SKIN PENETRATION ENHANCEMENT BY TAT-GKH 485 Modified Eagle's medium (DMEM), supplemented with 10% fetal bovine serum (FBS) and penicillin/streptomysin (all from Life Technologies, Gibco, BRL). The medium was changed every two to three days. After confluence, the 3T3-L1 cells were differentiated into adipocytes by the addition of 2 lag/ml of insulin, 2 laM of dexamethasone, and 111 lag/ml of methyl-isobutylxanthine (all from Sigma) to the basal medium. After 48 h, the medium was replaced by 10% FBS basal medium, DMEM, containing only 2 lag/ml insulin, for ten days, during which time the cells developed into mature adipocytes with typical histologic appearance. LIPOLYSIS MEASUREMENTS The lipolytic effect, i.e., the degradation of triglycerides to glycerol and fatty acids, was evaluated by determining the quantity of glycerol released by the cells. Lipolysis was measured as the percent of glycerol in TAT-GKH-treated adipocytes vs glycerol in nontreated adipocytes (basal lipolysis) using Krebs-Ringer bicarbonate buffer containing 1% FBS (pH = 7.4) as incubation medium. After the adipocytes were washed with phosphate buffer saline (PBS, pH = 7.4), the TAT-GKH (final concentration 10 -4 M - 10 -7 M) dissolved in the incubation medium was added to the adipocytes and incubated at 37øC for 2 h. The lipolytic agent isoproterenol (Sigma) was used as a positive control at 10 -6 M. After incubation, the reaction mixture was centrifuged at 100 g at room temperature for 30 sec to separate the medium and the adipocytes. The glycerol content of the incubation medium was determined using a colorimetric assay (GPO-Trinder, Sigma) (14). ANIMALS Male Sprague-Dawley rats weighing 370-420 g and 8-9-week-old female hairless mice weighing 27-33 g (DaeHan Biolink, Taejeon, Korea) were housed in a temperature- controlled room (22 ø + 2øC) and subjected to a 12-h light/dark cycle. Animals had free access to laboratory food and water and were carefully handled. IN VITRO LIPOLYSIS ON ADIPOCYTES FROM RATS For in vitro experiments, rats were sacrificed by cervical dislocation after an overnight fast, and epididymal adipose tissue was immediately removed. Isolated fat cells were obtained by collagenase digestion (1 mg/ml, 37øC) in Krebs-Ringer bicarbonate buffer containing 3.5 g/100 ml of bovine serum albumin (BSA) and 0.6 mM of glucose at pH 7.4 (KRBA), under continuous vigorous shaking (90 cycles/min) according to the method of Rodbell (15). Fat cells were filtered through nylon mesh and washed three times with the same incubation buffer (KRBA), to eliminate the stroma-vascular frac- tion and collagenase A. Measurements of lipolytic activity were performed by incubating isolated adipocytes in 200 pl of KRBA buffer with continuous gentle shaking (30 cycles/min). After two hours of incubation with TAT-GKH (10-4--10 -7 M) at 37øC, the reaction was stopped with ice and an aliquot (50 pl) was taken to determine glycerol release in the incubation buffer by the calorimetric assay (14). Basal lipolysis was determined in the absence of TAT-GKH. The metabolic activity was expressed as micromoles of glycerol released per milligram of total lipids, which were determined gravimetrically after solvent extraction (16).

486 JOURNAL OF COSMETIC SCIENCE CYTOTOXICITY ASSAY Preconfiuent 3T3-L1 preadipocytes were seeded in 96-well plates at a density of 5 x 103 cells/ml/well. The same doses of TAT-GKH were added to the culture medium. The cytotoxicity was evaluated by the MTT [3,(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide] test (17) and by measuring formazan formation spectro- photometrically at 570 nm. SKIN PERMEATION STUDIES Vertically assembled Franz-type diffusion cells (Microette transdermal diffusion system, Hanson Research Corporation, Chatsworth, CA) were used for in vitro skin permeation experiments. The system consisted of Franz-type diffusion cells with an effective diffu- sion area of 1.776 cm 2 and a receptor volume of 7.0 ml, an autosampler, and a cell drive system with rpm controller. The fundamental experiments were performed according to the method given in our previous report (18). Briefly, the excised skin of female hairless mouse was obtained from 8-9-week-old, 27-33-g animals. The dermal side of the skin was soaked in buffer with 15% ethanol solution containing 5 mM of phenylmethylsul- fonyl fluoride (PMSF) for 12 h at 10øC to inhibit the enzyme. The skin was mounted on a diffusion cell, and the receiver compartment was filled with 7 ml of 50 mM PBS with 15% ethanol containing 5 mM PMSF and maintained at 32øC by circulating water within a jacket around the lower chamber. PMSF was used to inhibit the enzyme and 15% ethanol was used to dissolve the PMSF. The ethanol concentration had no effect on the penetration of peptides, as reported by Ghanem et al. (19), who showed that ethanol at low levels (25%) had little or no effect on pore pathways. A 20% ethanol solution containing 1% TAT-GKH (w/v) and 1% GKH (w/v) was applied in the donor com- partment and was uniformly distributed with a micropipette on the skin surface (100 pl). The receptor fluid was mixed by a magnetic stirrer throughout the experiment and was collected from the receiver compartment at predetermined time (every 12 h after sample application) and replaced by fresh fluid. At the end of the experiment (24 h after sample application), the receptor fluid was collected and the donor compartment was washed with 500 pl of ethanol three times. After completion of the preset time (24 h), the skin samples were taken out of the diffusion cells. The skin was homogenized by 4 ml of PBS to extract TAT-GKH and GKH. After filtration on Millex filter FG (pore size: 0.2 pm, Millipore), the solutions were assessed by HPLC. Five hundred microliters of the receptor fluid withdrawn from the receiver compartment at predetermined times was treated with 10 pl of TFA. Following centrifugation (13,000 rpm), the amounts of TAT-GKH and GKH in the supernatants were determined by analytical HPLC. HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY (HPLC) The HPLC consisted of a solvent delivery pump (600 pump, Waters Co., MA), a C•8 column (HP ODS hypersil 5 !•m, 4.6 x 150 mm, Hewlett Packard, Germany), a 486 UV detector (Waters), and a data process system (Waters Millennium). The TAT-GKH was analyzed with the mobile phase of distilled water in 0.1% TFA. TAT-GKH was ana- lyzed with the mobile phase of 15% acetonitrile in 0.1% TFA and at a flow rate of 1 ml/min. Absorbance at 210 nm was measured for the assay of these peptides. The