Cistanche also has the function of promoting collagen production, which can increase the elasticity and luster of the skin and help repair damaged skin cells. Cistanche Phenylethanol Glycosides have a significant down-regulating effect on tyrosinase activity, and the effect on tyrosinase is shown to be competitive and reversible inhibition, which can provide a scientific basis for developing and utilizing the whitening ingredients in Cistanche. Therefore, cistanche has a key role in skin whitening. It can inhibit melanin production to reduce discoloration and dullness; and promote collagen production to improve skin elasticity and radiance. Due to the widespread recognition of these effects of cistanche, many skin whitening products have begun to infuse herbal ingredients such as Cistanche to meet consumer demand, thus increasing the commercial value of Cistanche in skin whitening products. In summary, the role of cistanche in skin whitening is crucial. Its antioxidant effect and collagen-producing effect can reduce discoloration and dullness, improve skin elasticity and luster, and thus achieve a whitening effect. Also, the wide application of Cistanche in skin whitening products demonstrates that its role in commercial value cannot be underestimated.

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Tyrosinase is the rate-limiting enzyme of melanin production and, accordingly, is the most prominent target for inhibiting hyperpigmentation. Numerous tyrosinase inhibitors have been identified, but most of those lack clinical efficiency because they were identified using mushroom tyrosinase as the target. Therefore, we used recombinant human tyrosinase to screen a library of 50,000 compounds and compared the active screening hits with well-known whitening ingredients. Hydroquinone and its derivative arbutin only weakly inhibited human tyrosinase with a half-maximal inhibitory concentration (IC50) in the millimolar range, and kojic acid showed a weak efficacy (IC50 > 500 mmol/L). The most potent inhibitors of human tyrosinase identified in this screen were resorcinol-thiazole derivatives, especially the newly identified Thiamidol (Beiersdorf AG, Hamburg, Germany) (isobutyl amido thiazolyl resorcinol), which had an IC50 of 1.1 mmol/L. In contrast, Thiamidol only weakly inhibited mushroom tyrosinase (IC50 =108 mmol/L). In melanocyte cultures, Thiamidol strongly but reversibly inhibited melanin production (IC50 =0.9 mmol/L), whereas hydroquinone irreversibly inhibited melanogenesis (IC 50 = 16.3 mmol/L). Clinically, Thiamidol visibly reduced the appearance of age spots within 4 weeks, and after 12 weeks some age spots were indistinguishable from the normal adjacent skin. The full potential of Thiamidol to reduce hyperpigmentation of human skin needs to be explored in future studies.

INTRODUCTION

Melasma, actinic and senile lentigines, and postinflammatory hyperpigmentation are major cosmetic problems for which many patients seek medical advice. Generally, those disorders affect populations with darker skin complexions with a greater frequency and severity (Stratigos and Katsambas, 2004). Many topical products are available to treat hyperpigmented disorders, and they contain diverse active ingredients to reduce melanin production and/or distribution. Although skin hyperpigmentation can be reduced by various mechanisms (Briganti et al., 2003), tyrosinase, the rate-limiting enzyme of melanin production, is the obvious target for inhibitors of hyperpigmentation (Kanteev et al., 2015; Lee et al., 2014; Ramsden and Riley, 2014). Many substances have been described in the literature as inhibitors of tyrosinase, but most of them lack clinical efficiency, and only a few compounds are currently used in topical dermatological products (Chang, 2009; Kim and Uyama, 2005; Rescigno et al., 2002). Among those, kojic acid, hydroquinone, and arbutin are the most common (Solano et al., 2006).

The unsatisfactory clinical efficiency of currently used tyrosinase inhibitors is large because those compounds were tested using only tyrosinase isolated from the mushroom Agaricus bisporus (mTyr) (Espin et al., 2000; Garcia-Molina et al., 2005), which is the only active tyrosinase readily commercially available. The catalytic activities and substrate specificities of mTyr are significantly different from the mammalian enzyme (Hearing et al., 1980). The three-dimensional structures of several tyrosinases were recently solved, among them the structures of mTyr (Ismaya et al., 2011) and of two bacterial enzymes from Streptomyces castaneoglobisporus (Matoba et al., 2006) and Bacillus megaterium (Sendovski et al., 2011). By contrast, very little kinetic or structural information is available for human tyrosinase (hTyr), mainly because of the substantial difficulties in obtaining sufficient amounts of hTyr from natural sources or by heterologous expression. hTyr has been transiently expressed in various animal cell lines (Olivares et al., 2002; Schweikardt et al., 2007; Tripathi et al., 1992; Wendt, 2006), but yields were always too low for a detailed characterization of the resulting hTyr preparations. More recently, several groups have developed more efficient expression systems for hTyr (Cordes et al., 2013; Fogal et al., 2015; Lai et al., 2016), but data on the three-dimensional structure of hTyr or kinetic data of hTyr inhibitors were still missing.

