JOURNAL OF COSMETIC SCIENCE 424 the reaggregation of dissociated corneocytes. These same authors reported on the ability of n-acetylglucosamine (NAG), n-acetylneuraminic acid, and n-acetylgalactosamine to affect the dissociation aggregate of extracted skin corneocytes. These amino sugars bind to the lectin-like (sugar-binding) glycoproteins that bind corneocytes together. This lectin- binding protein has more recently been shown to be CD44, the receptor for hyaluronic acid (6). Hyaluronic acid has been shown to be present in the epidermis (7) and even in the stratum corneum (8). We hypothesized that amino sugars, by disrupting corneocyte bonds, like alpha-hydroxy acids may work topically to promote desquamation. This desquamation will be similar to that caused by alpha-hydroxy acids, with the benefi ts associated with them but more gentle to the skin. METHODS CELL CULTURE AND VIABILITY HaCaT cells were grown to confl uence in six-well plates (Costar, Corning Corp., Corning, NY). These cells are a spontaneously immortalized human keratinocyte line (9), kindly supplied to us by Dr. Norbert E. Fusenig of the German Cancer Research Center, Heidel- berg. Cells were grown in Dulbecco’s Modifi ed Eagle’s Medium, DMEM, (Gibco BRL, Grand Island, NY). Cells were treated with 50 mM, 125 mM, and 250 mM NAG (Sigma) for 24 hours in whole media. MICROSCOPIC VISUALIZATION Cell cultures were viewed at ×100 magnifi cation by phase contrast and refl ectance mi- croscopy with an Olympus BX60 microscope (Olympus, Melville, New York). GEL ELECTROPHORESIS AND WESTERN BLOT Tissue culture plates were scraped 24 hours after treatment with NAG into media and centrifuged at 3,000 rpm for ten minutes. Harvested cells were then resuspended in lysing buffer (150 mM NaCl, 50 mM Tris pH 8.0, 1% NP40) and sonicated for three one-minute intervals with a cone attachment on a model W-225 sonicator (Heat Systems- Ultrasonics, Inc., Farmingdale, NY) set at 100 watts/minute. Samples were then directly applied to sodium lauryl sulfate polyacrylamide gels (Amersham Pharmacia Biotech, Inc., Piscataway, NJ) and electrophoresed in a Phastsystem gel electrophoresis unit (Am- ersham Pharmacia Biotech, Inc., Piscataway, NJ). Immediately after electrophoresis, the gels were overlaid with Immobilon-P transfer membranes (Millipore Corp., MA), wet with distilled water and two layers of wet Whatman fi lter paper. This sandwich was then heated to 50 degrees for 30 minutes to allow transfer to take place. The membranes were then immediately blocked by placing them in 3% milk protein (BioRad Laboratories, CA) in 50 mM Tris HCl pH 8.0, 0.138 M NaCl, 2.7 mM KCl (TBS) for 18 hours. Following this blocking step, the membranes were washed three times with TTBS (Triton-X100

NAG AS EXFOLIATION ENHANCER 425 containing TBS). These membranes were then incubated for 120 minutes with an anti- body for involucrin (Biomedical Techniques Inc., MA) and keratin K1 and K10 (Chemi- con, Temecula, CA). Following this incubation the membranes were again washed three times with TTBS. The membranes were then incubated with a secondary antibody (either goat or rabbit anti-mouse antibody) conjugated to alkaline phosphatase. The enzyme activity was then visualized by reaction with 5-bromo-4-chloro-3-indolyl phosphate and nitro-blue tetrazolium tablets (Sigma, St. Louis, MO). The blots were scanned with a Hewlett-Packard ScanJet IIc and analyzed with the densitometric software package SigmaGel 1.0. CLINICAL STUDY DESIGN The subjects included in this study were 45 females between the ages of 21 and 65 years, all meeting the screening criteria of good health and not pregnant or lactating. The sub- jects reported for testing without moisturizers or any other products on their faces and hands, and their baseline measurements were taken. They were given the product to take home and self-administer for four weeks to their right hand only, twice a day, in the morning after washing and in the evening at least 15 minutes before bedtime. The left hand served as the untreated control site. The subjects were only allowed to use the test product and specifi cally log its use in a daily diary we provided. At the end of two and four weeks the subjects returned for testing without applying the product for at least 12 hours and they were re-evaluated under the same conditions. SKIN EXFOLIATION VIA THE D-SQUAME DISCS METHOD AND IMAGE ANALYSIS Skin exfoliation was evaluated by measuring the amount of fl akes removed from the skin surface using D-Squame discs and analyzing them by image analysis. Four D-Squame discs were fi rmly and evenly pressed on the face and the back of each hand with a hand-held uniform pressure device and removed by gently pulling them away from the skin. The D-Squame discs were mounted on clear microscope slides and labeled according to the panelist’s name and date of visit. Desquamation was evaluated from the D-Squame discs using the image analyzer. Skin evaluation was carried out before treatment and after two and four weeks of treatment. The OPTIMA image analyzer (Optimas 6.5 from Media Cybernetics, Bethesda, MD) was used to evaluate skin fl akiness. The D-Squame samples containing the corneocytes were placed under a camera on top of a light table and each image was imported into the image analyzer. The average gray value corresponding to the sample density was measured: the denser the sample, the higher the gray value difference. ASSESSMENT OF SKIN MOISTURIZATION WITH A DERMAL PHASE METER Skin surface capacitance measurements were made with a NOVA dermal phase meter (DPM 9003 Nova Technology, Portsmouth, NH). The DPM is an electronic instrument that non-invasively measures skin capacitance in vivo. The capacitance readings are directly related to picoFarads of capacitance in the volume of skin that is effectively measured,