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

Preparationandstabilityof cosmeticformulationswith an anti-agingpeptide

M. A. RUIZ,

B. CLARES, M. E. MORALES,

S. CAZALLA,

and

V. GALLARDO, DepartamentodeFarmaciay Tecnolog/a

Farmacgutica,Facultad de Farmacia, Universidadde Granada,

10871

Granada,Spain.

Acceptedfor publicationNovember21, 2006.

Synopsis

Wrinkling of theskinis the mostobvioussignof deteriorationof the humanbodywith age.Thisprocess

involvesa numberof genetic,constitutional,hormonal,nutritional,andenvironmentalfactors,in addition

to the influenceof frequentlyrepeatedfacialmovementsduringlaughing,smoking,etc.Thisarticlereviews

the physiologicalbasisand mechanismof actionof the activecosmeticingredientacetylhexapeptide-8

(Argireline©).We preparedtwoformulations:anemulsionwithanexternalaqueousphasefornormaltodry

skin, and a gel for oily skin. Laboratoryanalyses,theologytestsand in vitroreleaseassayswereusedto

evaluatethe stabilityof theseformulationsfor cosmetictreatment.

INTRODUCTION

The searchfor new compoundsto preventor attenuateskin aging and enhanceself- image(1) is a priority of currentresearchon activecosmetics.Given the socialimpli-

cationssurroundingphysicalappearance,we haveundertakenwork to investigatethe treatmentof facialexpressionwrinkles.Favorableresultswith botulin toxin infiltration led to the developmentof a newactiveprinciplewith effectssimilarto the botoxeffect,

namedArgireline©, asan alternativeto botulinumtoxin.

Unlike othercreamsdevelopedto treatagingwrinkles,the formulastestedin thisstudy areintendedto treat expressionwrinkles.Substanceswith a botox-likeeffectact upon

the samephenomenonas botulin toxin, but via a different mechanismof action. To understandthe mechanismof actionof the formulaswe tested,a brief review of how

expressionwrinklesareformedmay be helpful.

Expressionwrinkles(2) formasa resultof repeatedmusclecontractioncausedby dermal atrophyand the appearanceof hypodermalfibrosis(3). Facialmovementscausecellsof the dermisto contractand relax,and subjectfibroblastsanchoredby the networkof

Addressall correspondenceto M. A. Ruiz Martinez.

157

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collagenand elastinfibersto similar stresses.As a result,the skin becomescontracted

into a permanentexpressionwrinkle, wherethe extracellularmatrix of collagenand

elastin has been found to break down (4).

Severalprocessesarevulnerableto alterationfromskinwrinkling in cosmeticterms(5):

ß Neuronalexocytosisinvolvesneurotransmitterreleasefromsynapticvesiclesinto the synapticspace.Synapticvesiclesbearingneurotransmittersaretakenup bythesoluble

N-ethylmaleimide-sensitivefactorattachmentprotein receptor(SNARE) complex

and fusedwith the cell membrane,releasingneurotransmittersin the process.The

receptorcomplexconsistsof three proteins:synaptobrevin(VAMP), syntaxin,and

synaptosomal-associatedprotein(SNAP-25) (6).

ß Contractionand relaxationof fibroblasts,the cellsthat producecollagenandelastin

and are responsiblefor maintainingthe extracellularmatrix, are transmittedto the connectivetissue,wheretheseforcessuccessivelystretchand relaxthe skin.

Specifically,typeA botulintoxinproducedby Clostridi•mbot•lin•mactsby irreversibly

destroyingSNAP-25 proteinin the SNARE complex,thus preventingthe releaseof

acetylcholineand paralyzingthe involvedmuscle(7). Between15 and 20 daysafter

infiltration,newnerveendingsareformed,andtheseendingsbecomeactivewithin two or threemonths.After threeto sixmonths,nervesignalsto the musclearecompletely

restored (8).

A novelaspectof Argireline© is its abilityto actvia topicalapplication,whichoffers

multiple advantagesin comparisonto formulationsbasedon botulin toxin. The main

advantageofArgireline© liesin its lowertoxicityperunitweight.Onegramofbotulin

toxinisenoughto kill onemillionpersons,whereasArgireline©isabout4000 timesless

potent,andthusconstitutesa saferalternativefor treatingwrinkles(9). With the latter,

injectionsto the face,whicharepotentiallyuncomfortableor painful,areunnecessary.

