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CYP3A4, CYP3A5, and MDR1 in Human Small and Large Intestinal Cell Llines Suitable for Drug Transport Studies

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ABSTRACT

The aim of this study was to find a cell culture model of the intestinal epithelium for use in studies of CYP3A4-mediated first-pass metabolism of drugs and also for studies of the interplay between CYP3A4 metabolism and P-glycoprotein efflux. For this purpose, the expression of CYP3A4, CYP3A5, and MDR1 mRNA was studied in three cell lines of the normal human intestinal epithelium and three transformed cell lines of colonic (Caco-2) origin. Surprisingly, only transformed cell lines were induced by 1α,25-dihydroxy vitamin D3 (D3) to express high amounts of CYP3A4. In contrast to the original findings for this model, the monolayer integrity was maintained during D3 treatment. Levels of CYP3A mRNA expression in Caco-2 and TC7 cells differed dramatically. The highest levels of CYP3A4 and lowest levels of CYP3A5 mRNA expression were observed in D3 treated Caco-2 cells of high passage numbers, resulting in a CYP3A4/3A5 expression ratio greater than fourfold higher than that seen in TC7 cells. Functional studies, using the CYP3A probe testosterone, showed that CYP3A activity was completely absent only in uninduced Caco-2 cells. After D3 induction, high levels of the metabolite were produced in both cell lines (149.4 ± 12.3 and 86.5 ± 3.8 pmol 6β-OH testosterone/min/mg cellular protein from 75 μM testosterone in Caco-2 and TC7 cells, respectively). The maximum velocity (Vmax) and the apparent Michaelis constant (Km) for the 6β-hydroxylation of testosterone by CYP3A4 in intact Caco-2 monolayers were similar to those obtained from human intestinal microsomes. Only minor changes in P-glycoprotein activity were observed when the metabolically stable P-glycoprotein substrate celiprolol was used. In conclusion, these results show that the features of the generally available Caco-2 cell line from American Type Culture Collection make it suitable for studies of CYP3A4-mediated first-pass metabolism and also for studies of the interplay between CYP3A4 and drug efflux mechanisms.

Section snippets

INTRODUCTION

The cytochrome P450 (CYP) enzyme CYP3A4, which is responsible for the oxidative metabolism of > 60% of all clinically used drugs,1 is found predominantly in the small intestine and the liver.2 Recent studies suggest that drugs such as cyclosporin can be metabolized to a comparable degree in the mucosal lining of the intestinal epithelium and in the liver.3 It has been hypothesized that the gene product of the multidrug resistance gene MDR1, P-glycoprotein (Pgp), and CYP3A4 can act

Materials

D3 was purchased from Solvay Duphar (Weesp, The Netherlands). Testosterone, 6β-hydroxytestosterone, clotrimazole, 9-cis-retinoic acid, and dexamethasone were obtained from Sigma Chemical Company (St. Louis, MO). Androstenedione and 2β-hydroxytestosterone were kindly provided by AstraZeneca R&D (Mölndal, Sweden). Recombinant human growth hormone was a generous gift from Dr. Jonas Fransson (Pharmacia & Upjohn, Stockholm, Sweden). [14C]celiprolol (37.3 μCi mg−1) was provided by Rhone-Poulenc Rorer

CYP3A4, CYP3A5, and MDR1 mRNA in Human Small and Large Intestinal Epithelial Cell Lines

The mRNA expression of CYP3A4, CYP3A5, and MDR1 was studied in the presence and absence of a laminin-containing ECM.30 RT-PCR analysis showed that CYP3A4 was expressed weakly in two of the three human duodenal cell lines (BN and LG) (Figure 1). In contrast, CYP3A4 was not expressed in the three variants of the Caco-2 cell line. However, CYP3A5 was expressed in two of the three investigated Caco-2 cell lines: the original Caco-2 population from ATCC (Caco-2) and the clone TC7 (Figure 1). The

