Absorption properties and P-glycoprotein activity of modified Caco-2 cell lines

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Abstract

Caco-2 cell line is extensively used as an in vitro model in studying small intestinal absorption but it lacks proper expression of efflux pumps and cytochrome P450 enzymes that are involved in absorption and first pass metabolism of drugs. We created two novel Caco-2 cell lines expressing orphan nuclear receptors pregnane X receptor and constitutive androstane receptor that regulate many genes involved in xenobiotic metabolism. We conducted a systematic study on expression of some metabolic genes, P-glycoprotein activity and absorption properties of several drugs with these new cell lines and previously described modified Caco-2 cell lines (MDR1 transfection, vincristine treatment and 1α,25-dihydroxyvitamin D3 treatment). A short culture time medium was also included in the study. Most modified cell lines formed tight differentiated monolayers. MDR1, CYP2C9 and CYP3A4 genes were upregulated in some cell lines. Elevated P-glycoprotein activities were observed by calcein-AM uptake experiments but this did not affect significantly the permeability of selected P-glycoprotein substrates. Some cell lines had similar passive and active permeability properties to Caco/WT cells while in few cell lines these were altered. Passive transcellular permeability was modestly elevated in all modified cell lines. In addition, several compounds showed pH-dependent permeability properties.

Introduction

The majority of orally administered drugs are absorbed in the small intestine. During absorption process the drug molecules interact with oxidizing cytochrome P450 (CYP) enzymes and ATP-driven efflux pumps of the ATP binding cassette transporter (ABC) family that are expressed in the small intestinal epithelium (Suzuki and Sugiyama, 2000). These proteins may lead to reduced bioavailability of drugs by metabolising them and pumping them back to the intestinal lumen from the enterocytes.

Colon carcinoma cell line Caco-2 is a widely used in vitro model for predicting intestinal absorption of drug molecules (Artursson et al., 2001) but it expresses only low levels of CYP enzymes, especially the important isoform CYP3A4 (Prueksaritanont et al., 1996, Nakamura et al., 2003). ABC transporters are expressed more abundantly but quantitative differences to the small intestinal epithelium exist (Taipalensuu et al., 2001). Cummins et al. (2004) have shown with Caco-2 cells transfected with CYP3A4 that oxidative metabolism is an important process during drug absorption. Thus, a cell line expressing both CYP enzymes and ABC transporters at high levels would be a valuable in vitro tool for studying drug absorption across the intestinal epithelium.

Our aim in the present study was to increase the CYP and efflux pump activities by transfecting Caco-2 cells with pregnane X receptor (PXR; NR1I2) and constitutive androstane receptor (CAR; NR1I3) genes. These orphan nuclear receptors have been found to regulate co-ordinately the expression of many metabolic genes in response to several xenobiotic and endogenous compounds in mice (Maglich et al., 2002, Rosenfeld et al., 2003) and in man (Synold et al., 2001). Nuclear receptors bind to certain DNA elements in the promoter regions of their target genes in response to ligands. They recruit co-activators and enhance mRNA synthesis of the target genes but can also repress transcription by recruiting corepressors (Aranda and Pascual, 2001). Known target genes for both PXR and CAR include MDR1, MRP2, CYP2B6, CYP2C9 and CYP3A4 (Honkakoski, 2003). Thus, in principle, many efflux pump and CYP genes may be regulated by transfecting only a single gene.

Previous attempts in increasing drug metabolism and efflux in Caco-2 cells have been focusing on modifying the activity of only a single gene product. As a result, several modified Caco-2 cell lines have been introduced. Transfection of Caco-2 cells with MDR1 cDNA has been shown to increase verapamil binding and decrease the apical-to-basolateral transport of quinidine and talinol (Döppenschmitt et al., 1999a). 1α,25-Dihydroxyvitamin D3 can induce functionally active CYP3A4 expression in Caco-2 cell lines (Schmiedlin-Ren et al., 1997) presumably via binding of Vitamin D receptor to PXR responsive elements of CYP3A4 promoter (Mäkinen et al., 2002, Drocourt et al., 2002). Culturing Caco-2 cells in the presence of vincristine increases P-glycoprotein activity which has been determined by calcein-AM extrusion assay (Eneroth et al., 2001). Cytotoxic vinca alkaloids are substrates of P-glycoprotein and prolonged exposure to vincristine leads to proliferation of only resistant subpopulations that express P-glycoprotein well (Hoskins et al., 1993). Also, a modified culture medium that accelerates the differentiation of Caco-2 cells has been developed (Lentz et al., 2000). Individual cell lines have been characterized to some degree but a systematic comparison is still lacking. Therefore, we studied the gene expression and transport properties of the modified Caco-2 cell lines (Table 1).

Section snippets

Chemicals

Atenolol, antipyrine, baclofen, cyclosporin A, ibuprofen, progesterone, propranolol hydrochloride and vincristine sulfate were from Sigma (St. Louis, MO). Cephalexin, rhodamine-123 and verapamil were purchased from MP Biomedicals (Irvine, CA). 3H-mannitol was from Perkin-Elmer (Boston, MA) and 1α,25-dihydroxyvitamin D3 from Leo Pharma (Denmark).

Caco/WT

Caco-2 cells (ATCC HTB-37) were obtained from American type culture collection (Manassas, VA). All cell culture reagents were from Bio-Whittaker

Monolayer integrity and cell differentiation

The apparent permeability of mannitol across Caco/WT cells was 1.29 × 10−6 cm/s and it was pH independent (Table 3). Caco/4d cells were more and Caco/VCR less permeable for mannitol than Caco/WT cells. Other cell lines were close to the Caco/WT cells. All modified cell lines had higher alkaline phosphatase activity than the Caco/WT cells indicating adequate cellular differentiation (Table 3).

mRNA levels of metabolic genes

The results of quantitative RT-PCR experiments are shown in Table 4. All modified cell lines, except in

Discussion

Our goal was to modify Caco-2 cells to better predict intestinal drug absorption. Specifically, we aimed at increasing cytochrome P450 and apically located efflux pump activities. This study introduces two novel Caco-2 cell lines (Caco/mCAR and Caco/hPXR) and compares their characteristics systematically with wild type cells and some previously described modified Caco-2 cells.

Acknowledgements

This study was financially supported by the National Technology Agency of Finland (TEKES) and the Academy of Finland. We thank Mr. Markku Taskinen for skilful technical assistance.

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