Biotransformation Capacity of Carboxylesterase in Skin and Keratinocytes for the Penta-Ethyl Ester Prodrug of DTPA

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Abstract
The penta-ethyl ester prodrug of the chelating agent diethylene triamine pentaacetic acid (DTPA), referred to as C2E5, effectively accelerated clearance of americium after transdermal delivery. Carboxylesterases (CESs) play important roles in facilitating C2E5 hydrolysis. However, whether CESs in human skin hydrolyze C2E5 remains unknown. We evaluated the gene and protein expression of CESs in distinctive human epidermal cell lines: HEKa, HEKn, HaCaT and A431. The substrates, p-nitrophenyl acetate (pNPA) and 4-nitrophenyl valerate (4-NPV), were used to access esterase and CES activity. C2E5 hydrolysis was measured by radiometric HPLC after incubating [14C]-C2E5 with S9 fractions prepared from skin cell lines with analysis. CESs specific inhibitors were used to access metabolism in human skin S9 fractions with analysis by LC/MS/MS. We identified the CES1 and CES2 bands in the western blot. The gene expression of these enzymes was supported by a real-time polymerase chain reaction (RT-qPCR). pNPA and 4-NPV assays demonstrated esterases and CESs activity in all the cell lines that were comparable to human skin S9 fractions. The prodrug C2E5 was hydrolyzed by skin S9 fractions resulting in a primary metabolite, C2E4. In human skin S9 fractions, inhibition of C2E5 hydrolysis was greatest with a pan CES inhibitor (benzil). CES1 inhibition (troglitazone) was greater than CES2 (loperamide), suggesting a primary metabolic role for CES1. These results indicate that human keratinocyte cell lines are useful for the evaluation of human cutaneous metabolism and absorption of ester-based prodrugs. However, keratinocytes from skin provide a small contribution to the overall metabolism of C2E5.

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