In tc rnr t io r r r l . |ournr l o f Dernr r to logy , Vo l . - j5 , No. 9 , Scptembcr I 996

PHARMACOLOGY ANI) THERAPEUTICS

TREATMENT OF SKIN AGING WITHTOPICAL ESTROGENS

JOLANTA B . SCHMIDT , M .D . ,

MARTINA B INDER, M .D . , GABRIELE DEMSCHIK , M .D . ,

CHRIST IAN B IEGI ,MAYER, PH .D . , AN I ) ANGEL IKA RE INER, M .D

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AbstractBackground. The coincidence of cl imacteric symptoms andthe beginning of skin aging suggests that estrogen deficiencymay be a common and impor tan t fac to r in the per imeno-pausal woman. Often hormones have been considered im-portant in endogenous aging of the skin, but their role hasnot been clearly defined. Therefore, we investigated, whethertopical treatment of the skin with estrogen could reversesome of the changes in the aging skin.

Materials and Methods. The effects oI 0.01/o estradiol and0.3% es t r io l compounds were compared in 59 prec l imac-teric women with skin aging symptoms. Monthly determi-nations of estrodiol (ez), fol l ic le-st imulat ing hormone (rss),and pro lac t in (pnr ) were done and the month ly c l in ica l mon-'rtoring was supplemented by measurements of skin hydra-t ion by corneometry and profi lometry. In 10 patients, skinbioosies were taken for immunohistochemical determina-t ion of col lagen types I and l l l .

f iesults. After treatment for 6 months, elast ici ty and f irm-ness o f the sk in had marked ly improved and the wr ink ledepth and pore sizes had decreased by 61 to 100% in bothgroups. Furthermore, skin moisture had increased and themeasurement of wrinkles using skin prof i lometry, revealedsignif icant, or even highly signif icant, decreases of wrinkledepth in the estradiol and the estr iol groups. respectively.0n immunohistochemistry, signif icant increases of Type l l lol lagen label ing were combined with increased numbersof col lagen f ibers at the end of the treatment period. As tohormone levels, only those of pnl had increased signif icant-lyand no systemic hormonal side effects were noted.

ftomthe Department of Dermatology, Divisiorr of Special andhr i ronmental Dermatology, Univers i ty of Vienna Medicalldrool, Vienna, Austria; the lnstitute for Medical and Chemi-olhboratory Diagnostics, Departnrent of Hormone Analysis,l lnivenity of Vienna, Vienna, Austr ia; and the Irrst i tute ofOinical Pathology, University of Vienr-ra, Vienna, Austria.

for correspondence: Jolanta B. Schmidt , M.D., De-. of Dermatology, Div is ion of Specia l and Environ-

Dermatology, University of Vienna Medical School,

Conclusions. Both estrogen compounds were found to behighly effect ive in preventing or treating skin aging in peri-menopausa l women, c l in ica l l y , by measurement da ta , andby an inc rease in co l lagen Type l l l .

Int J Dermatol 1996; 35:669-674

Topical tret inoin was the f irst substance with whichsignif icant improvements of skin appearancer could becor re la ted w i th h is to log ic and in u i t ro f ind ings .2-aTretinoin is a very effect ive agent against solar radia-t ion-induced exogenous skin aging.5 In contrast, so farno agents were found to be effect ive in endogenousaging. Although hormones often were considered asimportant factors in endogenous skin aging, the scien-t i f i c b a c k g r o u n d o f a h o r m o n a l r o l e i n t h e a g r n gprocesses of the skin st i l l remains poor.

In gynecology, the therapeutic effects of topical es-trogens on atrophy of the vaginal and vulvar epithel iahave been well described for many years.6'7 During thepast years, estrogen supplementation of the cl imactericwoman has opened new aspects on the wide variabi l i tyof estrogen effects in various t issues. Beneficial effectson serum cholesterol,8 on vascular act ions in contextwith the myocardial infarct ion rate,e and improvementand prevention of osteoporosisr0 are only some of theparagynecologic estrogenic effects.

