J. Cosmet. 54, 161-174 (March/April2003) Sci.

Effectof formulation the topical delivery on of o-tocopherol

MEERA RANGARAJAN andJOEL L. ZATZ, Organon USA, 3 75 Mt. Pleasant Avenue, West Orange, 07052 (M.R.), and Laboratory NJ of Cancer Research, Rutgers University, 41-D Gordon Road,Piscataway, mJ 08854 (J.L.Z.).

Accepted publication for August 2002. 19,

Synopsis

The objectiveof this research to investigate effectof concentration deliverysystemon skin was the and permeation (x-tocopherol of ((x-T). Also, the additionof sunscreens oleicacid on (x-T permeation and was studied using an in vitro micro-Yucatan pig skin model. Various delivery systems (x-T (1%) were of formulated,which includedsimplesolution,gels,emulsions, microemulsions. experimental and The design chosen this studywasa statistical for randomized complete blockdesign. (x-T delivery wasproportional to its concentration. hydroalcoholic deliveredsignificantly The gel higher amounts (x-T into the receptor of than the other gels used.A microemulsion containingisopropylmyristateemergedas the best delivery systemfor (x-T amongall the systems studied.Pig skin is a suitablein vitro model for studying the permeation (x-T and possibly of otherantioxidants, thoughin vivoexperiments humans requiredto in are
further corroborate the data.

INTRODUCTION

Research shownthat UV radiation damages has DNA and genetic material, oxidizes lipids and produces harmfulfree radicals, causes inflammationthat alsoproduces free radicals, disruptscell communication, causes expression stress of response genes,and weakens immune response the skin. o-Tocopherol the of (o-T) is the major lipophilic antioxidantin many biological systems Topicalapplication o-T hasbeenshown (1). of to protectagainstUV-induced cutaneous damage,carcinogenic mutagenicactivity and of ionizing radiation,and chemicalagents(2). o-T has been found to reducetumor incidence mice (3) and decrease lines,wrinkles,and sagginginducedby photoin fine aging (4,5). o-T is now considered essential the stabilization biologicalmemfor of branes, particularlythosecontaininglarge amountsof polyunsaturated fatty acids.The oxidation unsaturated produces of fats lipid peroxides, whichinterfere with the structure and functionof biological membranes. When dl-o-tocopherol addedto commercial was

Addressall correspondence Meera Rangarajan. to

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creamsin concentrations 0.1% or 1%, no markedincrease lipid peroxides of in was observed during a 96-hour exposure sunlight(6). The antioxidantactivity of c-toto copherol hasbeensummarized elsewhere (7). Varying concentrations c-T havebeenusedin differentstudies. of Beijersbergen van Henegouwen /. (8) haveused0.25% of c-TAc to studyits UV protectingactivityin et vivoin rat. c-T in low concentrations 0.05-2% wasusedasan antioxidant topical of in preparations. higherconcentrations In (2-10%) c-T seemed havebenefitin stratum to corneum hydration(9,10). Pure c-T and concentrated preparations c-T (10-20%) could irritate humanskin, and cases allergiccontactdermatitisand other typesof immuof nologic hypersensitivity reactionsare reported (11). However, there is no systematic studyin the literatureon the effectof concentration c-T on skin permeation. of Sunscreens the second are type of photoprotectants are commonly that usedalongwith antioxidants. Very little information,however, available the augmentation the is on of activityof eitherthe sunscreens the presence antioxidants vice versa. in of or Darr et/.
(12) studiedthe combinationof the two antioxidantvitamins, C and E, with commercial

UVA sunscreen (oxybenzone) foundgreaterthan additiveprotection and against phototoxicdamage. Theseresults emphasize importance combiningantioxidants the of with
commonsunscreens maximize photoprotection. to Alberts eta/. (13) reportedthat in a survey about62% of sunscreen formulations contained some form of c-T. They caution, however, that furtherresearch to be performed studythe potentialharmfuleffects has to of"other ingredients" (like c-T) that canbe addedto commercial sunscreens. Alteration of the skin transport chemicals be achieved meansof permeation-enhancing of can by