Moreover,Argireline© can be usedas an interim treatmentbetweenbotulin toxin

injections,sinceit prolongsthe effectsof Botoxand reducesthe frequencyof microin-

jections.The syntheticpeptideis cheaper,and is indicatedfor personswho havedevel-

opedimmunity to botulin toxin afterprolongeduse.

Argireline©(acetylhexapeptide-8),theactiveprinciplein theformulationsstudiedhere,

is a hexapeptideformedfrom a chainof sixaminoacidslinkedvia a syntheticprocess. This peptidehastwo main actions.One is musclerelaxationby inhibiting the SNARE

complex,but unlikebotulintoxin,Argireline©doesnotirreversiblydestroytheSNAP-

25 protein,but modifiesits conformationandcompeteswith it for sitesin the SNARE complex.The hexapeptide,an analogof the N-terminal end of the SNAP-25 protein,

doesnot completelydestroythe SNARE complexbut onlydestabilizesit slightly,such

that the synapticvesiclescannotbind and releaseacetylcholineefficiently(10). As a result,a degreeof neurotransmissionispreservedin equilibriumwith musclerelaxation,

musclecontractionis attenuated,andwrinkleformationisprevented(11). Argireline©

is alsoable to relaxfibroblaststhat relaxthe collagenandelastinmatrix, througha

mechanisminvolvingcalciumion uptake.

The main objectiveof this studywasto developa formulationthat ensuredtransfor-

mationof 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

damentalconsiderationfor useracceptability),heatstability,and rheologicalcharacter- isticsovertime and at differenttemperatures.

MATERIAL

MATERIALS

AND

METHODS

The productsusedascomponentsin our formulationswere:

ß Argireline© (acetylhexapeptide-8),BatchF1460/04,suppliedby Lipotec(Barcelona,

Spain).

ß 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).

ß Tefose2561 (PEG-6 stearate,Ceteth-20, glyceryl stearate,Steareth-20),Batch 0503697, suppliedby Roig Farma-Fagron(Terrasa,Spain).

ß Cyclomethiconepentamer(cyclopentasiloxane),purity99.26%, Batch0509565, sup-

plied by Roig Farma-Fagron(Terrasa,Spain).

ß Sorbitol70%, Ph. Eur., purity 70.1%, Batch0405020, suppliedby Roig Farma-

Fagron(Terrasa,Spain).

ß Glycerol,Ph. Eur., purity 99.8%, Batch05F0204, suppliedby Roig Farma-Fagron

(Terrasa,Spain).

ß Hispagel200©, Batch0409242,suppliedby RoigFarma-Fagron(Terrasa,Spain).

ß Propyleneglycol,Ph. Eur.,watercontent<0.1%, Batch04K23FP,suppliedby Roig

Farma-Fagron(Terrasa,Spain).

ß Phenonip© (phenoxyethanol,methylparaben,butylparaben,ethylparaben,propyl-

paraben),Batch0510592, suppliedby Roig Farma-Fagron(Terrasa,Spain).

ß KathonCG© (methylchloroisothiazolinone1.5%, methylisothiazolinone0.37%), pu- rity 75.2%, Batch0504885, pH 2.6.

ß Deionizeddistilledwater, suppliedby Interapothek(Murcia, Spain).

METHODS

Preparationofj•brmalations.We preparedanoil/wateremulsionfornormal-to-dryskinand

acreamforoilyskin.Argireline©issoldunderthebrandnameLipotec©asa transparent

aqueoussolutionthat contains0.5 g/1 acetylhexapeptide-8,0.5% Phenonip©, and

99.45% water.It wasrefrigerateduponarrivalat the laboratoryandkept at 4øC,and

addedcold to all formulationsat a concentrationof 5% Argireline© solution.The

compositionsof the creamandgel formulationsarelistedin Table I.

The creamwaspreparedbyweighingtheingredientsof theoil andwaterphases,heating

the oil phaseuntil all componentshad melted, heating the water phaseto the same

temperatureundergentleshakingto ensurehomogeneity,andobtainingthe emulsion

by addingthewaterphaseto the oil phase.The systemwasstabilizedby gentleshaking

while the formulationcooledto room temperature.

