Originl Pper Skin Phrmol Physiol 216;29:135 147 DOI: 1.1159/445955 Reeived: Deemer 29, 215 Aepted: April 4, 216 Pulished online: June 2, 216 The Effet of n Emollient Contining Ure, Cermide NP, nd Ltte on Skin Brrier Struture nd Funtion in Older People with Dry Skin Simon G. Dny Kirsty Brown Tim Higgs-Byliss John Chittok Lujin Alenli, Mihel J. Cork Ademi Unit of Dermtology Reserh, Deprtment of Infetion, Immunity nd Crdiovsulr Disese, Fulty of Mediine, Dentistry nd Helth, University of Sheffield Medil Shool, Peditri Dermtology Clini, Sheffield Children s Hospitl, nd Deprtment of Dermtology, Royl Hllmshire Hospitl, Sheffield, UK Key Words Skin rrier Emollient Xerosis Ure Cermide Fourier trnsform infrred spetrosopy Trnsepiderml wter loss Astrt Xerosis ffets up to 75% of older people nd develops s result of skin rrier defet. Emollients re widely used to tret xerosis; however, there is limited understnding of the differenes etween them nd their effets on the skin rrier in older people. This study imed to ompre the effet of ommerilly ville ontining 5% ure, ermide NP nd ltte (test ) to n lterntive without these dditives (ontrol ) on the properties of the skin rrier in older people. Two ohorts of 21 volunteers ged >6 yers with dry skin were reruited. The first pplied the test to one forerm nd no tretment to the other for 28 dys. The seond ompred the test to the ontrol oserving the sme prmeters. Effets on the skin rrier were determined y mesuring skin rrier funtion, hydrtion, skin surfe ph nd y nlysing Fourier trnsform infrred spetr efore nd fter tretment. A third ohort of 6 young dults ws re- ruited to investigte the effet of single tretment with the test on the moleulr struture of the skin rrier t greter depths y employing the tpe-stripping tehnique. The test hydrted the skin to signifintly greter extent nd for longer period of time ompred to the ontrol, n effet ssoited with signifint elevtion of roxylte groups ( mrker of nturl moisturizing ftor ontent) within the strtum orneum. Furthermore, the test imprted dditionl enefits to the struture nd funtion of the skin rrier not exhiited y the ontrol. In onlusion, the test ddressed the pthologil fetures of xeroti ged skin, supporting its use s first-line therpy for xeroti skin onditions in this popultion. Introdution 216 The Author(s) Pulished y S. Krger AG, Bsel Worldwide the prevlene of xerosis inreses with dvning ge, ffeting up to 75% of older people [1]. The development of xeroti onditions, suh s topi dermtitis (AD), stetoti ezem nd winter xerosis, is ssoited with skin rrier defet. This defet is hr- E-Mil krger@krger.om www.krger.om/spp 216 The Author(s) Pulished y S. Krger AG, Bsel 166 5527/16/293 135$39.5/ This rtile is liensed under the Cretive Commons Attriution- NonCommeril-NoDerivtives 4. Interntionl Liense (CC BY- NC-ND) (http://www.krger.om/servies/openaessliense). Usge nd distriution for ommeril purposes s well s ny distriution of modified mteril requires written permission. Simon G. Dny Ademi Unit of Dermtology Reserh, Deprtment of Infetion, Immunity nd Crdiovsulr Disese, Fulty of Mediine, Dentistry nd Helth University of Sheffield Medil Shool, Sheffield S1 2RX (UK) E-Mil s.dny @ sheffield..uk
Tle 1. Emollient rems used in this study Mnufturer Produt ph Ingredients Control Test Aqumol rem (Thornton & Ross Ltd., Huddersfield, UK) Blneum rem (Almirll Herml GmH, Reinek, Germny) 5.57 ±.5 Purified wter, white soft prffin, liquid prffin, eteryl lohol, PPG-5-eteth 2, disodium oomphodiette, polysorte 6, sodium hloride, hlororesol, itri id monohydrte 4.73 ±.2 Ure 5%, ermide NP, qu, glyine soy oil, propylene glyol, eteryl lohol, liquid prffin, isohexdene, sodium ltte, lti id, PEG-2 sterte, polysorte 6, squlne, steri id, disodium EDTA, leithin, toopherol, soryl plmitte, hydrogented plm glyeride itrte terized y redued nturl moisturizing ftor [NMF, omprising sodium pyrrolidone roxyli id (PCA), ure nd ltte mongst others] nd norml levels of interellulr lipids (holesterol, ermides nd free ftty ids) in the strtum orneum (SC) [2 5]. Additionlly, the ged skin rrier reovers more slowly fter exposure to irritnts [6]. The growing reognition tht xeroti skin onditions rise primrily s result of defetive skin rrier lls for greter ppreition of the effet of topil produts on this rrier [7, 8]. A puity of evidene on the mehnisti effets of s hs led mny to elieve tht s re ll the sme. We, nd others, hve demonstrted however tht s hve very different effets on the skin rrier depending on their formultion [9, 1]. For exmple, some optimlly designed s ontining humetnts meliorte skin dryness nd redue the severity of skin inflmmtion [11 13]. In strk ontrst, queous rem, ontining the hrsh nioni surftnt sodium luryl sulphte (SLS) nd no humetnt, ws found to dmge the skin rrier [14 17], n effet linked to high rtes of dverse retions nd exertion of AD symptoms [18]. In response to growing evidene nd improvements in formultion, reent onsensus