In skin t issue-an active target si te of estrogens asshown by estrogen receptor determinationrl l ' l l -s511s-

gens increase vascularization and show effects at vari-ous levels of dermal t issue. Improvement of the struc-tu re o f e las t i c f ibers a f te r use o f es t r io l c reaml ' r hasbeen descr ibed. Fur thermore , inc reases o f ac id mu-copolysaccharides and of hyaluronic acidl4'15 augmentthe dermal water content. Dist inct trophic effects of es-trogen compounds-and also of estr iol-on vaginal at-rophy by increasing the maturation index associatedwith an increase of the number of superf icial epithel ialcel ls, support the epithel ial act ions of estrogens.6'r6

These actions of estrogens prompted us to investi-ga te whether top ica l es t rogen t rea tment o f the sk incou ld reverse ag ing symptoms in es t rogen-def ic ien twomen. In a prel iminary study, the effects of a 0.3"/"topical estr iol compound on the facial skin of cl imac-te r ic women had been inves t iga tedrT and comparedGurtel 18-20. A-1090 Vienna. Austr ia.

669

I n t e r n : r t i o n l l J o u r n a l o f D e r m a t o l o g lV o l . 3 5 , N o . 9 , S c p t e m b e r 1 9 9 6

with those of a 0.017o estradiol compound. In anotherstLldy, the effects of topical estr iol iontophoresrs rrear-ment were s tud ied in a t roph ic acne scars and com-pared w i th those o f t re t ino in - ion tophores is . l8 Boths tud ies showed promis ing resu l ts . In the f i rs t s tudy ,cl inical effects were shown in 80 to 100Y" of the pa-t ients. For the acne scars, signif icant and persistent im-provement was demonstrated in al l cases.

These prel iminary results encouraged us to extendt h e i n v e s t i g a t i o n s o f e s t r o g e n c o m p o u n d s o n s k i naging to a larger number of patients. Moreover, thecl inical monitoring was substantiated by skin measure-ment data and by investigations of col lagen antibodiesus ing immunoh is tochemis t ry .

Materials and MethodsPat ien ts : Two groups o f women vo lun teers w i th sk inag ing symptoms, who d id no t take hormona l supp lements .were included in the study. Al l women were peri- or post-menopausal or had had a hysterectomy, three of them withan ovariectomy. Twenty-eight women with a mean age of54 years ( range 43-66 years ) were t rea ted w i th a 0 .01%est rad io l compound. Th i r ty women w i th a mean age o f 53years ( range 41-67 years ) used a 0 .3% es t r io l c ream. Theointment, at a dose of 1 g, was applied dai ly for 6 monthson the face and neck using an applicator. To investigate forposs ib le sys temic hormona l s ide e f fec ts , le venous b loodwas sampled at 8 AM before treatment and then at monthlyin te rva ls fo r de termina t ion o f fo l l i c le -s t imu la t ing hormone( rsu) , p ro lac t in (pn l ) , and es t rad io l (ez) by s tandard rad io im-munoassay (nrn) methods.

Monitoring of the condit ion of the skin before and at theend o f the t rea tment inc luded c l in ica l con t ro ls , sk in p ro-f i lomet ry , corneomet ry . and, in 10 cases , sk in b iops ies fo rimmunoh is tochemica l inves t iga t ion o f the co l lagen types .Ten women who had agreed to a sk in b iopsy vo lun tar i l y ,confirmed this by giving writ ten informed consent. For thec l in ica l eva lua t ion , wr ink l ing , sk in e las t i c i t y , pore s ize , andsk in mois tu re were assessed accord ino to an ind iv idua leva lua t ion scheme.

Skin Profi lometry: Wrinkles were measured and evaluat-ed in pa t ien ts o f the es t rad io l and the es t io l g roups . Wi ththe method, f ive fold measurements of the profi le of repl i-cas of identical corresponding wrinkles are performed withthe Hommel tes te T 2000 (Hommelwerke , Schwenn ingen,

Germany) . The mater ia l fo r the rep l i cas was Prov i l LCD(Bayer Dental), a rapidly drying material on a si l icone basis.In brief, a microsensor scans the print under a defined anghand at constant speed. The movements are transformed intounits of elast ic tension. The electr ic impulses are magnif iedand d ig i t i zed . The representa t ive parameter RzD is themean roughness depth of a single roughness depth outoff i ve measurement d is tances . The method has been de .scr ibed in de ta i l .2o

