KTTKS, GHK (Gly-His-Lys) and pal-KTTKS were obtained from Peptron (Daejon, Korea). Ethylenediaminetetraacetic acid (EDTA), DL-thiorphan (TP), thimerosal (TM), phenylmeth anesulfonylfluoride (PMSF), 1,10-phenanthroline (PNT), bovine serum albumin (BSA) and ascorbic acid 6-palmitate (pal-AA) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Pentafluoropropionic acid (PFPA) was obtained from TCI (Tokyo, Japan). All other chemicals were of analytical reagent grade.

Hairless mice were obtained from Orient Bio Inc. (Gyeonggi, Korea). Male CrlOri:SKH1-hr strain hairless mice, weighing 25 ± 3 g, were used throughout this experiment. Skin preparation process was carried out immediately when the mice were delivered. The animal experiments were approved by Kyungsung University Animal Care and Use Committee, and all procedures were conducted in accordance with the “Guide for the Care and Use of Laboratory Animals” published by the National Institute of Health.

Hairless mice were sacrificed by cervical dislocation and dorsal skin was removed from the nape of the neck and back. The adherent fat and subcutaneous tissue were removed. Hairless mouse skin without the stratum corneum was prepared by a tape stripping method (Benson et al., 2003; Kikwai et al., 2005). The prepared mouse skin without the stratum corneum was mounted on a side-by-side diffusion cell with the epidermal layer facing the donor compartment. The diffusion cell had approximately 0.8 cm² effective diffusion surface area. The donor and receptor compartments were filled with 2.5 ml of HEPES buffer (10 mM, pH 7.4) and kept at 37°C. The receptor and donor solutions were collected at 6, 10, and 24 h. After collection, the compartments were immediately refilled with 2.5 ml of fresh HEPES buffer (10 mM, pH 7.4). The total volume of skin extracts from the donor and receptor compartments for each diffusion cell study was 7.5 ml. The receptor solution extracted from the dermal side of the skin was used as the dermal skin extract, and the donor solution extracted from the epidermis without the stratum corneum was used as the epidermal skin extract for the stability tests. These extracts were filtered using Minisart RC 15 (pore size: 0.2 μm, Sartorius, Germany) and stored at 4°C prior to use.

Hairless mouse whole skin (0.8 cm²) was cut into small pieces. A skin homogenate was prepared using 1 ml of ice-cold sterilized distilled water for 15 min by using a microhomogenizer (High Intensity Ultrasonic Liquid Processors, Sonics & Materials, CT, USA). The skin homogenate was centrifuged at 10,000 rpm for 30 min at 4°C. The supernatant was collected and filtered with Minisart RC 15 (pore size: 0.2 μm, Sartorius, Germany). The filtrate was diluted to the final volume of 7.5 ml with the HEPES buffer solution (10 mM, pH 7.4).

A portion (200 μl) of KTTKS or pal-KTTKS (40 μg/ml in 10 mM HEPES buffer, pH 7.4, as peptide concentration) was incubated with 200 μl of the epidermal skin extract, dermal skin extract, or skin homogenates at 37°C for 120 min. The protein concentrations of epidermal skin extract, dermal skin extract, and skin homogenates determined by Lowry method using BSA as standard (Lowry et al., 1951) were 2, 15, and 100 μg/ml, respectively. At predetermined times, samples were taken from the incubation mixture. The amount of KTTKS or pal-KTTKS in the samples was analyzed using LC-MS/MS. The effects of various proteolytic enzyme inhibitors were also investigated. Five kinds of protease inhibitors, TM (0.5 mM), TP (0.25 mM), EDTA (0.5 mM), PMSF (5 mM), and PNT (1 mM), were added to KTTKS or pal-KTTKS in skin extracts or homogenates at 37°C for 120 min. In addition, the combination of PMSF (5 mM) and PNT (1 mM) was added to KTTKS or pal-KTTKS in skin extracts or homogenates.