sttement y helth re professionls highlighted the need to differentite s sed on their mehnism of tion [19]. The im of this study ws to diretly ompre the effet of n rem, ville for presription in the UK, ontining the skin-moisturizing ftors ure, ermide NP (previously ermide 3), nd ltte (test ), to n rem without these dditives (ontrol ) in volunteers with xerosis ged over 6 yers. Mteril nd Methods Sujets Three ohorts of volunteers were reruited. Volunteers sored their skin dryness on 5-point sle from 1 (no dryness) to 5 (severe dryness with rking nd lifting sles). Cohort 1 onsisted of 21 volunteers (17 women) with dry skin (men sore 3) nd men ge of 69 (6 89) yers. Eight prtiipnts (38%) reported previously hving AD. Cohort 2 omprised 6 volunteers (4 women) with helthy skin (no history of skin onditions or topy, men skin dryness sore ) nd men ge of 33 (21 45) yers. This seprte popultion (ohort 2) with no ge restrition ws reruited for prtil resons due to the length of the mesurement sessions nd the intensity of mesurements. For ohort 3, 21 volunteers (14 women) with dry skin (men sore 3) nd men ge of 68 (6 79) yers were reruited, 18 of whom ompleted the study. Three volunteers withdrew for resons unrelted to the study. Six prtiipnts (33%) reported previous history of AD. Reruitment ws open to mles nd femles with Fitzptrik skin type of I IV. Exlusion riteri onsisted of the use of systemi or topil ntiinflmmtory medition in the 6 months leding up to the study, pregnny, rest-feeding, nd eing under the ge of 18. Volunteers refrined from using topil produts (exept the study preprtion s direted) on the tretment sites for 7 dys prior to prtiiption nd for the durtion of the study. There were 2 test sites per volunteer, one on eh forerm (volr side, 3 m elow elow flexure to 3 m ove the wrist). Cohorts 1 nd 3 undertook 28-dy forerm-ontrolled oserver-lind studies involving self-tretment with the test (Blneum rem, tle 1 ) nd/or the ontrol (Aqumol rem, tle 1 ). In essene ohort 1 undertook diret omprison of the test to untreted skin, nd ohort 3 undertook diret omprison of the test to referene. The prtiipnts in ohort 1 treted one forerm with 2-fingertip units (1.24 ±.127 g) of test nd the other forerm with no tretment (rndomized) twie dily for 28 dys. Following the esstion of tretment (12 2 h) the iophysil properties of the test sites were ssessed to determine the effet of the tretment on the underlying ondition of the skin rrier (not their trnsient olu- 136 Skin Phrmol Physiol 216;29:135 147 DOI: 1.1159/445955 Dny/Brown/Higgs-Byliss/Chittok/ Alenli/Cork
5.75 p =.5 45 p =.3 24 Skin surfe ph 5.5 5.25 5. 4.75 p =.85 p =.48 Cpitne (AU) 4 35 p =.317 p =.271 Redness (AU) 23 22 21 4.5 Dy Dy 29 3 Dy Dy 29 2 Dy Dy 29 TEWL (g/m 2 /h) 12 11 1 9 8 p =.2 p <.1 TEWL (g/m 2 /h) 5 4 3 2 NTC TE 7 1 d 6 Dy Dy 29 e 4 8 12 Tpe strip numer 16 2 Fig. 1. The effet of 28-dy tretment with the test (TE), ompred to n untreted ontrol (NTC), on skin surfe ph ( ), hydrtion ( ), redness ( ), TEWL ( d ) nd SC integrity ( e ) 12 2 h following the lst pplition in people with dry skin over the ge of 6 (n = 21). Two-wy ANOVA reported signifint effet of test tretment (nd the intertion etween tretment nd tpe strip numer) on SC integrity. Asterisks indite signifint differenes (p <.5) reported using the Holm-Sidk posttest following one-wy ANOVA ( d ) nd Bonferroni posttest with p <.5, p <.1, nd p <.1 ( e ). sive properties). The prtiipnts in ohort 3 treted 1 forerm with 1.28 ±.126 g test nd the other forerm with 1.21 ±.12 g ontrol (rndomized llotion) twie dily for 28 dys. Before initition nd fter esstion (12 2 h following the lst pplition) of tretment, the iophysil properties of the test sites were ssessed. Cohort 2 undertook forerm-ontrolled study wherein single pplition of test (2 fingertip units) ws mde to 1 test site. Assessments were performed efore nd 3 h following tretment pplition. Biophysil Mesurements Trnsepiderml wter loss (TEWL) mesurements were performed using n AquFlux AF2 ondensing hmer proe (Biox Systems Ltd., London, UK). Redness, pitne nd skin surfe ph were mesured using Mexmeter MX18, Corneometer CM825 nd Skin ph Meter PH95, respetively (CK eletroni GmH, Cologne, Germny). All ssessments were performed in room mintined t 21 ± 2 C nd 38 5% reltive humidity ording to pulished guidelines [2]. All test sites were limtized to room onditions for 2 min efore ssessment. Tpe-stripping, to experimentlly disrupt the SC, ws performed s previously desried [21]. Prior to tpe-stripping the test sites were lened with otton wool nd wter, nd llowed to relimtize for 2 min. Extrpoltion of depth rehed y tpe-stripping ws sed on the infrred sorne (SqumeSn 85A, CuDerm, Dlls, Tex., USA) of tpe strips (to quntify protein) in ordne with pulished methodology [22, 23]. SC Protese Ativity Assessment of protese tivity ws mde on smples omprising 3 onseutive tpe strips s previously desried [22]. Cseinolyti, hymotrypsin-like nd trypsin-like tivities were determined using EnzChek (Life Tehnologies Ltd., Pisley, UK), MeOSu-Arg-Pro-Tyr-AMC (Peptide Protein Reserh Ltd., Funtley, UK), nd Bo-Phe-Ser-Arg-AMC (Bhem, Buendorf, Switzerlnd) sustrtes, respetively. Quntifition of PCA nd Ltte The levels of PCA in SC smples olleted y tpe-stripping (strips 4 6 pooled) were quntified s previously desried [25]. The Effet of n Emollient on Skin Brrier Struture nd Funtion Skin Phrmol Physiol 216;29:135 147 DOI: 1.1159/445955 137
