2.5. Penetration Assay with Finite Dosage Using Vertical Franz Diffusion Cells

OECD guidelines [21,24] and the published opinions of the Scientific Committee on Cosmetic Products and Non-Food Products (SCCS) [25] were adhered in the penetration protocol. The excised pig skin was placed in a static Franz diffusion cell (Lara Spiral, Couternon, France) (1.86 cm² and 3 mL) with the dermis side facing the acceptor chamber. The receptor chamber was filled with receptor fluid. The receptor fluid was the same used in the kinetic assay (Section 2.4). Air bubbles entrapped below the skin were carefully removed and the assembled cells were deposited in a temperature-regulated water bath (Telesystem HP 15, H+P Labortechnik GmgH, Oberschleissheim, Germany) on top of a water-resistant magnetic stirring plate (Variomag 15 and Telemodul, H+P Labortechnik GmgH, Oberschleissheim, Germany). The homogeneity of receptor fluid was maintained by continuously stirring at 700 rpm. The system was thermostatted at 43 ± 1 °C to provide a skin surface temperature of 32 ± 1 °C.

The integrity and permeability of membranes were evaluated with the trans-epidermal water loss (TEWL) value as detailed in Section 2.3. Lidocaine, diclofenac sodium, ibuprofen and caffeine were selected to be compared with the infinite kinetic results. The concentration of solutions of active drugs was the same as of kinetic assay.

The finite assay protocol was conducted as described in previous studies [26]. Briefly, 20 µL of each API solution was applied to the skin surface in the donor chamber (n = 3). According to the OECD methodology used [21], penetration studies were performed for 24 h. After the exposure time, the receptor fluid of all the membranes was collected to transfer to a 5 mL volumetric flask. The remaining API solution was removed from skin surface with specific washing procedure (W). Stratum corneum layers were obtained by tape stripping protocol with adhesive tape (D-Squame, Cuderm Corporation, Dallas, TX, USA) applied under controlled pressure (80 g/cm²). Viable epidermis (E) and the dermis (D) were separated after heating the skin at 80 °C for several seconds. All samples were extracted with adequate solvent (Table 1) and filtered with a 0.45 µm Nylon filter (Cameo, Sigma-Aldrich, St. Louis, MO, USA) before HPLC quantification (Section 2.6).

HPLC analytical conditions and parameters for diclofenac sodium (DS), ibuprofen (IBU), lidocaine (LIDO), and caffeine (CAF).

The resulting mass balance was acceptable (100 ± 15%) for each compound after an exposure time of 24 h. The results are presented as the normalized amounts (%) of penetrated substance and their standard deviations. The amount permeated in the skin was considered the sum of that in the epidermis, dermis, and receptor fluid [21].

Since permeability constants were obtained by the permeation kinetic assay and a mathematical model, a permeability constant with results from the penetration assay was also calculated for easy comparison. This following equation reported by Lian [27] assumes that the system was at the steady state, even though the system will hardly reach steady state. In addition, the equation underlines that the rate of the substance being transferred across the skin obeys Fick’s law of diffusion.

The steady-state flux through the skin (dMdt) was considered as the absorbed amount of substance permeating through the skin (equivalent to the sum of that in the epidermis, dermis, and receptor fluid) divided by the time (24 h). The surface area of exposure, A, was 1.86 cm². C1 is the concentration of the donor solution, and C2 is the receptor fluid concentration.