In Vitro Assessment of Irritation Potential of an Anti-Melanogenic Agent,PF3758309,in a Reconstructed Human Epidermis Model

Purpose:This study was designed to determine whether a novel anti-melanogenic agent,PF3758309,has the potential to cause acute cutaneous irritation using a human skin equivalent model (HSEM).Methods:Human skin equivalent was constructed through incubation of normal human derived epidermal keratinocytes (HEKs)on collagen matrix insert with proliferation media.Using constructed human skin equivalent,the irritation potential of PF3758309 were investigated whether the viability of this agent-treated HESM is under 50%(irritant)or not (non-irritant)using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)assay after applying the agent to the epidermal surface of the HSEM.Also,the PF3758309-mediated anti-pigmentation effects were analyzed using Fontana-Masson staining in the HSEM.Results:The integrity of the constructed HSEM was confirmed using immunohistochemical staining with differentiation markers of epidermis,and observed that Keratin 5,Keratin I and Involucrin were stained in basal,supra-basal and granular layers,respectively.In vitro irritation assay showed that the mean viabilities of the PF3758309 was 83.6%,78.8%and 77.8%at the treatment doses of 0.2,0.5 and I mg,respectively;however,the mean viability of the positive control (5%sodium dodecyl sulfate)-treated HESM was 2.8%.Also,in vitro corrosion assay showed that the mean viabilities of the PF3758309 was 95.3%,95.0%and 94.2% at the treatment doses of 0.2,0.5 and 1 mg,respectively.Furthermore,using a Fontana-Masson staining assay,the melanin levels in the PF3758309-treated HSEM was significantly decreased compared with the levels in control HSEM.Conclusion: The anti-melanogenic agent,PF3758309,has no skin irritation potential under the conditions used in this study.