2005 ANNUAL SCIENTIFIC MEETING 199 IMPROVING EFFICACY OF SKIN LIGHTENING PRODUCTS BY USING MULTIPLE PIGMENTATION INHIBITION MECHANISIMS Di Qu, Ph.D., Jesse C. Leverett and John V. Scimeca R&D - Skincare & Cosmetics, Access Business Group, Ada, MI Summary: The efficacy of skin lightening products containing multiple pigmentation inhibition mechanisms was examined via in-vitro and in-vivo studies. The results were compared with that of formulas containing only a single inhibition mechanism. The multiple mechanism products showed higher reduction in total melanin than the single mechanism ones in the in-vitro efficacy studies where cultures ofreconstructmed human epidermis were used. In- vivo studies confirmed the advantage of such an approach. More than 30% reduction in mottled pigmentation and 50% reduction in dark skin tone were achieved after a 12-week use period. This study shows that multiple inhibition mechanism improves the skin lightening efficacy of a product. Introduction: Skin lightening products take a large market share in Asian countries. Consumers continue to demand for more efficacious products which often marketed under an OTC-equivalent product category, such as quasi-drug cosmetics in Japan, special use cosmetics in China, and functional cosmetics in Korea. The levels and functions of ingredients under this category are usually regulated. To increase product efficacy, utilization of multiple pigmentation inhibition mechanisms provides a way [I]. Melanogcnesis of the skin involves multiple pathways and skin lightening can be achieved through many mechanisms other than tyrosinase inhibition [2]. In this study, we formulated products with ingredients exhibiting various pigmentation inhibition properties such as tyrosinase inhibition, rnelanosome transfer inhibition, a-MSH inhibition, eumelanin-pheomelenin ratio alteration, and existing melanin removal. In-vitro and in-vivo studies were conducted to confirm the efficacy. Material and Methods: Tyrosinase activity assay [3] was used to screen melanin inhibition activity of various ingredients. In-vitro efficacy was tested using a 3-D model of reconstructured human epidermis with melanocytes (Melanoderm from MatTek). The samples of skin model were incubated with various ingredients or products three times a week for two weeks. Total melanin, surface coloration, cell liability, and microscopic images were examined after that [ 4 ]. In-vivo studies were conducted via clinical tests in an independent testing lab on the face of 60 female Asian subjects. Skin lightening parameters such as skin clarity, skin tone, and mottled pigmentation were clinically graded based on a IO point scale. Color pictures were taken at the time of evaluation. Results: Various ingredients were screened for pigment inhibition activity. Some significant results are listed in Table I where tyrosinase inhibition activity (column 3) was compared with total melanin and surface coloration results (columns 4 & 5). Notice the trend in column 3 do not translate into column 4 indicating some ingredients worked under mechanisms other than tyrosinase inhibition. For instance, while citrus unshiu extract showed a much lower tyrosinase inhibition than bearberry extract and the botanical blend, their levels ofinhibition in melanin production in 3-D skin model were comparable. For wheat germ extract, it had a 10% inhibition in tyrosinase activity. However, it inhibited about 20% melanin production in 3-D skin model. Its coloration score was also very low indicating not much eumelanin was produced on the surface of the skin model. These results supported literature and vendor data, as citrus unshiu extract is known to inhibit melanin production in addition to tyrosinase inhibition. Wheat germ extract is believed to switch the melanin production from eumelanin (a dark/brown melanin) to pheomelanin (a light/yellow melanin) while the content of lectins in the material inhibits transfer ofmelanosomes from the melanocytes to the keratinocytes [5]. Comparative efficacy data were obtained when incorporating ingredients with various pigment inhibition mechanisms into some cosmetic formulas. Their efficacy of melanin inhibition was measured against the formulas with single inhibition mechanism. In both systems a 3% magnesium ascorbyl phosphate was used to establish an OTC-equivalent status. Figure I shows the total melanin content of the 3-D skin models after treated with various products for two weeks. It is seen that the single mechanism formulas had an average reduction in total melanin of Table 1 Cone. T� I�(%) Canlral (C&Aft medium) - - Mg-Ascabyl Phosphala 3% - Na-AsccrbylPholphale 3% - 8-b9r,yEldraf;I 2% 99.7 Bolanteal llend4 2% 91.0 BdalicalExtraC12 01%&0lids 703 Rasptiarry Ellhel 2% 393 Otnm Ulllliu EllllaCI 2% 24.2 'M.-GermEldra:I 2% 11.4 T01111Malril1 Coloration (%Conlrol) (1-5 scale) 1000 3 530 2 49.0 2 &20 2 690 3 450 2 87 0 4 62.0 1 81.0 1 Figure 1. Comparison of In-Vitro Elllcacr Control Toner Lotion Cream 0 20 40 60 80 Total Melanin (of% Control) 100 Cl Multiple mechanisms ■ Single mechanism

200 JOURNAL OF COSMETIC SCIENCE about 20% whereas the multiple mechanism formulas achieved about 35%. Figure 2 shows the images of surface melanin on the 3-D skin models where (A) is the control, (B) from a single mechanism formula, and (C) from a multiple mechanism formula. We see that much less melanin is shown on (C) than on (B) which indicates the formula with multiple mechanisms is more efficacious than that of a single one. The morphology of the melanocytes is shown in Figure 3. The melanocytes treated with the control (A) were full of melanosomes. Much less melanosomes and melanocytes were seen in the product treated samples. The shape of the melanocytes became thinner, and the color of their dendrites was more transluscent. The multiple-mechanism sample (C) had clearly less melanin and melanocytes than the single-mechanism one (B) indicating a better skin lightening efficacy. The in-vitro results were then confirmed through a couple of clinical studies. As shown in Figures 4- 6, the multiple mechanism formula out performed the single mechanism formula in a 12-week study in the U.S., and a 4-week study in Japan. Figure 7 shows the before and after photos comparing the overall skin conditions, such as mottled pigmentation, skin tone and clarity, of a panelist's face. Figure 4. Figure 5. IIMW � in u S.-Da1SlanTone 0% ,...., _____ -y-. -5% Figure 6 2....... 8-w.k 12-week II Single Mec:hanam ■ Multiple Mecnam..,. lnvlvo Elllcacy Study ,n J;apen {4 --■) Mottled P,grnenta!JOn Skin Tone II Sinfle Mechanism ■ Mulltple Mechaniaml 2-Wlillk IISuigla� Figure 7. Sample pictures from a 12-week in-vivo study in the U.S. 8-weak 12-week ...... MechaniarTa Discussion: It was noticed from the in vitro study that single ingredient performed better than a complete formula. We believe the phenomenon was caused by the interactions among various ingredients as we had seen certain ingredients promoted pigmentation in the in-vitro study and we might have used some ingredients in a formula for other functions without knowing their counteractive effects in pigment inhibition. Conclusion: Formulas with multiple pigment inhibition mechanisms show better efficacy than single mechanism ones in our in-vitro and in-vivo studies. The combined action is clearly better than a single action. This approach provides a meaningful way to increasing performance of skin lightening products in the OTC-equivalent category. Reference I. Petit, L., et al. International Journal of Cosmetic Science, 54( 4 ), p 169-181. 2003 2. Giuseppe Prata, Melanins and Melanogenesis. Academic Press. 1992. 3. Sigma-Aldrich Quality Control Test Procedure, Enzymatic Assay of Tyrosinase (EC.1.14.18.1) 4. Bessou-Touya, S., et al. J. Invest. Dermatol., 111 (6), p 1103-1108. 1998 5. Minwalla, L., et al. Pigment Cell Res., 14(3), pl85-194. 2001