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. Issue 1 (February 2017)

• Record Type: Journal Article
• Authors: Takenaka, Toru; Kazuki, Kanako; Harada, Naomoto; Kuze, Jiro; Chiba, Masato; Iwao, Takahiro; Matsunaga, Tamihide; Abe, Satoshi; Oshimura, Mitsuo; Kazuki, Yasuhiro
• Is Part Of: Drug metabolism and pharmacokinetics. Volume 32:Issue 1(2017)
• Page Start: 61
• Page End: 68
• Publication Date: 2017-02
• Subjects: CYP3A4 -- NADPH-cytochrome P450 reductase -- Caco-2 cells -- Human artificial chromosome vector -- Intestinal metabolism; ABT 1-aminobenzotriazole -- CLint intrinsic clearance -- CPR NADPH-cytochrome P450 reductase -- Ct threshold cycle -- CYP cytochrome P450 -- Eg intestinal extraction ratio in humans -- ER extraction ratio -- Fa fraction absorbed -- Fg intestinal availability -- FISH fluorescence in situ hybridization -- GFP green fluorescent protein -- HAC human artificial chromosome -- LC-MS/MS liquid chromatography–tandem mass spectrometry -- MMCT microcell-mediated chromosome transfer -- PAC P1 artificial chromosome -- Papp apparent permeability coefficient -- PCR polymerase chain reaction -- P-gp P-glycoprotein -- PPIA peptidylprolyl isomerase A -- RT-PCR reverse transcription-polymerase chain reaction -- TEER transepithelial electrical resistance -- TM transport medium; Drugs -- Metabolism -- Periodicals; Pharmacokinetics -- Periodicals; 615.7
• DOI: 10.1016/j.dmpk.2016.08.004
• Languages: English
• ISSNs: 1347-4367

Abstract

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 ( E g) from reported intestinal availability ( F g) was found for 17 substrates of CYP3A4 ( r 2 = 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.