The gel waspreparedby weighingall ingredients,slowlyaddingwater, and shaking

gently(to avoidtheformationofbubbles)until a gelhadformed.Theformulationswere

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Table

I

Formulasfor the Gel andCreamFormulationswith Argireline©

Formula

Gel

Cream

Composition

Hispagel 200 25%

Propyleneglycol3% + Argireline© 5%

Phenonip0.3%

Water

to a volume

Oil phase

of 100

ml

Neo PCL O/W 23%

Tefose 1.5 %

Cyclomethiconepentamere2%

Water phase

Sorbitol 4%

Glycerine4%

Kathon

0.1%

Water to a volumeof 100 ml + Argireline© 5%

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

propellerHeidolph RZR 1.

Organolepticcharacteristics.Organolepticcharacteristicswere classifiedwith descriptive

terms(12) asthick, hard,creamy,smooth,soft,dry, thin, spreadable,cool,or warm.The

creamandgelwerescoredforcolor,odor,texture,consistency,andappearance(exudates) 24 h afterpreparationandafterstorageat bothtemperaturesfor 30, 60 and90 days,six

months, and 12 months.

pH. ChemicalstabilitywasevaluatedaspH duringstoragefor threemonthsto predict

the behaviorof the formulationsin contactwith humanskin.To measurepH we used

a Crison501 digital pH/mV-meter with the electrodefor viscoussamples.

Rheologicalcharacteristics.The rheologicalpropertiesof the formulationswerestudiedas viscosity,a parametercloselyrelatedwith stability(13). Assayswererun at increasing

shearratesin a BrookfieldDV II+ viscosimeter(BrookfieldEngineeringLaboratories,

Stoughton,MA) connectedto a PC with theappropriatesoftware.Rheologicaldatawere

recordedperiodicallyduring a maximumperiodof 30 days.

Stability.TheactivityofArgireline©peptidewasstudiedastheeffectof temperatureon the stabilityof the activeprinciple.Samplesof the commerciallyavailableArgireline©

solutionwerestoredat 25øC,40øC,and60øCin anincubatorfor 24 h, andactivitywas

then determinedwith high-performanceliquid chromatography.

In

vitro release

Assayswith no membrane.To avoidmanipulationsandvehiclesthat might interfere

with the cutaneousreleaseof Argireline©, we studiedreleasefrom the gel andcream

formulationsin vitro.In this studywe testeddiffusionin a systemwith no membrane, in which the excipientand the receptorphasewere in direct contact(14,15). Both formulationswerealsostudiedin an in vitroreleasesystemthat simulatedthe physi- ologicalconditionsof drug desorption(16). To simulatetheseconditions,the formu- lationswereplacedin a 32øCbath at 60 rpm in the releasemediaphosphate-buffered solution(PBS)at pH 5.6. Releasewasmeasuredby spectrophotometryovertime andat

COSMETIC

FORMULATIONS

WITH

ARGIRELINE

©

161

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

sameformulationwith no activeprinciplewasassayedasa control.

Diffusionacrossa membrane.Most publishedstudiesinvolveFranz-typecells(17-19).

The FDC-400 cell (Vidra-Foc,Barcelona,Spain)consistsof two compartmentswith a

membraneclampedbetweenthe donorandreceiverchambers.As the receptorphasewe

useda phosphate-bufferedsolutionat pH 5.6 (normalskinpH). Threetypesof mem-

brane(all 47 mm in diameterwith 0.45-•m pore size)were tested:methylcellulose,

nylon,andpolysulfone(suppliedby Millipore, Madrid, Spain).

The concentrationof Argireline© in the receptor cell was measuredby UV- spectrophotometryat 260 nm(kmax).Themethodwaspreviouslyvalidatedandverified

for accuracy,precision,and linearity(20). A Perkin Elmer UV/Vis Lambda40 UV-

spectrophotometerwasusedfor all measurements.

RESULTS

AND

DISCUSSION

The dataaregivenasthe meanandstandarddeviationof sixdeterminationsmadewith samplesof eachformulationat eachtemperatureandaftereachstorageperiod.All results

werecomparedby analysisof variance(ANOVA) for a 95% confidencelevelto identify significantdifferences.

ORGANOLEPTIC

CHARACTERISTICS

TablesII andIII showthe changesin organolepticpropertieswith time in the gel and

creamformulations,respectively.The temperatureor durationof storagedid not sig-

nificantlyaffecttheexternalappearanceortextureofeitherformulationafter12 months.

