298 JOURNAL OF COSMETIC SCIENCE Table I The Mean Values for Light and Dark Human Melanocyte Viability at Different dA and HQ Concentrations [The concentration that allows 95% viability (bold) was chosen for each test compound (see Table II)} Treatment (µM) dA 25 dA 12.S dA 6.25 dA 3.125 dA 1.5625 dA 0.78 HQ25 HQ 12.S HQ 6.25 HQ 3.125 HQ 1.5625 HQ 0.78 HQ 0.391 HQ 0.195 Control Mean viable cell per control for light human melanocytes ± SD* 0.795 ± 0.012** 0.877 ± 0.063 0.963 ± 0.018 0.986 ± 0.011 1.002 ± 0.024 0.990 ± 0.021 0.851 ± 0.023 0.854 ± 0.025 0.909 ± 0.010 0.899 ± 0.035 0.953 ± 0.047 0.969 ± 0.019 0.975 ± 0.043 0.983 ± 0.036 1.01 ± 0.02 Mean viable cell per control for dark human melanocytes ± SD* 0.761 ± 0.076 0.854 ± 0.045 0.912 ± 0.031 0.949 ± 0.049 0.971 ± 0.027 0.814 ± 0.019 0.859 ± 0.035 0.910 ± 0.032 0.929 ± 0.006 0.932 ± 0.023 0.958 ± 0.034 0.979 ± 0.024 1.00 ± 0.00 * Values for light and dark human melanocytes represent the mean of cell lines developed from four and six separate foreskins, respectively. ** In each individual experiment, duplicate samples were measured for each duplicate flask/concentration. Table II Concentrations of Tyrosinase Inhibitors That Allow for 95% Viability per Control in Normal Human Melanocytes Derived from Dark (NHM-dk) and Light (NHM-lt) Skinned Persons Compounds NHM-dk (µM) NHM-lt (µM) HQ 0.391 1.5625 dA 1.5625 6.25 TBP so so KA so so AR so so the cell viability of keratinocytes and fibroblasts as compared to dA and HQ (Figure 2). In contrast, TBP demonstrated a significant reduction of viability in keratinocytes and fibroblasts compared to KA and AR (Figure 2). COMPARATIVE EFFECT OF TYROSINASE INHIBITORS ON TYROSINASE ACTIVITY AND MELANIN SYNTHESIS OF CULTURED HUMAN MELANOCYTES At concentrations that did not significantly effect viability (Table II), the efficacy of dA in inhibiting tyrosinase activity and reducing melanin content in dark human melano­ cytes was determined and compared with the efficacy of HQ, KA, AR, and TBP. Within intact dark human melanocytes, dA significantly inhibited tyrosinase activity with a concomitant significant reduction in melanin content (Figure 3a,b). KA and TBP also significantly inhibited tyrosinase activity and melanin content. In contrast, HQ exhib­ ited a significant reduction in tyrosinase activity without a significant reduction in

EFFICACY AND SAFETY OF DEOXYARBUTIN 12 g � : QB .. • 06 • 0 04 .! .! 02 0 +-------r-------,--------, 0 a 10 :a) an:.- 12 • 06 • 04 a Q2 * 0 +------.--------.--------, 0 b 10 2J QR( ..... 3l 299 Figure 1. Viability and morphology of normal human fibroblasts and keratinocytes were less significantly affrcreJ by JA compareJ to HQ. Human f"ibroblasts anJ keratinocytes cultures were treated daily with fresh media containing either dA or HQ in a dose-dependent manner for five clays. On day 6, cells were cletachecl and counted with a Coulter Counter. The cell number of the treated flask was divideJ hy the mean cell numbl'r of the vehicle-control flasks anJ plorceJ vs the test comrounJ concentrations. (a) Effect of· dA anJ HQ on viability of normal human fibroblasts. Duplicate samples were measured for each duplicate flask/ concentration. (b) Effect of dA ancl HQ on viability of normal human keratinocytes. Results are expressed as the mean of triplicate cultures/concentration. (c-e) Morphology of keratinocytes culturl's treateJ with (c) vehicle alone, or with 25 pM of (d) dA, or (e) HQ. * = Significant difference between the clA- and HQ-created groups at the same concentration at /1 0.05. melanin content (Figure 3a,b). AR did not alter either tyrosinase act1v1ty or melanin content. Within intact light human melanocytes, dA, KA, and TBP significantly in­ hibited tyrosinase activity (Figure 3c). In contrast, HQ exhibited no significant inhi­ bition of tyrosinase activity in melanocytes from light skin (Figure 3c). AR-treated melanocytes exhibited enhanced in situ tyrosinase activity as compared to the untreated control. REVERSIBILITY OF TYROSINASE INHIBITORS IN CULTURED HUMAN MELANOCYTES The time-dependent inhibition and the reversibility of inhibition on the tyrosinase activity and melanin content of dark human melanocytes treated with dA was assessed and compared with the effect of HQ and TBP at concentrations that did not significantly affect viability. Treatment of dark melanocytes with dA and TBP for five and ten days