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.

SKIN PENETRATION BY COPPER-PEPTIDE COMPLEXES 61 In the literature we may find that GSH has an activity of reducing free radicals and peroxides that are responsible for tyrosinase activation, melanin formation, and modu­ lation of the depigmenting abilities of melanocytotoxic agents. This leads to the skin lightening effect of GSH application and a possibility of its usage in the treatment of pigmentary disorders (17-20). Another important issue in skin protectioning is the anti-UV (UVA and UVB) radiation activity of the cosmetic ingredient. Glutathione is one of the ingredients that may play such a role (21-24). Due to the lack of data concerning the transport of the peptides and their complexes through the skin, we focused our research on this subject. The main goals of our experiments were to prove the ability of copper tripeptide complexes to penetrate the skin, to determine the permeability coefficient for these compounds, and to establish the form of the compound that actually penetrates through the membrane. Our previous study (25) proved that cooper peptides can migrate through the model lipophilic mem­ brane from an aqueous solution (25), which made us continue the investigation of the transport of copper peptide complexes, but this time through an emulsion. Since most of the cosmetic formulae used as a source of active ingredients, like peptides and their complexes, are O/W emulsions, we used them in our investigations. The in vitro penetration process was studied in the model system, a Franz diffusion cell (26-28) with a liposome membrane, where liquid crystalline systems of physicochemical prop­ erties similar to the ones of the intercellular cement of stratum corneum were used as a standard model of a skin barrier (29-32). MATERIALS AND METHODS TYPES OF APPARATUS The absorption spectra were recorded using a SPECOL 11 spectrophotometer (Zeiss, Jena, Germany) with 5-mm glass cells. The pH measurements were carried out using an Elmetron ES24 pH meter (Poland). The reversed-phase liquid chromatographic experiment (RPLC) was performed by a Perkin Elmer binary LC 250 computer-controlled pump (Norwalk, CT) and a Rheodyne model 7125 injection vial with injection loops (20 µl) (Cotati, Rheodyne, CA) with a Perkin Elmer model LC-95 UV/Vis spectrophotometric detector. Peptides were sepa­ rated on the Hypersil BOS C18 analytical column (4.0 x 125 mm) (Agilent Technolo­ gies, Wilmington, NC). The acquisition and handling of the data were carried out with a 1020 LC Plus (Perkin Elmer) computer program. The copper peptide complexes were characterized by an ESI mass spectrometer, LC-MSD ll00 (Agilent) with a quadrupole mass analyzer (HP7500A). REAGENTS • A stock Cu(II) solution, (1 mg/ml- 1 ) was obtained by dissolution of copper(II) chlo­ ride dehydrate (POCH, Gliwice, Poland) in water. • A GHK-Cu solution (0.01 M) was prepared by dissolution of Prezatide copper acetate (GHK-Cu) (ProCyte Corporation, USA) in water. • A GSH stock solution (l0mg/ml) was prepared by dissolution of glutathione (Sigma-