeptideor contactwith

the skin, and facilitated releasethroughoutthe 90-min assayperiod. Releasewas
greater from samplesstoredat room temperaturethan from refrigeratedsamples.

COSMETIC FORMULATIONS

120

WITH

ARGIRELINE

169

100
80

Cream

4C

[] Cream

25 C

'1-

60

[] Gel 4 C
40

[] Gel 25 C

20

15

3O

45

6O

9O

Time (min)
Figure 8. Release
withoutmembraneof the Argireline
samples
maintainedat 25 and4C.

The gel formulation showedevidenceof thermal gelling during the first 15 days
of storageafter preparation,and this reduceddiffusion of the peptide from samples stored at 25C. The resultsof in vitro assaysconfirmedthat the active principle penetratedthe artificial membraneand that it is a suitabledeliveryfrom both
excipients.

lOO

80

CREAM

---A--GEL
60

40
20
0

Time (hours)
Figure 9. Percentage
of ArgirelJne
released
fromthe gel andcreamasa functionof the time.

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JOURNAL OF COSMETIC SCIENCE

60

40
20

g
<E

-AM

Time (hours)
Figure 10. Amountof Argireline
released
asa functionof the timefor the two testedsamples.
ACKNOWLEDGMENTS

Part of thisworkwassupported
by the Spanish
Ministryof EducationandScience
and
byEuropean
Regional
Development
FundsunderProjectMAT2005-07746-C02-02
and
Projectof Excellence
FQM 410. We thank K. Shashok
for translatingpartsof the
original manuscriptinto English.

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