226 JOURNAL OF COSMETIC SCIENCE PUFFY EYES: A MULTI-FACTORAL COSMETIC PROBLEM NEEDS A MULTI-FACETED SOLUTION Karl Lintner, Ph.D., Claire Mas-Chamberlin, Philippe Mondon, Ph.D., Olivier Peschard, Ph.D. and Francois Lamy, Ph.D. Sederma, Le Perray en Yvelines, France Key words: lymphatic drainage, bradykinin, capillary fragility Introduction "Puffy eyes" are a well-known aesthetic problem for the ageing population. It is not possible to define a single cause for the slight swelling, the slackening of the skin, the occasional redness and sensitivity of the area immediately beneath the eyes where the skin is particularly thin (0.5mm on average). Several factors contribute to the emergence of bags under the eyes and their accentuation over time: a) CapillaIJ' fragility: The eyelid is crossed by a fine, dense network of arterial and venous capillaries. With age and outside influences, the vessel walls become fragile and plasma fluid leaks from the vascular bed b) Lymphatic drainage: The accumulation of interstitial fluids, when not eliminated by the lymphatic system, results in local overload and distension of the skin: defective drainage greatly contributes to the fonnation of bags under the eyes. c) Aging: The constant use of the eyelids and the fineness of their epithelium fairly rapidly result in tissue sagging with weakening of the supporting structure around the microvessels and in increases of dilated areas that retain edema. An inflammatory component generally accompanies infiltration of the tissues. The combination of those three factors is above all responsible for the phenomenon 'bags and rings under the eyes'. This paper describes an approach to treat these symptoms with a combination of several active substances intended to manage each of the major etiologies described above. Materials and Methods Hesperidine methyl chalcone is obtained by extraction and purification of hesperidine from citrus sinensis fruit, followed by selective methylation. The dipeptide Val-Trp (VW) and the tetrapeptide Pal-Gly-Gln-Pro­ Arg (Pal-GQPR) were obtained by liquid phase peptide synthesis and purified to 90%. Normal human keratinocytes were neo-cultured in standard medium until subconfluence, then exposed to increasing concentrations of Pal-GQPR IL6 secretion into the culture medium was quantified by ELISA method. In a second protocol, the cells were subjected to UVB,radiation (35 rnJ/cm2) after 24 hours of culture in the presence or absence of Pal-GQPR, then cultured with fresh medium for a further 24 hours. Angiotensin converting enzyme and its substrate kit were obtained from SIGMA. A.C.E. inhibition was measured at A= 340 nm. Clinical study (in vivo) was carried out on 20 female volunteers with chronic "bags" under the eyes. Measurement of skin profile used the fringe projection method (EoTech, France) coupled to image analysis (lnnov Metric Software INC, USA). Standard selection criteria applied, Student's t-test for paired series was used to analyze the data. Results and Discussion: In vitro studies: a) inhibition of bradykinin metabolism The contraction of lymphangions, necessary to ensure drainage, is dependent on the endogenous nonapeptide brndykinin (BK) which activates the 02-receptors of the lymphangion (vasa lymphatica), and increases the frequency of contractions [1,2,]. However, the lifespan of BK is relatively short it is rapidly fragmented by a degradation enzyme, angiotensin converting enzyme or A.C.E [4]: A.C.E, through its action, thus controls the frequency and intensity of the lymph pulsations. The dipeptide Val-Trp (VW) is a natural fragment of enzymatic hydrolysis of food proteins and was 100 BO 60 40 20 FAPGG Fig.1 Bnldyklnln described by Saito [3] as occurring in fermented sake and sake lee. It is a potent inhibitor of the angiotensin converting enzyme: incubation of the A.C.E. with a synthetic substrate (furyl-aryloyl-phenylalanyl-gly­ gly=F APGG) leads to release of F AP and GG fragments and a measurable UV signal. Figure I shows the concentration dependent inhibition of this reaction. Figure I shows also the inhibition of the bradykinin breakdown by Val-Trp. Inhibiting the metabolism of bradykinin, increasing the local BK pool is thus intended to stimulate the pumping frequency of the lymphatic canals and to improve drainage.

2003 ANNUAL SCIENTIFIC MEETING 227 In vitro studies: b) reduction of IL6 production The importance of cytokine balance in the control of the processes of immune and inflammatory reactions is well known. Interleukin 6, in particular, plays a key role in the inflammatory response induced by UV radiation or related to age (2-fold increase in circulating levels), locally creating chronic pro-inflammatory conditions. In our experiments, a marked decrease in basal IL6 secretion was observed at a concentration of 10 ppm. In the absence of Pal-GQPR, UV irradiation induces up to 20-fold increase in the IL6 levels. In the presence of the peptide, IL6 levels were reduced by 33 to 60%: Variation of basal and UV-induced IL6 level in the nresence ofnentide Pal-C-rOPR Pal-GQPR Decrease in IL6 (% basal level) Decrease in induced IL6 level (ppm) 10 -15% -33% 15 -20% -37% 30 -25% -60% The effects of hesperidine methyl chalcone on capillary fragility (leaking of serum through blood vessel walls) have been well documented in the literature [5,6] and were not repeated here. In vivo studies: The study had an open-label design and each subject acted as her own control. The results obtained at baseline (TO) were compared to those obtained at time points T28 and T56 days. The volunteers applied a gel containing 33 ppm of VW, 10 ppm of Pal-GQPR and 150 ppm of hesperidine methyl chalcone, morning and evening for 2 months. The product was applied by dabbing with the fingers, under each eye, from the lateral extremity to the medial extremity. The detenninations on T28 and T56 were conducted in the absence of the product the last am,lication having taken place the previous day. The measurements conducted were thus free from any interference related to the cosmetic application technique (drainage due to a mechanical effect) and taking into account the peri-orbital stasis that is known to be amplified after a night in the prone position. Decrease n = 20 of Mean altitude Significance TO Si · cance T56/f28 T28-TO -0.08 p0.05 (mm) T56-TO -0.20 p0.01 0.05 The pictures show the visible improvement after 2 months of product application. Conclusion: Three identified causes (capillary fragility, poor drainage, mild inflammation) of "puffy eyes" call for a combination of actives for treatment. Hesperidine chalcone, Val-Trp and Pal-GQPR each address at least one of the causes and, formulated in a topical product show visible improvements that appear to validate the underlying assumptions. ---�References Ill DOBBINS, 1990 Constriction of the perfused lymphatics by acetylcholine, bradykinin and histamine. Microcirc. Endothelium Lymphatics, dee, 6 (6), p409 [2] YOKOYAMA, 1996, Effects ofbradikinin on lymphatic pumping in rat mesentery, Am J Physiol, 270( 5 Pt 1), p G752 [3] SAITO1 et al., 1994 Structure and activity of angiotensin converting enzyme inhibitory peptides from Sake and Sake lee. Biosc. Biotechnol. Biochem, 58 (10), pl767 [4] WARREN, 1995 Captopril increases skin microvascular blood flow secondary to bradykinin Faseb J, 9(5), p41 l [5) RUDOVSKI, 1989 Improving venous tone and capillary sealing. Fortsch med, 107(19):52, p55 [6) GARG et al., 2001 Chemistry and pharmacology of the citrus bioflavonoid hesperidin Phytother Res, 15 (8), dee, p 655