Hong-Ju Shin

Different effects of five depigmentary compounds, rhododendrol, raspberry ketone, monobenzone, rucinol and AP736 on melanogenesis and viability of human epidermal melanocytes

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. This study assessed the in vitro effects of five depigmentary compounds including leukoderma‐inducing agents. 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 (UVB) radiation. Concentrations of RD, RK and MB that inhibit melanogenesis are similar to concentrations that are cytotoxic; however, concentrations of rucinol (RC) and AP736 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 RC and AP736. 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.

Oral and topical pharmacokinetic studies of a novel TRPV1 antagonist, PAC-14028 in rats and minipigs using liquid chromatography/tandem mass spectrometric method

PAC-14028 ((E)-N-((R)-1-(3,5-difluoro-4-methanesulfonylamino-phenyl)-ethyl)-3-(2-propyl-6-trifluoromethyl-pyridine-3-yl)-acrylamide) is a novel and potent transient receptor potential vanilloid type I (TRPV1) antagonist. We developed and validated a rapid, sensitive and selective liquid chromatography/tandem mass spectrometric method for determination of PAC-14028 in rat and minipig plasma. After protein precipitation PAC-14028 and internal standard (methylated analog, PAC-14026) were separated on a Symmetry C(18) column (4.6 mm × 75 mm, 3.5 μm) with an isocratic mobile phase, acetonitrile: water (8:2, v/v) containing 0.2% formic acid and monitored by electrospray positive ionization with multiple reaction monitoring mode (PAC-14028, 492→156; IS, 506→156, m/z). The calibration curve was linear over the range of 1.0-500 ng/ml (r(2)>0.999) and lower limit of quantitation (LLOQ) was 1 ng/ml. The precision and accuracy were within ± 15% and the stability was acceptable during bench-top, auto-sampler, 3 freeze-thaw cycles and 4-week storage in a freezer at -80°C. This method was successfully applied to the intravenous, oral and topical pharmacokinetic studies of PAC-14028 in rats and minipigs, which showed comparable pharmacokinetic parameters (T1/2, 2.1h and 3.8h; F%, 52.7% and 64.2% for rats and minipigs, respectively). Percutaneous absorption of PAC-14028 was negligible after topical application (F% 0.2-1.7%).

A novel adamantyl benzylbenzamide derivative, AP736, inhibits melanogenesis in B16F10 mouse melanoma cells via glycogen synthase kinase 3β phosphorylation

Recently, much effort has been made to develop effective dermatological depigmenting compounds. In this study, we investigated the novel candidate compound, AP736 (an adamantyl benzylbenzamide derivative), and its effects on melanogenesis in B16F10 melanoma cells, as well as the mechanisms involved. AP736 has been reported to exert anti-melanogenic effects in melanocytes in vitro and in artificial skin equivalents through the inhibition of key melanogenic enzymes and the suppression of the cAMP-protein kinase A (PKA)-cAMP response element‑binding protein (CREB) signaling pathway. Thus, we examined another pathway of melanogenesis involving the effects of AP736 on the glycogen synthesis kinase 3β (GSK3β) pathway. Melanin content and tyrosinase activity were measured using a spectrophotometer after the cells were treated with AP736. The AP736-induced activation of signaling pathways was examined by western blot analysis. We confirmed that AP736 decreased melanin production in a dose-dependent manner; however, it did not directly inhibit tyrosinase, the rate-limiting melanogenic enzyme. The expression of microphthalmia-associated transcription factor, tyrosinase, and related signal transduction pathways was also investigated. The Wnt signaling pathway is deeply involved in melanogenesis; therefore, phosphorylation by GSK3β was assessed following treatment with AP736. AP736 induced GSK3β phosphorylation (inactivation), but it did not alter the level of β-catenin. Furthermore, the expression of α-melanocyte-stimulating hormone-induced tyrosinase was downregulated by AP736. Our data suggest that AP736 exerts hypopigmentary effects through the downregulation of tyrosinase via GSK3β phosphorylation.

Effect of high advanced-collagen tripeptide on wound healing and skin recovery after fractional photothermolysis treatment

Collagens have long been used in pharmaceuticals and food supplements for the improvement of skin.

