Parallel evaluation of alternative skin barrier models and excised human skin for dermal absorption studies in vitro

Abstract

Skin permeation is a primary consideration in the safety assessment of cosmetic ingredients, topical drugs, and human users handling veterinary medicinal products. While excised human skin (EHS) remains the 'gold standard' for in vitro permeation testing (IVPT) studies, unreliable supply and high cost motivate the search for alternative skin barrier models. In this study, a standardized dermal absorption testing protocol was developed to evaluate the suitability of alternative skin barrier models to predict skin absorption in humans. Under this protocol, side-by-side assessments of a commercially available reconstructed human epidermis (RhE) model (EpiDerm-200-X, MatTek), a synthetic barrier membrane (Strat-M, Sigma-Aldrich), and EHS were performed. The skin barrier models were mounted on Franz diffusion cells and the permeation of caffeine, salicylic acid, and testosterone was quantified. Transepidermal water loss (TEWL) and histology of the biological models were also compared. EpiDerm-200-X exhibited native human epidermis-like morphology, including a characteristic stratum corneum, but had an elevated TEWL as compared to EHS. The mean 6 h cumulative permeation of a finite dose (6 nmol/cm²) of caffeine and testosterone was highest in EpiDerm-200-X, followed by EHS and Strat-M. Salicylic acid permeated most in EHS, followed by EpiDerm-200-X and Strat-M. Overall, evaluating novel alternative skin barrier models in the manner outlined herein has the potential to reduce the time from basic science discovery to regulatory impact.

Keywords: Dermal absorption; Franz cell; Human skin; In vitro permeation testing; Reconstructed human epidermis; Strat-M.

Figure 1.

Chemical structures, molecular weights (MW, g/mol), and n-octanol-water partition coefficients (Log K_ow) of radiolabeled caffeine (CAS 58-08-2), salicylic acid (CAS 69-72-7), and testosterone (CAS 58-22-0) as used for in vitro skin permeation experiments.

Figure 2.

Permeation of caffeine (Log K_ow = −0.07) through excised human skin (EHS) (A) and Strat-M (B) mounted on either 15 mm (black, circles) or 5 mm (red, squares) orifice Franz cells (FCs). A finite dose (6 nmol/cm²; 10 μL/cm²) was applied topically, and the cumulative amount permeated (top row), flux (middle row), and mass distribution (bottom row) were determined over 6 h. Mean cumulative amount across FC orifice sizes was statistically compared at each time point, for each model, by ANOVA with the maximum modulus test. Experiments performed in triplicate. Data shown as mean ± standard error of mean. No significant differences were observed. Mass distribution values were adjusted using an arcsin-square root transformation and the effect of FC orifice size within collection groups (i.e., donor, tissue, or receptor) was determined by ANOVA with the maximum modulus test. ns = no significance.

Figure 3.

Assessment of excised human skin (EHS) and EpiDerm-200-X reconstructed human epidermis morphological and barrier variability amongst donors/batches. EHS samples from three donors were fixed and processed for histological examination (A). Samples were stained with hematoxylin and eosin (H&E). Representative images are presented. Scale bar = 50 μm. Transepidermal water loss (TEWL) was measured on EHS samples mounted on 5 mm FCs (B). N = 6 – 20 samples per donor. The difference in mean TEWL was statistically compared by ANOVA with Tukey’s test; ns = no significance, ** = p < 0.01. Three independently received EpiDerm-200-X batches were examined by histology (H&E; C) and TEWL (D) in an identical manner as EHS. Representative H&E images are presented. 40X scale bar = 50 μm; 10X scale bar = 200 μm. ‘Center’ and ‘Edge’ denote the portion of the tissue closest to the center or the edge of the transwell support upon which it was manufactured, respectively. N = 8 – 22 samples per batch. Data shown as mean ± standard error of mean. The difference in mean TEWL was statistically compared by ANOVA with Tukey’s test; ns = no significance, * = p < 0.05.

Figure 4.

Permeation of caffeine (Log K_ow = −0.07), salicylic acid (Log K_ow = 2.26), and testosterone (Log K_ow = 3.32) through excised human skin (EHS) (A), Strat-M (B), and EpiDerm-200-X (C). EHS and alternative skin barrier models were mounted on 5 mm orifice Franz cells and a finite dose (6 nmol/cm²; 10 μL/cm²) of the caffeine (black, circles), salicylic acid (red, triangles), or testosterone (green, squares) was applied topically. Cumulative amount permeated (top row), flux (middle row), and mass distribution (bottom row) were determined over 6 h. N = 3 donors/batches per barrier for each chemical, each in triplicate. Data shown as mean ± standard error of mean. Mean cumulative amount across chemicals was statistically compared at each time point, for each model, by ANOVA with Tukey’s test. ‘a’ denotes p < 0.05 when comparing caffeine and salicylic acid; ‘b’ denotes p < 0.05 when comparing caffeine and testosterone; ‘c’ denotes p < 0.05 when comparing salicylic acid and testosterone. All other comparisons were determined to be not statistically significant. Mass distribution values were adjusted using an arcsin-square root transformation and chemicals within collection groups (i.e., donor, tissue, or receptor) were compared by ANOVA with Tukey’s test. * = p < 0.05, ** = p < 0.01, *** = p < 0.001. All other comparisons were determined to be not statistically significant.

Figure 5.

Evaluation of the vehicle effect on permeation of caffeine (Log K_ow = −0.07) through excised human skin (EHS) (A), Strat-M (B), and EpiDerm-200-X (C). EHS and alternative skin barrier models were mounted on 5 mm Franz cells and a finite dose of caffeine (6 nmol/cm²; 10 μL/cm²) in 33% or 100% ethanol (EtOH) was applied topically. Cumulative amount permeated (top row), flux (middle row), and mass distribution (bottom row) were determined over 6 h. N = 3 donors/batches per barrier for each formulation, each in triplicate. Data shown as mean ± standard error of mean. Mean cumulative amount across vehicles was statistically compared at each time point, for each model, by ANOVA with the maximum modulus test. * = p < 0.05, *** = p < 0.001. Mass distribution values were adjusted using an arcsin-square root transformation and chemicals within collection groups (i.e., donor, tissue, or receptor) were compared by ANOVA with the maximum modulus test. ns = no significance, *** = p < 0.001.

Figure 6.

Evaluating vehicle influence on permeation of salicylic acid (Log K_ow = 2.26) through excised human skin (EHS) (A), Strat-M (B), and EpiDerm-200-X (C). EHS and alternative skin barrier models were mounted on 5 mm Franz cells and a finite dose of salicylic acid (6 nmol/cm²; 10 μL/cm²) in 33% or 100% ethanol (EtOH) was applied topically. Cumulative amount permeated (top row), flux (middle row), and mass distribution (bottom row) were determined over 6 h. N = 3 donors/batches per barrier for each formulation, each in triplicate. Data shown as mean ± standard error of mean. Mean cumulative amount across vehicles was statistically compared at each time point, for each model, by ANOVA with the maximum modulus test. ** = p < 0.01, *** = p < 0.001. Mass balance values were adjusted using an arcsin-square root transformation and chemicals within collection groups (i.e., donor, tissue, or receptor) were compared by ANOVA with the maximum modulus test. ns = no significance, * = p < 0.05, *** = p < 0.001.