2005 ANNUAL SCIENTIFIC MEETING 4-(1-PHENYLETHYL) 1,3-BENZENEDIOL: A NEW HIGHLY POTENT LIGHTENING AGENT Gerhard Schmaus, Ph.D., Gabriele Vielhaber, Ph.D., Karin Jacobs and Helge Franke Symrise GmbH & Co. KG, Muhlenfeldstr. I, D-37603, Holzminden, Germany Introduction 197 There is an increasing worldwide demand for skin lightening active ingredients. Whereas a pale skin is the beauty ideal � Asian countries, Caucasian skin types from Europe and the US aim to treat pigment spots. Current skin lightening actives such as Kojic acid, Arbutin or Ascorbic acid derivatives have several disadvantages regarding their safety or stability. Since nature is without any doubt an inexhaustible source of inspiration for new actives for cosmetics, we recently focused within a continuously running natural product research program on different wood extracts and pure isolates, obtained thereof. The goal was to evaluate their tyrosinase inhibitory activity and to identify a new skin lightening agent, lacking those negative side effects described above. Materials and Methods Origin of test material: Phenolic compounds with different substitution patterns were either isolated from plant extracts by preparative liquid chormatography or they were synthesized. Mushroom Tyrosinase Assay: Tyrosinase reactions were performed in a 96well rnicroplate with 66.7 mM phosphate buffer (pH 6.8) containing 50 u/ml mushroom tyrosinase (EC.1.14.18.l, Fluka Chemie GmbH, Buchs, Switzerland) and test compounds. After pre-incubating at 37°C for IO min, L-3,4-dihydroxyphenylalanine (L-DOPA) was added to the mixture and the formed dopachrome was measured photometrically at 475 nm. The inhibitory effect of the test samples was expressed relative to the control and IC50 (50 % inhibitory concentration) values were calculated. Kojic acid was used as a reference. In vitro Toning Assay with B16V Mouse Melanoma Cells: B16V cells were seeded into 96well microplates. After adhesion (24 h) the medium was replaced by freshly prepared solutions of the test compounds at non cytotoxic concentrations (solvent: cell culture medium containing IO nM cx.-Melanocyte .Stimulating Hormone). After incubation for another 96 h, melanin was extracted with NaOH and the absorption at 400 nm was measured. In vitro Toning Assay with Pigmented 3D Epidermis Models: Freshly prepared solutions of the test compounds (solvent: PBS) were applied at non cytotoxic concentrations on the top of MelanoDerm™ MEL-300-B models (skin type IV). The test compounds were reapplied daily. After overall incubation time of 7 and 19 days, respectively, the melanin was extracted with soluene and the absorption at 400 run was measured. In Vivo Lightening Efficacy was studied in a human model on Asian skin. Test products and placebo formulation were applied 2 times daily. Subjects were requested to avoid any UV exposition during the test period. Measurement of the lightening efficacy was done by chromametry and visual assessment at t = 0 and after 28 days. Results and Discussion Within our natural products screening the tyrosinase inhibiting activity of ,,Scotch Pine" (Pinus sylvestris) heart wood extracts and pure isolates thereof attracted our specific interest. From this extract we isolated stilbene derivatives like Pinosylvin-3-O-methylether and Pinosylvin. Since we observed that such stilbene derivatives are fairly unstable under light condition, more stable partially hydrogenated derivatives like Dihydropinosylvin-3-O- methylether and Dihydropinosylvin, both also occurring in trace amouts in pine heart wood, as well as a couple of nature-derived substances with very similar structure like 4-benzyl-1,3-benzenediol, 2-( 1- phenylethyl) 1,3-benzenediol and 4-(1-phenylethyl) 1,3-benzenediol were synthesizd. The inhibitory activity of all compounds was measured with the same mushroom tyrosinase assay and their IC50 values relative to Kojic acid were calculated. Several compounds like Pinosylvin-3-O- methylether, Dihydropinosylvin-3-O-methylether, 4-benzyl-1,3- benzenediol and 2-(1-phenylethyl)I,3-benzenediol showed only weak to moderate tyrosinase inhibitory activity. 4-( 1-phenylethyl) I ,3- Figure 1: Structural formula of 4-( 1-phenylethyl)1,3-benzenediol