Cseinolyti tivity (nu/μg) 2. 1.5 1..5 p =.23 Chymotrypsin-like tivity (nu/μg) 4. 3. 2. 1. p <.1 Trypsin-like tivity (nu/μg) 4. 3. 2. 1. Fig. 2. The effet of 28-dy tretment with the test (TE) ompred to n untreted ontrol (NTC) on seinolyti ( ), hymotrypsin-like ( ) nd trypsin-like ( ) protese tivity. p vlues re the results of pired t test. Ltte smples were olleted using prewetted otton sw, whih ws rued ginst the skin nd then trnsferred to 1 ml PBS. Ltte onentrtions were determined using fluorometri L -ltte ssy (Am, Cmridge, UK) ording to the mnufturer s instrutions. Fourier Trnsform Infrred Spetrosopy Fourier trnsform infrred (FTIR) spetr were olleted using silver hlide fire-opti proe (FTIR Flexispe PIR 9, Art Photonis, Berlin, Germny) tthed to Niolet is5 FTIR spetrometer (Thermo Fisher Sientifi In., Wlthm, Mss., USA), equipped with ooled merury-dmium-telluride detetor nd purged with dry N 2. An verge of 32 sns ws olleted for eh mesurement t resolution of 4 wve numers. Integrtion of pek intensities ws performed using Omni 9. softwre (Thermo Eletron Corp., Mdison, Wis., USA). Pek intensities in the spetrl region from 4, to 2, m 1 were normlized to the seline t 3,8 m 1, nd intensities in the region from 2, to 1, m 1 were normlized to the seline t 1,85 m 1 to ount for ontt pressure. The mount of wter in the SC ws determined y dividing the re of the pek t 3,3 m 1, enompssing the virtions from the O H of wter nd the N H of proteins, y the re of the pek t 1,55 m 1, lso relting to virtions from N H onds. Lipid hin onformtion ws determined s previously desried [23]. Additionl spetr were olleted from solutions of lti id, itri id, PCA, nd ure in wter, minerl oil (liquid prffin), nd ermide (mixture from ovine rin) in hloroform (Sigm-Aldrih, UK). The ontriution of wter or hloroform ws removed y spetrl sutrtion of wter-only ontrol. Dt Anlysis The results were nlysed y Prism v6.1 (Grphpd Softwre In., Clif., USA). The signifine threshold ws p <.5. Results re presented s mens ± stndrd error of the men. Results Effet of the Test Emollient on the Skin Brrier with the test for 28 dys signifintly redued TEWL nd skin surfe ph y n verge of 1.7 ±.29 g/m 2 /h nd.15 ±.7 ph units ompred to the untreted ontrol site, respetively, in volunteers with dry skin ged >6 yers, suggesting positive effet on underlying skin rrier funtion ( fig. 1 ). Hydrtion ws inresed signifintly (+2.9 ±.95 units) ompred to the untreted ontrol despite the lst pplition eing 12 h previously. There ws no effet of the tretment on skin redness, inditing tht the test is mild on ged skin under norml use onditions. To further ssess the effet of the test on the integrity of the SC, tpe-stripping to experimentlly dmge the SC in ontrolled wy ws performed in onjuntion with TEWL mesurements ( fig. 1 e). TEWL onsistently inresed t redued rte on the site treted with the test with eh onseutive 4 tpe strips ompred to the untreted ontrol suggesting signifintly inresed SC integrity. The tivity of extrellulr proteses involved in desqumtion is n importnt determinnt of SC integrity. As suh, we quntified rod-spetrum nd peptide-speifi tivity in the superfiil lyers of the SC ( fig. 2 ). Brod-spetrum tivity ws signifintly redued following tretment with the test, s ws hymotrypsin-like tivity. Trypsin-like tivity ws lso deresed, ut not signifintly. 138 Skin Phrmol Physiol 216;29:135 147 DOI: 1.1159/445955 Dny/Brown/Higgs-Byliss/Chittok/ Alenli/Cork
TEWL (g/m 2 /h) 14 12 1 8 6 Before After Skin surfe ph 5.5 5. 4.5 4. Cpitne (AU) 6 5 4 3 2 Before pplition 1. After pplition (3 h).8.6.4 Bulk wter (3,3 AII pek re) d.2 5 1 15 2 25 3 Totl mount of SC removed (μg/m 2 ) Bulk wter (3,3 AII pek re) e 6 4 2 r =.7851 p =.25 2 3 4 5 6 Cpitne (AU) Chnge in intensity (m 2 ) f 35 3 25 2 15 1 5 Amide I (C=O) Amide II (N H) Lipid (CH 2 ) Ltte Croxylte ( COO) Ure Lipid esters 5 1 15 2 25 3 Totl mount of SC removed (μg/m 2 ) vsch2 pek COG g 2,852 2,851 2,85 2,849 Before pplition After pplition (3 h) 5 1 15 2 25 3 Totl mount of SC removed (μg/m 2 ).6 Before After d Asorne.4.2 e f g h h 3,75 3,5 3,25 3, 2,75 2,5 2,25 2, 1,75 1,5 1,25 1, 75 Wve numers (m 1 ) Fig. 3. The effet of single pplition of the test on the iophysil properties of the skin: TEWL ( ), skin surfe ph ( ), nd hydrtion ( ) efore nd 3 h following pplition. d The onentrtion of wter in the SC determined y FTIR spetrosopy efore nd 3 h following pplition. Mesurements were olleted in onjuntion with tpe-stripping