Proteomic profiling of metabolizing enzymes and transporters in 2 layers of Caucasian human skin.

Topical and transdermal drug and cosmetic development is advancing across industry; however, the efforts may in part be hindered by paucity of data on the fate of chemicals in human skin, which depend on protein-mediated transport and biotransformation of such chemicals. A label-free mass spectrometry-based proteomics approach was used to comprehensively quantify drug metabolizing enzymes and transporters in the fractionated epidermis and dermis of 17 healthy Caucasian human skin samples. Over 1000 proteins were identified for cytosolic and membrane components, and abundance were obtained using a modified HiN (high 3/2 ion intensity) approach (without using standards). Key findings included high interindividual variability in the expression of phase I and II enzymes (eg, aldehyde dehydrogenase 2, carboxylesterase 1, glutathione S-transferase P1, and glutathione peroxidase 3) and solute carrier transporters (eg, SLC25A5, SLC25A6). Notably, several metabolic enzymes, previously uncharacterized in human skin, were quantified in the membrane fractions, including glutathione S-transferases (GSTs) such as GSTM3 (12.2 pmol/mg protein) and GSTP1 (5.55 pmol/mg protein). Subcellular localization analysis revealed that many quantified proteins were associated with mitochondrial or membrane compartments, reinforcing the functional diversity and compartmentalized nature of dermal metabolism. Furthermore, strong correlations in enzyme and transporter expression were observed between the dermis and epidermis (Spearman's ρ > 0.85). This dataset provides the most detailed quantification to date of drug metabolizing enzymes and transporters in human skin and offers critical input parameters for dermal physiologically based pharmacokinetic modeling applications. These quantitative insights improve the accuracy and clinical relevancy of in vitro in vivo extrapolations related to dermal drug metabolism and disposition for chemicals applied to the skin or those that come into contact with the skin inadvertently. Integration of this proteomic dataset into physiologically based pharmacokinetic frameworks will enhance the scientific reliability, applicability across product applications and regulatory acceptance of skin-based models for drug development and safety evaluation. SIGNIFICANCE STATEMENT: Understanding drug metabolism and transport in human skin is essential for predicting dermal absorption and safety. This study provides the most comprehensive proteomic dataset of metabolizing enzymes and transporters in epidermis and dermis, revealing high interindividual variability, previously unquantified enzymes, and strong layer correlations, supporting improved dermal physiologically based pharmacokinetic model development.