RESULTS

Inhibition of hTyr

A screen of 50,000 compounds in the library, which spans a wide chemical space, yielded several hit series of active and effective hTyr inhibitors. Among them, derivatives of thiazolyl-resorcinol were the most promising group. This lead compound was then optimized to develop derivatives with high activity and physicochemical properties compatible with topical formulations. Thiamidol (isobutyl amido thiazolyl resorcinol, compound 1) (Figure 1) was identified as one of the most potent derivatives. In addition to Thiamidol, 4-butyl resorcinol (compound 2) and the classical tyrosinase inhibitors kojic acid (compound 5), hydroquinone (compound 6), and arbutin (compound 7), as well as rhododendron (compound 9), were also tested as inhibitors of the diphenols (L-dopa oxidase) activity of hTyr over a wide range of concentrations (up to 4 orders of magnitude). The results are summarized in Figure 2a and Table 1. Among these actives, Thiamidol was by far the most efficient inhibitor of hTyr, with a half-maximal inhibitory concentration (IC50) of 1.1 mmol/L, with almost complete enzyme inhibition of hTyr occurring at concentrations above 10 mmol/L. The resorcinol derivatives 4-butylresorcinol, 4-hexylresorcinol, and 4-phenylethylresorcinol had IC50 values of 21mmol/L, 94mmol/L, and 131 mmol/L, respectively (Table 1). With an IC50 of about 500 mmol/L, kojic acid was 500 times less potent than Thiamidol. Hydroquinone and arbutin were both very poor inhibitors of hTyr, with IC50 values in the millimolar range. Kojic acid, arbutin, and hydroquinone were not able to completely inhibit hTyr in the concentration range tested. Racemic rhododendron was also rather ineffective as an inhibitor of L-dopa oxidation, with an IC50 >1,200 mmol/L (Figure 2a).

A detailed kinetic analysis of the inhibition of hTyr by Thiamidol yielded a strictly competitive type of inhibition with an inhibitor constant (Ki) of 0.25 mmol/L (Figure 2b, Table 1). This value is in agreement with the IC50 value estimated from dose-response curves (1.1 mmol/L) (cf. Figure 2a) which, for competitive inhibition, should be about 3 times higher than the Ki. The Ki values for 4-butyl resorcinol (9 mmol/L), 4-hexylresorcinol (39 mmol/L), and 4- phenylethylresorcinol (24 mmol/L) were also markedly higher than the Ki value of Thiamidol (Table 1). These data illustrate that the thiazolyl amide moiety of Thiamidol conveys a much better inhibition of hTyr than do the hydrocarbon side chains present in three other derivatives of resorcinol (4-butyl-, 4-hexyl-, and 4-phenyl ethyl resorcinol). As noted, the efficacy is distinctively different in mTyr, where 4-butyl resorcinol, 4-hexylresorcinol, and 4-phenylethylresorcinol, and even kojic acid, are superior to Thiamidol in inhibiting the enzyme (Table 1). Thus, Thiamidol would not have been identified as positive in a screening using mTyr, and the efficacy of 4-phenylethylresorcinol would have been grossly overestimated.

Garcia-Jimenez et al. (2016) recently reported that mTyr slowly oxidizes certain resorcinols, provided that the prevailing met- form of the enzyme is previously converted to either the oxy- or deoxy-form by additives like H2O2 and ascorbate and that the reaction is sustained by o-diphenols. Therefore, we used quantitative high-performance liquid chromatography analysis (Ito and Wakamatsu, 2015) to ascertain whether Thiamidol might also be a substrate of hTyr. In our normal assay conditions (i.e., in the absence of the additives mentioned by Garcia-Jimenez et al., 2016), no detectable oxidation of Thiamidol took place within several hours of incubation with hTyr, whereas rhododendron was readily oxidized within that time frame (see Supplementary Figure S1 online). Thus, we assume that the reaction described by Garcia-Jimenez et al. is not relevant for Thiamidol and hTyr in physiological conditions.