Intact hairless mouse skin was mounted on Franz diffusion cells with the epidermal side facing the donor compartment. The receptor compartment was filled with HEPES buffer (10 mM, pH 7.4) mixed with 15% ethanol containing PMSF and PNT (final concentrations of 5 mM and 1 mM, respectively) as proteolytic enzyme inhibitors. The donor compartment was loaded with 1 ml of KTTKS or pal-KTTKS (100 μg/ml in 15% ethanol) solution. The receptor solution was taken out periodically and replaced with fresh buffer solution over a period of 48 h. The collected samples were centrifuged and the super-natants were analyzed by LC-MS/MS.

To assess the amount of the peptides retained in the skin layers, the skin was removed from the diffusion cell after 24 h of sample loading. Donor solution remaining on the surface of the skin was removed by washing with 1 ml of distilled water (4 times repeatedly). Lint-free absorbent wipes were used to dry the skin. The stratum corneum layer was carefully collected by raking with a blunt spatula. The epidermis was separated from the dermis by using a blunt knife. Each separated skin layer (stratum corneum, epidermis, and dermis) was minced using scissors. KTTKS or pal-KTTKS distributed in each skin layer was extracted using 1 ml of methanol for 24 h with continuous shaking. After extraction for 24 h, the samples were centrifuged and the supernatants were analyzed by performing LC-MS/MS.

LC-MS/MS was performed using an Agilent Series 1200 LC instrument with 6410 Triple Quad LC-MS/MS system (Agilent Technologies, Palo Alto, CA, USA). Analysis of KTTKS was performed using Acclaim 300 C₁₈ column (2.1×150 mm, 3 μm, Dionex, Sunnyvale, CA, USA) by isocratic elution using a mobile phase consisting of 5 mM PFPA aqueous solution and acetonitrile (87:13, v/v) as described previously (Park et al., 2012). Pal-KTTKS was analyzed using Capcell Pak C8 column (2.0×100 mm, 5 μm, Shisheido, Tokyo, Japan) by isocratic elution using a mobile phase consisting of 20 mM ammonium acetate solution and acetonitrile (30:70, v/v) containing 0.05% acetic acid. The flow rate was 0.2 ml/min. The GHK and the pal-AA were used as internal standards (IS) for KTTKS and pal-KTTKS, respectively. Data from mass spectrometry were collected and processed using Qualitative Analysis software version B.04.00 from Mass Hunter Workstation.

A standard stock solution of pal-KTTKS at a concentration of 1 mg/ml was prepared by dissolving pal-KTTKS in methanol. Then, dilutions were made to obtain calibration standards at concentrations of 0.5, 1, 2, 5, 10, and 20 μg/ml. The concentration of pal-AA used as IS was 10 μg/ml. The calibration curve was plotted logarithmically using peak-area ratios of pal-KTTKS to IS versus concentrations of pal-KTTKS. Intraand inter-day precision and accuracy of the LC-MS/MS method were determined by analyzing quality control (QC) samples of pal-KTTKS at concentrations of 0.5, 2, 5, and 20 μg/ml. Intra-day precision was determined by repeating the analysis of each QC sample three times a day. Inter-day precision and accuracy were determined by repeating this analysis on three consecutive days. Precision (expressed as the relative standard deviation) should be <15%, and accuracy should be within ± 15%. The lower limit of quantitation (LLOQ) is acceptable with precision <20% and accuracy within ±20%.

The LC-MS/MS method for the quantification of pal-KTTKS was developed by simultaneously monitoring the selected MS/MS transitions for pal-KTTKS (m/z 802.3→129.0) and pal-AA as IS (m/z 415.3→239.1). Fig. 1 shows the LC-MS/MS chromatograms of pal-KTTKS and IS (pal-AA) spiked with the dermal skin extract and after incubation in the dermal skin extract for 60 min at 37°C. The retention times of pal-KTTKS and IS were 1.6 and 3.5 min, respectively. LC-MS/MS of KTTKS was performed using the previously developed methods (Park et al., 2012).

