CYP3A4, CYP3A5, and MDR1 in human small and large intestinal cell lines suitable for drug transport studies

• Published in: Journal of pharmaceutical sciences Vol. 90; no. 11; pp. 1736 - 1751
• Main Authors: Engman, Helena A., Lennernäs, Hans, Taipalensuu, Jan, Otter, Charlotta, Leidvik, Brith, Artursson, Per
• Format: Journal Article
• Language: English
• Published: New York John Wiley & Sons, Inc 01.11.2001; Wiley; American Pharmaceutical Association
• Subjects: Animal cells; ATP Binding Cassette Transporter, Subfamily B, Member 1 - biosynthesis; bioavailability; Biological and medical sciences; Biological Transport; Biotechnology; Caco-2; Caco-2 Cells - drug effects; Caco-2 Cells - enzymology; Cell Line; Cholecalciferol - pharmacology; CYP3A; Cytochrome P-450 CYP3A; Cytochrome P-450 Enzyme System - biosynthesis; Establishment of new cell lines, improvement of cultural methods, mass cultures; Eukaryotic cell cultures; first pass extraction; Fundamental and applied biological sciences. Psychology; General pharmacology; Humans; Hydroxytestosterones - metabolism; intestinal epithelium; Intestine, Large - cytology; Intestine, Large - drug effects; Intestine, Large - enzymology; Intestine, Small - cytology; Intestine, Small - drug effects; Intestine, Small - enzymology; Medical sciences; Methods. Procedures. Technologies; Mixed Function Oxygenases - biosynthesis; P-glycoprotein; permeability; Pharmacokinetics. Pharmacogenetics. Drug-receptor interactions; Pharmacology. Drug treatments; RNA, Messenger - biosynthesis; Human; Cell culture; Digestive system; Enzyme; Isozyme; Cytochrome P450; Biological transport; P Glycoprotein; Gene expression; Metabolism; In vitro; Small intestine; Modeling; Enzymatic activity; Investigation method; Established cell line; Active ingredient; Colon; Pharmacokinetics
• ISSN: 0022-3549; 1520-6017
• DOI: 10.1002/jps.1123

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. © 2001 Wiley‐Liss, Inc. and the American Pharmaceutical Association J Pharm Sci 90:1736–1751, 2001