Inhibition of melanin production

We then tested the potential inhibitory effects of these compounds using a three-dimensional model for human skin. As observed with purified hTyr, arbutin showed only negligible efficacy at inhibiting melanin production in MelanoDerm (MatTek Corporation, Ashland, MA) skin models (IC50 > 4,000 mmol/L) (Figure 2c). Kojic acid inhibited melanin production with an IC50 of w400 mmol/L, showing a surprisingly steep dose-response curve, with concentrations below 200 mmol/L only slightly inhibiting melanin production (i.e., by 5% at 150 mmol/L). Rhododendron showed only marginal effects on melanogenesis, with an apparent IC50 for inhibition of w1,200 mmol/L. Hydroquinone inhibited melanin production in MelanoDerm skin models with an IC50 of 15 mmol/ L, suggesting that it has a mechanism other than tyrosinase inhibition. 4-Butylresorcinol inhibited melanin synthesis with an IC50 of 13.5 mmol/L. Again, Thiamidol was, by far, the most potent inhibitor of melanin production in MelanoDerm skin models, with an IC50 of 0.9 mmol/L, and in monolayer cultures, Thiamidol visibly reduced melanin formation (Figure 3a).

Hydroquinone and Thiamidol were then tested in long-term melanocyte monolayer cultures to check the potential reversibility of inhibition. Although 1 mmol/L Thiamidol reduced melanin production to less than 60% after 2 weeks, 1 mmol/L hydroquinone reduced melanin production only to approximately 85% (Figure 3b). However, upon further cultivation without the active compounds, melanocytes that had been inhibited by Thiamidol rapidly restarted their melanin production, reaching pretreatment levels within 1 week. In contrast, hydroquinone-treated cells did not recover their full capacity for melanin production within the 2-week culture period, and melanin production continued at 85% of pretreatment levels.

Molecular modeling

Possible binding modes of Thiamidol to hTyr were examined by virtual docking studies. Figure 4a shows the active site of the homology model of hTyr in the met- form, with a docked Thiamidol ligand in a lowest-energy conformation. The di-copper center with the bridging oxygen is visible on the left. Only amino acid residues immediately adjacent to the bound inhibitor are shown. (Residue numbering includes the signal peptide). The inner surface of the binding pocket is colored according to hydrophobicity on a scale from blue for hydrophilic to brown for hydrophobic. Although the environment of the di-copper center is distinctly hydrophilic, a strongly hydrophobic subpocket is formed mainly by the side chains of I368, V377, and F347. In the spatial orientation shown, the 1-hydroxy group of the aromatic ring of the ligand makes extensive contacts with the di-copper center, and the 3-hydroxy group is involved in hydrogen bonds with the side chain of S380 and the backbone carbonyl of M374. The thiazolyl ring is held in place by hydrophobic interactions with the nonpolar pocket (Figure 4b), formed by side chains of amino acids, most of which differ between mTyr and hTyr (Figure 4c).

Comparable results were obtained when Thiamidol was docked to the recently published x-ray structure of the structurally similar TRP1, a Zn2þ-containing melanogenic enzyme of yet unknown function in humans (Ghanem and Fabrice, 2011; Lai et al., 2017), suggesting that the TYRP1 enzyme is inhibited by Thiamidol as well (see Supplementary Figure S2 online).

Clinical studies

The in vivo efficacy of Thiamidol was then examined in clinical studies where elderly subjects treated age spots on their skin twice daily with a formula containing 0.2% Thiamidol or with the vehicle only as a control. Already after 4 weeks of treatment, the treated age spots were significantly lighter than the untreated control age spots (Figure 5a). Improvement continued over the entire treatment period, and after 12 weeks some of the age spots were indistinguishable from the surrounding normally pigmented skin (Figure 5b). EpiFlash (Canfifield Scientifific Inc., Parsippany, NJ) photographs showed visible improvement in the appearance of age spots, and the untreated control age spots remained unchanged (not shown). A follow-up study showed that concentrations of Thiamidol as low as 0.1% effectively reduced the visibility of age spots (see Supplementary Figure S3 online).

DISCUSSION

The safest and most effective way to treat cutaneous hyperpigmentation is to reduce melanin production by inhibiting tyrosinase activity. However, most tyrosinase inhibitors described in the literature lack clinical efficiency when incorporated into topical products. Almost all of them were tested only against mTyr (Espin et al., 2000; Garcia-Molina et al., 2005) and thus, although effective against mTyr, turned out to be poor inhibitors of hTyr. Commercially available mTyr is not a homogeneous preparation but rather is a mixture of several tyrosinase isoenzymes and small amounts of additional enzyme activities that may affect inhibition studies in unpredictable ways (Pretzler et al., 2017). Isoenzymes AbPPO3 and AbPPO4, the main components of commercially available mTyr, have amino acid sequences in the region of the active site that significantly differ from hTyr (Figure 4c). Both mTyr isoenzymes contain extra loops between Asn371 (one of the glycosylation sites of hTyr) and Gly372. Several of the residues interacting with Thiamidol in hTyr (cf. Figure 4b) are not conserved in mTyr, for example, Ile368, Ser375, and Ser380. Phe207 is structurally conserved in mTyr, whereas Phe347 is not. Because even small changes in enzyme-ligand interactions may have dramatic effects on binding affinities, the diverse inhibition profiles of hTyr and mTyr (summarized in Table 1) did not come as a surprise.

