J. Cosmet. Sci., 60, 423–428 (July/August 2009) 423 The effect of N-acetyl-glucosamine on stratum corneum desquamation and water content in human skin T. MAMMONE, D. GAN, C. FTHENAKIS, and K. MARENUS, Estee Lauder Research Laboratory, Melville, NY 11747. Accepted for publication February 11, 2009. Synopsis Alpha-hydroxy acids have been used topically to treat skin for both dermatological and cosmetic problems for many years. Though there are many known benefi ts of the use of alpha-hydroxy acids on skin, there have been recent reports that topical treatments with alpha-hydroxy acids increase skin damage resulting from UVB. Additionally, high concentrations of alpha-hydroxy acids by themselves have also been found to cause skin irritation. In order to fi nd alternatives to alpha-hydroxy acids, we investigated a variety of amino sugar compounds that were previously reported to inhibit the reaggregation of dissociated corneocytes by modulating cellular adhe- sion. In vivo, we observed that topical treatments with a formulation containing N-acetyl-glucosamine (NAG) led to an increase in skin moisturization, a decrease in skin fl akiness, and the normalization of stratum cor- neum exfoliation. In vitro, we observed an upregulation of differentiation markers, keratin 10 and involucrin, in keratinocytes treated with NAG. CD44 is a lectin cell adhesion molecule that is also expressed in kerati- nocytes. Amino sugars such as NAG may competitively bind to CD44, modulating keratinocyte cellular adhesion. We hypothesize that these amino sugars modulate keratinocyte cellular adhesion and differentia- tion, leading to the normalization of stratum corneum exfoliation. We propose the use of amino sugars such as NAG as alternative compounds to replace the use of alpha-hydroxy acids in skin care. INTRODUCTION Alpha-hydroxy acids have been used for a number of years to treat skin for both dermato- logical and cosmetic problems (1). Topical treatments with alpha-hydroxy acids have clearly demonstrated an anti-aging effect (2), photoprotection (3), and anti-infl ammatory activity (3). However, the mechanism by which alpha-hydroxy acids operate is largely unknown. One hypothesis for their mechanism of action is that they lower corneocyte cohesion (4) and thereby enhance the stratum corneum exfoliation process. This mechanism of action suggests, however, a purely denaturing effect on the proteins of the epidermis and repre- sents a low specifi c functionality. This is demonstrated by the fact that high concentra- tions, 5–10%, of alpha hydroxy acids have a direct irritating effect. In order to develop alternatives to the use of alpha-hydroxy acids with higher specifi c activity and fewer side effects, we have been investigating a variety of materials. One such class of compounds is amino sugars. These have been reported by Brysk et al. (5) to inhibit

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