agents suchasoleicacidand dodecylazacycloheptan-2-one (laurocapram, azone) (14). Bhatia et/. (15) useda combination ethanoland 10% oleic acid pretreatment of to significantly increase permeability the coefficient cholecystokinin-8. of KitagawaandLi (16) foundthat 1% 1-menthol plus 15% ethanol increased permeability benzoic the of acid but decreased of its higher alkyl substituents. that Morgan et/. (17,18) studieda new classof penetrationenhancers, octyl salicylate(OSal) and padimate O (Pad O), which have been usedas effectivesunscreens, along with oleic acid, to enhance the permeabilityof the sexhormones testosterone, estradiol, and progesterone. They found OSal to give the highestenhancement permeability. in The effects padimateO and of oleic acid were comparable eachother, albeit better than the control. to Previous work doneby the authors usingthe prodrugesterof c-T, c-tocopheryl acetate (c-TAc),demonstrated metabolism c-TAc to c-T in pig skin (19). Permeation the of of c-TAcand its metabolism werefoundto be a functionof the deliverysystem (20). An emulsion system containing isopropyl myristate (IPM) emerged the mostdesirable as
formulation in terms of skin delivery of c-TAc.

The objective this work was to (i) formulatedifferentdeliverysystems c-T, (ii) of for delineatethe effectof both biphasicand uniphasic deliverysystems the permeation on of c-T, (iii) studythe effectof concentration c-T permeation, on (iv) characterize the effect of sunscreens c-T permeation,and (v) formulateand study c-T permeation on with oleic acid,which is believedto functionas a penetration enhancer.
MATERIALS
CHEMICALS AND

AND

METHODS

INSTRUMENTS

D-o-Tocopherol obtainedas a gift from Archer DanielsMidland Company(IL). was

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163

The followingchemicals wereobtained directlyfrom the manufacturer usedwithout and purification:SD alcohol,Eastman(TN); isopropylmyristate(IPM) and mineral oil, SigmaChemical Company (NJ); diisopropyl adipate(DIA), Ceraphyl 230, isocetyl

alcohol, Ceraphyl ISP Vandyk(NJ); carbomer, and , Carbopol B F Goodrich , (OH);

DEA-cetyl phosphate, RocheVitamins and Fine Chemicals(NJ); diazazodinylurea and

germall,SuttonLaboratories (NJ); ethomeen C/25, Akzonobel (IL); hydroxypropyl cellulose,Klucel Hercules , Co., Germany; Tween20,polyoxyethylene sorbitan (20) monolaurate, Tween80, and polyoxyethylene sorbitan (20) monooleate, Surfactants ICI (DE); and Transcutol,diethyleneglycol monoethylether, Gattefosse (France).Scintillation fluid Scintiverse, reagent alcohol, and glacialaceticacidwereof HPLC gradeand wereobtained from FisherScientific (Springfield, NJ). Water refers freshlydeionized to water.Tissue solubilizingfluid, Solvable waspurchased TM, from Packard InstrumentCo. (Meriden, CT). Transparent tape # 800 was purchased from 3M PackagingSystems
Division (St. Paul, MN). All other materials were obtained from standardsources.

TOPICAL

FORMULATIONS

Formulations ot-Tpermeation All formulations for study. wereprepared a weight/weight on basis. The compositions the formulations of usedin this studyareshown TablesI and in II. Gels 1, 2, and 3 differed their alcohol in content. Gels 1 and2 werepurelyalcoholic, with gel 2 containinga surfactant. Gel 3 wasa hydroalcoholic The oils in the case gel. of the macroemulsion formulation(emulsion1) were kept at a constant ratio of diisopropyl adipate:mineral 1:1, throughoutthe experiment.Emulsions oil, were made by heating the oily and aqueous phases separately (55C) and shearmixing the two phases (aqueous addedto oily) to yield creamyemulsions. Simplevortexingwassufficientto give clearmicroemulsions.

Formulations concentration The concentrations for study. chosen the concentration for dependency studywere 0.25%, 1%, and 4% o-T. Prototype formulations includingthe IPM solution,gel 3, and emulsion1 were madeat thesethree concentrations o-T. of Higher or loweramounts o-T were compensated by decreasing increasing of for or the solventfor o-T used in the corresponding formulations(IPM for solutions,SD 1%
alcoholfor gels, and the oils for emulsions).

