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A Novel Adamantyl N-benzylbenzamide Derivative (AP736TM) for Regulation of Epidermal Melanogenesis

A Novel Adamantyl N-benzylbenzamide Derivative (AP736TM) for Regulation of Epidermal Melanogenesis
Baek, Heung Soo
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The color of the skin is determined relatively by melanin which is a pigment existing in the epidermis. Melanin is synthesized in a special organelles called melanosomes, which contain key enzymes such as tyrosinase and tyrosinase related proteins (TRPs). Melanin has a variety of roles, but the most important thing is the role of protecting human skin from ultraviolet rays. Differences in race and skin color are related to the number, size, shape, distribution and disintegration of melanosomes. Despite the convenient function of protecting the skin from UV radiation, excessive pigmentation can cause aesthetic problems such as melisma, post-inflammatory pigmentation (PIH), age spots, etc. Upon exposure of the skin to UV radiation, melanogenesis is enhanced by the activation of the key enzyme of melanogenesis, tyrosinase. Tyrosinase is a glycoprotein located in the membrane of the melanosome, a mini-factorial vesicle inside the melanocyte. Therefore, the main therapeutic strategy for the pigmentation disorders is to inhibit tyrosinase activity. Thus, there is considerable interest in the development of new tyrosinase inhibitors with improved potency for use as de-pigmenting agents. However, it has proven difficult to develop tyrosinase inhibitors that have sufficient clinical efficacy as well as in vitro efficacy. Indeed, most known depigmenting agents currently in use have unsatisfactory efficacy in humans, in addition to low stability, and skin irritation. In order to obtain a significantly more effective agent, we sought to develop a small molecule possessing both anti-melanogenic activity and tyrosinase inhibition, which we anticipate will possess substantial clinical efficacy in skin depigmentation. In chapter 2, we synthesized a series of potent anti-melanogenic agents while preserving tyrosinase inhibitory activity by modulating the physicochemical properties of the molecules. We determined that modulation of molecular lipophilicity by modification via hydrophobic functional groups is important for enhanced depigmenting activity without a concomitant increase in cytotoxicity, and the selection of the position and numbers of OH is critical for tyrosinase inhibitory activity. In addition, we confirmed the presence of an adamantyl group at the 5-position on the A ring seems not only to affect melanogenesis through its relative lipophilicity, but also reinforces binding interactions between the resorcinol moiety (B ring) of the ligand and the active site of tyrosinase. Among these analogues, 5-adamantyl-N-(2,4-dihydroxybenzyl)-2,4-dimethoxybenzamide (AP736TM) was selected as the final whitening candidate in consideration of anti-melanogenesis and cell viability. In chapter 3, we investigated the depigmenting effect and biological depigmenting mechanism of AP736TM, a whitening candidate derived from chapter 2. Here we found that a tyrosinase inhibitor, AP736TM potently suppresses tyrosinase expression, and the mechanism underlying was elucidated. AP736TM attenuated the melanin production induced by diverse melanogenic stimuli in murine and human melanocytes. It suppressed the expression of key melanogenic enzymes
tyrosinase, tyrosinase-related protein-1 and -2. The expression of microphthalmia-associated transcription factor (MiTF), a major promoter of melanogenesis was also decreased. AP736TM inhibited the activation of cAMP response element-binding protein (CREB) and phosphokinase A (PKA), and cAMP elevation, reflecting that cAMP-PKA-CREB signaling axis was suppressed, resulting in the down-regulation of MiTF and tyrosinase. Along with the previously reported tyrosinase inhibitory activity, the suppression of cAMP-PKA-CREB-mediated MiTF and tyrosinase expression by AP736TM may be efficient for the treatment of hyperpigmentation. In chapter 4, we demonstrated the anti-melanogenic and anti-aging efficacy of AP736TM and identify modulation of miR-125b expression as a possible mechanism of action.AP736 TM induced miR-125b expression in human primary melanocytes. Using a UVB-stimulated artificial skin model, we found that AP736 TM exerted whitening effects that were accompanied by increased miR-125b expression. We also found that AP736 TM increased miR-125b expression in UVB-stimulated keratinocytes. Notably, AP736 TM scavenged reactive oxygen species and inhibited the expression of MMP-1 and -3 mRNA and protein expression in UVB-stimulated keratinocytes. These results suggest that AP736 TM not only has whitening ability but also anti-aging activity in UVB-stimulated human epidermal cells, effects that are attributable, at least in part, to the action of AP736 TM as a miR-125b inducer. In chapter 5, we conducted a study on the safety evaluation of AP736TM, a whitening candidate substance. Currently numerous medications are used to treat hyperpigmentation. However, several reports have indicated that repeated application of some agents, such as rhododendrol(RD), raspberry ketone(RK), and monobenzone(MB), can be toxic to melanocytes. Although these agents had severe side effects in human trials, no current in vitro methods can predict the safety of such drugs. The present study assessed the in vitro effects of five leukoderma-inducing compounds. In particular, we determined the effects of different concentrations and exposure times of different depigmentary agents on cell viability and melanogenesis in the presence and absence of ultraviolet B radiation. Concentrations of RD, RK and MB that inhibit melanogenesis are similar to concentrations that are cytotoxic
however, concentration of AP736TM that inhibit melanogenesis are much lower than concentrations that are cytotoxic. Furthermore, the concentrations that cause toxic effects depend on exposure duration, and prolonged exposure to RD, RK and MB had more cytotoxic effects than prolonged exposure to AP736TM. The cytotoxic effects of RD and RK appear to be mediated by apoptosis due to increased expression of caspase-3 and caspase-8
UVB radiation increased the cytotoxicity of these agents and also increased caspase activity. Our results indicate that different leukoderma-inducing compounds have different effects on the viability of normal epidermal melanocytes and suggest that the in vitro assay used here can be used to predict whether an investigational compound that induces leukoderma may lead to adverse effects in human trials. In chapter 6, we conducted a clinical trial to compare the whitening effects of a topical formulation containing AP736TM with that of a vehicle control formulation to verify the findings obtained using the in vitro model. To estimate the clinical effectiveness of AP736TM for the treatment of facial hyperpigmentation, we examined the efficacy and safety of a topical formulation containing AP736TM compared with a vehicle formulation in human facial skin. we demonstrated that a formulation containing a novel skin whitening ingredient, AP736TM, effectively reduced pigmentation and was well tolerated by study subjects.
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