Corneometry: The hydration of the stratum corneum wasdetermined by corneometry between 11 and noon at a roomtemperature ol 21'C and a humidity of 40 to 45%. The instru.ment used was the corneometer CM 420 (SchwarzhauptMedizintechnik, Cologne, Germany), which acts by measur.ing capacitance with a plane capacitor. Data are displayednumerical ly. One unit represents 0.02 ng per cm2 watercon.tent of stratum corneum corresponding to a level of 20 nm.21

lmmunohistochemistry: Biopsy specimens were fixed rou-t ine ly in 10% buf fe red fo rmaldehyde and embedded inparaff in. Paraff in sections, 4 pm thick, were cut and subiected to immunocytochemistry according to the ABc-technique.Pr imary an t ibod ies used were d i rec ted aga ins t co l lagen I( A B 7 4 5 , C h e m i c o n , T e m e c u l a , C A ) , d i l u t e d 1 : 8 0 0 , a n daga ins t co l lagen l l l (PS 49, Monosan, Uden, The Nether .lands). di luted 1:10. The secondary antibody consisted of bi.o t i n y l a t e d a n t i r a b b i t l g G a n d w a s f o l l o w e d b y t h e A B csolut ion (both from Vectastain, Burl ingame, CA). Al l incuba-t ions were performed for 30 minutes at room temperature.For development of the peroxidase reaction, diaminoben-zidinetetrahydrochloride (oas) was used. The preparationswere evaluated semiquanti t ively by estimating the degree ofthe immunoh is tochemica l s ta in ing .

RESUI,TS

At the end of treatr lent, marked improvement of skinaging symptoms was noted. The cl inical f indings wereconfirmed by the measurement data. The Vilcoxon-test was used for stat ist ical analysis. No signif icant dif-ferences between Ez and psH levels were found betweenpre t rea tment and f ina l va lues . Pro lac t in showed ahighly signif icant increase in the estradiol, and a signif-icant increase in the estr iol groups (Table 1), but lnL

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Tablc 1, Init ial and Final Hormone Levels in Perimenopausal Women Treated with Topical Estrogens

Estradiol Group Estriol Grou|t

N lnitial Leuel Final lauel P Normal Range N lnitidl Leuel Final Lcuel

FSH (rnU/rnl)

PRl. (ng/mL)

E1 (pg/ml)

P > . 0 5N SP = .0005HSP > . 0 5NS

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0-5.5

22

2 l

21

24

24

l 8

.55 .9 r 38 .9" 57 .0 t 41 .7

5 . 1 t 2 . 0 8 . 3 x . 4 . 4

60.0 t I 09 .5 78 . l t 729 .5

66.9 x. 33.2 72.5 = 30.0 > .05NS

6 . 5 t 4 . 1 8 . 4 = 3 . 7 P = . 0 2 9S

2 3 . 4 t 1 9 . 4 2 2 . 9 = 2 9 . 8 P > . 0 5NS

"Values are mcrrn r st r rndard c lcv i l t ion.N=nun rbe ro fpa t i en t s ;E :=cs t r i r d i o l ;FSH= fo l l i c l es t i n ruh t i ngho r r . none ;PR l -=p ro l ac t i n ;NS=no ts i gn i f i can t ;S=s i€l n i f i can t lHS=h igh l is ien i f icent .

670

Treatment of Skin Aging with Topical EstrogensSchmidt et al.

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Table 2. Grading of Clinical Effects of Topical EstrogenTreatment on Facial Skin

n Minor effects consisting of increased vascularizatronand moisture of skin

** Minor decrease of wrinkle depth or improvement offirmness and elasticity of skin

*** Clear improvement of wrinkles, evident by monitoringand photodocumentarion

**no Significant decrease of wrinkle depth

levels stayed within the normal range in both treatmentSroups.

The clinical improvement of specific skin parame-ters was evaluated according to a personal evaluationscheme (Table 2); it was seen after 9 to 19 weeks in theestradiol and after 7 to 17 weeks in the estriol group.Improvement of vascularization, elasticity, and skinmoisture ranged between 100% and 61t/. in the twogroups (Table 1).

Wrinkle measurements by profilometry showed sig-nificant decreases in the estradiol and highly significantdecreases in the estriol group (Figs. 1A, 18, Table 3).Skin moisture by corneometry showed insignificant in-creases in both groups (Table 3). For immunohisto-chemistry, the specific staining was localized to thedermal connective tissue.