Formulations sunscreens. with Sunscreen formulations containingo-T includedgel 3 and emulsions and 3. Two sunscreens 1 were studied,octyl methoxycinnamate (OMC) and octyl salicylate (OSal),at concentrations 7% and 5%, respectively. formulations of The
Table I

Topical Gel Formulations Used in the Study:o-Tocopherol Gels

Ingredient
tx-T

Gel 1 (% w/w)
1

Gel 2
1

Gel 3
1

SD alcohol

96

91
-5

69
14
5
3
8

Isocetylalcohol
PEG-15 cocamine

---

Hydroxy propyl
cellulose
Water

3
--

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Table II

TopicalEmulsionFormulations Used in This Study

Ingredient
Emulsion ot-T 1a 1.00

ot-T (% w/w)

Diisopropyladipate
Mineral oil

5.38
5.38

DEA-cetyl phosphate Diazazodinylurea

Carbomer Water

2.00 0.30
0.30 85.64
1

Emulsion 2b
ot-T

Isopropylmyristate Polysorbate 80
Sorbitol Water Emulsion 3 c
ot-T

10 16
30 43
1

Benzylalcohol Diethyleneglycolmonoethyl ether

Tween 20 Water a o/w macroemulsion.

21.3 16.9
18.1 42.7

b,c Microemulsion containing orbenzyl IPM alcohol oilyphases. as

remained similarto gel 3 (Table I) and emulsions and 3, given in Table II, with the 1 difference the addedsunscreen of agents. The addedingredients werecompensated for by a decrease the concentration SD alcohol the gelsand the oily phases the in of in in
microemulsions. The ratio of the two oils for emulsion 1 was still maintained the same.

Oleic acidstudy. the penetration For enhancer study,the IPM solution, emulsion1, and gel 3 werestudied,containing additionto the ingredients in listedin Table I and II, 1% oleic acid. A gel 3 formulation containing 5% oleic acid wasalsoincludedfor study purposes. controlexperiment A with ot-T wasrun simultaneously with all three formulations without the added oleic acid.

All ot-T formulations were observed stability for a periodof two weeks.Also, to for ensure that ot-T was not oxidizedduring manufacture itself, cold formulations immediatelyuponmanufacture two weeks and laterwereassayed drug contentby HPLC. for Detailsof the HPLC procedure given elsewhere are (19).
RECEPTOR FLUID

The receptor fluid wasanaqueous solution 0.1% polyoxyethylene ether(PEG-20 of oleyl oleyl ether),a nonionic surfactant with an HLB of 16. This receptor fluid, thoughnot physiologic, maintains adequate solubility ot-Twithoutaffecting of skinbarrierfunction (21). The receptor pumpedat a rate of 1.5 ml/h. The apparent was solubilityof ot-T in 0.1% PEG-20 oleyl ether at 37C wasmeasured be 1.08 mg/ml, substantially to in excess the maximumconcentration the receptor of in solution,therebyassuring maintenance of the sink condition at all times.

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TISSUE

AND

PREPARATION

Micro-Yucatanpig skin obtainedfrom CharlesRiver Laboratories (Wilmington, MA) wasusedas the biologicalmembrane studyin vitropercutaneous to absorption. Upon receiptthe freshlyexcised skin waswashed gently with 1% (w/w) aqueous detergent, rinsedwith deionized water, and patted dry with a papertowel. A 250-300-1am-thick layerof the skin wascut from the surface with a PadgettElectrodermatome instruTM ment (PadgettInstrument,Kansas City, MO). The skin pieceswere then rinsedand

driedwith paper towels before storage plastic in bags 4C.Skinwasremoved at fromthe refrigerator kept in isotonic and solution hydrate roomtemperature hourbefore to at one
startingthe experiment. The dermatomed skin wascut into 10-mm circularpieces with a brass punchandplaced epidermis-side-up Bronaugh in diffusion cells.The skin treated in this fashionfrom the stageof receiptuntil use retainedits original permeability characteristics four weeksafter dermatoming(22). for

RADIOLABELING

OF o-T

D-alpha-[3H]tocopherol was custom synthesizedAmersham by Pharmacia Biotech, England.This wasreceived a toluene:ethanol solution as (9:1) with a specific activityof 19 Ci/mmol (molecular weight432, at this specific activity).o-T formulations werespiked
with the radiolabeled o-T suchthat eachfinite doseof 5-1al formulationappliedon the skin containedapproximately300,000 dpm (disintegrations per minute). Gels were spikedbeforeaddingthe gelling agent.Biphasic o/w formulations werespikedbefore additionof the aqueous phase. Analyses the radiolabeled for o-T throughoutthis experimentwere carriedout with a liquid scintillationcounter(LSC, BeckmanInstruments).

