# Skin Hyperpigmentation: Understanding the Exposome Effect

SymBright® 2036 & SymBright® PLUS

The complete environmental exposure of an individual was defined as 'exposome' in 2005 (by CP Wild). Since then, this definition has been adapted to skin exposure as all external factors causing skin aging (including solar radiation, pollution, tobacco, temperature and stress). In our research programme, the effect of selected exposome factors on skin pigmentation was investigated in order to develop an active to protect skin against dark spot formation caused by the exposome, one of the skin's aging processes. UV radiation, visible light radiation and diesel exhaust particles were used as external stressors. The active molecule SymBright 2036® (INCI: Sclareolide) developed within the project scope is a sesquiterpene lactone that can be obtained from clary sage (Salvia sclarea L.).

Introduction

The list of external factors damaging our skin is growing. All of them appear to trigger a common reaction sequence in the skin involving inflammation and melanin production. While this sequence is entirely part of the skin's natural defense system, it subsequently typically results in pigmentation disorders and dark spots that cause excessive reactions. Skin brightening has come a long way from a basic 'whitening' approach aimed at darker-skinned ethnic consumers, to new targets such as targeted spotting to brighten the skin and reduce the appearance of brown/age spots. Uneven skin tone can be a source of embarrassment for many consumers. 58% of consumers feel bad about themselves due to uneven skin tone [1]. Demand for skin perfection is increasing among women of all ages. Understanding the pigmentation mechanisms in the epidermis is important for these purposes. It is accepted that tyrosinase inhibitors in cosmetic products contribute to reducing skin pigmentation. However, the stimuli causing hypopigmentation in keratinocytes and melanocytes to prevent freckles and melasma formation are diverse: UV radiation is a well-known pigmentation enhancer; acne scars, epilation or shaving lesions are increasingly demanding more effective products [2]; but air pollution has also recently been identified as a major inducer of lentigines [3,4]. All these stimuli converge on the underlying mechanism of keratinocytes releasing a group of cytokines that trigger melanocytes to proliferate, differentiate, produce melanin and transfer melanosomes to keratinocytes, with the most common mechanism known as post-inflammatory hyperpigmentation (PIH). Since pigment spots can only be resolved over a long period of time if left untreated [6], protection against PIH along with depigmentation effects is the most promising cosmetic treatment. In recent years, the view on sun-induced pigmentation has diversified from just the UV portion to the additional contribution of visible light. Since approximately 52% of the sun contributes to visible light at sea level [7] and this portion of sunlight penetrates deep into the skin, interest in the effects of visible light on skin physiology has been growing in recent years [8]. An important observation was skin tanning despite the use of UV protection products, which led to the need for a deeper look at the parts of solar radiation beyond UV that are related to pigmentation. Duteil (et al. [9]) nicely demonstrated that pigmentation induced by the blue/violet portion of visible light is more persistent than UV-induced pigmentation. Interest in products that provide protection against this persistent hyperpigmentation is also increasing. In vitro models are being developed to investigate mechanisms and identify potent actives.

