title: Establishment and Characterization of a Novel Caco-2 Subclone with a Similar Low Expression Level of Human Carboxylesterase 1 to Human Small Intestine.
abstract: Caco-2 cells predominantly express human carboxylesterase 1 (hCE1), unlike the human intestine that predominantly expresses human carboxylesterase 2 (hCE2). Transport experiments using Caco-2 cell monolayers often lead to misestimation of the intestinal absorption of prodrugs because of this difference, as prodrugs designed to increase the bioavailability of parent drugs are made to be resistant to hCE2 in the intestine, so that they can be hydrolyzed by hCE1 in the liver. In the present study, we tried to establish a new Caco-2 subclone, with a similar pattern of carboxylase expression to human intestine, to enable a more accurate estimation of the intestinal absorption of prodrugs. Although no subclone could be identified with high expression levels of only hCE2, two subclones, #45 and #78, with extremely low expression levels of hCE1 were subcloned from parental Caco-2 cells by the limiting dilution technique. Unfortunately, subclone #45 did not form enterocyte-like cell monolayers due to low expression of claudins and β-actin. However, subclone #78 formed polarized cell monolayers over 4 weeks and showed similar paracellular and transcellular transport properties to parental Caco-2 cell monolayers. In addition, the intestinal transport of oseltamivir, a hCE1 substrate, could be evaluated in subclone #78 cell monolayers, including P-glycoprotein-mediated efflux under nonhydrolysis conditions, unlike parental Caco-2 cells. Consequently, it is proposed that subclone #78 may provide a more effective system in which to evaluate the intestinal absorption of prodrugs that are intended to be hydrolyzed by hCE1.
authors.affiliation: Graduate School of Pharmaceutical Sciences, Kumamoto University, Kumamoto, Japan.
authors.forename: Kayoko
authors.initials: K
authors.lastname: Ohura
authors.affiliation: Graduate School of Pharmaceutical Sciences, Kumamoto University, Kumamoto, Japan.
authors.forename: Hikaru
authors.initials: H
authors.lastname: Nishiyama
authors.affiliation: Graduate School of Pharmaceutical Sciences, Kumamoto University, Kumamoto, Japan.
authors.forename: Saori
authors.initials: S
authors.lastname: Saco
authors.affiliation: Graduate School of Pharmaceutical Sciences, Kumamoto University, Kumamoto, Japan.
authors.forename: Keisuke
authors.initials: K
authors.lastname: Kurokawa
authors.affiliation: Graduate School of Pharmaceutical Sciences, Kumamoto University, Kumamoto, Japan iteruko@gpo.kumamoto-u.ac.jp.
authors.forename: Teruko
authors.initials: T
authors.lastname: Imai
mesh_terms: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000378:metabolism; D000199:Actins / Q000378:metabolism; D001682:Biological Availability; D018938:Caco-2 Cells; D043182:Carboxylesterase / Q000378:metabolism; D002265:Carboxylic Ester Hydrolases / Q000378:metabolism*; D045744:Cell Line, Tumor; D006801:Humans; D006868:Hydrolysis; D007408:Intestinal Absorption / Q000502:physiology; D007421:Intestine, Small / Q000378:metabolism*; D008099:Liver / Q000378:metabolism; D053139:Oseltamivir / Q000378:metabolism; D011355:Prodrugs / Q000378:metabolism
chemical_list: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D000199:Actins; D011355:Prodrugs; D053139:Oseltamivir; D002265:Carboxylic Ester Hydrolases; C474609:CES1 protein, human; C517204:CES2 protein, human; D043182:Carboxylesterase