STATISTICAL

DESIGN

OF THE

EXPERIMENT

The applicationof formulations the pig skin was carriedout using a statistically on approved model.A randomized completeblock designwaschosen the designfor the as experiment. The statistical modelappeared shownin Table III. For the otherstudies, as theapplication each of formulation completely was randomized the number cells over of and daysof the study.

DOSING

Finite dosingwasusedto simulatethe actualuseconditions all the in vitropermeation in and metabolismexperiments. The smallestvolume of the formulation required, to

obtain complete uniform and coveragethediffusion surface (0.636cm2), of cell area was
determined be 5 lal, corresponding a weight of about4 mg for eachformulation. to to After application,the preparation was uniformly spreadon the stratum corneum(SC) sideof the skin with the help of a glassrod, and the tip of the rod waswashed into a vial containing ml of ethanolin orderto account the materiallost on spreading. 1.5 for With this technique,the exactamount of material applied to the skin surface was determined. estimatethe amountof o-T in the original formulation,5 lal of the To

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Table III

Statistical Randomized CompleteBlock Designfor the Applicationof Formulations

Formulation
IPM solution
Gel 1

Days 1
W1 b
X1 c
W2 X2

Day 2
Y1 d
Z1 e
Y2 Z2

Gel 2

W3 X3

Y3 Z3

Gel 3
Emulsion 1

W4
X4 W5 X5 W6 X6

Y4
Z4 Y5 Z5 Y6 Z6

Emulsion 2 Emulsion 3

W7 X7

Y7 Z7

Two consecutive days, 1 and 2.

b,c Replicates day1 forIPM solution. on d,e Replicates day2 forIPM solution. on

formulation counted its radioactive was for counts afterequilibration each of formulation for a periodof 24 hours.
IN VITRO SKIN PERMEATION/METABOLISM METHODOLOGY

A flow-throughsystem wasusedfor conducting vitropermeation in experiments. The total system consisted a receptor of fluid reservoir; variable a flow rateperistaltic pump, Cassette (Manostat, New York); a circulating water bath, Lauda (BrickmanInstru ment, Westbury,NY); anda two-cell-holding heatingblock,14 Teflon flow-through
diffusion cells, and a Retriever IV fraction collector (ISCO Inc., Lincoln, NE) to collect

effluentfractions overthe adjusted time period.Eachdiffusion cell hadan innerdiameter

of9 mmand surface of0.636cm exposedthereceptor Thereceptor a area 2 to fluid. fluid

waspumpedat a flow rate of 1.5 ml/h from the reservoir the diffusion to cellsplacedin the holdingblocks. The skin surface temperature maintained 32Cby adjusting was at the circulatingwater bath temperature 39C (23). The effluentfrom the diffusion to cellswas collecteddirectly into glassscintillationvials everyfour hoursfor a period of
24 hours.

SKIN

TREATMENT

The liquid scintillationcountingtechnique was usedto analyzeall the in vitropermeation samples.In each experiment a minimum of four replicateswere used. At the conclusion the experiment,scintillation fluid was added to the effluent samples of collecteddirectly into the vials and the amount of ot-T penetratedwas estimatedfrom the counts radioactivity of presentin the samples. Countswere obtained dpm, which as werethen converted into micrograms activeby taking into account spikingratio of the for eachformulationand specific activity of the active.

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After 24 hours donor the compartment washed was threetimeswith 1 ml of acetonitrile. Washeswere collectedand analyzedfor the amount of ot-T remainingon the skin surface. Washed skinsamples wereremoved fromthe cells. The tape-stripping technique wasusedto separate SCfromthe restof the epidermis get an estimate material the to of remaining in the barrier layer of the skin. In this technique,seventeen strips of the active-treated sideof the skin, usinga 3M Scotch tape, were taken as two + fifteen TM strips.The first two stripsrepresented activesuperficially the adheringto the surface (and so were included in the wash) and the next 15 strips represented the active recovered from the SC. Scintillation fluid wasaddedto the vials containing tape the stripsandallowedto stand roomtemperature at least24 hours enable at for to extraction of ot-T in orderto performscintillation countingon the samples. The remainder the of skinwasdigested 3 ml of tissue in solubilizer 50Cfor 24 hoursin an oven.This was at doneto get an estimate materialin the viabletissues the skin.After skindigestion, of of
the samples were neutralizedwith glacial aceticacid, and scintillationfluid was added for counting.

