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Construction of a Functional Transporter Analysis System Using MDR1 Knockdown Caco-2 Cells

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Purpose

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.

Methods

The effective sites of RNAi were selected using siRNA libraries and single siRNAs and MDR1 stable knockdown Caco-2 cells were constructed using a tRNAval-shRNA expression vector.

Results

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.

Conclusions

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.

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Abbreviations

MDR1 :

multidrug resistance 1

P-gp:

p-glycoprotein

RNAi:

RNA interference

ShRNA:

short hairpin RNA

SiRNA:

small interference RNA

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Acknowledgments

We thank BD Biosciences for helpful comments. This study was supported by Grant-in-Aid for Young Scientist (B) 16790100 from the Ministry of Education, Culture, Sports, Science and Technology.

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Authors and Affiliations

  1. Department of Molecular Pharmacokinetics, Graduate School of Pharmaceutical Sciences, The University of Tokyo, Bunkyo-ku, Tokyo, 113-0033, Japan

    Tomoko Watanabe, Reiko Onuki & Yuichi Sugiyama

  2. Department of Biopharmacy, Faculty of Pharmaceutical Sciences, Setsunan University, Hirakata, Osaka, 573-0101, Japan

    Shinji Yamashita

  3. Department of Chemistry and Biotechnology, School of Engineering, The University of Tokyo, Bunkyo-ku, Tokyo, 113-0033, Japan

    Kazunari Taira

  4. Gene Function Research Center, National Institute of Advanced Industrial Science and Technology (AIST), Central 4, Tsukuba, 305-8562, Japan

    Kazunari Taira

Authors
  1. Tomoko Watanabe
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  2. Reiko Onuki
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  3. Shinji Yamashita
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  4. Kazunari Taira
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  5. Yuichi Sugiyama
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Corresponding author

Correspondence to Yuichi Sugiyama.

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Watanabe, T., Onuki, R., Yamashita, S. et al. Construction of a Functional Transporter Analysis System Using MDR1 Knockdown Caco-2 Cells. Pharm Res 22, 1287–1293 (2005). https://doi.org/10.1007/s11095-005-5270-z

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  • DOI: https://doi.org/10.1007/s11095-005-5270-z

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