Materials and Methods

Skin pigmentation in vitro: B16V melanoma cells were seeded in 96-well microtitre plates. After adhesion, various concentrations of test substances and 10 nM α-MSH were added. After 96 hours of incubation, cellular melanin was extracted with alkaline solution, and absorbance was measured at 400 nm. Skin pigmentation ex vivo: Full-thickness human skin explants obtained from abdominal plastic surgery (7 x 3 mm; Ø x thickness) were transferred to culture plates and placed on a cotton pad immersed in culture medium. 4-6 skin explants per test concentration and placebo were used. SymBright 2036® formulated as a hydrodispersion gel was applied topically once daily. Histological sections were prepared on day 6 and melanin quantity was determined by image analysis after Fontana-Masson staining. Kojic acid served as a positive control and was applied at a concentration of 0.1% in DMSO. In vitro visible light-induced hyperpigmentation: Human epidermal melanoma cells A375 were cultured in 6-well plates. Cells were exposed to visible light (480 J/cm2) with a Hydrosun 750 equipped with a KG1 filter (to filter infrared A radiation) and HBM1 (Hydrosun) (Medizintechnik GmbH, Müllheim, Germany). Cells were treated with test compounds at non-cytotoxic concentrations 48 hours before and after irradiation. Melanocytes were lysed in a NaOH solution. Melanin was quantified by absorbance measurement at 405 nm and calculation based on a melanin standard curve. Visible light-induced hyperpigmentation ex vivo: Finished formulations were applied to ex vivo human skin explants obtained from abdominal surgery of a donor with phototype IV (Fitzpatrick scale). 48 hours after removal of formulations with a cotton pad, skin explants were exposed to visible light (480 J/cm2) with a Hydrosun 750 equipped with a KG1 filter. Formulations were then reapplied. After 48 hours, skin sections were prepared and melanin was stained with Fontana-Masson. Melanin analysis was performed semi-quantitatively by image analysis.

Pollution-Induced Hyperpigmentation

Model DEP: Standard reference materials (SRMs) SRM1650b (mean particle diameter 0.18 um) from the National Institute of Standards and Technology (Gaithersburg, MD, USA) were used as suitable surrogates for authentic street particulate matter. SRMs were suspended in phosphate-buffered saline and subjected to 1 minute of sonication. They were then added directly to keratinocytes at a concentration of 1.5 ug/cm2. RNA Isolation and PCR: Total RNA was prepared as previously described [10]. Cyp1A1 and MMP-1 genes were measured 6 hours after DEP application; IL-6 and POMC genes were measured 24 hours after DEP application by qRT-PCR. Three samples per condition were processed, each with 2 determinations.

Clinical Study

24 Asian adult subjects with phototypes II to III applied a placebo formulation and the same formulation with 0.2% SymBright® to their forearms twice daily from day 0 to day 48. They were exposed to progressive UV doses (0.7 MED to 1 MED) on days 0, 1, 2 and 3 to induce pigmentation. Maximum pigmentation was achieved on day 7. Therefore, pigmentation variation is normalized to day 7.

Results

SymBright 2036® showed a potent brightening effect when tested in vitro on B16V melanoma cells with an IC50 of 10.2 uM (Figure 1A). In the next step, SymBright® was tested on ex vivo human skin (phototype intermediate) dosed at 0.1% in a hydrodispersion gel formulation. A significant brightening effect was observed with a 41% reduction compared to placebo (Figure 1B). By comparison, the positive control kojic acid applied at 0.1% in DMSO reduced melanin content by 30%. [caption id="attachment_137889" align="aligncenter"] Figure 1: Melanogenesis inhibition: SymBright 2036® inhibited melanin production in vitro in B16V cells. Melanin in human ex vivo skin explants was reduced at 0.1% SymBright® applied in a gel formulation after 6 days; positive control kojic acid was applied at 0.1% in DMSO (B).[/caption] Due to in vitro experiments on B16V cells and also qRT-PCR of ex vivo explants, it was shown that the brightening activity is based on a direct effect on melanocytes, as well as an altered cross-talk between keratinocytes and melanocytes, including POMC as one of the regulated genes involved. Protection against visible light-induced hyperpigmentation was demonstrated in vitro in the human A375 cell line and significantly inhibited 70% of induction at 0.00079% active (data not shown). [caption id="attachment_137890" align="alignleft"] Figure 2: Ex vivo inhibition of visible light-induced melanin synthesis.[/caption] [caption id="attachment_137891" align="alignnone"] Figure 3: POMC gene expression stimulation from air pollution: SRM1650b significantly stimulates POMC gene expression. SymBright 2036® significantly blocks this increase.[/caption] Hyperpigmentation from urban pollution was investigated in vitro on HaCaT cells by application of diesel exhaust particles SRM1650b, which resulted in increased secretion of POMC protein. Active dose 0.00025%; this succeeded in reducing this increase by 49%. In an in vivo study, the active was applied at 0.2% to the forearm of Asian subjects. Hyperpigmentation was induced by consecutive 4 days of UV irradiation. The active succeeded in significantly reducing hyperpigmentation within 48 days compared to placebo. [caption id="attachment_137892" align="aligncenter"] Figure 4: In vivo UV-induced pigmentation — 24 Asian adult subjects application — exposure from 0.7 MED to 1 MED on days 0, 1, 2 and 3 maximum pigmentation achieved on day 7. Therefore, pigmentation variation is normalized to day 7. SymBright 2036® shows a good trend in preventing UV-induced pigmentation with significant results after 48 days of application.[/caption]