Receptor solutionwascollected glass in scintillationvialseveryfour hours.Scintillation fluid wasaddedto the vialsand they werecounted hourslater. Thus, the amountof 24 ot-T wasestimated the followingfourlocations the in vitropermeation in in experiment: (a) receptor fluid, (b) washes, stratum corneum,and (d) viable tissues the skin. (c) of

STATISTICAL

ANALYSIS

Statistical differences between the formulations

and all the other formulations

with the

simple solution were estimatedusing a Student'st-test. In the oleic acid study, the resultswith the oleic acid were compared the controlformulations, to which did not contain oleicacid.The design these any of studies enabled to compare permeation us ot-T with ot-tocopheryl acetate (ot-TAc)permeation a studythat waspublished in earlier(19).

RESULTS

COMPARISON

OF VEHICLES

Figure 1 shows permeation ot-T in termsof the amountof activein the stratum the of corneum,the viable skin, and the total amount permeated, which is inclusiveof the amountof ot-T in the SC,viableskin,and receptor. IPM solution yieldedsignificantly higheramounts ot-T (or= 0.05) in the SCthan gels1 and2 andemulsion The other of 1. formulations not differfromeach did otherin termsof thisparameter. the viableskin, In emulsion hadhigherpermeation, 2 whichdifferedsignificantly from that of emulsion3 (or = 0.05). IPM solutionhad a higheramountof total ot-T permeation than emulsion 3 and gel 1. However,this amountwassignificantlylower than the amountpermeated with emulsion 2. Emulsion 2 had the highest permeationcomparedto all the other formulations. wassignificantly It higherthan for the otheremulsion formulations, IPM solution, andgel 1. Gel 3 alsohadsignificantly highertotal permeation compared gel to 1 in this study.In termsof the amountof o-T in the receptor 24 hours,gel 3 fared at the best,havingsignificantly higherpermeation compared the othergels. to

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Figure 1. Effectof formulations the skindeliveryof t-T. Values percent on are applieddose+ SEM (n = 4). I, Stratumcorneum. Viable skin.[], Total amountpermeated. Amount in receptor. B, l-q,

Comparison oftx-Tandtx-TAcpermeation. Results ot-Tpermeation of studywerecompared to ct-TAc permeation studies performed earlier(19). ct-TAc is a commonly usedprodrug precursor ct-T andit hasto undergo of metabolism skinenzymes release active by to the antioxidant,ot-T. Table IV shows the comparative valuesfor the permeationin viable skin and receptorand total skin and receptorfor ct-T and ct-TAc, as a function of the deliverysystem.ct-T permeation studiesshowed lower permeationof activethan the a ct-TAc studies throughall the parameters measured. find the extentof this decrease, To a ratiowastakenof the average amountof ot-TAcpermeated the ot-T permeated. to The amountof activepermeated viableskinandreceptor the ct-T permeation in in studywas lower by about 2.22 times than the activethat permeated from the ot-TAc permeation study.The ct-TAc permeation studyhad valuesabout2.98 times higher than the ct-T study when measured total skin plus receptor,which is inclusiveof the stratum as corneum.Although the valuesobtainedin studiesusing either ot-TAc or ot-T were numerically different,certaintrends observed both the studies in remained same. the For
Table IV

Comparison c-T and c-TAc Permeation 24 Hours of at

Viable skin + receptor (% applied dose)
Formulation cz-T cz-TAc cz-TAc/cz-T cz-T

Total skin + receptor (% applied dose)

