Permeation studies

Permeation studies were carried out using static Franz-type diffusion cells (effective diffusional area 1.1 cm²), utilizing skin samples obtained from five patients (four females and one male; mean age: 49 years old, ranging 37–61 years old) undergoing abdominoplasties in Perth Hospitals (Curtin Human Ethics Approval number PH-18-11). Written informed consent was obtained from all of the volunteers. Abdominal skin samples collected were immediately placed at 4°C. The subcutaneous fat was removed within 24 hours of surgery, and the full-thickness skin samples were stored at −20°C for no longer than 6 months. The epidermis was detached from the dermis by a standard heat separation method.10 Briefly, the epidermal layer was carefully stripped from whole-skin samples, following submersion in 60°C water for ~2 minutes, and then carefully peeling back the top epidermal layer. The epidermis was mounted to the Franz cell with the stratum corneum facing toward the donor compartment. The receptor compartment (containing a magnetic stirrer) was loaded with PBS (~3 mL at pH 7.4) and allowed to equilibrate to 35°C for 15 minutes before the donor compartment was loaded. Keeping the temperature of the receptor compartment at 35°C enabled the skin surface temperature to be kept at 32°C. Skin integrity was assessed by observing PBS movement from donor to receptor for 20 minutes before the start of experimentation. Any skin membranes suspected of having holes, before or during the experiment, by closely monitoring the donor and receptor volumes, were eliminated as they were detected.

It is important to note that there is variability between skin samples from the same individual as well as between individuals. Site differences including structural variability of skin (eg, lipid content) and previous skin care/treatment may contribute to this within the same patient as well as between patients.11 Thus, we assessed triplicate samples from five different individuals (all are abdominal sections) for each assay (ie, five separate experiments each using triplicate skin samples from a different donor each assessing the three different peptides individually within the same experiment: L-AAPV, L-C₇-AAPV, and L-AAPV-C₇).

The experiments began with the donor compartment loaded with 300 μL PG or 300 μL PG containing 3 mg of L-AAPV, L-C₇-AAPV, or L-AAPV-C₇, and sealed. The amounts were completely soluble in PG at these concentrations. Aliquots (200 μL) were withdrawn from the receptor compartment at 0, 0.5, 1, 2, 4, 8, and 24-hour time points and replaced with an equivalent amount of PBS pre-equilibrated at 35°C. The temperature of the receptor compartment was maintained at 35°C for the duration of the experiment, and this enabled a skin surface temperature of 32°C. Collected aliquots from each experiment were kept at 4°C in the injecting tray of the autosampler while awaiting analysis by HPLC-UV.

Transdermal flux was calculated as the cumulative amount in the receptor over the first 2 hours (the slope of the graphs in Figure 2) divided by the effective diffusional area of each of the skin samples.

Cumulative amounts of parent peptide “L-AAPV (●)” and N- or C-acyl lipidated peptide derivatives “L-C₇-AAPV (■) and L-AAPV-C₇ (○)” in the receptor compartment after 24 hours.

Notes: Results shown are the mean (± SD) of five separate experiments, each conducted in triplicate. Each experiment utilized triplicate skin sections from a different skin donor.

Abbreviations: L-AAPV, L-Ala-L-Ala-L-Pro-L-Val; L-C₇-AAPV, C₇-L-Ala-L-Ala-L-Pro-L-Val; L-AAPV-C₇, L-Ala-L-Ala-L-Pro-L-Val-C₇; SD, standard deviation.