As demonstrated in Figure 2A, a difference in im-rnunohistochemical staining for collagen III could bedemonstrated in biopsies taken before and after treat-ment. In biopsy specimens after treatment, there ap-peared to be a more intense color reaction as com-pared to biopsies taken before treatment, indicating anincrease in collagen III after rreatment (Fig. 2B). Noclear difference in staining for collagen I could bedemonstrated before and after therapy (Table 4).

Side Effects

The side effects were less in the estriol than in theestradiol group. Breast tension, naturally occurring inperiods of hormonal changes, was noted by 12 of 30patients treated with estriol compared to 7 of 28 pa-tients of the estradiol group. One patient treated with

Figure 1, /, Wrinkles prior to estriol rreatment. B, Wrinklesat the end of a 6-month treatmenr period with topical 0.3%estriol.

estriol complained of itching, one patient treated withestradiol complained of local reddening of the face. Ofthe 28 women treated with estradiol, three observedincreased facial pigmentarion.

DISCUSSION

Estrogen compounds where shown to be effective in thetreatment of skin aging symptoms of perimenopausalwomen. Both estradiol and estriol, at the concentra-tions used, exhibited comparable results. In a previouspilot study we had shown the lack of systemic hormon-

Table 3' Cl inical Evaluation of Symptoms of Aging in 58 women Treated with Topical Estradiol Compared to Estr iol

Incidence ofImprouement

(%)

Onset of Effects in'\Xleeksafter Start of Treatment

(Mean)

Estradiol 0.01% (N = 30) Estriol 0.3%" (N = 28)lncidence of Onset of Effects in \yeeksImprouement after Start of Treatment29 (%) (Mean)

r5VascularizationSkin elasticity and firmness

1001001008 7/ . )

91 39

1 61,9

9696968 96 1

MoistureVrinkle depth

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67)

5-

Trcirtncnt of Skin Aging rvith Topical FistrogensSchnr ic l r e r r l .

Iable 5. Distr ibution of Type I and Type II I Col lagen in the Dermis and at the Basal Membrane before and at th€End of Treatment with Topical Estrogen in 10 Women

Collagen I Collagen III

PretredtmentDernt is BM"

End of TreatmentDermis BM

PretreatmentDermis

End of TreatmentDermis

+

a

+

a

+

+

+

+

+

+

+

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+

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lagen IIL These f indings support the role that estrogensplav in the dermis and may serve as an explanation forthe cl inical observations of increased f irrnness and sig-nif icantly less wrinkl ing during treatment. Both typesof col lagen change during the agir.rg process. Type Icol lagen represents the predomir-rant col lagen type inadu l t human sk in , lT whereas type I I I co l lagen, a lsowidely distr ibuted throughout the body, predominaresin t issues of fetuses.r8 For the f irst t ime, increases ofmainly type II I col lagen indicate dist inct modulatoryeffects of estrogen on col lagen t issue.

The posit ive effects of estrogens on the water con-tent of the skir.r , that were observed in al l patients, mayb e d u e t o d e r m a l a n d e p i d e r m a l c o m p o n e n t s . T h eestrogen-st imulated increases of acid mucopolysaccha-rides and of hyaluronic acidl4'15 contr ibute to an rncreasewater content in the dermis. The increased epidermalwater content may be due to increased epidermal skinthicknessle"r0 with subsequently elevated amounts of anatural moisturizing factor.

The present study supports the effect iveness of es-t rogen compounds in the t rea tment o f sk in ag ing inper imenopausa l women. Both , es t rad io l and es t r io lshowed good and con.rparable effects in the concentra-t ions used and a lack of systemic side effects wherr ap-pl ied only to the face and neck. Obviously, no predic-t ions about systemic side effects can be made about thesarre hormonal substances Llnder dif ferent condit ions(e .g . , i r . rc reased concent ra t ions or s ize o f app l i ca t ionfield). Dose-f inding studies in both direct ions are there-fo re abso lu te ly necessary , in o rder to f ind the min i -muf f r concent ra t ion o f an es t rogen compound tha tcombines the best possible cl inical effects without sys-temic honnonal side effects. In general, estr iol seems tobe the preferable estrogen for topical use. Lack of sys-ter.nic side effects and promising local effects after ex-ternal use suggest that estr iol wi l l gain importance fortopical use in dermatology in the future.

