In vitro human eyelid permeation of Timolol and Dorzolamide distinctly differs from analogue analyses with thigh skin

Human eyelids are a unique and underexplored biological barrier in view of drug delivery. Preliminary clinical evidence supports direct eyelid drug application, underscoring the value of refined in vitro models for advancing targeted ocular delivery. Thus, this study investigated drug permeation and penetration in human eyelid skin. Owing to the limited availability and small size of eyelid tissue donations, comparisons were made with thigh skin under equivalent experimental conditions, using Timolol maleate (TIM) and Dorzolamide hydrochloride (DZA) as model compounds. Both full thickness skin and isolated epidermis were tested, fresh and stored frozen. Skin structure was characterized by histology and microscopic imaging, thickness measurements, and transepidermal water loss (TEWL). Eyelid skin consistently showed 1.5-fold higher TEWL than thigh skin, reflecting its thinner stratum corneum (SC). Frozen storage did not affect TEWL in thigh or full thickness eyelid skin, but increased TEWL 2.7-fold in isolated eyelid epidermis, indicating barrier damage. After 6 h, permeation was increased in fresh eyelid epidermis (TIM: 17 ± 14 μg/cm 2 ; DZA: 28 ± 28 μg/cm 2 ) compared to fresh thigh epidermis (TIM: 1.13 ± 0.83 μg/cm 2 ; DZA: 0.43 ± 0.29 μg/cm 2 ) and markedly higher in epidermis from once-frozen eyelid tissue, reflecting barrier disruption. The results emphasize that thicker-SC membranes are unsuitable surrogates in eyelid formulation development. Frozen storage of eyelid skin should be carefully considered for in vitro permeation studies. • Eyelid TEWL is approx. 1.5× higher than thigh skin, consistent with thinner SC • In vitro/ex vivo eyelid studies must use eyelid skin, not thicker surrogates • Frozen storage may compromise eyelid barrier and modify permeability • Depth-cut profiling enables penetration assessment without uniform skin thickness