Antioxidant Effects of N-acetylcysteine Amide and NPI-002 on Human Skin or Equivalents

Purpose: Nacuity Pharmaceuticals, Inc., a leading pharmaceutical developer of ocular small molecule antioxidants, sought to explore the antioxidant potential of N-acetylcysteine amide (NACA) and another new chemical entity, NPI-002, on skin, or equivalents. NACA and NPI-002 were evaluated for their effects on melanogenesis in reconstructed human epidermal (RHE) cell cultures, as well as glutathione levels in healthy human skin explants after ultraviolet irradiation.

Methods: Skin lightening: The skin lightening assessment was conducted using RHE cultures containing melanocytes from African American descent (MelanoDerm^TM).  NACA or NPI-002 were individually added directly into the culture media every other day at a final concentration of 10mM. Test articles were compared to an untreated sample (negative control) and a sample pre-treated with 2% glutathione disulfide (GSSG) positive control.  Samples were harvested for skin lightening assessment after 7 and 14 days of culture, with day 0 samples retained for comparison.  At harvest, samples were photographed and extracted for melanin quantification/ content.
Melanin quantification: Two of the 6 replicates per treatment were retained for melanin quantification.  At harvest, samples were submerged for 30min in 1% sodium bicarbonate solution and transferred to Eppendorf tubes with Solvable™ reagent for overnight incubation at 60 °C.  A synthetic melanin standard curve was generated in Solvable™ reagent and transferred to a 96-well plate and read at 490nm for quantification (expressed as mg/ml melanin content).
Tyrosinase activity: Tyrosinase activity was assessed per kit instructions (BioVision cat#K742-100). Tissue was either non-treated (negative control) or pre-treated with either NACA (0.1, 1 or 10 mM of each), NPI-002 (0.1, 1 or 10 mM of each), or 2% kojic acid (positive control). Tissue were lifted from the Transwell^® for a 30min incubation in 1% sodium bicarbonate solution and transferred to ceramic bead homogenization tubes containing chilled tyrosinase assay buffer. Tissue was stored at -80°C until time of assay. After homogenisation, a bicinchoninic acid assay was performed for protein quantification and 10µg lysate was used for the tyrosinase assay. Untreated tissue served as negative control. A kinetic read was taken (510nm absorbance minus background control) at 10min to 30min intervals out to 120min and graphed as nmol/ min.
Antioxidant potential: The protective effects of NACA or NPI-002 against UVA radiation-induced oxidative skin damage was evaluated. Ex vivo human skin from a single light-skinned Caucasian donor was exposed to UVA radiation (24 J/cm²) with and without pre-treatment by topical application of NACA and NPI-002 applied at a final concentration of 100 mM each, solvated in 10% DMSO/water.  The treatments were applied approximately 30min prior to UVA exposure and tissue was harvested approximately 24hrs following exposure.  Samples were heat separated into epidermal and dermal layers, weighed, and submerged into a 150µM 2-chloro-1-methylpyridinium iodide (CMPI) derivatization solution, and analyzed for glutathione content. Tissue, with and without pre-treatment with 100 mM NACA,or 100 mM NPI-002 was exposed to UVA followed by assessment of glutathione and N-acetycysteine (NAC) content by LCMS. 

Results: NACA treatment at 10mM successfully inhibited the pigmentation of MelanoDerm™ cultures over a 14-day period to a greater extent than either 2% GSSG or 10mM NPI-002 based on both visual scoring and melanin extraction.
Compared to the day 14 untreated control, tyrosinase activity decreased greater than 50% with 1mM NACA or NPI-002.  NACA or NPI-002 at 10mM decreased tyrosinase similarly to 2% Kojic acid positive control. 
Tissue, with and without pre-treatment with 100 mM NACA,or 100 mM NPI-002 and exposed to UVA was assessed for glutathione and N-acetycysteine (NAC) content by a qualified LCMS method. Both 100mM NACA and NPI-002 pretreatment significantly (p< 0.05) increased glutathione and NAC content compared to untreated and UVA-exposed tissue.

Conclusion: Melanin, the primary component of skin pigmentation and secreted by melanocytes, protects the skin from harmful UV radiation, however, accumulation of melanin pigment in sun-damaged skin results in various hyperpigmentation disorders which can be a source of distress. 
Antioxidants, NACA and NPI-002, inhibited melanin production in human melanocytes and increased glutathione and NAC in healthy human skin explants exposed to UV-radiation. Taken together, these findings support the theory that reactive oxygen species are involved in melanogenesis in RHE cultures as well as skin exposure to UV irradiation.  
Glutathione (GSH) inhibits melanogenesis by suppressing the activity of tyrosinase, and oral administration of GSH in humans reduces melanin production in the skin. Indeed, significant inhibition of tyrosinase activity was observed in a dose responsive manner with NACA and NPI-002.
Based on these results, additional research is warranted to further elucidate the effects of NACA and NPI-002 for their potential therapeutic utility as antioxidants, i.e., potential therapeutics, for skin.