Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Protocol
  • Published:

Determination of drug permeability and prediction of drug absorption in Caco-2 monolayers

Nature Protocols volume 2, pages 2111–2119 (2007)Cite this article

Abstract

Permeability coefficients across monolayers of the human colon carcinoma cell line Caco-2, cultured on permeable supports, are commonly used to predict the absorption of orally administered drugs and other xenobiotics. This protocol describes our method for the cultivation, characterization and determination of permeability coefficients of xenobiotics (which are, typically, drug-like compounds) in the Caco-2 model. A few modifications that have been introduced over the years are incorporated in the protocol. The method can be used to trace the permeability of a test compound in two directions, from the apical to the basolateral side or vice versa, and both passive and active transport processes can be studied. The permeability assay can be completed within one working day, provided that the Caco-2 monolayers have been cultured and differentiated on the permeable supports 3 weeks in advance.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on SpringerLink
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1
Figure 2: Application of the technique in absorption and drug metabolism studies.
Figure 3: Morphology of Caco-2 cell monolayers.
Figure 4: Anticipated results.

Similar content being viewed by others

References

  1. Artursson, P., Palm, K. & Luthman, K. Caco-2 monolayers in experimental and theoretical predictions of drug transport. Adv. Drug Deliv. Rev. 46, 27–43 (2001).

    Article  CAS  PubMed  Google Scholar 

  2. Stenberg, P., Norinder, U., Luthman, K. & Artursson, P. Experimental and computational screening models for the prediction of intestinal drug absorption. J. Med. Chem. 44, 1927–1937 (2001).

    Article  CAS  PubMed  Google Scholar 

  3. Artursson, P. & Karlsson, J. Correlation between oral drug absorption in humans and apparent drug permeability coefficients in human intestinal epithelial (Caco-2) cells. Biochem. Biophys. Res. Commun. 175, 880–885 (1991).

    Article  CAS  PubMed  Google Scholar 

  4. Food and Drug Administration. FDA Guidance for industry, waiver of in vivo bioavailability and bioequivalence studies for immediate-release solid oral dosage forms based on a biopharmaceutics classification system. (Food and Drug Administration, Baltimore, MD, 2000). http://www.fda.gov/cder/guidance/3618fnl.htm (2007).

  5. Hubatsch, I., Lazorova, L., Vahlne, A. & Artursson, P. Orally active antiviral tripeptide glycyl-prolyl-glycinamide is activated by CD26 (dipeptidyl peptidase IV) before transport across the intestinal epithelium. Antimicrob. Agents Chemother. 49, 1087–1092 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Schmiedlin-Ren, P. et al. Expression of enzymatically active CYP3A4 by Caco-2 cells grown on extracellular matrix-coated permeable supports in the presence of 1alpha, 25-dihydroxyvitamin D3. Mol. Pharmacol. 51, 741–754 (1997).

    Article  CAS  PubMed  Google Scholar 

  7. Engman, H.A. et al. CYP3A4, CYP3A5, and MDR1 in human small and large intestinal cell lines suitable for drug transport studies. J. Pharm. Sci. 90, 1736–1751 (2001).

    Article  CAS  PubMed  Google Scholar 

  8. Neuhoff, S., Artursson, P., Zamora, I. & Ungell, A.L. Impact of extracellular protein binding on passive and active drug transport across Caco-2 cells. Pharm. Res. 23, 350–359 (2006).

    Article  CAS  PubMed  Google Scholar 

  9. Neuhoff, S., Ungell, A.L., Zamora, I. & Artursson, P. pH-dependent bidirectional transport of weakly basic drugs across Caco-2 monolayers: implications for drug–drug interactions. Pharm. Res. 20, 1141–1148 (2003).

    Article  CAS  PubMed  Google Scholar 

  10. Neuhoff, S., Ungell, A.L., Zamora, I. & Artursson, P. pH-dependent passive and active transport of acidic drugs across Caco-2 cell monolayers. Eur. J. Pharm. Sci. 25, 211–220 (2005).

    Article  CAS  PubMed  Google Scholar 

  11. Fallingborg, J. et al. pH-profile and regional transit times of the normal gut measured by a radiotelemetry device. Aliment. Pharmacol. Ther. 3, 605–613 (1989).

    Article  CAS  PubMed  Google Scholar 

  12. Yamashita, S. et al. Optimized conditions for prediction of intestinal drug permeability using Caco-2 cells. Eur. J. Pharm. Sci. 10, 195–204 (2000).

    Article  CAS  PubMed  Google Scholar 

  13. Yamashita, S. et al. New and better protocols for a short-term Caco-2 cell culture system. J. Pharm. Sci. 91, 669–679 (2002).