The main objective of this study was to compare the effects of arbutin, hydroquinone, and kojic acid with various resorcinol derivatives on the catalytic function of hTyr and on melanin production in vivo. Except for Thiamidol, all of the tested substances have been described as tyrosinase inhibitors (Kim et al., 2012); however, their reported inhibitory activities are extremely divergent. In the medical literature, hydroquinone is considered the criterion standard for the treatment of skin hyperpigmentation, although there are severe concerns regarding its safety. Hydroquinone is banned in the European Union from use in cosmetics, but it is still sold in the United States as an over-the-counter drug in formulations containing up to 2% hydroquinone. Recently, the US Food and Drug Administration (2006) expressed concern about hydroquinone; however, a final ruling is still pending. The published IC50 values for hydroquinone inhibition of mTyr cover a range from 1.1 mmol/L (Kang et al., 2003) to 680 mmol/L (Abu Ubeid et al., 2009). In our analysis, hydroquinone was remarkably ineffective against hTyr, inhibiting it only slightly, reaching just 50% inhibition at approximately 4,000 mmol/L. Although hydroquinone has been considered a tyrosinase inhibitor since the early 1990s (Palumbo et al., 1991), our results suggest that its cytotoxic properties are more important, not only for its adverse effects on melanocytes but also for its efficacy as an inhibitor of melanogenesis (Jimbow et al., 1974; Penney et al., 1984; Smith et al., 1988). This view is substantiated not only by our results with hTyr and the fact that hydroquinone significantly reduced melanin production in skin models but also by our experiments with melanocyte cultures. Here, hydroquinone reduced melanin production, but the treated cells did not regain the full capacity to produce melanin after the removal of the active.

Although arbutin is generally considered an effective tyrosinase inhibitor, the published IC50 values of arbutin for mTyr range from 40 mmol/L (Ying et al., 1999) to more than 30,000 mmol/L (Sugimoto et al., 2005). In our test system, we found very high IC50 values (>4,000 mmol/L) for arbutin with both purified hTyr and the MelanoDerm skin model. Data on the efficacy of both a-arbutin and b-arbutin have been published (Garcia-Jimenez et al., 2017). However, both compounds are hydroquinone pro-drugs, with their biological activity dependent on the release of hydroquinone from the molecule (Briganti et al., 2003). The European Union Scientific Committee on Consumer Products (2008) published a critical opinion on arbutin. Given the release of hydroquinone from the molecule, it regards the use of arbutin in cosmetic products is unsafe.

The published IC50 values for tyrosinase inhibition by kojic acid range from 6 mmol/L (Curto et al., 1999) to more than 100 mmol/L (Jeon et al., 2005). As an inhibitor of hTyr, kojic acid is much less efficient, with an IC50 of about 500 mmol/L. Kojic acid exhibits a mixed type of inhibition, with a Ki of 145 mmol/L, indicating that it binds to the deoxyform of tyrosinase (Sun et al., 2014). When used to treat the MelanoDerm model, kojic acid shows an exceptionally steep dose-response curve, with relative inhibition increasing from 5% at 150 mmol/L to more than 75% inhibition at 900 mmol/L (see Figure 2c). This fact may be the main reason for the very limited efficiency of kojic acid in vivo. Concerning the safety of kojic acid, the European Scientific Committee on Consumer Safety (2012) now considers kojic acid at concentrations up to 1.0% to be safe for cosmetic products when applied to healthy skin, a view shared by the Cosmetic Ingredient Review Expert Panel (Burnett et al., 2010).

Rhododendron was granted quasi-drug status in Japan in 2008 and was used as a whitening ingredient in cosmetic products. It was assumed to be a competitive inhibitor of tyrosinase. However, in 2013, rhododendron-containing products were recalled in 10 Asian countries when close to 20,000 consumers developed leukoderma after using the products. It was shown that rhododendron is not only an inhibitor but is also a substrate of both hTyr (Ito et al., 2014a) and mTyr (Ito et al., 2014b). The tyrosinase-dependent accumulation of endoplasmic reticulum stress and/or activation of the apoptotic pathway may contribute to the melanocyte cytotoxicity of rhododendron (Sasaki et al., 2014).

