LG 106kV AND INCLUSION COMPLEXES 229 flitrate was diluted with 50% v/v methanol-water and injected to HPLC. The experi- ments were carried out in triplicate. The HPLC consisted of a solvent delivery pump (Waters 626, Waters Co., MA), a C18 column (Hewlett Packard BDS), a UV detector (Waters 486), and a data processing system (Millennium 32, Waters). The mobile phase was composed of 58% v/v methanol and 42% v/v water that contained phosphoric acid to adjust to a pH 2.5. A wavelength of 283 nm was selected, and the temperature of the column was kept at 30øC. The correction of concentration against each sample point was undertaken. An apparent 1:1 stability constant, Kc, was calculated from the initial straight line portion of phase solubility diagrams according to the following equation (9): Koe -- slope intercept-(1-slope) PREPARATION OF INCLUSION COMPLEXES For the complex preparation, two methods were used, depending on the physical prop- erties of CyDs: 1. In the case of preparation of solid complexes, an aqueous solution of •3-CyD was prepared at room temperature. An equimolar amount of the guest compound was added, and the mixture was stirred at ambient temperature for 24 hr. The •3-CyD inclusion complex formation was indicated by the precipitation of a white solid. The solid complex was isolated by filtration, washed with distilled water, and dried under vacuum. 2. To prepare the complexes with CyD derivatives, an excess amount of the guest compound was added to an aqueous solution of the CyD derivatives, and the mixture was stirred at ambient temperature for 24 hr. After the excess of guest compound was removed by filtration, the complex powder was obtained by spray-drying the flitrate. DETERMINATION OF PHYSICOCHEMICAL PROPERTIES OF CyD COMPLEXES The microscopic aspect of the raw materials was compared with that of the products obtained by precipitation and simple physical mixture, by examination under the scan- ning electron microscope (JSM 840A, Jeol, Japan). The reality of the inclusion was confirmed by subjecting the raw materials, the simple physical mixture, and the precipitated product to differential scanning calorimetry (Setaram TG-DSC, Caluire, France). The samples were analyzed in an open capsule to allow the evaporation of water lost by CyD. Determinations were carried out on 10 mg of LG106W or on the corresponding quantities. The different samples were heated from 25øC to 250øC at a rate of 10øC/min. The powder X-ray diffraction patterns were also taken by a Rigaku Rint-2500 diffrac- tometer (Tokyo, Japan). The complexes were prepared as following: The complex of LG106W with HP-[3-CyD in a molar ratio of 1:1 was prepared by the kneading method, i.e., LG106W (30 mg) was dissolved in a small amount of ethanol, and the solution was kneaded thoroughly with HP-•3-CyD (124 mg) and water (about 1 ml) for about 40 min. The solid complex was dried under reduced pressure at room temperature for two days, and subjected to powder X-ray diffraction. The operation conditions were: X-ray of

230 JOURNAL OF COSMETIC SCIENCE Ni-filtered Cu-K o• radiation (1.542 A), a voltage of 40 kV, a current of 40 mA, and a goniometer angular increment of lø/min. STABILITY STUDIES To evaluate temperature stability, aqueous 1,3-butylene glycol solution was prepared and divided into two parts. LG106W in free or complex form was added to each solution. The solutions were moved to 5.0-ml Teflon (FEP) centrifuge tubes (Nalgene, Nalge Company, New York). The tubes were sealed with parafilm and kept at 25øC, 40øC, and 50øC in temperature-controlled chambers. Periodically, 0.1-ml aliquots of these were pipetted out and then diluted with 50% v/v methanol. The solutions were analyzed for LG106W by HPLC. In the case of the pH stability study, a universal buffer solution was prepared and the experiment was conducted under the same conditions as those of the temperature test. INHIBITION OF MELANIN SYNTHESIS AND CYTOTOXICITY B16 mouse melanoma cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% fetal calf serum in a humidified incubator at 37øC under 5% CO 2. The cells were seeded into a 6.0-cm petri dish at a density of 5 x 105 cells per dish. After the cells were attached, the medium was replaced with fresh medium containing various concentrations of LG106W and its CyD inclusion complexes. The cells were cultured for two days, and the medium was replaced with fresh medium once more, further incubated for a day. Then the cells were harvested with a cell scraper (10). The inhibition of melanin synthesis was evaluated by an expert's sight. In order to screen the potential toxicity of raw materials, an MTT (3-[4,5- Dimethylthiazol-2-yl]2,5-diphenyltetrazolium bromide thiazoyl blue) reduction test was carried out. First, V79-4 cells (Chinese hamster, continuous cell line of lung tissue fibroblast) were cultured, and then the serial dilutions of the test materials were applied to the cultured cells for 24 hr. For a viability check, the MTT test was performed according to the method described previously (11). MTT-formazan extracts were mea- sured by reading the absorbance at 570 nm. IN VITRO SKIN PERMEATION The skin permeability of LG106W was determined in vitro. Female hairless mice (8- week-old) were sacrificed by cervical dislocation, and their full-thickness skins were removed and placed on Franz-type diffusion cells with an effective diffusion area of 1.766 cm 2 and a receptor volume of 7.0 ml (Microette, Hanson Research Co., CA). The receptor compartment consisted of isotonic aqueous 50-mM phosphate buffer (pH 7.4) containing 1.0% w/v HP-IB-CyD. The receptor compartment was kept at 32øC by circulating water through an external jacket and stirring with a magnetic bar. The vehicle compositions of the donor compartment selected for the incorporation of LG106W and of the inclusion complex were of two types: water and an oil-in-water (O/W) emulsion base. The formula of the emulsion was selected for its viscosity, spread- ability, and stability. The following was adopted: cetostearyl alcohol 0.5% w/w glyceryl