Functional Analysis of Modified Caco-2 Cells Carrying CES2A1 as a Model for Intestinal Absorption of Prodrugs.

Carboxylesterase (CES) plays an important role in the metabolism of ester-containing drugs such as prodrugs and is highly expressed in the human intestine and liver. The ideal prodrug is barely hydrolyzed by human intestinal CES (CES2A1) but is extensively converted to an active drug by human hepatic CES (CES1A). It is, therefore, important to evaluate CES2A1-mediated hydrolysis during intestinal absorption. Unfortunately, Caco-2 cells, the most common enterocyte model for drug permeability, are not suitable for permeability studies of prodrugs due to their high and extremely low expression of CES1A and CES2A1, respectively. Previously, we have prepared CES2/Caco-2^CES1KD cells with reduced human CES1A and highly expressed CES2A1. In the present study, the metabolic and transport properties of CES2/Caco-2^CES1KD cells were characterized. The expression of transporters and metabolizing enzymes other than CESs was similar in CES2/Caco-2^CES1KD and Caco-2 cells. However, the expression of CES2A1 in CES2/Caco-2^CES1KD was about 7-10 fold higher than that of CES1A in Caco-2 cells and comparable to levels found in the human intestine. Hydrolysis during transport across cell monolayers was analyzed using ethyl and butyl esters of p-aminobenzoic acid (PABA). Ethyl PABA, a better substrate for CES1A than CES2A1, was similarly hydrolyzed in Caco-2 and CES2/Caco-2^CES1KD cell monolayers due to the high expression of CES2A1 in CES2/Caco-2^CES1KD cells. Butyl PABA, a good substrate for CES2A1, was substantially hydrolyzed in CES2/Caco-2^CES1KD cell monolayers, in contrast to negligible hydrolysis in Caco-2 cell monolayers. N-Acetylation of PABA derived from PABA esters showed similar activity in Caco-2 and CES2/Caco-2^CES1KD cell monolayers.