cz-TAc cz-TAc/cz-T

IPM solution Gel 1 Gel 2 Gel 3 Emulsion 1 Emulsion 2 Emulsion 3 Average

9.74 6.47 6.62 8.55 6.72 12.24 6.95

+ 0.89 + 0.54 _+1.26 + 0.55 _+1.4 _+0.98 + 1.28

15.93 17.54 9.99 19.69 16.02 31.67 16.82

-+ 2.18 + 4.79 + 0.95 -+ 2.28 + 4.27 + 5.87 + 4.97

1.64 2.71 1.51 2.30 2.38 2.59 2.42 2.22

3.17 2.24 3.16 3.42 3.46 5.83 2.30

+ 0.49 _+0.24 + 0.88 + 0.37 _+1.22 -+ 0.77 _+0.32

6.66 8.73 6.42 9.65 9.85 15.22 10.49

+ 1.27 -+ 3.19 _+0.26 -+ 1.10 -+ 1.98 + 2.27 _+3.10

2.10 3.89 2.03 2.82 2.85 2.61 4.56 2.98

Valuesare percentapplieddose _+ SEM (n = 4).

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169

example,in termsof the total amountof activepermeated both the studies, in permeation from emulsion wasstatistically 2 significantly greaterthan from emulsions and 1
3, whereas emulsions 1 and 3 did not differ from each other. In terms of the amount

permeated viableskin and receptor, in againemulsion2 emerged the bestformulaas tion. Permeation activewassignificantly of highercompared emulsion3 in the ot-T to permeationand compared the solutionin the ot-TAc permeation.There were no to differences betweenthe other formulations both the studies. in Thus, althoughthe two setsof experimentwere conducted different times and under differentexperimental at conditions, usingdifferentanalytical techniques (HPLC for the ot-TAcassay LSCfor and the ot-T assay), similartrendin permeation a functionof formulation a as wasobserved. There is very little structuraldifferencebetweenot-TAc and ot-T. They have similar lipophilicities andaredevoidof functional groups, whichcandissociate give different to ionicformsat skinpH. They may be expected behave to similarlywith respect skin to
penetration(7).
CONCENTRATION STUDY

Representative receptor profiles the concentration for dependency studyof IPM solution areshown Figure2. The linearityof the plotssuggests achievement steady in the of state.

Theslopes these of straight lines, which thefluxvalues, are along withtheirr2 values
for all three formulations, are shown in Table V. As the concentrationincreased,there

wasa proportional increase flux, showing existence dose in the of proportionality ot-T for permeation. Interestinglythe permeation values the activein the stratumcorneum for at different concentrations showed deviationfrom linearity, which was unique to the a stratumcorneumand not observed the permeation in profilesfor the rest of the skin tissue. Figure3 shows permeation ot-Tin viableskinwhenthe amount the of permeated

was plotted pg/cm as 2.

1.4E+00
1.2E+00

1.0E+00 8.0E-01 6.0E-01

4.0E-01
2.0E-01

O.OE+00

10

15

20

25

30

Time (hours)
Figure 2. Plot of receptor profiles IPM solutionin the ot-T concentration for study.Valuesare concen-

tration (pg/cm + SEM(n = 4). Equations thestraight areshown. 0.25%IPM solution. -2) of lines O, [, 1% IPM solution. 4% IPM solution. /, Regression equations:= 0.0033x- 0.0084,r2 = 0.9899;y = y 0.0146x- 0.0242,r2 = 0.9919; y = 0.0485x- 0.0946,r2 = 0.993. and

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Table V

Slopes r2 Values theReceptor o-TConcentration and for in Study

IPM solution Gel

Emulsion

Concentration
0.25 1 4

Flux
0.0033 0.014 0.0485

r2
0.990 0.992 0.993

Flux
0.0041 0.011 0.0397

r2
0.999 0.984 0.960

Flux
0.0012 0.0065 0.0327

r2
0.994 0.993 0.980

Fluxvalues in pg/cm-2hr are -1.

35-

30

20

Concentration ofalpha-T (%)

Figure3. Permeation profile o-Tin viable fortheconcentration when of skin study expressed aspg/cm 2
+ SEM(n = 4). ', IPM solution. Gel 3. ', Emulsion Regression l, 3. equations:= 7.9944x+ 0.0225, y r2 = 0.9997;y = 3.2494x+ 0.1042,r2 = 0.999;andy = 1.3517x+ 1.2034,r2 = 0.8756.
EFFECT OF SUNSCREENS