to remove the uppermost lyers of orneoytes nd hieve greter depths. Two-wy ANOVA reported signifint effet of test tretment (nd the intertion etween tretment nd tpe strip numer) on SC wter ontent. AII = Amide II. p <.5. e Correltion etween wter onentrtion nd pitne (Person oeffiient shown). Open irles orrespond to mesurements tken efore tretment nd filled irles to mesurements tken fter tretment. AII = Amide II. f The hnge in onentrtion of key funtionl groups (3 h posttretment sutrted y pretretment mesurements) throughout the SC s determined y FTIR. g The position of the pek t 2,85 m 1, relting to the symmetri strething of CH 2 lipid groups, throughout the depth of the SC. A two-wy ANOVA reported no effet of tretment. ν s = Asymmetri strething of the CH 2 ond of lipids; COG = entre of grvity. h Exmple spetr olleted efore nd fter tretment. Regions identified on the spetr inlude: = 3,3 m 1 O H of wter; = 2,92 nd 2,85 m 1 CH2 symmetri nd symmetri strething of lipids; = 1,74 m 1 lipid esters; d = 1,65 nd 1,55 m 1 relting to mide I nd mide II; e = 1,465 m 1 CH 2 sissoring of lipids; f = 1,41 m 1 COO roxyltes; g = 1,175 m 1 N H ending; h = 1,124 m 1 speifi to ltte. The Effet of n Emollient on Skin Brrier Struture nd Funtion Skin Phrmol Physiol 216;29:135 147 DOI: 1.1159/445955 139
The Short-Term Effets of the Test Emollient on SC Hydrtion Hving determined tht 28 dys of tretment with the test exert positive effets on the underlying ondition of the skin rrier, we next investigted the short-term properties of the formultion. Three hours following single pplition of the test to the skin of dults <5 yers of ge with no skin omplints (ohort 2), TEWL deresed nd SC pitne inresed inditive of skin olusion ( fig. 3, ). Skin surfe ph lso deresed y.45 ±.75 units, nd SC hydrtion inresed y 15.17 ± 2.4 units on verge ( fig. 3 ). Using FTIR, the moleulr properties of the skin surfe, to depth of 1.5 μm, were determined efore nd fter tretment. Tpe-stripping ws employed to hieve greter depth profile, spnning pproximtely 4.5 μm SC depth in totl (ssuming n SC density of 1 μg/μm 3 ). Overll there were numer of hnges to the spetr olleted fter tretment ompred to efore tretment, with the gretest hnges oserved t the skin surfe ( fig. 3 h, without tpe-stripping). The mount of wter in the SC, sed on sorne t 3,3 m 1 (orresponding to OH ond of wter) ws elevted throughout most of this depth following tretment pplition ( fig. 3 d, signifint to depth of pprox. 2.74 μm sed on mesurement fter removing 124.23 ± 9.26 μm SC y tpe-stripping). The men intensity of the 3,3 m 1 nd ross the depth of the SC diretly orrelted (Person s r =.785) with pitne mesurements of SC hydrtion ( fig. 3 e, whih mesures ross 1 2 μm of the skin depth). Due to the redued penetrtive depth of FTIR ompred to pitne mesurements, there is greter sensitivity for quntifying lolized surfe hnges in wter ontent using the former method. Three hours following single pplition of the test, SC lipid levels were elevted, s indited y the inresed sorne t 2,92 nd 2,85 m 1 orresponding to symmetri nd symmetri CH 2 strething of lipids. In the spetrl region etween 1, nd 1,75 m 1, there were further hnges to the sorne spetr of the skin tht mthed the spetr of the test (online suppl. fig. 1; for ll online suppl. mteril, see www.krger.om/doi/1.1159/445955). The sorne spetrum of the test is omposite of ll its ingredients; the spetr of the mjor ingredients re presented in online supplementry figure 1. The hnges in sorne (fter vs. efore tretment) t speifi spetrl regions highlighted in online supplementry figure 1 hve een presented in figure 3 f. Asorne is inresed t regions entered on: 1,74 m 1, orresponding to lipid esters suh s those in seum nd nturl oils; 1,65 m 1, orresponding to the C = O ond of proteins (mide I) for exmple; 1,54 m 1, orresponding to the N H ond of proteins (mide II), ermides nd ure; 1,41 m 1, orresponding to roxylte groups ( COO); 1,175 m 1, orresponding to ure, nd 1,124 m 1, orresponding to ltte. No signifint differene in the hin onformtion of SC lipids s result of these ompositionl hnges ws oserved ( fig. 3 g). Effet of the Test Emollient Compred to n Emollient without Ure, Cermide NP nd Ltte on the Skin Brrier Hving determined tht the test delivers moisturizing ftors to the SC, nd tht this orresponds with inresed wter levels in the skin, we sought to diretly ompre its effet to n without these dded moisturizing ftors, the ontrol ( tle 1 ). The effet of 28 dys of tretment with the test ompred to the ontrol on the iophysil properties of the skin of volunteers ged over 6 yers with self-reported dry skin is presented in figure 4 (ohort 3). There were no hnges in skin redness inditing tht neither formultion indues erythem under the onditions tested. Skin surfe ph ws signifintly inresed on the site treted with the ontrol ompred to the test (+.23 ±.5 units, p =.7) nd ompred to the seline mesurements (+.3 ±.7 units, p =.15). TEWL ws