Yucatan micropig (YMP) skin sets frozen at −80 °C were purchased from Charles River

Japan, Inc. (Kanagawa, Japan). Skin was thawed at 20–25 °C for approximately 30 min, followed by removal of the adhering fat layer using scissors and a grater, and cut into appropriate sizes (intact skin). YMP intact skin has the stratum corneum (SC) consists of about 20-layer, a part of SC was removed from intact skin with adhesive tape (Scotch®313, 3 M, Tokyo) 15 times (stripped skin) to make a model of damaged skin. Skin penetration was measured in a modified Franz diffusion cell apparatus [effective area, 1.1 cm2; receptor, 16 mL isotonic phosphate buffered solution (pH 7.1) maintained at 37 °C mixed with a star-head magnet at 600 rpm]. For the infinite dose condition, skin was mounted directly on the cell, GDC-0973 manufacturer a 2.0-mL aliquot of EL was poured into the donor phase, and the donor phase was occluded. At predetermined times, 200-μL aliquots were withdrawn from the receptor compartment. selleck chemical The same volume of fresh solution was added to the receptor compartment after withdrawal to maintain constant volume. At 27 h after application, skin was removed from the cell, washed with purified water, gently dried, and used for further testing. For the practical dose, EL was spread on the skin at 2 μL/cm2, and the skin was mounted on the cell. The donor phase was not occluded. At 4, 14, and 24 h after

application, 200-μL aliquots were withdrawn from the receptor compartment and skin was removed from the cell and used for further tests without washing to determine the mass balance of DPH. After the skin permeation study, skin was Thymidylate synthase stripped 10 times (intact skin) or 5 times (stripped skin) with adhesive tape (Scotch CC1820-Bx-J, 3 M) to determine the amount

of DPH near the surface of skin, followed by soaking in methanol. The skin was then separated into the epidermis and dermis by the heat separation method [7]. Methanol was added to each part, and the epidermis and dermis were homogenized and centrifuged at 3000 rpm for 5 min, and the supernatant filtered with a membrane filter (0.45 μm for epidermis and 0.20 μm for dermis). The DPH concentration in the solutions obtained was determined using HPLC. Rabbits (Japanese white, males, body weight ca. 3 kg) were used for the in vivo skin permeation study. The hair of the back was removed using an electric hair clipper followed by depilatory cream the day before application. The EL was spread at 2 μL/cm2. After 4-h application, SC was stripped 5 times with adhesive tape, followed by sacrifice of the rabbit and isolation of the skin. The skin was separated into the epidermis and dermis using the heat separation method. The remaining process followed was the same as that of the in vitro study. This study was approved by the Ethics Committee of Showa Pharmaceutical University.