After 30 days,refrigeratedsamplesshowedbetter consistencythan samplesstoredat

room temperature.Consistencytended to decreasein the gel formulation after 12

months,with no differencesbetweensamplesstoredunder refrigerationor at room

Table

II

Changesin OrganolepticCharacteristicsof the Gel FormulationDuring Storage

Storageconditions

Organolepticcharacteristics

Time

Temp. (øC)

Color

Texture

Odor

Consistency

Exudate

0

days

4

Transparent Smooth,thin, cool

Noticeable

Viscous,easy

No

 

to spread

 

25

Transparent

Smooth,thin

Noticeable

Viscous,easy

No

 

to spread

30

days

4

Unchanged

Unchanged

Unchanged Unchanged

No

 

25

Unchanged

Unchanged

Unchanged

Thinner

No

60

days

4

Unchanged

Unchanged

Unchanged Unchanged

No

 

25

Unchanged

Unchanged

Unchanged

Unchanged

No

90

days

4

Unchanged

Unchanged

Unchanged Unchanged

No

 

25

Unchanged

Unchanged

Unchanged

Unchanged

No

6

months

4

Unchanged

Unchanged

Unchanged Unchanged

No

 

25

Unchanged

Unchanged

Unchanged

Unchanged

No

12

months

4

Unchanged

Unchanged

Change

Decrease

Yes

 

25 Unchanged

Unchanged

Change

Decrease

Yes

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JOURNAL OF COSMETIC SCIENCE

Table

III

Changesin OrganolepticCharacteristicsof the CreamFormulationDuring Storage

Storageconditions

Time

Temp. (øC)

Color

Organolepticcharacteristics

Texture

Odor

Consistency

Exudate

0

days

4

White

Smooth,creamy Noticeable

Viscous,easy

No

 

to spread

 

25

White

Smooth,creamy

Noticeable

Viscous,easy

No

 

to spread

30 days

4

Unchanged Unchanged

Unchanged Viscous,harder

No

25

Unchanged

Unchanged

Unchanged

Viscous,softer

No

60 days

4

Unchanged Unchanged

Unchanged Unchanged

No

25

Unchanged

Unchanged

Unchanged

Unchanged

No

90

days

4

Unchanged Unchanged

Unchanged Unchanged

No

 

25

Unchanged

Unchanged

Unchanged

Unchanged

No

6

months

4

White

Smooth,creamy Unchanged Viscous,harder

No

 

25

White

Smooth,creamy

Unchanged

Viscous,harder

No

12

months

4

White

Creamy,hard

Unchanged Viscous,harder,

Yes

 

crust formation

 

25

White

Smooth,creamy

Unchanged Viscous,harder

No

temperature.In the creamformulation,consistencyincreasedafter six months,and a crusthadformedafterstoragefor 12 monthsat 4øC.

pH

The datain Table IV showthat pH wasacidicin bothof the freshlymadeup formu- lations,but washigherin the gel thanin the cream.No significantchangesovertime

wereseenin eitherformulationregardlessof storagetemperature,a finding that makes

theseformulationssuitablefor topicalapplication(only in regardto pH value).

RHEOLOGICAL

CHARACTERISTICS

Rheologicalassaysto measureviscosityunderdifferentstorageconditionsandat differ- enttimesindicatedthat bothformulationsshowedpseudoplasticbehavior.Figures1 and

2 plot themeanvaluesforthecreamformulationafter24 h, sevendays,and30 daysof

storageat 4øCand25øC,respectively.Storagetemperaturehadno significanteffecton

pH

Table

IV

Changesin pH During Storage

0

Time (days)

30

60

90

Gel

4øC

6.18

6.1

6.18

6.12

25øC

6.36

6

6.25

6

Cream

4øC

4.1

4

4.2

4.1

25øC

3.92

4

3.95

4.05

COSMETIC

FORMULATIONS

WITH

ARGIRELINE

©

163

80000

 

--•-25

øC 24

h

C 25øC7days

• 25øC 30 days

60000

ß-• 40000

o

20000

0

0.5

2.5

10

Speed (rpm)

100

Figure 1. Viscosityvsspeedof Argireline© creamsmaintainedat 25øCasa functionof storagetime.

viscosity,and shearrateswere the samein both samplesat all assaytimes,a resultthat

suggeststhat the creamformulationcanbe safelystoredat roomtemperature.