unhnged following tretment with the test (11.27 ±.19 g/m 2 /h efore nd 11.58 ±.23 g/m 2 /h fter). Following tretment with the ontrol, TEWL inresed from 11.15 ±.23 to 11.94 ±.26 g/m 2 /h (p <.1) suggesting smll redution in skin rrier funtion. Moreover, the integrity of the SC ws signifintly deresed t the site treted with the ontrol, indited y elevted TEWL throughout tpe-stripping ( fig. 4 e), when ompred to oth pretretment ssessments of the test re (djent test site) nd the sites treted with the test t the sme time point. Beuse the s differentilly ffeted SC integrity nd skin surfe ph, ssessment of SC proteolyti tivity ws lso performed ( fig. 5 ). In line with ohort 1, the test redued rod-spetrum tivity y.36 ±.16 nu/μg ompred to the ontrol, whih exhiited similr tivity ompred to the untreted sites in ohort 1 ( fig. 2 ). There ws no differene in hymotrypsin-like tivity etween the test nd ontrol treted sites, suggesting tht oth tretments inhiit this 14 Skin Phrmol Physiol 216;29:135 147 DOI: 1.1159/445955 Dny/Brown/Higgs-Byliss/Chittok/ Alenli/Cork
5.5 45 26 Skin surfe ph 5. 4.5 Cpitne (AU) 4 35 Redness (AU) 24 22 4. Test Control 3 Test Control 2 Test Control TEWL (g/m 2 /h) 14 13 12 11 1 9 Chnge in TEWL (g/m 2 /h) 15 1 5 5 1 Test Control d 8 Test Control e 15 4 8 12 16 2 Tpe strip numer Fig. 4. The effet of 28-dy tretment with the test, ompred to the ontrol on skin surfe ph ( ), hydrtion ( ), redness ( ), skin rrier funtion ( d ) nd SC integrity ( e ) 12 2 h following the lst pplition in people with dry skin over the ge of 6 (n = 18). For d, one-wy ANOVA found signifint differenes etween the groups for TEWL (p =.18), hydrtion (p =.26) nd skin surfe ph (p =.2). Asterisks indite the results of Bonferroni posttest: p <.5, p <.1, p <.1. e SC integrity is displyed s the hnge from seline (TEWL fter TEWL efore ). A two-wy ANOVA reported signifint differene etween the test nd ontrol -treted sites. Asterisks indite the results of Bonferroni posttest: p <.1, p <.1. tivity (see lso fig. 2 ). Trypsin-like tivity ws signifintly inresed y 1.2 ±.59 nu/μg on the sites treted with the ontrol ompred to the test, whih exhiited similr tivity to untreted skin ( fig. 2 ). Elevted proteolyti tivity is therefore potentil mehnism y whih SC integrity is redued following tretment with the ontrol. Skin hydrtion inresed y 12.2 ± 5.4% following tretment with the test nd 6.6 ± 3.51% following tretment with the ontrol (12 2 h following lst pplition); however, the inrese ws only signifint for the test ( fig. 4 ). To etter understnd how the s lter hydrtion, levels of two NMF omponents in the SC were quntified ( fig. 6 ). Ltte is redily detetle in superfiil skin smples nd ws used s mrker for the delivery of exogenous moisturizing ftors from the test. PCA, not present in either of the formultions tested, is useful iomrker for totl filggrinderived NMF omponents nd ws employed to determine the effet of the tretments on endogenous moisturizing ftor levels [24]. Ltte ws signifintly elevted (y 57% on verge) in the SC of test - ut not ontrol -treted skin >12 h following the lst pplition. The mount of ltte in the skin ws lso found to signifintly orrelte with skin surfe ph, helping explin the differene in skin surfe ph etween the sites The Effet of n Emollient on Skin Brrier Struture nd Funtion Skin Phrmol Physiol 216;29:135 147 DOI: 1.1159/445955 141
Cseinolyti tivity (nu/μg) 2. 1.5 1..5 Test Control Chymotrypsin-like tivity (nu/μg) 2. 1.5 1..5 Test Control Trypsin-like tivity (nu/μg) 5. 4. 3. 2. 1. Test Control Fig. 5. The effet of 28-dy tretment with the test ompred to the ontrol on seinolyti ( ), hymotrypsin-like ( ) nd trypsin-like ( ) protese tivity. Asterisks indite the results of pired t test: p <.5. Ltte (nmol/smple) 1, 8 6 4 2 6. 1, Skin surfe ph 5.5 5. 4.5 r =.4635 p =.44 PCA (μmol/g protein) 8 6 4 2 TEWL (g/m 2 /h) 2 15 1 r =.4295 p =.112 Control Test 4. 5 1, 1,5 2, Ltte (nmol) Control Test d 5 2. 2.5 3. 3.5 log PCA onentrtion 1,124 m 1 nd intensity (ltte) e 1.25 1..75.5.25 Control Test 1,124 m 1 nd intensity (ltte) f 2. 1.5 1..5 r =.7792 p.1 5 1, 1,5 2, Ltte (nmol) 1,41 m 1 nd intensity (roxylte) g.6.4.2 Control Test 1,41 m 1 nd intensity (roxylte) h.5.4.3.2.1 r =.5835 p =.5 2. 2.5 3. 3.5 log PCA onentrtion Fig. 6. The effet of 28-dy tretment with the test ompred to the ontrol on ltte levels quntified from skin sws ( ), PCA levels quntified from tpe strip smples ( ), FTIRdetermined ltte levels sed on sorne t 1,124 m 1 ( e ), nd FTIR-determined roxyli id levels sed on sorne t 1,41 m 1 /mide II ( g ) 12 2 h following the lst pplition in people with dry skin over the ge of 6 (n = 18). A one-wy ANOVA found signifint differenes etween the groups for ltte (p =.154), PCA (p =.2), 1,124 m 1 (p =.275), nd 1,41 m 1 /mide II (p.1). Asterisks indite the results of Bonferroni posttest: p <.5. Correltions etween the prmeters nd iophysil mesurements re presented in, d, f, nd h. 142 Skin Phrmol Physiol 216;29:135 147 DOI: 1.1159/445955 Dny/Brown/Higgs-Byliss/Chittok/ Alenli/Cork
Fig. 7. Correltion etween FTIR-determined roxyli ids in the SC nd mesures of hydrtion, inluding pitne ( ) nd FTIR-determined wter ontent ( ). Open irles represent mesurement tken efore tretment, grey irles tht following tretment with the ontrol nd lk irles tht following tretment with the test. Dshed lines represent the liner reltionship etween the prmeters for ll groups nd the solid line the one for the test -treted group only. 1,41 m 1 nd intensity (roxylte).6.5.4.3.2 r =.6336 p =.5 r =.3368 p =.63.1 25 3 35 4 45 5 55 6 Cpitne (AU) 1,41 m 1 nd intensity (roxylte).6.5.4.3.2 r =.763 p =.1 r =.7553 p <.1.1 1 2 3 4 5 6 7 8 3,3 m 1 nd intensity (wter) treted with the test nd ontrol s ( fig. 6 ). Interestingly PCA ws lso signifintly elevted (y 43%) following tretment with the test ut not the ontrol, suggesting tht tretment with the test promotes endogenous NMF prodution (PCA not present in either topil formultion). The levels of PCA were orrelted with skin rrier funtion ( fig. 6 d). FTIR spetrosopy ws onduted t the skin surfe nd following 3 onseutive tpe strips to determine the moleulr properties of the skin to depth of 1.5 μm nd to depth of.56 2.6 μm, respetively (sed on the removl of 55.71 ± 2.35 μm protein y tpe-stripping no differene etween the protein removed from the test sites ws found using one-wy ANOVA). Exmple sorne spetr of the skin efore nd fter tretment re presented in online supplementry figure 2, nd the men hnges in the spetrl fetures identified re detiled in online supplementry tle 1. The mount of wter in the skin inresed signifintly on the sites treted with the test ut remined unhnged on the sites treted with ontrol. The mount of lipid (vch 2 nd δch 2 ) groups on nd within the skin inresed signifintly on oth the test nd ontrol -treted sites. In ontrst, the intensity of the peks t 1,74 m 1 (lipid esters), 1,65 m 1 (mide I), 1,55 m 1 (mide II), 1,41 m 1 (roxyl group), 1,175 m 1 (ure), nd 1,124 m 1 (ltte) re ll signifintly higher on the sites treted with the test ompred to the ontrol. Eh of the peks reltes to speifi moleulr onds, s opposed to speifi ompounds, so it is not possile to determine the hnges in speifi skin omponents. The intensity of the pek t 1,124 m 1, relting to ltte, did orrelte with ltte levels quntified from the skin smples ( fig. 6 e, f). The pek t 1,41 m 1 orresponding to the roxyl group of skin humetnts, inluding PCA, is lso ontriuted to y ermides nd ure. When normlized to the mide II pek, to enrih for roxyli ids (i.e. to remove the ontriution of moleules ontining the N H group suh s ermides nd ure), the intensity of the pek t 1,41 m 1 orreltes well with PCA levels quntified in the skin ( fig. 6 h). In greement with HPLC-derived PCA levels, signifint inrese in roxyli ids ws indited y the hnge in the 1,41 m 1 /mide II rtio following tretment with the test ( fig. 6 g). Inreses in ermide, ure nd/or protein levels following tretment with the test re lso suggested y the inrese in mide I nd 1,175 m 1 pek intensities. Notly tretment with the ontrol tully redued the levels of mide onds within the SC, most likely euse the ermide, ure nd/or protein frtion of the SC hd een displed y the inrese in lipids. To determine whether hnges in roxyli id nd ermide/ure/protein levels ffet skin hydrtion, they were orrelted with pitne mesurements ( fig. 7 ). The 1,41 m 1 /mide II rtio orrelted wekly with pitne nd strongly with FTIR-determined SC wter ontent. The lower orreltion with pitne n e linked to the different penetrtion depths of the tehniques. Following tretment with the test, where hnges in wter nd roxyli id levels our predominntly in the uppermost lyers of the SC from where FTIR mesurements re gthered, the orreltion with pitne improved. A signifint orreltion etween mide II pek intensities nd pitne or FTIR SC wter ontent ws not oserved. The Effet of n Emollient on Skin Brrier Struture nd Funtion Skin Phrmol Physiol 216;29:135 147 DOI: 1.1159/445955 143
Disussion The finding tht queous rem BP dmges the skin rrier highlighted the need to etter understnd the effet of s on the skin [15, 18]. In ontrst to queous rem, the test, fter 28 dys of tretment, improved skin rrier funtion (deresed TEWL) nd SC integrity ompred to n untreted ontrol in volunteers with dry skin ged over 6 yers. The negtive effets of queous rem hve een ttriuted to the use of SLS s n emulsifier [17, 25]. SLS is stndrd skin irritnt, eliiting multiple negtive effets on the skin inluding elevtion of skin surfe ph nd redution in skin rrier funtion (elevted TEWL) [26]. The use of reltively (reltive to SLS) milder non-ioni surftnts with redued irritnt potentil likely ontriutes to the improved effet of the test [27]. These sme enefiil effets were not oserved when the ontrol, lso formulted with milder (reltive to SLS) surftnts, ws used to tret dry skin for 28 dys. Whilst not s dmging s queous rem, the