300 12 a 0 100 191 On:(114 JOURNAL OF COSMETIC SCIENCE :ax, 29) 1.4 - 12 � 1L--��-----=--.._� 0 • 0.8 'i 0.6 u ! 0.4 Q2 0 +--"""'T"--�----.---�--, 0 SI b 1a:J 151 OR� 3D ZIJ Figure 2. Viability of normal human fibroblasts and keratinocytes was unaffected by AR and KA and significantly affected by TBP. Human fibroblast and keratinocyte cultures were treated daily with fresh media containing AR, KA, or TBP in a dose-dependent manner for five days. On the sixth day, cells were detached and counted with a Coulter Counter. The cell number of the created flask was divided by the mean cell number of the vehicle-control flasks and plotted v. the test compound concentrations. (a) Effect of AR and KA on viability of normal human fibroblasts as compared to the effect of TBP. Duplicate samples were measured for each duplicate flask/concentration. (b) Effect of AR and KA on viability of normal human keratinocytes as compared to the effect of TBP. Results are expressed as the mean of triplicate cultures/ concentration. * = Significant difference from both KA- and AR-treated groups at the same concentration at p 0.05. 1JII C * - i'II � IQ dA IQISO "IP5l - 0.311 1.56 Figure 3. At a concentration that did not affect viability, dA significantly inhibited in situ tyrosinase activity of cultured dark and light human melanocytes, with concomitant significant reduction in melanin content. Triplicate cultures at a density of 1 x 105 cells/well of dark and light skin human melanocytes were treated daily for five days with fresh media containing the previously determined concentrations that did not significantly affect viability of each test compound. On the fifth day, cells were fed with fresh media containing 1 µCi/ml of H 3 -tyrosine and the appropriate compound. After 24 hours, the radioactivity of the tritiated water was counted in a Packard 1900 CA liquid scintillation analyzer, and the melanin and protein content were determined for each culture as previously mentioned in Materials and Methods. Tyrosinase activity for cells in a specified concentration of test compound was expressed as DPM/24 hours/µg protein divided by the mean DPM/24 hours/µg protein of the vehicle-control flasks. (a,b) The effect of five days treatment with dA, HQ, AR, KA, and TBP on in situ tyrosinase activity and melanin content of dark human melanocytes, respectively. (c) In situ tyrosinase activity of light human melanocytes. Results are expressed as the mean of triplicate cultures ± SD. * = Significant difference from the control group at p 0.05. significantly reduced tyrosinase activity (Figure 4a) and melanin content (Figure 46). In contrast, HQ was similar, except that the melanin content was not significantly reduced at day 5 (Figure 46). After halting treatment, the dA-treated melanocytes returned to statistically normal values of tyrosinase activity, whereas HQ- and TBP-treated mela­ nocytes remained statistically reduced from the control group. Treatment with dA, HQ, or TBP resulted in a concomitant reduction in melanin content. The reduction in melanin was detected by five days of treatment for both dA and TBP and by ten days of

EFFICACY AND SAFETY OF DEOXY ARBUTIN :g 12 �8 ��OB o­ m·i o.s �2 en a. 0.4 0 0) �.2_ 02 a Q. 0 :g 12 C 0 - 0 1 C ,_ � � 08 oa5 .£ 0 06 � a. (I) O 0.4 �E � 02 Q. 0 b Control dA 1.56 HQ 0.39 TBP 50 Control dA l .56 HQ 0.39 TBP 50 301 Figure 4. dA is a reversible inhibitor in which significant recovery of tyrosinase activity and melanin content occurred five days after halting treatment. Dark human melanocytes, in six triplicate groups at a density of 1 x 10 5 cells/well, were treated daily with fresh growth media containing each test compound at the concentration that did not affect viability. After five days, one treated group and one control group were assessed for inhibition of tyrosinase activity and melanin content reduction. At day 5, one of the treated groups had treatment halted and was fed, along with one of the control groups, fresh media for another five days. On day 11, the four remaining groups (test compound treated for ten days, vehicle treated for ten days, test compound treated for five days and then untreated for five days, and vehicle treated for five days and then untreated for five days) were assessed for tyrosinase and melanin content as described in Materials and Methods. (a) Tyrosinase activity, expressed as DPM/24 hours/µg protein divided by the mean DPM/24 hours/µg protein of the vehicle-control flask, and (b) melanin content, expressed as µg melanin/mg protein divided by the mean µg melanin/mg protein of the vehicle-control flasks, for cells treated with test compound for five days (stippled bars), ten days (striped bars), or for five days and untreated for five subsequent days (solid bars). * = Significant difference from the control group at p 0.05. treatment for HQ. Only the inhibitory effect of dA on melanin synthesis was shown to be reversible. ANIMAL STUDY RESULTS The effectiveness of dA on reducing hyperpigmentation compared to HQ in a xenograft model with human skin grafted onto SCID mice was assessed and compared with the effect of HQ and TBP. Three mice per group were treated five days per week for eight weeks with dA, HQ, and TBP at 5% concentration. Photos for each graft were taken every two weeks using the Charm View™ (Moritex, Japan) system and then assessed using Photoshop software for L (lightening) and a (redness) values. L and a values were