Exploratory study to evaluate two clinical methods for assessing moisturizing effect on skin barrier repair

Abstract Background Two clinical methods of assessing a moisturizer's effect on stratum corneum (SC) barrier repair were evaluated in female subjects with dry skin, to identify an assessment method for future studies. Methods In this single‐centre, split‐body study, women with dry skin applied moisturizer before (method A) or after (method B) SC barrier perturbation using D‐Squame® stripping discs. Transepidermal water loss (TEWL) and residual protein on D‐Squame discs were assessed over 14 days. Results Twenty‐four subjects were included. For method A, the mean slope values of plots of 1/TEWL vs cumulative protein removed decreased over time for both treated and untreated areas, indicating improved SC barrier quality. There were no significant differences between treated and untreated areas, although a trend to a more negative slope was observed by Day 14 in the treated areas (P = 0.082), suggesting treatment improved barrier quality. For method B, using pre‐ and post‐stripping as covariates, no statistical differences/trends were observed between treated and untreated areas for change in TEWL from post‐stripping to any evaluation from Days 3‐14. TEWL values returned towards pre‐stripping values for treated and untreated areas by the initial Day 3 evaluation. Conclusion For method A, there were trends suggesting the moisturizing treatment improved SC barrier quality. For method B, there were no significant differences/trends between treated and untreated areas. Further assessment with different methodologies is warranted to design appropriate clinical protocols for evaluating accelerated skin barrier repair. These data are insufficient to conclude whether the product or methodology was responsible for the results.

Recently, Lu and colleagues developed a novel clinical and data analysis procedure based on sequential tape stripping with TEWL measurement and SC protein analysis, in the course of a study investigating the SC barrier and the hygroscopic properties of normal and cosmetic dry skin. This pro cedure was thought to be a more robust method than those previously used for SC barrier analysis. 3 BuildingonthefindingsofLuetal, 3 the present study explored two different clinical methods of assessing the effect of a moisturizing product on SC barrier repair in female subjects with dry skin, with the objective of identifying an assessment method for accelerated barrier repair for use in future studies.

| Study design
This was a single-cen tre, non -rand omized, split-body study to explore two clinical meth ods of a ssessi ng SC barrier repair, using a marketed cosmetic moisturizer (Curel, a registered trademark of Kao Corporation, Tokyo, Japan) containing glycerine, isopropyl palmitate, petrolatum, and Butyro spermum parkii (shea) butter in female subjects with dry skin. The primary objective was to identify an accelerated method of assessing SC barrier repair. The study was conducted between8Apriland22May2015atasinglecentreinIrving,TX, USA,inaccordancewithapplicablelocalethicalandregulatoryrequirements.Allsubjectsprovidedwritteninformedconsent.

| Subjects
Eligiblesubjectswerehealthyfemal eCaucasians,aged≥18years, withaminimumdrynessgradingof2(moderateflaking/scaling;0-4 gradingscale)atbaseline(Day0)onbothlowerlegsand≤0.5-point difference between the right and left leg and the upper and lower areas on each outer leg , as det ermine d by the visual grading of a trained examiner.

| Methods and assessments
Eligible subjects underwent a 7-day washout period, using only the provided soap to cleanse the lower legs (Ivory® Original; Procter & Gamble, Cincinnati, OH, USA). This soap was used from screening until study completion.
The intervention included two methods that were defined as method A and method B. A subject's right lower leg was used for methodA,andasubject'sleftlowerlegwasusedformethodB.For both methods, subject visits/assessments took place at baseline and onDays3,5,7,10,12and14.
At the baseline visit (Day 0), the outer aspects of a subject's lower legs were each marked into an upper test area to be treated with moisturizer and a lower test area to be left untreated.
FormethodB,twosmallareasweremarked,oneonthetreated upper area (#15) and one on the untreated lower area (#16) where thestrippingandTEWLmeasurementsweretaken.Atbaselinevisit, TEWL measurements were taken at the designated skin areas before and after skin barrier perturbation, with D-Squame® stripping discs applied and removed 20 times. No skin stripping was performed on the left leg during subsequent visits, only TEWL was measured.
Following baseline assessments, subjects were instructed to apply 240mg moisturizer (approximately 2mg/cm 2 equivalent) to the marked upper area of both legs twice daily (morning and evening)for14days.
For both methods, moisturizer was applied 30 minutes after the last TEWL measurement on the upper test area only. The last mois-

| Statistical analyses
AsdescribedinthemethodofLuetal, 3 the relative barrier quality among the subjects (or subject groups) can be obtained by comparing the slopes of the regression of the 1/TEWL vs cumulative protein (Cp) removed for each subject (or subject group). The relative SC thickness can be determined from the values of Cp removal that can be quantified according to where the regression lines intercept the x-axis. It was planned to recruit 25 subjects so that at least 20 evaluable subjects would complete the study. With 20 subjects, the study had 90% power to show a difference between treated and untreated groups of 32% in SC barrier quality and 22% in SC thickness.

| Method A
The mean slope values of plots of 1/TEWL vs Cp decreased over time for both treated and untreated areas, indicating an improvement in SC barrier quality (Figure 1 and Figure 2). Slope values were   not significantly different between treated and untreated areas at any time; however, a trend was observed for values of treated areas to be morenegativethanuntreatedareasbyDay14(P = 0.082) ( Table 1a).
The total amount of protein removed by tape stripping on untreated areas was higher than for the treated areas (with statistically significant differencesseenatDays7,12and14;Table1b).Thiscouldpossiblybe These findings suggest that the moisturizer treatment did not produce physiological improvement in the SC barrier under the conditions of this study.

| Safety
One AE of dermatitis following contact with poison ivy was reported; this was mild and not considered to be related to the study product.NoseriousAEswerereported.

| CON CLUS IONS
Although the study by Lu and colleagues demonstrated a method that was thought to be more robust than those previously used for SC barrier analysis, 3 the present investigation did not manage to secure a significant exploratory method for the purposes of accelerated repair.
Findings from method A suggest that the moisturizing treat- The results of this study precluded validation of the accelerated barrier repair methodology. In addition, it was not possible to determine whether the product or the methodology was responsible for the results. Further exploratory work would be required to develop and validate a clinical method to assess accelerated skin barrier repair.

ACK N OWLED G EM ENTS
The authors thank all of the subjects for their participation and John Healthcare.

CO N FLI C T O F I NTE R E S T
Jane Snatchfold provides independent consulting services to GlaxoSmithKline Consumer Healthcare. Darren Targett provided independent consulting services to GlaxoSmithKline Consumer Healthcare at the time the analysis was conducted.