    Article  CAS  PubMed  Google Scholar 

  14. Delie, F. & Rubas, W. A human colonic cell line sharing similarities with enterocytes as a model to examine oral absorption: advantages and limitations of the Caco-2 model. Crit. Rev. Ther. Drug Carrier Syst. 14, 221–286 (1997).

    Article  CAS  PubMed  Google Scholar 

  15. Karlsson, J. & Artursson, P. A method for the determination of cellular permeability coefficients and aqueous boundary layer thickness in monolayers of intestinal epithelial (Caco-2) cells grown in permeable filter chambers. Int. J. Pharm. 71, 55–64 (1991).

    Article  CAS  Google Scholar 

  16. Avdeef, A. et al. Caco-2 permeability of weakly basic drugs predicted with the double-sink PAMPA pKa(flux) method. Eur. J. Pharm. Sci. 24, 333–349 (2005).

    Article  CAS  PubMed  Google Scholar 

  17. Tavelin, S., Grasjo, J., Taipalensuu, J., Ocklind, G. & Artursson, P. Applications of epithelial cell culture in studies of drug transport. Methods Mol. Biol. Vol. 188 (ed. Wise, C.) 233–272 (Humana Press, Totowa, New Jersey, 2002).

    CAS  PubMed  Google Scholar 

  18. Osth, K., Grasjo, J. & Bjork, E. A new method for drug transport studies on pig nasal mucosa using a horizontal Ussing chamber. J. Pharm. Sci. 91, 1259–1273 (2002).

    Article  CAS  PubMed  Google Scholar 

  19. Bergstrom, C.A. et al. Absorption classification of oral drugs based on molecular surface properties. J. Med. Chem. 46, 558–570 (2003).

    Article  PubMed  Google Scholar 

  20. Hidalgo, I.J., Raub, T.J. & Borchardt, R.T. Characterization of the human colon carcinoma cell line (Caco-2) as a model system for intestinal epithelial permeability. Gastroenterology 96, 736–749 (1989).

    Article  CAS  PubMed  Google Scholar 

  21. Konsoula, R. & Barile, F.A. Correlation of in vitro cytotoxicity with paracellular permeability in Caco-2 cells. Toxicol. In Vitro 19, 675–684 (2005).

    Article  CAS  PubMed  Google Scholar 

  22. Bentz, J., Tran, T.T., Polli, J.W., Ayrton, A. & Ellens, H. The steady-state Michaelis–Menten analysis of P-glycoprotein mediated transport through a confluent cell monolayer cannot predict the correct Michaelis constant Km. Pharm. Res. 22, 1667–1677 (2005).

    Article  CAS  PubMed  Google Scholar 

  23. Gatlik-Landwojtowicz, E., Aanismaa, P. & Seelig, A. Quantification and characterization of P-glycoprotein–substrate interactions. Biochemistry 45, 3020–3032 (2006).

    Article  CAS  PubMed  Google Scholar 

  24. Korjamo, T., Kemilainen, H., Heikkinen, A.T. & Monkkonen, J. Decrease in intracellular concentration causes the shift in Km value of efflux pump substrates. Drug Metab. Dispos. doi: 10.1124/dmd.107.016014.

    Article  CAS  PubMed  Google Scholar 

  25. Matsson, P. et al. A global drug inhibition pattern for the human ABC transporter BCRP (ABCG2). J. Pharmacol. Exp. Ther. doi: 10.1124/jpet.107.124768.

    Article  CAS  PubMed  Google Scholar 

  26. Adibi, S.A. The oligopeptide transporter (Pept-1) in human intestine: biology and function. Gastroenterology 113, 332–340 (1997).

    Article  CAS  PubMed  Google Scholar 

  27. Sai, Y. et al. Predominant contribution of organic anion transporting polypeptide OATP-B (OATP2B1) to apical uptake of estrone-3-sulfate by human intestinal Caco-2 cells. Drug Metab. Dispos. 34, 1423–1431 (2006).

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

This work has been supported by the Swedish Research Council, the Swedish Animal Welfare Agency and EU-grant no. LSHB-CT-2004-512051.

Author information

Authors and Affiliations

  1. Department of Pharmacy, Uppsala University, Box 580, Uppsala, SE-751 23, Sweden

    Ina Hubatsch, Eva G E Ragnarsson & Per Artursson

Authors
  1. Ina Hubatsch
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Eva G E Ragnarsson
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Per Artursson
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Per Artursson.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Hubatsch, I., Ragnarsson, E. & Artursson, P. Determination of drug permeability and prediction of drug absorption in Caco-2 monolayers. Nat Protoc 2, 2111–2119 (2007). https://doi.org/10.1038/nprot.2007.303

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nprot.2007.303

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing