title: Development of Caco-2 cells co-expressing CYP3A4 and NADPH-cytochrome P450 reductase using a human artificial chromosome for the prediction of intestinal extraction ratio of CYP3A4 substrates.
abstract: The Caco-2 cells co-expressing cytochrome P450 (CYP) 3A4 and NADPH-cytochrome P450 reductase (CPR) were developed using a human artificial chromosome (HAC) vector. The CYP3A4 and CPR genes were cloned into the HAC vector in CHO cells using the Cre-loxP system, and the microcell-mediated chromosome transfer technique was used to transfer the CYP3A4-CPR-HAC vector to Caco-2 cells. After seeding onto semipermeable culture inserts, the CYP3A4-CPR-HAC/Caco-2 cells were found to form tight monolayers, similar to the parental cells, as demonstrated by the high transepithelial electrical resistance (TEER) value and comparable permeability of non-CYP3A4 substrates between parent and CYP3A4-CPR-HAC/Caco-2 cell monolayers. The metabolic activity of CYP3A4 (midazolam 1'-hydroxylase activity) in the CYP3A4-CPR-HAC/Caco-2 cells was constant from 22 to 35 passages, indicating that HAC vectors conferred sufficient and sustained CYP3A4 activity to CYP3A4-CPR-HAC/Caco-2 cells. The strong relationship between the metabolic extraction ratios (ER) obtained from the CYP3A4-CPR-HAC/Caco-2 cells and calculated intestinal extraction ratios in humans (Eg) from reported intestinal availability (Fg) was found for 17 substrates of CYP3A4 (r2= 0.84). The present study suggests that the CYP3A4-CPR-HAC/Caco-2 cell monolayer can serve as an in vitro tool that facilitates the prediction of intestinal extraction ratio (or availability) in humans.
authors.affiliation: Discovery and Preclinical Research Division, Taiho Pharmaceutical Co. Ltd., Tsukuba, Ibaraki, Japan.
authors.forename: Toru
authors.initials: T
authors.lastname: Takenaka
authors.affiliation: Chromosome Engineering Research Center, Tottori University, Tottori, Japan.
authors.forename: Kanako
authors.initials: K
authors.lastname: Kazuki
authors.affiliation: Discovery and Preclinical Research Division, Taiho Pharmaceutical Co. Ltd., Tsukuba, Ibaraki, Japan.
authors.forename: Naomoto
authors.initials: N
authors.lastname: Harada
authors.affiliation: Quality and Reliability Assurance Division, Taiho Pharmaceutical Co. Ltd., Tokushima, Japan.
authors.forename: Jiro
authors.initials: J
authors.lastname: Kuze
authors.affiliation: Discovery and Preclinical Research Division, Taiho Pharmaceutical Co. Ltd., Tsukuba, Ibaraki, Japan.
authors.forename: Masato
authors.initials: M
authors.lastname: Chiba
authors.affiliation: Department of Clinical Pharmacy, Graduate School of Pharmaceutical Sciences, Nagoya City University, Nagoya, Japan.
authors.forename: Takahiro
authors.initials: T
authors.lastname: Iwao
authors.affiliation: Department of Clinical Pharmacy, Graduate School of Pharmaceutical Sciences, Nagoya City University, Nagoya, Japan.
authors.forename: Tamihide
authors.initials: T
authors.lastname: Matsunaga
authors.affiliation: Biomedical Science, Institute of Regenerative Medicine and Biofunction, Graduate School of Medical Science, Tottori University, Tottori, Japan.
authors.forename: Satoshi
authors.initials: S
authors.lastname: Abe
authors.affiliation: Chromosome Engineering Research Center, Tottori University, Tottori, Japan.
authors.forename: Mitsuo
authors.initials: M
authors.lastname: Oshimura
authors.affiliation: Chromosome Engineering Research Center, Tottori University, Tottori, Japan; Biomedical Science, Institute of Regenerative Medicine and Biofunction, Graduate School of Medical Science, Tottori University, Tottori, Japan. Electronic address: kazuki@grape.med.tottori-u.ac.jp.
authors.forename: Yasuhiro
authors.initials: Y
authors.lastname: Kazuki
mesh_terms: D018938:Caco-2 Cells; D022222:Chromosomes, Artificial, Human / Q000235:genetics*; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007413:Intestinal Mucosa / Q000378:metabolism*; D009251:NADPH-Ferrihemoprotein Reductase / Q000235:genetics / Q000378:metabolism*; D013379:Substrate Specificity
chemical_list: D003577:Cytochrome P-450 Enzyme System; D009251:NADPH-Ferrihemoprotein Reductase
keywords: CYP3A4; Caco-2 cells; Human artificial chromosome vector; Intestinal metabolism; NADPH-cytochrome P450 reductase