Figures3 and4 showthefindingsforthegel formulationafterstoragefor up to 30 days at the two temperatures.Viscositywasslightly lower in refrigeratedsamplesthan in sampleskept at roomtemperature,asa resultof thermalgelling(seenat low shearrate) (21). In samplestestedafter 30 daysof storage,viscositywasthe sameat both tem-

peratures.

At both storagetemperatures,viscositywaslowerin the gel than in the creamformu- lation. However,in general,temperaturedid not affecteither formulationunderour

study conditions.No significant changesin rheologicalcharacteristicswere seenin

eitherformulationduring the 30-dayperiodin which viscositywasstudied.

STABILITY

The chromatographicdataareshownin TableV. Figures5 to 7 arechromatogramsof acetylhexapeptide-8at roomtemperature(25øC)andafterbeingheatedto 40øCand 60øC for 24 h. The presenceof the activeprincipledecreasedto 58.8% and 41%,

respectively,makingextremetemperaturesa factorto take into considerationin efforts

to improvethe stabilityof the activeingredientduringstorageandduringheating,if

this is requiredin the processof formulation.

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80000

 

--I•

4 øC 24

h

0

4 øC7 days

=

4øC30days

60000

40000

20000

o

0.5

2.5

1o

Speed (rpm)

1oo

Figure2. Viscosityvsspeedof Argireline© creamsmaintainedat 4øCasa functionof storagetime.

IN

VITRO

RELEASE

Releaseassayswith nomembrane.Figure8 showsthe percentageof acetylhexapeptide-8 releasedfromthe creamandgel excipientinto the mediumwith time in samplesstored

at 4øCand 25øC.In the creamformulation,releasewasgreaterfromsamplesstoredat

roomtemperaturethanfrom refrigeratedsamples.The viscosityof the creamformula- tion at 25øCwaslower than at 4øC; hencethe fasterreleaseof the activeprinciple.

However,in the gel formulation,percentagereleasewaslowerfrom samplesstoredat

25øCthanfromrefrigeratedsamples,becauseofgellingasnotedabovein therheological

assays(22).

The data showedan increasein releasefrom both excipientswith time at both tem-

peratures,with maximalreleaseafter90 min. The rateof releaseof theactiveprinciple

wasconsideredsuitablefor usein topicalpreparationssinceit did not interferewith otherprocessesthat take placewhenthe activeprincipleis placedin contactwith the

skin.

Diffusionacrossthemembrane.We selectedas the most suitablemembranethat which

offeredthe leastresistanceto diffusionof the activeprinciple,in orderto minimizethe

COSMETIC

FORMULATIONS

WITH

ARGIRELINE

©

165

 

60000

 

--•--

25

øC 24

h

--•--

25 øC7 days

"

25 øC30 days

 

40000

o

 

20000

0.5

2.5

10

Speed (rpm)

100

Figure3. Viscosityvsspeedof Argireline© gel maintainedat 25øCasa functionof storagetime.

influenceof themembraneon theresults.Forthisstudy,a 5 mg m1-1solutionof

Argireline© wasusedasthedonorphase,andPBS(pH 5.6)wasusedasthesolventto

preparethedrugin thedonorphase.Thisbufferwasalsousedasthereceptorphase.We

previouslyverifiedthat sink conditionsweremaintained.Diffusionwasslightlymore rapidwith the nylonmembrane,andwe thereforeusedthis membranefor subsequent

studies.Both preparationsweresubjectedto i, vitrodiffusionassaysin a Franzcell.All

assayswere performedunder the sameconditionsasdescribedabovein the sectionon

membrane

selection.

Releaseassaysto measuretheamountandpercentageof activeprinciplein the receptor

cell(Figures9 and10) showedthat releasewasgreaterfromthe creamformulation(50%

afterfive hours)than from the gel (20% afterfive hours).The differencemay reflect thermalgelling of the latter formulationupon contactwith the dispersionmedium

i, vitro,an effectthatwouldbeexpectedto increaseviscosity.Diffusionof the peptide

wasfirst detectedten minutesafter the essaywasstarted.

