E. A. Jewell-Motz, Ph.D., J. R. Kaczvinsky, Ph.D., K. M . Lammers, M.S., S. Xie, Ph.D., R. M. Osborne, Ph.D P&G Be

1. Stratum basale Fold Change in AQP Protein Stratum corneum * ● Identify compounds that stimulate aquaporin-3 expression and activity in skin cells. ● Determine whether identified materials promote a skin moisturizing benefit in humans when formulated into product. * In vivo Dry Skin Grade Improvements (vs. moisturizer alone) 130 Objective 120 % Increase in Glycerol Transport 110 100 90 80 Control 100uM Modulation of Aquaporins to Deliver Consumer Benefits: Applications for Skin Care E. A. Jewell-Motz, Ph.D., J. R. Kaczvinsky, Ph.D., K. M . Lammers, M.S., S. Xie, Ph.D., R. M. Osborne, Ph.D P&G Beauty , Cincinnati, Ohio USA Caffeine Stimulates AQP3 Protein Expression in vitro Background Conclusions • AQP3 is expressed in human skin • An age-associated decrease in AQP3 correlates with an age-associated increase in dry skin. • Caffeine increases both the protein expression and glycerol transport activity of AQP3 as determined in vitro. • Moisturizer formulated with caffeine delivers a moisturization benefit to dry skin in clinical testing. • Modulation of aquaporins may be beneficial for a variety of consumer products applications including, but not limited to, anti-aging, dry mouth relief, urinary incontinence, or relief for diarrhea. AQP3 levels decrease with age Aquaporins (AQP) are an important class of proteins that regulate the transport of water and other small solutes across plasma membranes. First discovered in the early 1990’s (1), there are now 13 distinct AQP family members that have been identified in a variety of mammalian tissues, many of which are relevant to the consumer products industry. In particular, two aquaporins (AQP3 and AQP9) are expressed in human skin cells (2, 3). AQP3 has been implicated as playing a key role in the transport and distribution of epidermal water and glycerol (3,4) and the regulation of keratinocyte differentiation (5), both critical processes for maintaining skin hydration, barrier function and overall skin health. Product Formulated with CaffeineDelivers Skin Moisturization Benefit in Human Clinical Studies • ELISA for In Vitro AQP Expression • Cultured human neonatal keratinocytes were cultured in 96 well plates and treated at 37°C for 48-96 hrs • Fixed cells were incubated with anti-AQP3 antibody. • A peroxidase coupled secondary antibody was added, and AQP3 protein content was determined after treatment with Horseradish Peroxidase Chromogen TMB. • In vivo Expression of AQP 3 • 20 Caucasian female subjects, 18-55 years old • RNA isolated from hip biopsies & 1 leg biopsy • AQP RNA expression quantitated via GeneChip™ assay Caffeine Stimulates AQP3 Activity in vitro • References • Preston G. M., Carroll T.P., Guggino W. P. and Agre P., Science 256, 385-387 (1992). • Sougrat R., Morand M., Gondran C., Barre P., Gobin R., Bonte F., Dumas M., and Verbavatz J., J. Invest. Derm., 118(4), 678-685 (2002). • Sugiyama Y., Ota Y., Hara M., and Inoue S., Biochim. Biophys. Acta 1522, 82-88 (2001). • Hara-Chikuma M. and Verkman A. S., Biol Cell, 97(7), 479-486 (2005). • Zheng X. and Bollinger Bollag W., J. Invest. Derm., 121(6), 1487-1495 (2003). Clinical Design ●40 female subjects ●4 test sites per leg (8 total per subject), 5x4 cm2 area, 2 ul/cm2, total 40 ul treatment twice/day except weekends- 1x per day ●Assessments performed at baseline, 1, 2, and 3 weeks then 2, 7, and 11 day regression assessments ●Technical measures via expert visual grading for dryness and redness, ●Panelists were asked not to consume caffeine or herbal drinks Glycerol Transport Assay ● MatTek’s Epiderm cultures were treated with test compound for 48 hours before adding glycerol to the medium in the lower chamber. ● Glycerol levels within the upper chamber medium were determined 24 hours later (Free Glycerol Reagent, Sigma, St. Louis, MO). ● Measurement in the glycerol assay was converted into a percentage increase over the control (where the control is equal to 100%). Each mean and SEM were derived from at least three separate experiments, performed in duplicate. Aquaporin channel (Science 17 October 2003:Vol. 302. no. 5644, pp. 383 – 384) AQP3 is localized to human skin