j. Cosmet. Sci., 54, 483-491 (September/October 2003) Penetration enhancement in mouse skin and lipolysis in adipocytes by TAT-GKH, a new cosmetic ingredient JUN MAN LIM, MIN YOUL CHANG, SUN GYOO PARK, NAE GYU KANG, YOUNG SOOK SONG, YOUNG HWA LEE, YOUNG CHANG YOO, WAN GOO CHO, SOO YOUNG CHOI, and SHE HOON KANG, Cosmetic ROD Center, LG Household O Healthcare Research Park 84, Jang-dong, Yusong-gu, Taejon 305-343 (J.M.L., M.Y.C., S.G.P., N.G.K., Y.S.S., Y.H.L., Y.C.Y., W.G.C., S.E.K.), and Division of Lij• Sciences, Hallym University, Chunchon 200-702 (S. Y.C.), Korea. Accepted for publication November 15, 2002. Synopsis Since the basic domain of human immunodeficiency virus type I (HIV-1) transactivator of transcription (TAT) protein was reported to possess the ability to traverse biological membranes efficiently, various therapeutic proteins have been attached to TAT for the purpose of therapy. In this study, the tripeptide GKH (glycine-lysine-histidine) derived from parathyroid hormone (PTH), known as lipolytic peptide, was attached to 9-poly lysine (TAT) to be used as a cosmetic ingredient in slimming products. TAT-GKH at 10 -5 M induced approximately 37.6% and 41.5% maximal lipolytic effects in cultured 3T3-L1 differen- tiated adipocytes and in epididymal adipocytes isolated from rats, respectively, compared with basal lipoly- sis. The lipolytic effect of GKH was not changed by TAT-GKH fusion. In cytotoxicity tests, there was no cytotoxicity in any dose concentration of TAT-GKH. We confirmed that TAT-GKH induced lipolytic activity by GKH without cytotoxicity and with the possibility of its use as a safe cosmetic ingredient. TAT-GKH elevated penetration into excised hairless mice skin 36 times more efficiently than GKH. TAT-GKH can be used as a cosmetic ingredient in slimming products, with both penetration enhancement and lipolytic effect without cytotoxicity. INTRODUCTION In the effort to overcome the limiting barrier of drug delivery to skin, various physical and chemical vehicles such as micelies, liposomes, microemulsions, and nanoparticles have been used in cosmetic science as well as in pharmaceutical science. However, these approaches have been observed not only to be dependent on the applied constituents of Address all correspondence to Jun Man Lim. 483

484 JOURNAL OF COSMETIC SCIENCE the vehicle but also drastically on the composition/internal structure of the phase, which may hamper drug diffusion in the vehicles (1-3). Recently, the use of peptide and protein domains with amphipatic sequences for drug and gene delivery is getting increasing attention (4). The basic domain of human immunodeficiency virus type I (HIV-1) transactivator of transcription (TAT) protein was reported to possess the ability to traverse biological membranes efficiently in a process termed "protein transduction" (5-7). Furthermore, TAT peptide, chemically attached to various proteins, including horseradish peroxidase, [3-galactosidase, and ovalbumin, was able to deliver these proteins to various cells and even in mice tissues, with high levels in the heart, lung and spleen (8,9). Although the actual mechanism of TAT has not yet clearly been established, common structural features of TAT include the presence of basic amino acids (arginine and lysine) as well as the ability to adopt an alpha helical conformation (7). Parathyroid hormone (PTH) is a peptide hormone, a long chain containing 84 amino acids. Although its principal activity is calciotropic, published investigations have re- vealed that it also shows lipolytic activity in human adipose tissue (10,11). Recently, PTH-derived tripepride (GKH, glycine-lysine-histidine) was reported to stimulate [3-adrenergic receptor coupled to adenylyl cyclase via the stimulatory Gs protein, an increment of cAMP production that leads to activation of protein kinase A and phos- phorylation of hormone-sensitive lipase (HSL), resulting in glycerol release by fat cells (12). The short fragment of GKH that is nonpermeable into the cell was successfully attached to TAT (9-polylysine) in order to have both lipolytic and penetration effects on the skin, without affecting the lipolysis activity of GKH. In this study, we investigate the possibility of TAT-GKH as delivery vehicles without the loss of the lipolytic effect of GKH through TAT-GKH fusion, and discuss its usefulness as a cosmetic ingredient in slimming products. MATERIALS AND METHODS PREPARATION OF TAT-GKH PEPTIDE TAT-GKH peptide (KKKKKKKKK-GKH) was prepared with an automated peptide synthesizer (Applie Biosystem 433A) by using standard solid-phase fluoenylmethoxy- carbonyl (Fmoc) chemistry with HATU as the peptide-coupling agent. Cleavage from the resin was achieved by using a mixture of trifluoroacetic acid (TFA/H20/ ethanedithiol/phenol/thioanisole). Removal of the solvent gave a precipitant that was triturated with cold diethyl ether. The crude mixture obtained was centrifuged, then removed by decantation, and the resulting orange solid was purified by HPLC (Shimazu LC-8A) in the linear gradient of 28-30% of CH3CN in 0.1% TFA for 15 min. The product was isolated by lyophilization and characterized by MALDI-TOF mass spec- trophotometry (Perseptive Biosystems Voyager linear mass spectrophotometer) by using ot-cyano-4-hydroxy-cinnamic acid as a matrix. The purity of the peptide was 95% as determined by analytical grade HPLC (13). CELL CULTURE 3T3-L1 cells (mouse preadipocytes) were obtained from American Type Culture Col- lection (Rockville, MD), and were grown to confluence in a basal medium, Dulbecco's