Conclusion

SymBright 2036® produced in high purity is a natural, safe and sustainable product. Sage stems, leaves and flowering parts contain sclareol. Biotransformation of sclareol results in sclareolide. Through subsequent extraction and concentration processes, sclareolide is obtained in high purity. The safety profile of sclareolide is well known as it has been used for fragrance production and in fragrance blends for decades. SymBright 2036® is an approved and registered cosmetic ingredient in China. SymBright 2036® best protects the skin on the face and body against excessive reactions to harmful environmental effects that can lead to persistent hyperpigmentation disorders. With visible results after only a few weeks of use, it has been found to protect the skin against the harmful effects of air pollution and UV rays and blue light. The skin's natural radiance is preserved and even returns. Protection against hyperpigmentation caused by UV or air pollution, respectively, along with depigmentation effects is the most promising cosmetic treatment for spots and uneven pigmentation. By bringing these properties together in a single active, SymBright 2036® makes it an attractive material for deodorant, epilation and acne applications, as well as highly suitable for day creams that provide protection against harmful effects throughout the day. It brightens the skin and reduces the appearance of brown/age spots.

A Bright Partner for Bright Skin

As a pioneer investigator of the exposome, Symrise is continuously developing new solutions for pigmentation. Dark spots are now perceived like new wrinkles, and Symrise is searching for a bright partner with optimized efficacy by exploring ways to overcome this particular concern. To leverage our record-breaking active SymBright® 2036, we sought an intelligent and efficient combination. Niacinamide — vitamin B3 — was used to develop SymBright® PLUS, a ready-to-use liquid mixture that is a perfect match to enhance the efficacy of SymBright® 2036.