CONCLUSIONS

By considering the results of topical estrogen treatmentin skin aging in women, a better insight can be gainedon the hormonal aspects of endogenous aging of thesk in . Var ious s t ruc tu res invo lved in sk in ag ing areur.rder hormonal control. I f decreased estrogen levelscontr ibute to decreased functions of the skin, local es-trogen treatment of the skin would in turn represent alocal hormone substi tut ion therapy of the skin. So far,estrogen compounds and, in part icular, estr iol repre-sent a new and promis ing therapeut ic approach to -wards skin aging in perimenopausal women.

REFERENCES

1. Kl igman AM, Grove GL, Hirose R, Leyden J. Topicalt re t ino in fo r photoaged sk in . J Am Acad Dern ' ra to l1 9 8 6 ; l 5 : 8 3 6 - 8 - 5 9 .

2. Veiss J\V, E,l l is CN, Heirdington jT, Vorhees JJ. Topi-cal tret inoin in the treatment of aging skin. . f Am AcadDermatol 1 9881 1 9: 169-17 5.

3 . Tong PHS, Horowi tz MS, Vhe l le r [ .A . Tr i rnsre t ino icrrcid enhzrnces the growth responses of epidermal ker-atinocytes to epidermal growth factor ar-rd transforminp;growth factor beta. J Invest Dermatol 1990'87:663-667.

4. Harper RA, Burgoon T. Differential effects of ret inoic: rc id on the growth o f nor rna l f ib rob las t - l i ke ce l l s lnuitro from hurnrrn, swine irnd rabbit skin. Cell Biol IntRep 1982; 6 :163-170.

-5. Kl igman LH, Kl igman AM. Skin changes in photoag-ing: characterist ics, preventiorr and rep:r ir . In: Bal in AK,K l i g m a n A M , e d s . A g i n g a n d t h e s k i n . N e w Y o r k :R:rven Press, 1989:.331-346.

6. Artner J, Gitsch E. Uber Lokir lrvirkr.rngen vor-r Ostr iol.Gebur tsh i l fe und Frar . renhe i lkd 19591 l9 :8 l2 -819.

7. Bosell i F, Volpe A, Genazzani AR. Topical therapy: es-t r i o l v a g i n a l c r e a m i n p o s t m e n o p a u s a l w o m e n . I n :Genazzani AR, Volpe A, Farcchinett i F, eds. Gynaecolog-

I-Jr

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1 5 .

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ical endocrinology. Carrrforth: Parthenon Publishrng,687-694.R i e d c l M , R a f f e n b e u l V , L i c h t l e n P . O v a r i a n s e xs t e r o i d s a n d a t h e r o s c l e r o s i s . C l i n I n v e s t 1 9 9 3 ; 7 1 :406412.Falkeborn M, Persson I, Adami HO, et al. The r isk ofacutc n"tyocardial infarction after oestrogen and oestro-gen and progestogen replacement. Br J Obstet Gynecol1992'99:821-828.Ga l lagher JC, Nord in BEC. E f fec ts o f oes t rogen andprogcs t ( )gen therapy on ca lc ium metabo l ism in pos t -n tenoD: rusa l wonren. ln : F ron t ie rs o f Hormone Re-search 3. Basel: Karger : 1.50-176.Schmidt JB, Spona J. Ostrogen- und Androgenrezep-tren bei Patienten mit Akne vulgaris. Arch DermatolRes 1980;268:207-215.Schmidt JB, Lindmaier A, Spona J. Hormone receptorsin pubic skin of premenopausal and postnrenopausal fe-m:1les. Gynecol Obstet Invest 1990; 30:97-100.Punnonen R, Vaa ja lah t i P , Te isa la K . l .oca l oes t r io ltreatment improves the structure of elast ic f ibres in thesk in o f pos tmenopausa l women. Ann Ch i r Gynaeco l(SLrpp l ) 1 987 ; 202:3941.Grosnran N, Hridbey E, Schon J. The effect of oestro-genic treatnent on the acid mucopolysaccharide patternin skin of mice. Acta Pharmacol Toxicol 1971l '30:458-464.Grosman N. S tud ies on the hya lu ron ic ac id p ro te lnconrplex the molecular size of hyaluronic acid and theexchirngeabil i ty of chloride in skin of nrice before andaf te r oes t rc lgen t rea tment . Ac ta Pharmaco l Tox ico l1973t 33:201-208.I-Lrisi M, Franchi F, Kicovic PM. A group-comparatives tud ,v o f e f fec ts o f Oves t in c ream versus Premar incream in post-menop:lusal women with vaginal atro-phy . Matur i tas 1980; 2 :31 l -319.Schmidt JB, Binder M, Macheiner \W, et al. Treatmentof skin aging sympt()ms in perimenopausal females withes t rogen compounc ls -a p i lo t s tudy . Matur i tas 1994;20:2.5-30.Schnridt. fB, Binder M, Macheiner W, Bieglmayer CH.Ncw treatment of ir trophic acne-scars by iontophoresis