DISCUSSION

Schmiedlin-Ren et al.18 recently showed that D3 induces CYP3A4 expression in the Caco-2 cell line. This finding expanded the applicability of Caco-2 cells to include studies of the intestinal first-pass metabolism33 and of potentially important interactions between CYP3A4 and Pgp activities.35 However, the D3 induced Caco-2 cell model has not been fully characterized and can, therefore, be improved. Firstly, this cell line originates from the colon, where the expression of CYP is lower than in

ACKNOWLEDGEMENTS

This work was supported by AstraZeneca R&D, Mölndal, Sweden, and The Swedish Medical Research Council, grant 9478. We thank Dr. Edward L. LeCluyse, University of Chapel Hill, and Drs. Tommy B Andersson, Ulf Bredberg, Carl-Gunnar Regårdh, and Anna-Lena Ungell at AstraZeneca R&D Mölndal, Sweden, for valuable discussions.

REFERENCES (53)

  • A.R. Giuliano et al.

    Characterization of the vitamin D receptor from the Caco-2 human colon carcinoma cell line: effect on cellular differentiation

    Arch Biochem Biophys

    (1991)
  • S.A. Kliewer et al.

    An orphan nuclear receptor activated by pregnanes defines a novel steroid signaling pathway

    Cell

    (1998)
  • P. Maurel

    The CYP3A family

  • P.B. Watkins et al.

    Identification of glucocorticoid-inducible cytochromes P-450 in the intestinal mucosa of rats and man

    J Clin Invest

    (1987)
  • C.Y. Wu et al.

    Differentiation of absorption and first-pass gut and hepatic metabolism in humans: studies with cyclosporine

    Clin Pharmacol Ther

    (1995)
  • L.-.S.L. Gan et al.

    CYP3A-like cytochrome P450-mediated metabolism and polarized efflux of cyclosporin A in Caco-2 cells: Interaction between the two biochemical barriers to intestinal transport

    Drug Metab Dispos

    (1996)
  • V.J. Wacher et al.

    Overlapping substrate specificities and tissue distribution of cytochrome P-450 3A and P-glycoprotein have implications for drug delivery and activity in cancer chemotherapy

    Mol Carcinog

    (1995)
  • E.G. Schuetz et al.

    Modulators and substrates of P-glycoprotein and cytochrome P-450 3A coordinately upregulate these proteins in human colon carcinoma cells

    Mol Pharmacol

    (1996)
  • M. Pinto et al.

    Enterocyte-like differentiation and polarization of the human colon carcinoma cell line Caco-2 in culture

    Biol Cell

    (1983)
  • J. Hunter et al.

    Drug absorption limited by P-glycoprotein-mediated secretory drug transport in human intestinal epithelial Caco-2 cell layers

    Pharm Res

    (1993)
  • T. Hirohashi et al.

    Function and expression of multidrug resistance associated protein family in human colon adenocarcinoma cells (Caco-2)

    J Pharmacol Exp Ther

    (2000)
  • T. Prueksaritanont et al.

    Comparative studies of drug-metabolising enzymes in dog, monkey, and human small intestine, and in Caco-2 cells

    Drug Metab Dispos

    (1996)
  • S.D. Raeissi et al.

    Interplay between CYP3A-mediated metabolism and polarized efflux of terfendine metabolites in intestinal epithelial Caco-2 (TC7) cell monolayers

    Pharm Res

    (1999)
  • S.A. Wrighton et al.

    Studies on the expression and metabolic capabilities of human liver cytochrome P450IIIA5 (HLp3)

    Mol Pharmacol

    (1990)
  • Q.-.Y. Zhang et al.

    Characterization of human small intestinal cytochromes P-450

    Drug Metab Disp

    (1999)
  • M.F. Paine et al.

    Characterisation of interintestinal and intraintestinal variations in human CYP3A-dependent metabolism

    J Pharmacol Exp Ther

    (1997)

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