TOPICAL DELIVERY OF ANTI-INFLAMMATORY COMPOUNDS 345 UV-1700 Pharma Spec Shimadzu, Milan, Italy). Calibration curves for validated UV as- says of SG were performed on fi ve solutions in the concentration range 8—80 mg/ml. The correlation coeffi cient was greater than 0.990. Each point represented the average of three measurements, and the error was calculated as standard deviation (±SD). SG incorporation effi ciency was expressed as active recovery and calculated using equation (1): Mass of active in nanoparticles Drug recovery % 100 Mass of active fed to the system = × (1) Possible lipid interferences during UV determination of SG were also investigated by comparing the standard curve of the substance alone and in the presence of lipids. The differences observed between the standard curves were within the experimental error, thus inferring that no lipid interference occurred (data not shown). IN VITRO STUDIES Skin membrane preparation. Samples of adult human skin (mean age 36 ± 8 years) were obtained from breast reduction operations. Subcutaneous fat was carefully trimmed and the skin was immersed in distilled water at 60 ± 1°C for 2 min (16), after which SCE was removed from the dermis using a dull scalpel blade. Epidermal membranes were dried in a desiccator at approximately 25% relative humidity. The dried samples were wrapped in aluminum foil and stored at 4 ± 1°C until use. Previous research work showed the maintenance of SC barrier characteristics after storage in the re- ported conditions (17). Besides, preliminary experiments were carried out to assess the bar- rier integrity of SCE samples by measuring the in vitro permeability of [3H] water through the membranes using the Franz cell method described below. The value of calculated per- meability coeffi cient (Pm) for [3H] water agreed well with those reported earlier (18). In vitro skin permeation experiments. Samples of dried SCE were rehydrated by immersion in distilled water at room temperature for 1 h before being mounted in Franz-type diffusion cells supplied by LGA (Berkeley, CA). The exposed skin surface area was 0.75 cm2 and the receiver compartment volume was 4.5 ml. The receptor compartment was fi lled with a water–ethanol solution (50:50) (to allow the establishment of sink conditions and to sustain permeant solubilization), stirred at 500 rpm, and thermostated at 32 ± 1°C during all experiments (19,20). Approximately 100 mg of each formulation (A–H, SLN-IN, and SLN-OUT) was placed on the skin surface in the donor compartment and the latter was covered with Parafi lm® (Pechiney Plastic Packaging Company, Chicago, IL). Each experiment was run in duplicate for 24 h using three different donors (n = 6). At predetermined in- tervals, samples (200 μL) of receiving solution were withdrawn and replaced with fresh solution. The samples were analyzed for SG and DG content by HPLC as de- scribed below. The results were expressed as cumulative amount of SG and DG per- meating the SCE membranes after 24 h. Statistical analysis of data was performed using student’s t-test.

JOURNAL OF COSMETIC SCIENCE 346 IN VIVO STUDIES To investigate the relationship between in vitro skin permeation data and in vivo topical anti-infl ammatory activity, we evaluated the ability of the formulations that showed the best in vitro profi le, to inhibit the UV-induced skin erythema on healthy human volunteers. Volunteers recruitment. In vivo experiments were performed on a group of ten volunteers of both sexes in the age range 25–35 years. They were recruited after medical screening, including the fi lling of a health questionnaire, followed by physical examination of the application sites. After they were fully informed on the nature of the study and on the procedures involved, they gave their written consent. The participants did not suffer from any ailment and were not on any medication at the time of the study. They were rested for 15 min prior to the experiments and room conditions were set at 22±2°C and 40– 50% relative humidity. In vivo anti-infl ammatory activity. UVB-induced skin erythema was monitored by using a refl ectance visible spectrophotometer X-Rite model 968 (X Rite Inc., Grandville, MI), calibrated and controlled as reported earlier (12,21). Refl ectance spectra were obtained over the wavelength range 400–700 nm using illumi- nant C and 2° standard observer. From the spectral data obtained, the erythema index (EI) was calculated using equation (2) (22): ª § · § ·º = + + - + « ¨ ¨ ¸» « © ¹ © ¹» 560 540 580 510 610 1 1 1 1 1 EI 100 log 1.5 log log 2 log R R R R R (2) where 1/R is the inverse refl ectance at a specifi c wavelength (560, 540, 580, 510, and 610). The skin erythema was induced by UVB irradiation using a UVM-57 ultraviolet lamp (UVP, San Gabriel, CA) whose specifi c parameters are reported elsewhere (12). The minimal erythemal dose (MED) was preliminarily determined, and an irradiation dose corresponding to twice the value of MED was used throughout the study. For each subject, seven sites on the ventral surface of each forearm were defi ned using a circular template (1 cm2) and demarcated with permanent ink. One of the seven sites of each forearm was used as control, three sites were treated with 300 mg of formulation D, and the remaining three with 300 mg of formulation G. The preparations were spread uni- formly by means of a solid glass rod and then the sites were occluded for 6 h using Hill Top Chambers (Hill Top Research, Cincinnati, OH). After the occlusion period, the chambers were removed and the skin surfaces were gently washed to remove the gel and allowed to dry for 15 min. Each pretreated site was exposed to UVB irradiation 1, 3, and 6 h (t = 1, t = 3 and t = 6, respectively) after gel removal and the induced erythema was monitored for 52 h. EI baseline values were taken at each designated site before applica- tion of gel formulation and they were subtracted from the EI values obtained after UVB irradiation at each time point to obtain ΔEI values. For each site, the area under a curve (AUC) was computed using the trapezoidal rule. The volunteers were again recruited to complete the experimentation after a washout period of 2 weeks and the same experimen- tal procedure was repeated for the formulations SLN-IN and SLN-OUT.