with estriol and tretinoin-ar pilot study. Int J Dermatol1995;34 53-57.Reinthal ler A, Kirchheimer JC, Deutinger J, et al. Plas.minogen ac t iva tors , p lasminogen ac t iva tor inh ib i to rand f ibronectin in hurntrn granulosa cel ls and fol l icularf luid related to oocyte maturation and intrafol l iculargonadotropin levels. Fert i l Steri l 1990; 54:1045-1051Binder M, Macheiner .W, Schmidt JB. Die Profi lome-tr ie-eine Dokumentationsmethode der Hautstruktur,Hautarzt 1 994; 4 5:546-5 50.Schmidt JB, Hobisch G, Lindmaier A. Epidermal mois.ture and skin surface l ipids throughout l i fe as parame-ters fcrr cosmetic care. Appl Cosmetol 1990;8:17-22.Kainz C, Gitsch G, Stani J, et al. When applied to facialsk in , does es t rogen o in tment have sys temic e f fec ts?Arch Gynecol Obstet 1 993 25371-74.Laur i tzen C. Er fahrungen mi t e iner Ost r io l -Vag ina tcreme. Therapie Gegenw 1979 1,1,8:567-577.Tzingouinis VA, Aksu MF, Greenblatt RB. The signif i-cance r>f oestriol in the management of the post meno-pause. Acta Endocrinol Suppl 1980; 233:45-50.Frost P. Weinstein GD. Hsia SL. Metabolism of oesrra-diol and oestrogen in human skin. J Invest Dermatol1 .966; 46 :584-585.'Wendker H. Schaefer H. Zesch A. Penetrationskinetikund Vertei lung lokal appl izierter Ostrogene. Arch Der-matol Res 1.97 6; 256:67-7 4.Epstein EH, Munderloh NH. Isolat ion and characterrzati()n ()f CNBr peptides of human (alpha 1 [I I I ] 3) col-lagen and t issue distr ibution of (alpha 1 [ 2), alpha 2and (alpha 1 UIII 3) col lagens. J Biol Chem 7975;2509304-9312..Epstein EH. (Alpha 1 I3l) human skin col lagen. Releaseby pepsin digestion and preponderance in fetal l i fe. JBiol Chem 1 97 4; 249:3225-3231,.Rauramo L, Punnonen R. Wirkungen von Kastrat ionund Ost rogentherap ie au f d ie Haut e r Frau . TherapUmschau 1974 l ' 31 :173-17 5 .Brincat M, Moniz CJ, Studd JW. Long-term effects ofthe nrenopause and sex hormones on skin thickness. BrJ Obstet Gynaecol 1985; 92:256-259.

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Comparative Medical Research

Legions of aquatic animals-whose biodiversity is virtually untapped in biomedicalresearch-have been enlisted in the war against human disease. The zebrafish is animportant new model for developing transgenic techniques, for genetic and embry-ological studies, and for spawning a growing "zebrafish community" of researchers.National Center for Research Resources (Ncnn) also supports studies of abalone,cephalopods, clams, hydra, lobster, sea slugs, sea squirts, sea urchins, and trout.

Nonhuman primates continue to be widely-used animal models. Researchers atone of NCRR's seven Regional Primate Research Centers have isolated the first em-bryonic stem cells produced from rhesus monkeys and marmosets, opening the wayfor transgenesis and gene therapy in primate systems within the next five years.From Vaitukaitis JL. Looking ahead at comparatiue medicine research. Compard-tiue Patbol Bull 1995: XXVII(2):1.

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