Construction of a functional transporter analysis system using MDR1 knockdown Caco-2 cells

• Published in: Pharmaceutical research Vol. 22; no. 8; pp. 1287 - 1293
• Main Authors: Watanabe, Tomoko, Onuki, Reiko, Yamashita, Shinji, Taira, Kazunari, Sugiyama, Yuichi
• Format: Journal Article
• Language: English
• Published: United States Springer Nature B.V 01.08.2005
• Subjects: Bioavailability; Biological Transport, Active; Blotting, Western; Caco-2 Cells; Calcium Channel Blockers - pharmacology; Drugs; Gene Library; Genes; Genes, MDR - genetics; Genetic Vectors; Glycoproteins; Humans; Intestinal Absorption; Kinases; Membrane Transport Proteins - metabolism; Models, Biological; Organisms, Genetically Modified; Pharmaceuticals; Pharmacokinetics; Proteins; Reverse Transcriptase Polymerase Chain Reaction; RNA Interference - physiology; RNA, Messenger - biosynthesis; RNA, Messenger - genetics; RNA, Small Interfering - chemical synthesis; RNA, Small Interfering - genetics; RNA, Transfer, Val - metabolism; Transfection; Verapamil - pharmacology; St Louis Missouri; United States > US; Japan
• ISSN: 0724-8741; 1573-904X
• DOI: 10.1007/s11095-005-5270-z

The efflux transporter, P-glycoprotein (P-gp), located in the brush-border membrane of intestinal absorptive cells, reduces the bioavailability of a wide range of orally administered drugs. Using P-gp inhibitors in transport experiments in Caco-2 cell monolayers is widely accepted as an efficient way to estimate the contribution of P-gp to the intestinal absorption of drugs. However, there still remain some arguments that the inhibitors might affect the function of other proteins. Multidrug resistance 1 gene (MDR1) specifically inhibited Caco-2 cells were constructed, therefore, as a better in vitro evaluation system of intestinal drug absorption. The effective sites of RNAi were selected using siRNA libraries and single siRNAs and MDR1 stable knockdown Caco-2 cells were constructed using a tRNA(val)-shRNA expression vector. In siRNA stably expressed Caco-2 cells, the expression level of MDR1 was reduced at mRNA and protein levels. Transcellular transport studies using digoxin revealed that the P-gp function was suppressed completely, similar to that in verapamil-treated cells. MDR1 stable knockdown Caco-2 cells were successfully constructed by RNAi technology. This will consequently allow the development of a selection system for candidate drugs with improved absorption properties.