The 4-substituted resorcinol motif has been known for some time as an efficient chemical moiety that inhibits tyrosinase (Khatib et al., 2005). Many natural compounds that have been identified as whitening agents, mainly flavonoids, contain this motif (Shimizu et al., 2000, 2011). Because the bioavailability of flavonoids is generally low, our goal was to identify resorcinol derivatives with better effectiveness and bioavailability. 4-Butylresorcinol had already been identified as an inhibitor of mouse and human tyrosinase (Kim et al., 2005; Kolbe et al., 2013) and of the dihydroxy indole carboxylic acid oxidase activity of mouse TYRP1 (Katagiri et al., 2001), and it is commercially available for the medical and cosmetic treatment of hyperpigmentation (Bohnsack et al., 2012; Jimenez and Garcia-Carmona, 1997; Kim et al., 2005). Nevertheless, detailed kinetic data of 4-butyl resorcinol were still missing. In the hTyr assay, we found a strictly competitive type of inhibition by 4-butyl resorcinol with a Ki of 9.1 mmol/L, which is in excellent agreement with the IC50 value determined (Table 1).

In our in vitro experiments, Thiamidol, with an IC50 of 1.1 mmol/L in the hTyr enzyme assay and 0.9 mmol/L in the MelanoDerm skin model, was by far the most effective of all substances tested. Further experiments confirmed that Thiamidol is a strictly competitive inhibitor (Figure 2b) and not a substrate for tyrosinase (see Supplementary Figure S1), and, thus, Thiamidol is not converted to a toxic and potentially leukoderma-inducing quinone. Therefore, Thiamidol was selected for clinical studies to assess its efficacy in vivo. A study of Thiamidol using a spot applicator showed continuous improvement in the appearance of age spots over the entire 12-week treatment period, reaching statistical significance as early as 4 weeks. These results show a strong pigment-reducing efficacy of the Thiamidol test product and a clear clinical benefit in the management of skin hyperpigmentation.

MATERIALS AND METHODS

Human tyrosinase

A truncated, His-tagged form of hTyr (hTyr-DHis) comprising the catalytic domain of hTyr was expressed in HEK 293 cells and puri- verified by metal affinity chromatography on Ni2þ-Sepharose (GE Healthcare, Munich, Germany) as described elsewhere (Cordes et al., 2013). The resulting preparation had the same catalytic properties as wild-type hTyr.

Sources of inhibitors

From the Evotec compound library (Evotec, Hamburg, Germany), 50,000 compounds, covering a wide chemical space, were selected to conduct an HTS for hTyr inhibitors, assessed using the Tyr assay described in the next section. Derivatives of promising lead compounds were then synthesized for further optimization. The other inhibitors were purchased from various suppliers (see Supplementary Materials online for details).

Tyrosine assay and HTS procedure

Full details of the L-dopa oxidase activity and the HTS screening procedures used can be found in the Supplementary Materials.

Molecular modeling

In silico docking was based on a new homology model of hTyr, described elsewhere (Mann et al., 2017). The simulations were performed using Molegro Virtual Docker (Molegro, Aarhus, Denmark). Discovery Studio Visualizer 4.0 (Accelrys, San Diego, CA) was used for visual data analysis and presentation. The sequences were taken from the UniProt database (UniProt Consortium, 2017).

Skin model assays

Full details of MelanoDerm tissues used as a skin model and the quantity n of their melanin content can be found in the Supplementary Materials.

Melanocyte cultures

Full details of melanocyte cultures and the quantitation of their melanin content can be found in the Supplementary Materials.

Clinical studies

Two randomized in vivo studies (blinded for the test products, open for the untreated control) were conducted. One study enrolled 18 female subjects (56e71 years of age), with 17 subjects completing the study. The second study was performed with 19 subjects (18 females, 1 male; 58e70 years of age), with all 19 subjects completing the study. Each subject applied two different formulations twice daily to age spots on their volar forearms using a spot applicator. The formulations differed only in the active ingredient: 0.2% Thiamidol versus vehicle in the first study, and 0.1% Thiamidol versus vehicle in the second study. One age spot per subject was treated with a formula containing the active ingredient, and a control spot was treated with the vehicle only. The pigmentation of the age spots was analyzed as described in the Supplementary Materials. The in vivo studies were conducted according to the recommendations of the current version of the Declaration of Helsinki and the guidelines of the International Conference on Harmonization of Good Clinical Practice. All participants in these studies provided written informed consent. In addition, the studies were approved and cleared by the institutional review board of Beiersdorf AG (Hamburg, Germany).

CONFLICT OF INTEREST

SUPPLEMENTARY MATERIAL

Supplementary material is linked to the online version of the paper.

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