Development and evaluation of topical formulations for a novel skin whitening agent (AP736) using Hansen solubility parameters and PEG-PCL polymers

ABSTRACT AP736 itself is a novel skin whitening agent reported to exhibit anti‐melanogenic and tyrosinase inhibitory activity. However, formulating a topical product has been difficult because AP736 is insoluble in water as well as in many oils. In this study, we aimed to develop a new topical delivery system in which AP736 is not only physically stable, but also suitably delivered to the skin. By calculating each HSP (Hansen Solubility Parameters), ethylenedioxy moiety‐containing compounds could be easily selected for the formulation ingredients of AP736. Although diethylene glycol monoethyl ether with the highest solubility of AP736 enalbes to make AP736‐incorporated water‐in‐oil emulsions well, the recrystallization of AP736 was observed in oil‐in‐water emulsions. Therefore, we fabricated polymeric nanoparticles (PNPs) in order to encapsulate AP736 to prevent its recrystallization. We used three different PEG‐PCL polymers with various chain lengths and ethylenedioxy moiety‐containing surfactants (i.e. Choleth) for fabricating PNPs. The prepared PNPs had a mean particle size from 50nm to 200nm. Most of PNPs showed the good encapsulation efficiency up to 90%. In particular, Choleth‐24 had a significant role in encapsulating AP736 in PNPs. After encapsulation of AP736, no significant changes were observed in the sizes of tested PNPs within 4weeks. Further, the recrystallization of AP736 was not observed in oil‐in‐water emulsions after 24weeks of storage at 40°C. In vitro permeation study using Strat‐M showed that PNPs containing Choleth‐24 has the faster release pattern compared to PNPs using Tween 80 and saturated in D.I. water. These results are demonstrating that PNPs might be an effective vehicle for stabilization in oil‐in‐water emulsions and topical application of AP736.

AP736 induces miR-125b expression for the efficient whitening and anti-ageing action in human epidermal cells

Whether specific Tcell clones are present in tumor infiltrating lymphocytes (TILs) in BCC is unknown. We employed deep sequencing of mRNA coding for the Tcell receptor (TCR) chains αand β to characterize the repertoire of TILs in BCC. V and J gene-usage and CDR3 length were computed to determine the clonality of TCR and degree of overlap in TCR repertoires between skin resident T-cells and TILs. We found high diversity of the TCR repertoire in BCC and control skin with random VJ gene usage and similar CDR3-length distribution. Lack of TCR repertoire restriction indicates absence of tumorspecific TIL clones in BCC. 1 | BACKGROUND Basal cell carcinoma (BCC) of the skin is the commonest cancer worldwide with a locally invasive growth pattern.[1] The tumor microenvironment is characterized by abundant tumorinfiltrating lymphocytes (TILs) thought to be involved in cancer immunoediting,[2] the process in which early interaction between tumor cells and lymphocytes favours tumor inhibition followed by disequilibrium promoting tumor growth in later stages.[3] This disequilibrium may be further perturbed by immunosuppression, as documented by significantly increased risk of BCC in solid organ transplant recipients and allogeneic hematopoietic stem cell transplant recipients.[4,5] We have recently studied TILs in BCC and identified recruitment of Tregs to BCC causing local immunosuppression.[6] Whether specific Tcell clones are present in BCC and whether TILs

Antimelanogenic activity of a novel adamantyl benzylbenzamide derivative, AP736: a randomized, double-blind, vehicle-controlled comparative clinical trial performed in patients with hyperpigmentation during the summer

AP736 is a novel compound with an adamantyl benzylbenzamide moiety that has shown antimelanogenic activity in melanocytes in vitro and in artificial skin equivalent through the inhibition of key melanogenic enzymes and suppression of the cAMP‐phosphokinase A‐cAMP response element‐binding protein signaling pathway. To estimate the clinical effectiveness of AP736 for the treatment of facial hyperpigmentation, we examined the efficacy and safety of a topical formulation containing AP736 compared with a vehicle formulation in human facial skin. To evaluate the degree of whitening when used in a real‐life situation, subjects with hyperpigmentation conditions were selected and the trial was performed from mid‐May to the end of June, when there are strong UV rays in Korea.