166

 

60000

40000

o

u

 

20000

0

0.5

JOURNAL OF COSMETIC SCIENCE

2.5

4

øC 24

h

• 4 øC7 days

',','

,,,

4

oC

30 days

10

Speed (rpm)

100

Figure4. Viscosityvsspeedof Argireline© gelmaintainedat 4øCasa functionof storagetime.

Table

V

ChromatographicParametersof Stabilityat DifferentTemperatures

Temperature(øC)

RT

AUC

i•m/ml

%

25

18.5

1899098

500

100

40

18.5

1119000

294

58.80

60

18.5

781954

205

41

Althoughtherateofabsorptionbelow50% with bothformulationsmayappearlow,the resultsin generalshowthat both the creamand the gel formulationssatisfiedthe requirementsfor cosmeticproductsintendedfor topicalapplication,sincethe cosmeti-

callyactivesubstance,8-acetylhexapeptide,is targetedto treat the mostsuperficial

layersof the skin (23).

 

COSMETIC

 

FORMULATIONS

 

WITH

 

ARGIRELINE

 

©

167

020

0•00'

o•o•

 

Figure 5. Chromatogramof Argireline© at initial time.

 

-o,o• ,',

,

,

,

,

,

,

,

,

,

,

,

,

,

,

,

,

,

,

,

,

,

,

,

,

,

,

,

,

,

,

,

,

,

,

,

,

,

 

5•00

 

iO,00

 

15,00

 

20,00

 

25,00

 

30=00

 

15,00

 

Figure6. ChromatogramofArgireline© 24 hoursafterbeingsubjectedto temperatureof40øC.

168

JOURNAL OF COSMETIC SCIENCE

Figure7. Chromatogramof Argireline© 24 hoursafterbeingsubjectedto temperatureof 60øC.

Its mechanismof actiondiffersfrom that of botulinumtoxin (24). It penetratesthe

stratumcomeurnbut doesnot penetratethe dermis(5). Its sitesof actionare the

nociceptors,thermoreceptors,andmechanoreceptorsconnectedto the nervoussystemvia

afferentfibers,whichin turn areconnectedto the underlyingmusculature.This enables

8-acetylhexapeptideto act upon musclefiberswithout penetratingthe muscletissue

(25).

CONCLUSIONS

The formulationswe testedshowedgoodthixotropyanda slightlyacidpH, andtheir

rheologicalbehaviorandorganolepticpropertieswerestablefor the mostpartunderthe

temperatureand storageconditionsreportedhere(4øC and 25øC). Interestingly,we foundevidenceof activityof the activeprincipleunderextremetemperatureconditions

(40øC and 60øC).

The excipientsdid not impedethe releaseof 8-acetylhexapeptideor contactwith

the skin, and facilitatedreleasethroughoutthe 90-min assayperiod. Releasewas

greaterfrom samplesstoredat room temperaturethan from refrigeratedsamples.

120

100

80

60

40

20

-

COSMETIC

FORMULATIONS

'1-

15

3O

45

Time (min)

WITH

6O

ARGIRELINE

©

169

ß

Cream

4 ø C

[]

Cream

25 ¸ C

[]

Gel

4 ¸

C

[]

Gel

25 ¸ C

9O

Figure 8. Releasewithoutmembraneof the Argireline© samplesmaintainedat 25ø and4øC.

The gel formulation showedevidenceof thermal gelling during the first 15 days

of storageafter preparation,and this reduceddiffusionof the peptide from sam- ples storedat 25øC. The resultsof in vitro assaysconfirmedthat the active prin- ciple penetratedthe artificial membraneand that it is a suitabledeliveryfrom both

excipients.

lOO

80

60

40

20

0

0

CREAM

---A--GEL

1

2

3

4

5

Time (hours)

6

Figure9. Percentageof ArgirelJne© releasedfromthegel andcreamasa functionof thetime.

170

60

g

40

20

<E

o

o

JOURNAL OF COSMETIC SCIENCE

1

2

3

4

Time (hours)

-••AM

5

6

Figure 10. AmountofArgireline© releasedasa functionof thetimeforthetwotestedsamples.

ACKNOWLEDGMENTS

Partof thisworkwassupportedby theSpanishMinistryof EducationandScienceand

byEuropeanRegionalDevelopmentFundsunderProjectMAT2005-07746-C02-02and

Projectof ExcellenceFQM 410. We thankK. Shashokfor translatingpartsof the

original manuscriptinto English.

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