Estimating Intestinal Mucosal Permeation of Compounds Using Caco‐2 Cell Monolayers

Jinnian Gao1, Erin D. Hugger1, Melissa S. Beck‐Westermeyer1, Ronald T. Borchardt1

1 University of Kansas, Lawrence, Kansas
Publication Name:  Current Protocols in Pharmacology
Unit Number:  Unit 7.2
DOI:  10.1002/0471141755.ph0702s08
Online Posting Date:  May, 2001
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Abstract

Step‐by‐step protocols are provided in this unit for the measurement of apparent permeability coefficients of compounds using Caco‐2 cell monolayers as an in vitro model of the intestinal mucosa. Procedures for culturing the cells and transmonolayer transport studies are also included. Critical issues for successfully estimating intestinal mucosal permeation of drugs are discussed. Step‐by‐step protocols are provided in this unit for the measurement of apparent permeability coefficients of compounds using

     
 
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Table of Contents

  • Basic Protocol 1: Measurement of the Apparent Permeability Coefficient of a Compound Across Caco‐2 Cell Monolayers
  • Support Protocol 1: Testing Caco‐2 Monolayer Integrity: Mannitol Flux Experiment
  • Support Protocol 2: Growing Caco‐2 Cell Monolayers on Filters
  • Support Protocol 3: Starting and Maintaining Caco‐2 Cells in Culture
  • Reagents and Solutions
  • Commentary
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Measurement of the Apparent Permeability Coefficient of a Compound Across Caco‐2 Cell Monolayers

  Materials
  • Test compound: e.g., 1.0 mM L‐carnosine (Aldrich) dissolved in EBSS (see recipe), 37°C
  • HBSS (see recipe), 37°C
  • 0.05 N and 0.01 N HCl
  • Caco‐2 cells, grown 21 to 28 days in Transwells and validated for monolayer integrity (see protocol 2)
  • 37°C shaking water bath (e.g., Model 25, Precision Scientific)
  • Vacuum pump (e.g., Precision Scientific, model DD20)
  • High‐performance liquid chromatography system (e.g., Shimadzu LC‐6A)
  • Reverse‐phase HPLC column (e.g., Vydac C18, 4.6 × 250 mm)
NOTE: More applications of reverse‐phase HPLC columns can be found in the literature (e.g., Tamura et al., , and Camenisch et al., ).NOTE: The integrity of the cell monolayers must be validated prior to experimentation (see protocol 2).

Support Protocol 1: Testing Caco‐2 Monolayer Integrity: Mannitol Flux Experiment

  Materials
  • D‐[14C]Mannitol (51 mCi/mmol; NEN Life Sciences)
  • HBSS (see recipe), 37°C
  • Transwells of Caco‐2 cell monolayers grown 21 to 28 days (see protocol 3)
  • Scintillation cocktail (e.g., Research Products International)
  • 6‐ml scintillation vials and caps (Fisher)
  • 6‐well cell culture cluster (Costar)

Support Protocol 2: Growing Caco‐2 Cell Monolayers on Filters

  Materials
  • Ethanol/collagen solution (see recipe)
  • Incomplete DMEM (see recipe)
  • Complete DMEM‐10 (see recipe), 37°C
  • Caco‐2 cells in 150‐cm2 flask, >80% confluent, growing in complete DMEM‐10 (see protocol 4, step or )
  • 6‐well Transwells (3‐µm pore size, 24‐mm diameter; Costar)
  • Vacuum pump (e.g., Precision Scientific, model DD20)
  • Hemacytometer (e.g., Reichert Bright‐Line, Fisher)
  • Additional reagents and equipment for passaging cells (see protocol 4)

Support Protocol 3: Starting and Maintaining Caco‐2 Cells in Culture

  Materials
  • Frozen Caco‐2 cells (ATCC #HTB‐37)
  • Complete DMEM‐20 (see recipe), 37°C
  • EDTA/PBS solution (see recipe), 37°C
  • Trypsin solution: 0.25% (w/v) porcine trypsin (JRH Biosciences) in HBSS (see recipe), 37°C
  • Complete DMEM‐10 (see recipe), 37°C
  • 50‐ml plastic centrifuge tubes (Fisher)
  • 25‐ and 150‐cm2 tissue culture flasks (Corning)
  • Vacuum pump (e.g., Precision Scientific, model DD20)
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Figures