The LC-MS/MS method for analysis of pal-KTTKS was validated (Table 1). In terms of specificity, no interfering peaks were found with the same retention times as pal-KTTKS and IS in the skin extracts and homogenates. The calibration curve showed good linearity in the concentration range of 0.5?20 μg/ml with a slope of 1.5265, an intercept of ?1.844, and r² value of 0.9982. Intra- and inter-day precision and accuracy were determined by analyzing three replicates at each of 0.5 (LLOQ), 2, 5, and 20 μg/ml of pal-KTTKS. At LLOQ, intra- and inter-day accuracies were 97.8% and 116.4%, respectively, and intraand inter-day precisions were 14.3% and 10.3%, respectively. Overall, the intra- and inter-day accuracies were 97.8?101.4% and 98.0?116.4%, respectively, and intra- and inter-day precisions were 1.7?14.3% and 1.2?10.3%, respectively.

Stability of KTTKS and pal-KTTKS in skin extracts and homogenates was measured using LC-MS/MS. Fig. 2 shows the remaining amounts of KTTKS and pal-KTTKS in dermal skin extract, epidermal skin extract, and skin homogenates over 120 min at 37°C. Prior to the incubation in skin extracts and homogenates, both KTTKS and pal-KTTKS were found to be stable in the HEPES buffer (10 mM, pH 7.4) used as the incubation medium in stability and permeation studies. KTTKS was rapidly degraded in skin extracts and homogenates, as shown in Fig. 2(A). In the dermal skin extract and skin homogenate, KTTKS was almost degraded within 30 min, with 3.2% remaining in the dermal skin extract and at 60 min in skin homogenate with 1.5% remaining. The degradation of KTTKS in the epidermal skin extract was slower than that seen in dermal skin extract and skin homogenate. This may be attributed to lower amounts of proteolytic enzymes in the epidermal skin extract than in the dermal skin extract and the skin homogenate. Determination of protein concentration by the Lowry method showed that the epidermal skin extract contained 7.5-fold less protein than the dermal skin extract and 50-fold less protein than skin homogenate. As shown in Fig. 2(B), pal-KTTKS was more stable than KTTKS. In the epidermal skin extract, the concentration of pal-KTTKS throughout the 120-min incubation period was almost similar to its initial concentration. In dermal skin extract, 9.7% of pal-KTTKS remained after 120 min and 11.2% of pal-KTTKS remained at 60 min in the skin homogenate.

Fig. 3 shows the effects of various proteolytic enzyme inhibitors on the stability of KTTKS and pal-KTTKS in the dermal skin extract and the skin homogenates. PMSF and PNT showed strong protective effects against the degradation of KTTKS in the dermal skin extract and the skin homogenates. This indicates that KTTKS is susceptible to enzymatic degradation by serine proteases such as trypsin and chymotrypsin, and metalloenzymes. The combination of PMSF and PNT completely prevented the degradation of KTTKS in skin, as shown in Fig. 3A, B. The stability of pal-KTTKS was also significantly improved by protease inhibitors. While TM did not show a substantial protective effect, the other proteolytic inhibitors strongly inhibited the degradation of pal-KTTKS as shown in Fig. 3C, D.

The skin permeability of KTTKS and pal-KTTKS was studied using intact skin from a hairless mouse mounted on Franz diffusion cells with the epidermal layer facing the donor compartment. In the skin permeation experiments, no detectable levels of KTTKS and pal-KTTKS were observed in the receptor solution over a period of 48 h. Although a trace of pal-KTTKS appeared in the receptor solutions after 24 h by LC-MS/MS analysis, it was below the LOQ (<0.5 μg/ml). Therefore, it was concluded that neither KTTKS nor pal-KTTKS could permeate through full-thickness hairless mouse skin over the time period used in these experiments.

Fig. 4 presents the retention profiles of pal-KTTKS in the di fferent skin layers (stratum corneum, epidermis and dermis) over a period of 24 h. KTTKS was not detected in any skin layer. On the other hand, pal-KTTKS was observed in every skin layer: 4.2 ± 0.7 μg/cm² in the stratum corneum, 2.8 ± 0.5 μg/cm² in the epidermis, and 0.3 ± 0.1 μg/cm² in the dermis. Totally, 14.6% of applied pal-KTTKS was retained in the skin: 8.3% in the stratum corneum, 5.6% in the epidermis, and 0.6% in the dermis.