The formulations included this studyweregel 3, emulsion and emulsion for two in 1, 3 differentsunscreens, OMC and OSal.Figure4 shows profileof sunscreens viable the in skinandthe total amountpermeated, whichrefers the amountof activein the stratum to corneum, viableskin, and receptor, and the amountpermeated into the receptor % as applieddose. Alsoincluded the same in grapharethe values the c-T emulsion gel for 1, 3, and emulsion3, which are the control formulationsdevoid of sunscreens. Emulsion 1 containing sunscreens not showanysignificant did difference permeation in compared to the control.However,both the gels bearingthe sunscreens deliveredhigher amounts of c-T in the receptor. general, In therewasno significant difference between formulationscontainingsunscreens controlformulations. and
EFFECT OF OLEIC ACID (OA)

The formulations usedin the studywere IPM solutionwith and without oleicacid, gel
3 with and without oleic acid, and emulsion 1 with and without oleic acid. Also

includedwasa 5% oleic acid gel 3 to study the effectof increasing oleic acid concen-

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171

25

20

o 15

OMC OSal Control OMC gel OSalgel Control OMC OSal Control emulsionemulsionemulsion 3 3 gel3 emulsionemulsionemulsion
1 1 1 3 3 3

Figure4. Profiles sunscreen for formulations. Values percent are applied dose SEM(n = 4). [, Amount + of ot-Tin viable skin. Amount ot-Tpermeated SC + viable [], of in skin+ receptor. Amount ot-T I, of
permeated receptor. in

trations. Figure5 shows profilefor o-T permeation the skinfrom oleicacidand the in

control formulations. Included values the viableskinandreceptor the total are in and
amount permeated,which have been describedbefore. Oleic acid formulationswere

compared controlo-T formulations to usinga Student's t-test(o = 0.05). The 5% oleic

acidgel 3 wascompared boththe 1% oleic with acidgel 3 andwith control o-Tgel 3.

In each caseno statisticallysignificantdifferencewas obtainedbetweenthe test and
control formulation at o -- 0.05.

DISCUSSION

This studyshows o-Tcanbe delivered viableskinandreceptor that into fluid from various formulations. Topicalo-T treatment results a significant in increase o-T in
3.5E+01

3.0E+01 2.5E+01 2. 0E+01 1.SE+01 1.0E+01 5.0E+00

0. 0E+00

OA Control OA gel 3 solution solution

Control gel 3

5% OA gel 3

OA Control emulsion emulsion

1 1

Figure 5. Skinprofiles oleicacidstudy. for Values percent are applied dose SEM(n = 4). [, Viableskin e + receptor. SC + viableskin + receptor. l,

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concentrations the viable skin, the stratumcorneum, in and the receptor fluid. Earlier studies (24) had shown absence detectable the of amounts baseline of ot-T in pig skin without any exogenous application,using an HPLC equippedwith a UV detector (detection limit 0.25 lag/ml). Traberetal. (25) studied penetration distribution the and of ot-Tapplied mouse on skin.Theyfoundthat the largest fraction skinot-Tfollowing of topicalapplication not foundon the surface in the deeper was but subcutaneous layers. We also found that topical ot-T applicationmarkedly increased skin ot-T content. Lopez-Torres al. (26) foundtopicalot-T treatment significantly et to increase levels ot-T in boththe epidermis (62-fold)andthe dermis (22-fold)24 h afteradministration. Two routesof skinabsorption ot-T havebeensuggested: of from the stratumcorneum into the epidermis and dermisand throughthe hair folliclesby way of the pilosebaceous canal and into the outer root sheaths and eventuallyinto the dermal tissue(27).
This is the first systematic study delineatingthe effect of formulationfactorson the

permeation ot-T. All our formulations of werestudied stabilityusinga previously for established HPLC procedure immediatelyafter formulationand two weeksof storage underambientconditions (24). The formulations werealsofoundto be visuallystable. All the systems we usedhadthe ot-T in solution that eitherin the alcoholic phase (gels)
or in the oily phase(emulsions, solution). Emulsion 2, which was a microemulsion system containing IPM, showed highesttotal permeation ot-T. This wasfollowed the of by IPM solution. Therehavebeenreports IPM beinga possible of penetration enhancer. That couldhavebeenoneof the possible reasons better skin deliveryby formulations for containingIPM. However,we do not havesufficientevidence this point to makea at definite conclusion. highestpermeabilityof felodipine The from benzylalcoholmicroemulsions foundfor that system was that gavethe highest solubilityof the activeboth in microemulsion in the apparent and externalphase (28). The IPM-containingmicroemulsion performed better than the benzylalcohol-containing microemulsion.

