Abstract
In vitro permeability assays are a valuable tool for scientists during lead compound optimization. As a majority of discovery projects are focused on the development of orally bioavailable drugs, correlation of in vitro permeability data to in vivo absorption results is critical for understanding the structural–physicochemical relationship (SPR) of drugs exhibiting low levels of absorption. For more than a decade, the Caco-2 screening assay has remained a popular, in vitro system to test compounds for both intestinal permeability and efflux liability. Despite advances in artificial membrane technology and in silico modeling systems, drug compounds still benefit from testing in cell-based epithelial monolayer assays for lead optimization. This chapter provides technical information for performing and optimizing the Caco-2 assay. In addition, techniques are discussed for dealing with some of the most pressing issues surrounding in vitro permeability assays (i.e., low aqueous solubility of test compounds and low postassay recovery). Insights are offered to help researchers avoid common pitfalls in the interpretation of in vitro permeability data, which can often lead to the perception of misleading results for correlation to in vivo data.
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References
Artursson P, Palm K, and Luthman K (2001) Caco-2 monolayers in experimental and theoretical predictions of drug transport. Adv Drug Deliv Rev 46:27–43
Li AP (2001) In vitro approaches to evaluate ADMET drug properties. Curr Top Med Chem 4(7):701–706
Smith DA, van de Waterbeemd H (1999) Pharmacokinetics and metabolism in early drug discovery. Curr Opin Chem Biol 3(4):373–378
van de Waterbeemd H, Smith DA, Beaumont K, Walker DK (2001) Property-based design: optimization of drug absorption and pharmacokinetics. J Med Chem 44(9): 1313–1333
Matsson P, Bergstrom C, Nagahara N, Tavelin S, Norinder U, Artursson P (2005) Exploring the role of different drug transport routes in permeability screening. J Med Chem 48 (2): 604–613
Balimane PV, Chong S (2005) Cell culture-based models for intestinal permeability: a critique. Drug Discov Today 10(5):335–343
Yee S (1997) In vitro permeability across Caco-2 cells (colonic) can predict in vivo (small intestinal) absorption in man – fact or myth. Pharm Res 14(6):763–766
Chan S, Lowes S, Hirst BH (2004) The ABCs of drug transport in intestine and liver: efflux proteins limiting drug absorption and bioavailability. Eur J Pharm Sci 21:25–51
Hoffmann U, Kroemer HK (2004) The ABC transporters MDR1 and MRP2: multiple functions in disposition of xenobiotics and drug resistance. Drug Metab Rev 36(3–4): 669–701
Palm K, Luthman K, Ros J, Grasjo J, Artursson P (1999) Effect of Molecular Charge on Intestinal Epithelial Drug Transport: pH-Dependent Transport of Cationic Drugs. J Pharmacol Exp Ther 291(2):435–443
König J, Nies AT, Cui Y, Leier I, and Keppler D (1999) Conjugate export pumps of the multidrug resistance protein (MRP) family: localization, substrate specificity, and MRP2-mediated drug resistance. Biochim Biophys Acta 1461(2):377–394
Makhey VD, Guo A, Norris DA, Hu P, Yan J, Sinko PJ (1998) Characterization of the regional intestinal kinetics of drug efflux in rat and human intestine and in Caco-2 cells. Pharm. Res 15(8):1160–1167
Gao J, Murase O, Schowen RL, Aubé J, Borchardt RT (2001) A functional assay for quantitation of the apparent affinities of ligands of P-glycoprotein in Caco-2 cells. Pharm Res 18(2):171–176
Balimane PV, Han YH, Chong S (2006) Current industrial practices of assessing permeability and P-glycoprotein interaction. AAPS J 8(1):E1–E13
Fisher JM, Wrighton SA, Calamia JC, Shen DD, Kunze KL, and Thummel KE (1999) Midazolam metabolism by modified Caco-2 monolayers: effects of extracellular protein binding. J Pharmacol Exp Ther 289(2):1143–1150
Neuhoff S, Artursson P, Zamora I, Ungell AL (2006) Impact of extracellular protein binding on passive and active drug transport across Caco-2 cells. Pharm Res 23(2):350–359
Saha P, Kou JH (2002) Effect of bovine serum albumin on drug permeability estimation across Caco-2 monolayers. Eur J Pharm Biopharm 54(3):319–324
Lakeram M, Lockley DJ, Pendlington R, Forbes B (2008) Optimization of the caco-2 permeability assay using experimental design methodology. Pharm Res 25(7):1544–1551
Saha P, Kou JH (2000) Effect of solubilizing excipients on permeation of poorly water-soluble compounds across Caco-2 cell monolayers. Eur J Pharm Biopharm 50(3):403–411
Acknowledgments
The author would like to thank Deanna Di Grandi for her contributions and engaging discussions in optimizing and troubleshooting various aspects of the assay techniques described herein.
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Press, B. (2011). Optimization of the Caco-2 Permeability Assay to Screen Drug Compounds for Intestinal Absorption and Efflux. In: Turksen, K. (eds) Permeability Barrier. Methods in Molecular Biology, vol 763. Humana Press. https://doi.org/10.1007/978-1-61779-191-8_9
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DOI: https://doi.org/10.1007/978-1-61779-191-8_9
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