ontrol ws ssoited with elevted TEWL nd redued SC integrity. The response of prtiipnts with history of AD (33%) ws different to those without history of AD; however, the size of the sugroups ws not lrge enough to permit proper sugroup nlysis. Nevertheless, evidene lredy shows tht individuls with history of AD re more prone to skin rrier dmge from topil produts [15, 17]. Neither tretment dversely ffeted lipid hin onformtion determined y FTIR [23] ; however, the ontrol ws ssoited with signifintly elevted skin surfe ph nd protese tivity ompred to the test. A ph-indued elevtion of protese tivity is n estlished mehnism of skin rrier rekdown, hrterized y degrdtion of orneodesmosoml juntions in the SC [28]. The differentil effet of the tretments on SC ph nd protese tivity my therefore explin the different effets of the tretments on skin rrier funtion nd SC integrity. Whilst the surftnt/emulsifition system employed ould ount for the elevtion of skin surfe ph following tretment with the ontrol, it is unlikely to ount for the redution in skin surfe ph rought out y the test. The long-term redution of skin surfe ph using topil tretments hs reently een found to improve oth skin rrier funtion nd hydrtion, in greement with the results presented here [29]. Skin surfe ph is regulted vi severl mehnisms, e.g. y the genertion of idi nturl moisturizing ftors suh s uroni id nd lti id [3, 31]. The skin surfe ph of the sujets reruited in this study displyed signifint diret reltionship with the level of ltte in the skin. Moreover tretment with the test, unlike with the ontrol, elevted ltte levels in the skin for more thn 24 h following the lst pplition. Aged skin ontins signifintly redued levels of some roxyltes, inluding ltte nd PCA, whih my ount for the elevted skin surfe ph nd propensity for xerosis [5, 32, 33]. It is interesting therefore tht the test mintins skin surfe ph y replenishing the roxylte pool. The gretest differene etween the s tested ws their effet on hydrtion. While the ontrol ws poorly hydrting, the test imprted linilly relevnt levels of elevted hydrtion (similr in sle to the differene in hydrtion etween helthy nd AD skin [34, 35] ) for more thn 24 h fter esstion of tretment. The inrese in hydrtion outlsted the trnsient olusive effet of the rem nd n therefore e ttriuted to n inresed ility of the SC to hold onto moisture. Humetnts, inluding ure nd ltte found in the test, re nturlly present in the SC nd ontriute to its wter-holding pity [5]. Cermide, lso present in the test rem, is omponent of the lipid mtrix tht ontriutes to SC permeility rrier funtion [36]. Emollients ontining ermide NP hve een shown to oth hydrte the skin nd improve TEWL [37]. Following tretment with the test ut not the ontrol, signifint inreses in the levels of humetnts nd nturl skin lipids were oserved y FTIR spetrosopy. Using this tehnique it ws not possile to distinguish etween speifi ompounds; however, the SC pool of roxyli ids nd the omined levels of ure, ermides, nd protein inresed signifintly following single pplition of the test nd remined elevted for t lest 24 h fter 28-dy tretment regimen. The roxylte pool in prtiulr ws signifintly orrelted with the inresed wter-holding pity of the skin. Ure is the predominnt humetnt in the test nd so lso likely ounts for signifint proportion of the SC s improved wter-holding pity [38]. The hydrting effet of ure hs een demonstrted in rnge of ses previously (reviewed in Pn et l. [39] nd Loden [4] ). While humetnts like ure nd ltte hve very positive effets on the skin, exessive hydrtion used y exessive humetnt levels n led to enhned proteolyti degrdtion of the SC [41, 42], inditing tht the orret humetnt onentrtion is ruil to rrier repir. We oserved no redution in SC integrity in onjuntion with deresed protese tivity, inditing tht n pproprite ure onentrtion ws used to lne 144 Skin Phrmol Physiol 216;29:135 147 DOI: 1.1159/445955 Dny/Brown/Higgs-Byliss/Chittok/ Alenli/Cork
positive nd negtive effets in this study popultion. The omintion with lti id mintining skin surfe ph my ontriute to the overll positive effet oserved in this regrd [43]. In ddition to imprting skin moisturiztion, ure hs reently een shown to signifintly improve skin rrier funtion in helthy volunteers when pplied one dily for 4 weeks in 2% rem preprtion [44]. This improvement ws ssoited with the elevted expression of genes involved in SC homeostsis, inluding the FLG gene enoding filggrin. A 1% ure preprtion inresed FLG expression to the sme extent. Conentrtions less thn 1% were not tested in this prtiulr study; however, formultions ontining 5% ure hve een shown to strengthen the skin rrier in series of other studies [45, 46]. Moreover the rrier-strengthening properties of the s were linked to the ility of the s to dely relpses of oth AD nd hnd ezem in young dults [13, 47 49]. The results of this study further demonstrte tht distint 5% ure preprtion with ermide NP nd ltte lso imprts