Based the results the ot-Tpermeation on of study,we chose 3, an IPM solution, gel and the emulsion formulation prototype as formulations the concentration for dependency study.The formulation difficulties experienced makingmicroemulsions in precluded their incorporation the study. in
To studythe effectof concentration, times lowerand higheramounts ot-T than four of the normallyused1% wereformulated. The 1% formulations werealsotestedagainin this study.A detectable amountof the activewasfoundevenat 0.25% concentration as earlyasfour hoursin the receptor. The skinprofiles exhibited linearityand consequent dose proportionality. the concentration ot-Twasincreased the formulations As of in from 0.25% to 4%, proportionally greateramountsof activepermeated into the skin. In additionto the numerous benefits that havebeenreported with ot-T administration, the direct sunscreening effect of ot-T is now being added to the growing list (29). Sunscreens alongwith antioxidants used havebeenfoundto give an addedbenefitof photoprotection. We usedthree prototypicformulationsincludinggel 3, emulsion1, emulsion3, and two different sunscreens, OMC and octyl salicylate. Also, somesunscreens havebeenusedaspenetrationenhancers themselves. PadimateO was shownto improvepercutaneous absorption testosterone estradiol swine.Padimate was of and in O shownto lower the transitiontemperature the stratumcorneumlipids, which was of postulatedto result in significantincreases drug diffusivityacross skin (17,18). in the Theseauthorsalsousednovel topical sprayvehicles containingPadimateO and octyl

TOPICAL

DELIVERY

OF ot-TOCOPHEROL

173

salicylate study the transdermal to deliveryof sexhormones. Octyl salicylate gave the highestenhancement ratio, and other penetration enhancers oleic acid and laurolike capramgaveslightly lower ratios,albeit better than the control.

We did not find oleic acid to significantly enhance permeationof ot-T. A major the reasonfor this differencecould be the lipophilicty of the ot-T molecule. ot-T is a lipophiliccompound (octanol/water partitioncoefficient 480), log (P) = 2.68. A log = (P) > 2 is a goodindicatorof a highly lipophilic molecule.When 1-mentholalongwith ethanolwasusedasa penetration enhancer benzoic for acidand its 4-alkyl substituents, Kitagawa andLi (16) foundincreased permeability benzoic for acidbut decreased values for the higheralkyl substituents. additionof alkyl groups The madethe parentmolecule more lipophilic. Further analysisshowedthat addition of enhancers made the skin relatively more hydrophiliccomparedto the vehicle,which inducedan increase the in permeability coefficient benzoic of acidand decreases thoseof its lipophilicsubstituin ents.The effectof penetration enhancers the permeation [3-blockers studied on of was
(30). The authorsfound azoneto be a better enhancer than oleic acid. Their effectswere found to be more pronounced with hydrophilicdrugsthan with lipophilic [3-blockers. The increment lipid fluidity and the resultingenhanced of waterpermeabilityis thought to be the reason this phenomenon. for Possibly our studythe high lipophilicityof ot-T in was responsible the lack of effectof oleic acid. for

Pig skin is a suitable modelfor carrying in vitropermeation out studies ot-T andother of antioxidants. However,we noticedthat althoughstudiescarriedout on the samepiece of pig skinarecomparable whenpig skinwaschanged, thereis a possibility a variation of in values,thoughthe generaltrend tendsto remain the same.Thus pig skin serves a as goodindicator theeffect of that maybeobtained with different formulations, further but in vivostudiesare requiredto arrive at definite conclusions.
In summary,we haveshownthat topicalapplicationof ot-T significantlyincreased its concentration skin and receptor in fluid. High amountsof the activepermeated the in receptor from a hydroalcoholic Doseproportionality gel. was observed when different concentrations ot-T (up to 4%) were applied to the skin. We have shown that of micro-Yucatan skin is a suitablemodelto studythe effectof formulationfactors pig on the permeation actives. of Therearedifferences obtained between each pieceof skinwith respect permeation. to However,the generaltrend observed with the formulations is maintained. IPM-containingmicroemulsion An served an effective as delivery vehicle for ot-T. Oleic aciddid not significantly enhance permeation or-T,probablydueto the the of mechanism actionof oleicacid and the lipophilicity of ot-T. of
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