98 JOURNAL OF COSMETIC SCIENCE A LIE-DETECTOR INVESTIGATION TO MEASURE NEUROMODULATING ACTIVITY OF A TOPICALLY APPLIED PEPTIDE: DOUBLE BLIND CLINICAL STUOY OF N-ACETYL-TYR-ARG-CETYLESTIR Vlmsus PLACEBO Karl Lintner ', Ph.D., Claire Mas-Chamberlain •, Philippe Mondon •, Ph.D., Olivier Peschard •, Ph.D, Patrick Beau 2, Ph.D., Charlotte Musnier 2 and Arnaud AuberP, Ph.D. ZSederma, Le Perray en Yvelines, France 2Spincontrol, Tours, France SUniversity of Tours, France Key words :sensitive skir• neuropeptides. endorphin• Introduction Sensitive skin, real or perceived, is a subject of intensive research in cosmetic science. It is a fact that physical, chemical and psychological stress can be felt at the skin level by an increasing number of consumers. According to Lacharri•re [!], a clear correlation between the subjective feeling "I have sensitive skin" and an objectively measurable skin reaction to various forms of stress (climate, chemicals) can be established in a large portion of the population. Three levels of severity must then be distinguished: "sensitized skin" is a truly allergic reaction that implies the immune response. Irritation and redness are caused by the release of cytokinins and subsequent vasodilatation itching, burning, stinging and more or less painful sensation involves neurological molecules and mechanisms that particularly upset the state of well- being of the individual. In cosmetic terms, the three levels cited are approached in different ways: everything possible is done to avoid truly allergic, sensitizing substances in the formulations. The risks of irritation and local inflammation may be reduced by using only ingredients known to be mild themselves and by the addition of anti-irritant substances (allantoin, bisabolol, bacosides, darutoside and others). To reduce the unpleasant feeling of itch and sting, however, requires ingredients that act on the signal transmitting cells in the skin, i.e. the nerve endings (Merkel Cells, Meissner Bodies, C-fibers etc. [2]). The dipeptide Tyr-Arg has been shown to have analgesic potency in mouse brain via the release of enkephaline, an endogenous opiate peptide [3]. The closeness of developmental origins of brain and skin led us to investigate the potential use of this peptide in cosmetic application. This paper describes the neuromodulating activity of the derivatised peptide (N-Ac-Tyr-Arg-hexadecylester=Ac-YR-OHex) topically applied to the skin in view of reducing the cutaneous neural response to disagreeable stimuli. Materials and Methods Ac-YR-OHex is obtained by classical liquid phase peptide synthesis and purified to 98%. The peptide was formulated into a O/W emulsion at a level of 300 ppm. In vivo tests are carried out on a panel of 20 volunteers with informed consent. Each person served as her own control as vehicle and active emulsions were tested by application to the forearm until complete penetration. To measure an objective signal related to "unpleasantness", the electrodermal (or galvanic skin-) response was determined with the help of a physiograph MK III from Narco Bio System ("lie-detector"). The Ohmic Duration Response (ODR) is the parameter of the recorded signal that best reflects the emotional charge induced by the stimulus [4]. The stimulus consisted of sandpaper of different graininess being passed over the inside of the open hand. On the contra-lateral hand, the EDR probe is applied to the fingertips. The first measurement is done before application of the products (TO), the procedure is then repeated 1, 2 and 3 hours after product application. Simultaneously, the panelists express a subjective score (on a scale from 0 to 10) of 'unpleasantness' during the sand paper movement. Placebo and active product were randomly applied to either right or left hand. Statistical analysis by unilateral Student's test for paired series at the 5% level. Results: Electrodermal response in vivo Preliminary studies with the lie-detector equipment, carried out as a training phase for the panelists, allowed us to demonstrate that the correlation between subjective discrimination of different graininess and the electrodermal response was excellent. Thus the ODR signal could be used as a measure of the strength of emotional response to the chosen stimuli. Would the sensitivity of the method allow for sufficient discrimination between placebo and active product? Figure I shows that over the entire span of the experiment, a decrease of up to 20% in the response time (ODR) of the skin can be observed, both for the placebo and the active cream. Up to three hours afler product application, the signal attenuation is greater on

2002 ANNUAL SCIENTIFIC MEETING 99 the treated side than on the placebo side. This difference is significant at T=lhour (p0.05) and not significant, but still measurable, thereafter. • •0. o 70 T:,0 T•I h T,:2h T"3h Fig.l: decrease of ODR signal vfith time. *= p•O.05 The remarkable effect to be noted is, however, the perfect coincidence of the objective and the subjective result at T = 1 hour (fig. 2 and 3). Conclusion: The data presented confirm previous studies on the skin soothing and calming effects of the Tyr-Arg peptide on the skin [5]. These studies, also carried out in vivo on panels of 16 resp. 20 panellists, had shown that 300 ppm of N-Ac-Tyr-Arg-Ohex significantly reduced the sensitivity of skin to heat and to chemical irritation by capsaicin. These effects observed in vivo, where the purely perceptional, neural link from the unpleasant stimulus to the recorded electric signal is clearly In vivo: sensorial (subjective) perception The analysis of the scores indicated by the panelists after each stimulus on either hand leads to the results summarized in Figure 2 . Clearly, the volunteers perceive a decrease in the unpleasantness of the sand paper effect this is particularly evident one hour after application, and again persists until the end of the experiment. There is a significant (p0.05) difference at the T=lh time point, the trend continues until T=2h however, after 3 hours, the two sides seem to experience both "habituation" to the stimulus and no difference is observed. 80 T• T•h T•h T•h EI Ac-YR-Ohex ß Place bo [ Fig. 2: change in tactile perception of unpleasantness over time * =p0.05 established and the sensitivity of the skin surface is reduced in significant amount by the Ac-YR-Ohex molecule are probably explained by the release of endogenous opiate peptides endorphin and enkephaline in the epidermis, the presence of which has been documented in keratinocytes and Merkel cells. Ongoing in vitro studies are slated to confirm this hypothesis. Everyday unpleasant sensations such as chemical stinging, climatic aggression from heat, wind or cold or mechanical stress on the skin from the environment (clothes, surfaces) can be reduced with topical application of this neuromodulating peptide. These effects do not qualify as anesthetic nor strongly analgesic but are designed to participate in the generation of sensorial pleasure and well-being so actively sought today. Rarely do objective in vivo data correlate so successfully with subjective perception of so subtle effects on the skin. References [ 1 ] O. de Lacharri•re in: Proc. XIXth Journees Europ. Dermocosmet., Lyon, 1997 [2] N. Johnson, J. Clin. Neurophysiology 17, 539 (2000) [3] H. Takagi, H. Shiomi, H. Ueda and H. Amano, Eur. J. Pharmacol. 55, 109-111 (1979) [4] N. Vernet-Maury, J. Autonomic Nervous System 75 176-179 (1999) [5] K. Lintnet & O. Peschard Int. J. Cosmet. $ci. 22 207-218 (2000)