positive effets on skin rrier funtion nd SC integrity in n older popultion. In ddition, signifint rise in PCA levels, produt of filggrin tolism, in the SC following tretment with the test (whih does not ontin PCA) ut not the ontrol ws oserved. PCA levels tend to oinide with uroni id nd free mino id levels, euse they re ll derived from filggrin rekdown [24]. TEWL ws lso found to orrelte with PCA levels in this study, in line with the reltionship etween filggrin levels nd TEWL. Given tht PCA is not n ingredient of the s tested, this finding supports the ility of topil ure preprtions to indue filggrin gene expression, when formulted into presription t 5%. Moreover, it demonstrtes tht this inrese in expression leds to inresed filggrin levels nd susequently inresed NMF levels. This new finding further reinfores the potentil rrier-strengthening properties of rems. In ddition the level of ltte, non-filggrin-derived NMF omponent, ws lso speifilly inresed following tretment with the test. Ltte eliits iologil effets on the skin, resulting in enhnement of lipid synthesis, nd s suh my ontriute to the positive effets of the test on skin rrier homeostsis [5]. The test lso ontins ermide NP, omponent of the lipid lmelle. Cermides, the levels of whih derese with dvning ge, re essentil for optimum lipid struture nd permeility rrier funtion [3, 23, 51]. In this study we were unle to speifilly show whether ermide NP ws tken up y the skin due to the overlpping spetrl profiles of ermides, ure nd protein. Lipid esters were elevted following tretment with the test ; however, their penetrtion ws limited. Seum is soure of triglyerides (lipid esters) on the skin surfe, nd its prodution ws found to deline in women with dvning ge [52]. The level of totl lipids, whih inludes non-physiologil lipids (of white soft prffin nd liquid prffin), inreses signifintly following tretment with oth topil s. The omintion of lipid hnges following tretment with the test did not pper to ffet lipid hin onformtion; however, it remins possile tht the physiologil lipids, suh s ermide NP, ontriuted to the derese in TEWL nd improvement in SC integrity found following tretment with the test [37, 5]. With respet to SC hydrtion, the reltive proportion of moisturizing ftors to lipids ws importnt, wherein the size of the roxylte pool ws linked to SC wter ontent. Tken together, the dded humetnts (ltte nd ure) nd the inresed levels of endogenously produed humetnts (PCA) re importnt in imprting the signifint inrese in skin hydrtion following tretment with the test nd for the redution of skin surfe ph nd improvement in the struture nd funtion of the skin rrier. Importntly the levels of these metolites re depleted in ged nd xeroti skin, nd orrelte with the severity of xeroti skin onditions suh s AD [5, 24, 53, 54]. Low NMF levels ssoited with n FLG gene muttion re mjor risk ftor for the development of AD, nd y extension lso stetoti ezem [1, 55, 56]. The test therefore ddresses n importnt pthologil proess in xeroti skin onditions, setting it prt from s without humetnts nd skin lipids like the ontrol tested here. In onlusion, the test (ontining 5% ure, ermide NP nd ltte) signifintly hydrted the skin to greter extent, nd for longer period of time, ompred to n ontining no skin-moisturizing ftors, nd imprted dditionl enefits to the struture nd funtion of the skin rrier. As suh there is onvining rtionle for differentiting s sed on their omposition of SC onstituents [19]. In this se the test ws well tolerted in people with dry skin ged over 6 yers nd ddressed the pthologil fetures of dry skin, supporting role for this lss of s first-line tretment for xeroti skin onditions in the ged popultion. The struturl/iophysil nd iologil (enhned FLG expression) effets of ure nd ltte in the test, nd omintion of nturl skin lip- The Effet of n Emollient on Skin Brrier Struture nd Funtion Skin Phrmol Physiol 216;29:135 147 DOI: 1.1159/445955 145
ids, men tht generlizility with other humetnt s is tenuous [57]. As suh n evidene-sed pproh is lwys reommended for seleting s, s not ll rem formultions re the sme. Aknowledgements We re grteful to ll our volunteers who hve given up their time to tke prt in our studies. Thnks lso go to Les Hunter for the reruitment of volunteers who prtiipted in this study nd Jon Kily nd Ro Hnson for their tehnil ssistne. This study ws supported y reserh funding from Almirll SA, Brelon. Sttement of Ethis Written informed onsent ws otined from ll prtiipnts nd reord kept in the study site file. The NHS Trent Reserh Ethis Committee pproved the study, inluding the onsent proedure employed (No. 4/MREC/7). Dislosure Sttement M.J.C. nd S.G.D. hve reeived fees for giving letures for Almirll SA. nd ttending dvisory ords. M.J.C. nd S.G.D. hve reeived funding for investigtor-led reserh from Almirll, Astells, Byer, MSD, nd Stiefel-GSK, ll of whom re mnufturers of s. 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