]. Cosmet. Sci., 59, 59-69 Qanuary/February 2008) Biological activities of selected peptides: Skin penetration ability of copper complexes with peptides LENAMAZUROWSKA and MIROSLAW MOJSKI, Warsaw University of Technology, Faculty of Chemistry, Noakowskiego 3, 01-664 Warsaw, Poland. Accepted for publication August 15, 2007. Synopsis This study concerning the permeability through skin barriers of copper complexes with peptides is an important part of the research on their biological activity. The transport of copper complexes through the skin is essential in treatment of dermatological dysfunctions connected to the deficiency of these elements in the skin. During the last several years, a special interest in transepidermal copper delivery has been observed. This is the reason why copper compounds have been used as active compounds in care cosmetics. Yet, the transport process of copper complexes with tripeptides, glycyl-histidyl-lysine GHK, or )'-glutamyl­ cysteinyl-glycine GSH through the stratum corneum has received very little attention in the literature so far. The penetration ability of GHK-Cu and GSH-Cu through the stratum corneum and the influence of the complexes with tripeptide on the copper ion transport process is the key factor in their cosmetic and pharmaceutical activity. The in vitro penetration process was studied in the model system, a Franz diffusion cell with a liposome membrane, where liquid crystalline systems of physicochemical properties similar to the ones of the intercellular cement of stratum corneum were used as a standard model of a skin barrier. The results obtained demonstrated that copper complexes permeate through the membranes modeling the horny lipid layer and showed the influence of peptides on the dynamics of copper ion diffusion. INTRODUCTION The existence of metal ions is essential for all living organisms because they are con­ stituents of a large group of enzymes responsible for different physiological processes. Thus, they determine the proper functioning of the whole body, including the skin tissue. One of these essential metals is copper, which, according to its antiradical activity, the potential of regulating the melanogenesis process, and the synthesis of collagen, elastine, and GAGs (1,2), is widely used as a cosmetic ingredient. Despite the fact that copper is one of the most important metals for normal skin activity and growth, not all of the copper compounds, because of their toxicity, may be used as Address all correspondence to Lena Mazurowska. 59

60 JOURNAL OF COSMETIC SCIENCE cosmetic ingredients. Therefore, the simplest form of copper, an inorganic salt, cannot be a possible source of delivery of the metal ions to the low layers of skin because of its general toxicity to the organism. This is why other ways of transporting copper to the deep layers of skin tissue have to be found. One of the widely used methods of delivering metal ions into the skin is its complexation with different ligands, among which amino acids and peptides play a main role. In our investigation, small biological active peptides were used because, beyond their transport potential, they may function as an active ingredient in the cosmetic formulae. Active peptides show cosmetically interesting activities such as stimulation of collagen synthesis, chemotaxis, and antistaining effects (3 ). Among many possible natural ligands, GHK and GSH are mainly used due to their properties. Both peptides are intensively investigated because of their existence in the human organism and the different biological effects that they may show. The complexes of GHK and GSH with copper are widely known as protection and repair agents for skin tissue and because of this are often used as cosmetic ingredients. Originally, GHK-Cu glycyl-histidyl-lysine-Cu(II) was found in human plasma, and all of its properties were drawn on the basis of these investigations. GHK-Cu was isolated from human plasma by Pickart and Thaler in 1973 (4). (see also references 5 and 6). Formerly, GHK peptides found an application in medicine. This peptide was first described as a growth factor for a variety of differentiated cells. What is more, recent data suggest its physiological role is related to the process of wound healing and tissue repair (7-10). In further studies it was recognized that GHK is endowed with a wide range of more systemic biological activities including angiogenesis (blood vessel formation) (11), ac­ celeration of bone repair (12), and superoxide dismutase-like activity (13). GHK may also have other activities when it is complexed with the Cu metal ion, like the secretion of the tissue inhibitors of metalloproteinase (14). GSH is the next peptide that was isolated from a human body and enjoyed many researchers' attention. The tripeptide ')'-glutamyl-cysteinyl-glycine (GSH) is the major nonenzymatic regulator of intercellular redox homeostasis and is ubiquitously present in all cell types at millimolar concentrations. This cystein-containing tripeptide exists either in a reduced (GSH) or oxidized (GSSG) form, better referred to as glutathione disulfide, and participates in redox reactions by the reversible oxidation of its active thiol (15). Glutathione in the reduced (GSH) and oxidized (GSSG) forms is the main intra­ cellular non-protein thiol that performs the important biological functions involved in active transport of amino acids (-y-glutamyl cycle), operating enzymes (glutathione S-transferase, glutathione peroxidase, and glutathione reductase), complex formation with microelements (Zn2+, Cu2+), and functioning of the redox couple Cu2+ -Cu+ (16). GSH has many ascribed biological functions for skin, and one of them is implicated in skin lightening. In vivo and in vitro studies in the literature show the evidence of its involvement in the melanogenic pathway and shed light on its anti-melanogenic effect. The proposed mechanisms of action include the direct inactivation of the enzyme ty­ rosinase by binding with the copper-containing active site of the enzyme and by me­ diating the switch mechanism from eumelanin to phaeomelanin production.