198 JOURNAL OF COSMETIC SCIENCE benzenediol (Fig. I) was by far the most active compound within this compound class. It exhibited an ICso of 0.50 µM, thus reducing tyrosinase activity approximately 22 times more effectively than Kojic acid with an ICso= 11.05 µM under identical test conditions. Because of its excellent tyrosinase inhibitory activity, further detailed in vitro studies in melanocyte cultures and on 3D skin models as well as first human in vivo studies were performed with 4- ( l-phenylethyl)l,3-benzenediol. In the cellular lightening assay on B16V mouse melanoma cells, a pronounced efficacy of 4-( 1-phenylethyl) 1,3-benzenediol was found. It was by far the most potent inhibitor of melanin synthesis with an IC50 of 2.1 µM, whereas the ICso of 8-arbutin and Kojic acid was 67 µM and 440 µM, respectively, under identical test conditions. This means that 4-(1-phenylethyl)l,3-benzenediol was approx. 200 times stronger than Kojic acid in the cellular assay. In the pigmented 3D skin model, 4-(1-phenylethyl)l,3-benzenediol reduced the melanin content of pigmented 3D epidermis models at least IO times more efficiently than Kojic acid within a period of 19 days (Fig. 2). To confirm that 4-( 1-phenylethyl) 1,3-benzenediol sufficiently penetrates into skin and is effective also under human in vivo conditions a clinical study was performed with Asian subjects during a duration period of 4 weeks (Fig. 3). Product GS05048SL-A and -B (0.5 % 4-( 1-phenylethyl)1,3-benzenediol each) efficiently lighten the natural tone of Asian skin in vivo after 2x daily treatment for 28 days. 0.5 % 4-(1-phenylethyl)l,3-benzenediol is more efficient than 1.0 % Kojic acid. A more pronounced effect is achieved when 4-( 1-phenylethyl) 1,3-benzenediol is directly incorporated into an aqueous gel formulation with low oil phase content (GS05048SL-A). Pre-solubilising 4-(1- phenylethyl)l,3-benzenediol in a formulation with high oil phase content (GS05048SL-B) was less effective. Despite the considerable concentration gradient between the applied formulation and the skin itself, 4-(1- phenylethyl)l,3-benzenediol is obviously only poorly released from the oil phase because of it high lipophilicity Conclusion Systematic screening of plant extracts, isolated natural products and nature-derived synthetic derivatives thereof for potential skin lightening activity showed, that 4-(1-phenylethyl)l,3-benzenediol, a dihydroxylated diphenylmetbane derivative, possesses potent tyrosinase inhibitory activity. Further investigations on the lightening activity in a cellular lightening assay (Bl6V mouse melanoma cells) and on pigmented 3D skin models (MelanoDerm.™ skin type N). delivered the unequivocal proof in vitro, that 4-(1-phenylethyl)l,3-benzenediol is one of the most potent lightening agents ever reported. First studies on Asian skin confirmed that it also shows good lightening activity in a human in vivo situation. Figure 2: Lightening activity of 4-(1-phenylethyl) 1,3-benzenediol on pi � ented 3D epidermis models (MelanoDerm , skin type N) photos were taken after 19 days of daily treatment with Kojic acid and 4-(1-phenylethyl) 1,3-benzenediol (Bio377), respectively. Reduction oflMlanln ReducllonL.....---..,__.,;;;.._.,._.__oiliiiii__, oflMlanln Figure 3: In vivo efficacy of 4-(1-phenylethyl) 1,3- benzenediol on non-irradiated skin (mean chromameter readings to baseline after 28 days) 4 cosmetic formulations were studied: GS05048SL-A (0,5% 4-(1-Phenylethyl)l,3- benzenediol) GS05048SL-B (5% of a 10% solution of 4- (1-Phenylethyl)l,3-benzenediol in neutral oil) GS05048SL-C (1 % Kojic acid) GS05048SL-D (placebo formulation). 1.1•----------- .... ... • .... lr 11 2.11 ,: , .. i· .... ..• •UI - ... u, 'lM