This single site split body investigator blinded study enrolled 53 healthy women of Fitzpatrick skin types I-VI who signed informed consent (Concordia Institutional Review Board, Beach Haven, NJ). Twenty-six subjects aged 30-50 years were enrolled and twenty-seven subjects aged 51-65 years were enrolled with dry to very dry skin, skin texture roughness (visual/tactile), desquamation/ flakiness, lack of radiance/dull skin, and erythema on the lower legs. Following the completion of informed consent and photography consent, the subjects completed an itch assessment on an ordinal scale. Twenty-nine subjects were enrolled with mild to moderate self-perceived itchy skin on both legs with a score of 2 to 3 on a 5-point ordinal scale (0=none, 1=minimal, 2=mild, 3=moderate, 4=severe). Subjects were dispensed a diary and asked to follow their regular hair removal routine to include consistency in type of products (waxing, shaving, depilatory, etc.) used and the frequency of hair removal. Subjects recorded their hair removal activities in the diary. Subjects were dispensed a glycerin cleansing bar (Neutrogena®, Johnson & Johnson™, Skillman, NJ) for cleansing of the legs and body to replace their usual body soap and asked to return to the research center in 2 weeks. No other cleansers were allowed for the duration of the study.
Subjects were re-qualified after 2 weeks and asked to shower in the morning, apply no moisturizers, and present to the clinic between 3pm to 5pm. Subjects underwent dermatologist investigator clinical grading for skin dryness, skin texture/roughness (tactile), skin texture/roughness (visual), desquamation/flakiness, luminosity/radiance, erythema, and overall appearance of healthy skin for each leg separately. The investigator also assessed skin itching, stinging, and burning separately for each lower leg, defined as the subjective sensory assessment. All investigator assessments were on a 5-point ordinal scale (0=none, 1=minimal, 2=mild, 3=moderate, 4=severe). A noninvasive instrumental assessment of corneometry was obtained for each lower leg using a template to locate the same site for sampling at each visit. A lower leg target site was identified on each leg for standard visible light photography with a Slue Nikon D90 camera. Finally, surface biomarkers were collected using the DSquame ® technique separately for each lower leg.
The D-Squame technique involved placing a transparent sticky tape with clean forceps holding the side tab onto the templated location and pressing with a constant pressure plunger onto the skin for 10 seconds. The side tab was again grasped with the clean forceps and placed into a specimen bottle for transport to the laboratory (Synelvia, Labège, France). The D-Squames were only handled by the side tab to avoid contamination. The first tape strip was discarded and the 2 subsequent tapes collected from the same location were submitted for analysis. Subjects were dispensed the study product (Moisturizing Cream, CeraVe, New York, NY) and asked to apply it to one randomized lower leg, between the knee and ankle. Immediately after application dermatologist expert grading, subject selfassessment questionnaire, and subjective sensory assessment were performed along with target site photography separately for each lower leg.
Subjects were asked to apply the study product twice daily to the randomized leg from the knee down and to shower with the provided cleanser the morning of their return visit on day 3 between 3pm to 5pm. No study product or other products were applied to the legs following showering. At day 3, subjects underwent clinical expert grading, subject self-assessment questionnaire, subjective sensory assessment, and corneometry instrumental assessment separately for each lower leg. The lower leg target site on each leg was photographed and subjects returned to the research center for the same activities at week 4. Again, subjects were asked to shower with the provided cleanser the morning of their visit between 3pm to 5pm and apply no study product or other products to the legs. The target site selected on each leg underwent D-Squame collection of skin surface biomarkers with the first tape discarded and subsequent 2 tapes submitted for analysis.
Subjects discontinued study product use at this time and returned to the clinic at 48 hours for a regression analysis to determine the level of ceramides present in the skin after product use ceased. Dermatologist expert grading, D-Squame collection, subject self-assessment questionnaires, and subjective sensory assessment were performed along with target site photography separately for each lower leg. An instrumental assessment of corneometry was obtained separately for each lower leg.
A Mann-Whitney two-tailed t-test was used to analyze the nonparametric investigator and subject data. A t-test was used to analyze the numerical noninvasive data.
Lipid D-Squame Analytical Methods
Lipids were extracted from each D-Squame by a multi-step method using chloroform and methanol solvents to collect the neutral lipid, fatty acid and ceramide fractions. Cholesterol and free fatty acid levels in these extraction samples were analyzed using gas chromatography system coupled with mass spectrometry. Ceramide content was analyzed using a liquid chromatography-mass spectrometry method (LC-MS).