A statistical approach to design in vitro skin permeation studies according to the current regulatory framework

Transdermal patches and medicated plasters are designed to sustain efficacious systemic or loco-regional drug concentrations, respectively. To define the set-up of the in vitro skin permeation experiments, the selection of the diffusion cell, the relative operative conditions and the biological membrane are crucial. In 2014, the EMA introduced the "Guideline on the quality of transdermal patches", in which the importance of equivalence of drug fluxes in in vitro skin permeation study using Franz cells was particularly emphasized for the marketing authorization or postapproval changes of the dossier. This work aimed to design in vitro skin permeation experiments to compare two transdermal patches or medicated plasters on the basis of the variability of the drug permeation profiles through the epidermis. In particular, the impact of the inter-sample variability on the equivalence assessment was deeply investigated by using patches and plasters containing propranolol, diclofenac or nitroglycerin. The in vitro permeation studies were performed by Franz diffusion cell (permeation area: 0.636 cm2; receptor volume ≈ 3 mL) using human epidermis as membrane. The skin integrity was determined according to an internal standard procedure based on electrical resistance [1]. The results confirmed that the number of replicas and the acceptability interval for assessing equivalence should be rationalized based on the intrinsic variability of drug permeation flux. As an example, the equivalence of two propranolol patches (flux variability lower than 25%) can be assessed with six replicas, maintaining the confidence limit within the 0.8–1.25 range (α=0.05; power 90%). However, even if the drug has a low intrinsic variability, the fluxes of nitroglycerin patches result not equivalent using Franz diffusion cell because of those patches have different drug content and size. Indeed, in the case of transdermal patches, the in vivo permeation flux is modulated by both the patch dimension and the drug content. In these cases, fluxes obtained by in vitro permeation studies should be normalized for the effective patch area rather than the permeation area of the Franz diffusion cell. In contrast, the equivalence of diclofenac plasters (flux variability near the 50%) can be demonstrated only increasing the number of replicas for each formulation (i.e., 20 skin samples) and widening the acceptance range (i.e., 0.69-1.43), following the same statistical approach accepted by the EMA for bioequivalence study of high variable drug products. Literature: [1] U.M. Musazzi, Data in Brief 2018, 21, 1258-1262