Study 1: Transepidermal Water Loss
Enrolled subjects were healthy white males and females, aged 18–55 years, with Fitzpatrick skin type I, II or III. Subjects were excluded if they had any of the following: type I or II diabetes; current tobacco use; any skin condition on the volar forearms other than dry skin (e.g., psoriasis, eczema, atopic dermatitis); marks, scars, moles, scratches or other blemishes on the volar forearm test sites that would interfere with the study; or known allergies or sensitivities to any topical corticosteroid product or cosmetics, soaps or fragrances.
Participants visited the test site on 2 consecutive days, each day wearing a short-sleeved shirt and having not showered within the hour prior to their visits. Participants were required to discontinue use of all moisturizing products (e.g., soaps, lotions, sunscreens, insect repellents, etc.) on the volar forearms for the 3 days prior to the start of the study and for the duration of the study.
On day 1, volunteers were dry shaved with a disposable razor to create four 5 by 5-cm test sites on both volar forearms (two sites per forearm). Dry shaving disrupts the integrity of the stratum corneum and creates a damaged skin barrier. Evaporimeter measurements (RG1 Evaporimeter system, cyberDERM, Broomall, PA) with transepidermal water loss (TEWL) probes (Cortex Technology, Hadsund, Denmark) were conducted after shaving to confirm elevated water loss and provide a measure of skin barrier function. Both probes of the Evaporimeter contain sensors that measure the temperature and relative humidity at two fixed points along the axis perpendicular to the skin surface. Water loss data were collected at a rate of four inputs/s over a 20-s interval once steady-state conditions had been achieved to provide an average of evaporative water loss rate in gm/m2/h.
On day 2, the four test sites were evaluated with the evaporimeter for post-shaving TEWL. Measurements were taken following a minimum 25-min acclimatization period in a controlled environment with the relative humidity maintained at < 50% and temperature maintained between 19 and 22 °C. Capacitance, which measures epidermal hydration, was recorded at the same time as TEWL with a Corneometer® CM 825 (Courage + Khazaka Electronic GmbH, Cologne, Germany). Following these baseline readings, DFD-01 or vehicle (0.1 ml) were each randomly and blindly rubbed into a different test site using a clean finger cot, leaving one site per arm as a damaged, nontreated control. TEWL and capacitance measurements were repeated at 1, 2 and 4 h after treatment. Vehicle contained water, sorbitan monostearate, polyoxyl 20 cetostearyl ether, cetostearyl alcohol, mineral oil, propylparaben, methylparaben, butylated hydroxyl toluene, and hydroxyethyl cellulose and oleyl alcohol. DFD-01 was the same formulation with the addition of betamethasone dipropionate, 0.05%.
Participants were required to wear nonocclusive, noncontact, protective arm guards in between product application and each of the follow-up measurements to minimize product transfer and site contact with foreign material. Participants were instructed to refrain from exercising or drinking hot beverages or cold/iced coffee or tea during the 2 h prior to each measurement on day 2.
Study 2: Skin Flexibility
Study 2 enrolled healthy white males and females, aged 18–55 years. Unlike Study 1, there were no Fitzpatrick skin type restrictions. Pre-study requirements regarding skin product use and study inclusion and exclusion criteria were similar to those in Study 1. The use of arm guards between measurements and the restrictions on exercise and consumption of hot and cold drinks between readings were also similar to Study 1.
Each volunteer had six 5 by 5 cm sites (three sites per volar forearm) outlined (Fig. 1), and baseline cutometer measurements were taken from each site. Skin flexibility was tested with a Cutometer® MPA 580 (Courage + Khazaka Electronic GmbH). In this study, the instrument specifications used were a 2-mm-diameter aperture probe, mode 1 (constant negative pressure during on period), vacuum 450 mbar, 2 s on, 2 s off, three cycles. The instrument technician took one measurement (three cycles) from each site.
DFD-01 and vehicle (0.1 ml) were each randomly and blindly rubbed into one test site on each arm using a clean finger cot; the central site on each arm remained untreated as a control. Flexibility measurements were repeated at 1, 2 and 4 h after treatment, with the maximum extension of the third cycle (R3) used for analysis. Change in control sites subtracted from change in treated sites determined treatment effects.
Compliance with Ethics Guidelines
Ethical consent for Study 1 and Study 2 was received from IntegReview IRB prior to enrollment. For both studies, volunteers provided written informed consent and Health Insurance Portability and Accountability Act (HIPAA) authorization prior to participation. Studies were conducted in compliance with the Declaration of Helsinki, current International Council for Harmonisation guidelines for Good Clinical Practice (GCP), Food and Drug Administration (FDA) regulations and other applicable laws and regulations.
For both Study 1 and Study 2, repeated-measures ANOVA was used to compare the two test products and the nontreated controls. If differences were significant, multiple post hoc comparisons were made using a Tukey-Kramer multiple comparison test. For all analyses, the level of significance was P < 0.05.