Development of Caco-2 cells expressing four CYPs via a mammalian artificial chromosome

Abstract

Background Oral administration is the most common way to deliver drugs to the systemic circulation or target organs. Orally administered drugs are absorbed in the intestine and metabolized in the intestine and liver. In the early stages of drug development, it is important to predict first-pass metabolism accurately to select candidate drugs with high bioavailability. The Caco-2 cell line derived from colorectal cancer is widely used as an intestinal model to assess drug membrane permeability. However, because the expression of major drug-metabolizing enzymes, such as cytochrome P450 (CYP), is extremely low in Caco-2 cells, it is difficult to predict intestinal metabolism, which is a significant factor in predicting oral drug bioavailability. Previously, we constructed a mouse artificial chromosome vector carrying the CYP (CYP2C9, CYP2C19, CYP2D6, and CYP3A4) and P450 oxidoreductase (POR) (4CYPs-MAC) genes and increased CYP expression and metabolic activity in HepG2 cells via transfer of this vector. Results In the current study, to improve the Caco-2 cell assay model by taking metabolism into account, we attempted to increase CYP expression by transferring the 4CYPs-MAC into Caco-2 cells. The Caco-2 cells carrying the 4CYPs-MAC showed higher CYP mRNA expression and activity. In addition, high metabolic activity, availability for permeation test, and the potential to assess drug–drug interactions were confirmed. Conclusions The established Caco-2 cells with the 4CYPs-MAC are expected to enable more accurate prediction of the absorption and metabolism in the human intestine than parental Caco-2 cells. The mammalian artificial chromosome vector system would provide useful models for drug development.

Generation of tetracycline-controllable CYP3A4-expressing Caco-2 cells by the piggyBac transposon system

“…It is assumed that by using piggyBac transposon, more exogenous genes were inserted into the various regions of the host chromosomes as compared with other gene transfer methods, and thus the Caco-2 cells with high CYP3A4 activity could be established. To the best of our knowledge, CYP3A4-Caco-2 cells in the present study showed the highest CYP3A4 activity among the previously published studies 11 – 15 . Takenaka et al co-expressed NADHP-cytochrome P450 reductase as well as CYP3A4 in Caco-2 cells using a human artificial chromosome 14 .…”

“…It has been known that stable transfectants derived from Caco-2 cells cannot be obtained even by using virus vectors such as retrovirus, lentivirus, etc., as well as by a conventional plasmid transfection and drug selection. This is the reason why, though quite simple, a few studies about CYP3A4-expressing Caco-2 cells have been published 11 – 15 , 18 . To overcome these difficulties, we used piggyBac transposon system to forcibly integrate and express human CYP3A4 gene under the control of a tetracycline-controllable promoter into Caco-2 cells.…”

“…Since CYP3A4 is a dominant drug-metabolizing enzyme in the human small intestine, accounting for approximately 80% of total intestinal CYP content 10 , development of a Caco-2 cell model that can predict CYP3A4-mediated drug metabolism in the small intestine is desirable. Many groups have tried to establish Caco-2 cells stably expressing CYP3A4, but only a few were reported, which used viral vectors, episomal vectors, and artificial chromosomes 11 – 15 . Brimer-Cline and Schuetz used adenoviral vectors to induce CYP3A4 expression in Caco-2 and LLC-PK1 cells 11 .…”

“…In other reports, introduction of the CYP3A4 gene into Caco-2 cells using episomal vectors not only markedly affected cell growth, but resulted in unstable expression of CYP3A4 12 , 13 . Other groups developed Caco-2 cells co-expressing cytochrome P450 (CYPs) and NADPH-cytochrome P450 reductase (CPR) by using human artificial chromosome (HAC) vectors 14 , 15 . However, the metabolic activity for CYP3A4-substrate, midazolam, in these cells was equal to or slightly lower than that in human intestinal mucosa 14 .…”

Analyzing the metabolic fate of oral administration drugs: A review and state-of-the-art roadmap

“…Figure 2A clearly demonstrates the use of human artificial chromosome (HAC) vectors to develop Caco-2 cells co-expressing CYP3A4 and CYP450 reductase (CPR) (Takenaka et al, 2017). Specifically, CYP3A4 and CPR genes were cloned into HAC vectors in CHO cells using the Cre-loxP system, and then CYP3A4-CPR-HAC was transferred to Caco-2 cells by chromosomal transfer technology (Ohta et al, 2020). PiggyBac transposon isolated from Trichoplusiani also serves as a tool to overexpress CYP3A4 in Caco-2 cells (Ichikawa et al, 2021).…”