Test Efficacy of SymBright® PLUS

UV light can induce melanogenesis in the skin in two main different ways: direct activation of melanocytes or an indirect pathway by generating Reactive Oxygen Species (ROS) (for example in surrounding keratinocytes). These keratinocytes respond to ROS by producing stress signals that are subsequently secreted and then trigger melanogenesis. [caption id="attachment_137893" align="aligncenter"] Figure 5: Schematic representation of direct and indirect pathway for melanogenesis induction with UV light.[/caption] The newly launched component SymBright® PLUS was developed as a skin brightener. We therefore evaluated its efficacy on human skin explants exposed to UV radiation. Since ROS production is a very rapid process as the starting point for the indirect activation pathway, we examined SymBright® PLUS's ability to reduce the amount of ROS in skin explants irradiated with 60 J/cm2 UVA. In Figure 6, cross-sections of skin explants are shown with a green fluorescence signal, which is an indicator of ROS presence in the tissue. [caption id="attachment_137894" align="aligncenter"] Figure 6: Fluorescence staining (DCFH-DA) of histological sections from human skin explants (female donor, age 51, ITA angle 36° "medium"); fluorescence intensity is related to ROS presence in the tissue. Scale bars represent 100 μm.[/caption] Quantification of fluorescence allows evaluation of the ROS-reducing activity of test samples. SymBright® PLUS resulted in a 66% reduction in ROS score compared to UV-irradiated control. Furthermore, the components comprising SymBright® PLUS were tested at their individual concentrations, and none of the individual components achieved the efficacy of SymBright® PLUS in ROS reduction. This result demonstrates the superiority of SymBright® PLUS over its single components in terms of ROS reduction in human skin. In a second experiment, we analyzed the effect of SymBright® PLUS on UV-induced pigmentation. Human skin explants (female donor, age 43, ITA angle 24° "stratified") were exposed to UVB radiation at a dose of 75 mJ/cm2 for 7 days. Quantitative evaluation of melanin content in skin explants was performed using Fontana-Masson staining. SymBright® PLUS reduced melanin in skin explants by 26% compared to UV control (see Figure 7). [caption id="attachment_137895" align="aligncenter"] Figure 7: Evaluation of melanin content by Fontana Masson staining in human skin explants after 7-day treatment with test items and subsequent exposure to 75 mJ/cm2 UV B radiation. Black staining is indicative of melanin presence and was measured by image analysis. Scale bars represent 100 μm.[/caption] Changes in melanin content may result from degradation of existing melanin or reduced pigment biosynthesis. To assess the effect of SymBright® PLUS on melanogenesis, we analyzed the abundance of Melan-A as a marker of de novo formation and maturation of melanosomes, the organelles in which melanogenesis occurs. Treatment of UVB-irradiated skin explants with SymBright® PLUS resulted in a 64% reduction in Melan-A signals compared to UV-applied vehicle control. Furthermore, SymBright® PLUS performed better than its single components in reducing Melan-A signals, an indicator of de novo melanogenesis. [caption id="attachment_137896" align="aligncenter"] Figure 8: Representative image of Melan-A signals in sections of human skin explants after 7-day treatment with test items and daily irradiation with 75 mJ/cm2 UVB. The image shown is the UV-irradiated vehicle control. Cells with dark red coloration (examples shown by black arrows) contain Melan-A and are therefore considered to be engaged in de novo melanogenesis. Scale bar represents 100 μm.[/caption] Taken together, our findings demonstrate that SymBright® PLUS achieves not only very good reduction in UV-induced ROS species in human skin explants but also significant reduction in melanogenesis, thus potentially exerting an effect on both direct and indirect pathways for UV-induced melanogenesis. The product performs better than its single components in terms of ROS inhibition and de novo melanogenesis.

Conclusion

SymBright® PLUS is a versatile and in vivo proven effective solution for prevention of irregular skin pigmentation and hyperpigmented spots. Thanks to its easy-to-use liquid form, it can be used in many applications such as daily facial and body care, spot-preventing cosmetic products and sheet masks. References: 1. CMI Data Source: Symrise Cosmetic Ingredients Consumer database 2012-2013 (36,000 consumers across 10 markets) 2. Ortonne and Bissett (2008) J Invest Dermatol Symp Proc 13(1):10-4 3. Vierkötter et al. (2010) J Invest Dermatol 130(12): 2719-2726 4. Nakamura et al. (2015) Expert Dermatol 24(6): 407-11 5. Yamaguchi and Hearing (2009) Biofactors 35(2): 193-9 6. Davis and Callender (2010) J Clin Esthet Dermatol 3(7):20-31 7. CIE Technical Report No 85, 1989, table 4 'Global solar irradiance at sea level' 8. Dupont, E., Gomez, J., Bilodeau, D., Int J Cosmet Sci 35(3): 224-32 (2013) 9. Duteil, L., Cardot-Leccia, N., Queille-Roussel, C., Maubert, Y., Harmelin, Y., Boukari, F., Ambrosetti, D., Lacour, JP, Passeron, T., Pigment Cell Melanoma Res 27(5): 822-826 (2014) 10. Grether-Beck et al. (2008) Exp Dermatol 17: 771-79

Authors: I. Meyer, M. le Maire, M. Knupfer, A. Rousseau, Dr. J. Hans

Compiled by: Özgür Çelen Sales Manager Symrise