Videos

Literature Cited

Literature Cited
   Anderle, P., Niederer, E., Rubas, W., Hilgendorf, C., Spahn‐Langguth, H., Winderli‐Allenspach, H., Merkle, H.P., and Langguth, P. 1998. P‐Glycoprotein (P‐gp) mediated efflux in Caco‐2 cells: The influence of culturing conditions and drug exposure on P‐gp expression levels. J. Pharm. Sci. 87:757‐62.
   Artursson, P. and Borchardt, R.T. 1997. Intestinal drug absorption and metabolism in cell cultures: Caco‐2 and beyond. Pharm. Res. 14:1655‐1658.
  Artursson, P. and Karlsson, J. 1991. 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.
  Artursson, P., Palm, K., and Lathman, K. 1996. Caco‐2 monolayers in experimental and theoretical predictions of drug transport. Adv. Drug Delivery Rev. 22:67‐84.
  Bailey, C.A., Bryla, P., and Malick, A.W. 1996. The use of the intestinal epithelial cell culture model, Caco‐2, in pharmaceutical development. Adv. Drug Delivery Rev. 22:85‐103.
  Borchardt, R.T. 1995. The application of cell culture systems in drug discovery and development. J. Drug Targeting 3:179‐182.
  Borchardt, R.T., Wilson, G., and Smith, P., (eds.) 1996. Model Systems Used for Biopharmaceutical Assessment of Drug Absorption and Metabolism. Plenum, New York.
  Brayden, D. 1997. Human intestinal epithelial cell monolayers as prescreen for oral drug delivery. Pharm. News 4:11‐15.
  Burton, P.S., Conradi, R.A., Ho, N.F.H., Hilgers, A.R., and Borchardt, R.T. 1996. How structural features influence the permeability of peptides. J. Pharm. Sci. 85:1336‐1340.
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   Camenisch, G.P., Wang. W., Wang, B., and Borchardt, R.T. 1998. A comparison of the bioconversion rates and the Caco‐2 cell permeation characteristics of coumarin‐based cyclic prodrugs and methyl ester‐based linear prodrugs of RGD peptidomimetics. Pharm. Res. 15:1174‐1181.
  Gan, L.S.L. and Thakker, D.R. 1997. Application of the Caco‐2 model in the design and development of orally active drugs: Elucidation of biochemical and physical barriers posed by the intestinal epithelium. Adv. Drug Delivery Res. 23:77‐98.
  Ganapathy, V. and Leibach, F.H. 1983. Role of pH gradient and membrane potential in dipeptide transport in intestinal and renal brush‐border membrane vesicles from the rabbit. Studies with L‐carnosine and glycyl‐L‐proline. J. Biol. Chem. 258:14189‐14192.
  Hidalgo, I.J. and Li, J. 1996. Carrier‐mediated transport and efflux mechanisms in Caco‐2 cells. Adv. Drug Delivery Res. 22:53‐66.
  Hidalgo, I.J., Borchardt, R.T., and Raub, T. 1989. Biochemical, histological and physicochemical characterization of human adenocarcinoma cells (Caco‐2) as a model system for studying mucosal transport and metabolism of drugs. Gastroenterology 96:736‐749.
  Hillgren, K.M., Kato, A., and Borchardt, R.T. 1995. In vitro systems for studying intestinal drug absorption. Med. Res. Rev. 15:83‐109.
  Hosoya, .I., Kim, K.J., and Lee, V.H. 1996. Age‐dependent expression of P‐glycoprotein gp170 in Caco‐2 cell monolayers. Pharm. Res. 13:885‐890.
  Knipp, G.T., Ho, N.F.H., Barsuhn, C.L., and Borchardt, R.T. 1997. Paracellular diffusion in Caco‐2 monolayers: Effect of perturbants on the transport of hydrophilic compounds that vary in charge and size. J. Pharm. Sci. 86:1105‐1110.
  Leibach, F.H. and Ganapathy, V. 1996. Peptide transporters in the intestine and the kidney. Annu. Rev. Nutr. 16:99‐119.
  Lennernas, H., Palm, K., Fagerholm, U., and Artursson, P. 1996. Correlation between paracellular and transcellular drug permeability in the human jejunum and Caco‐2 monolayers. Int. J. Pharm. 127:103‐107.
  Navia, M.A. and Chaturvedi, P.R. 1996. Design principles for orally bioavailable drugs. Drug Discovery Today 1:179‐189.
  Nerurkar, M.M., Burton, P.S., and Borchardt, R.T. 1996. The use of surfactants to enhance the permeability of peptides through Caco‐2 cells by inhibition of an apically polarized efflux system. Pharm. Res. 13:528‐534.
  Schmiedlin‐Ren, P., Thummel, K.E., Fisher, J.M., Paine, M.F., Lown, K.S., and Watkins, P.B. 1997. Expression of enzymatically active CYP3A4 by Caco‐2 cells grown on extracellulara matrix‐coated permeable supports in the presence of 1α,25‐dihydroxyvitamin D3. Mol. Pharmacol. 51:741‐754.
  Tamura, K., Bhatnagar, P.K., Takata, J.S., Lee, C.P., Smith, P.L., and Borchardt, R.T. 1996. Metabolism, uptake and transepithelial transport of the diastereomers of Val‐Val in the human intestinal cell line, Caco‐2. Pharm. Res. 13:1222‐1227.
  Tang, A.S., Chikhale, P.J., Shah, P.K., and Borchardt, R.T. 1993. Utilization of a human intestinal epithelial cell culture system (Caco‐2) for evaluating cytoprotective agents. Pharm. Res. 10:1620‐1626.
  Wacher, V.J., Salphati, L., and Benet, L.Z. 1996. Active secretion and enterocytic drug metabolism barriers to drug absorption. Adv. Drug Delivery Rev. 20:99‐112.
  Walle, U.K. and Walle, T. 1998. Taxol transport by human intestinal epithelia Caco‐2 cells. Drug Metab. Dispos. 26:343‐346.
Key References
   Artursson and Borchardt, 1997. See above.
  A review and perspective on the application of the Caco‐2 cell culture system.
   Gan and Thakker, 1997. See above.
  A review of 84 articles on applications of Caco‐2 cell monolayers, with an emphasis on the use of this in vitro cell culture model for mechanistic studies of drug absorption.
   Hidalgo et al., 1989. See above.
  A research article on the morphological and biochemical characteristics of Caco‐2 cell monolayers.
Internet Resources
  http://www.fda.gov/cder/guidance/index/htm
  Food and Drug Administration. 1999. Guidance for industry: Waiver of in vivo bioavailability and bioequivalence studies for immediate release solid oral dosage forms containing certain active moieties/active ingredients based on a biopharmaceutics classification system (Draft).
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