2.3. In vitro skin permeation test (IVPT) studies

The formulations were applied to excised abdominal porcine skin mounted in diffusion cells with the dermal side in contact with a physiological buffer. The skin was not occluded and the formulations were not removed from the skin. The two sets of IVPT studies performed used the same receptor solution, which was stirred with a magnetic bar, of phosphate-buffered saline (pH 7.4) with (set 1) or without (set 2) sodium azide 0.01% w/v.

Vertical flow-through cells (Laboratory Glass Apparatus, Berkeley, CA, USA) with an exposed skin area of 3.14 cm² were used. Formulations were applied using the flat base of a 2 mL glass vial, and the mass of product applied was determined by simple weight difference. Four replicates were performed per formulation using skin from donor ‘Pig A’. The receptor solution volume was ∼7.7 mL, and was thermostatted via recirculating water maintained at 37°C. The receptor solution flow rate was ∼1 mL/h (the average for each cell was measured) and samples were collected on a fraction collector every 4 hours.

Static, Franz cells (Permegear, Hellertown, PA, USA) with an exposed skin area of 2.01 cm² and a receptor volume of ∼7.4 mL were used. Formulations were applied using a cotton bud (i.e., similar to the in vivo tape-stripping experiments), and the mass of product applied was again determined by weight difference. Eight replicates were performed per formulation, 4 using skin from donor ‘Pig B’, and 4 with skin from donor ‘Pig C’. Throughout the experiment, the diffusion cells remained in an oven at 32°C and 40% relative humidity, except for brief excursions when the receptor solution was sampled (1 mL) at 6, 8, 10, 12, 23, 26 and 30 hours. The concentrations of diclofenac measured in the receptor phase samples were never more than 2 μg/mL, i.e., far below the aqueous solubility of the drug at pH 7.4 (which is, in fact, about 10³-fold higher; Maitani et al., 1993), therefore ensuring that sink conditions were maintained throughout the experiment.