Minireviews
Biological, Pharmaceutical, and Analytical Considerations with Respect to the Transport Media Used in the Absorption Screening System, Caco-2

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ABSTRACT:

During the evaluation and selection of drug candidates, the Caco-2 cell culture system is commonly used for the determination of intestinal transport characteristics and to anticipate permeability limited drug absorption. Although classic HBSS-like buffered salt solutions are commonly used to perform Caco-2 transport experiments, different shortcomings (e.g., adsorption and low solubility) have been associated with the use of plain aqueous buffers. As transport experiments performed with unoptimized conditions may compromize the value of the Caco-2 model as a permeation screening tool, many efforts have been made to optimize the experimental conditions of Caco-2 transport assays. In this minireview, the hurdles associated with the use of saline aqueous buffers in Caco-2 transport experiments are summarized and the different options, which have been proposed to overcome these issues, are reviewed and discussed. Biologically, pharmaceutically, as well as analytically relevant media affecting the outcome of the transport experiments are described. Unfortunately, up to now, no systematic studies comparing the different experimental conditions have been performed, jeopardizing the possibility to define a (single) optimal solution to overcome the different issues associated with the use of saline aqueous buffers. Based on the reported options it can be proposed to use DMSO (≤1%) in standard screening procedures for the ranking of compounds based on their apical to basolateral transport. If compounds are not soluble in DMSO 1%, dimethylacetamide (3%) or N-1-methyl-pyrrolidone (2.5%) are good alternatives. However, these options do not imitate the in vivo situation. If one wants to take into account the physiological relevance of the media, the use of a biologically relevant apical medium (e.g., FASSIF) in combination with an analytically friendly, sink condition creating basolateral solvent (e.g., containing a micelle forming agent) can be suggested. © 2003 Wiley-Liss, Inc. and the American Pharmacists Association

Section snippets

INTRODUCTION

The transepithelial transport of compounds is an important characteristic, commonly assessed during the evaluation and selection of potential drug candidates.

To avoid the loss of promising compounds at later stages due to insufficient intestinal absorption, adequate screening systems are implemented at an early stage in drug discovery to assess and/or predict the ability of a drug to be absorbed. Different approaches and models of varying complexity have been developed and proposed to determine

BUFFERED SALT SOLUTIONS

Transport experiments using the Caco-2 cell culture model are usually performed with salt solutions [e.g., Hanks' Balanced Salt Solution (HBSS)] supplemented with glucose (final concentration of 25 mM) and buffered with 10 mM HEPES (pH 7.4) or 10 mM MES (pH 6.0) (Table 2). Commonly, no pH gradient over the cell monolayer is established, apical and basolateral solvents being buffered at pH 7.4. As summarized in Figure 1, different shortcomings have been associated with the use of HBSS-like buffers

ADSORPTION AND NONSPECIFIC BINDING

Adsorption to the culture device and/or nonspecific binding (in)to the cell monolayer can lead to an erroneous estimation (mostly underestimation) of the apparent permeability coefficient (Papp) of compounds as well as to poor recovery of the compound and a low mass balance. A low mass balance is often considered as problematic, and will invalidate the determination of reliable Papp values.

The need to minimize nonspecific binding to the cell monolayer during in vitro tests is not fully

SOLUBILITY ENHANCING ADDITIVES

The very low solubility, often observed for new drug candidates, impedes the determination of their absorption potential using the standard in vitro cell culture model. A multitude of approaches to increase the solubility (based on cosolvency, complexation, and micellar encapsulation) have been used in Caco-2 experiments for the assessment of the absorption potential of poorly soluble drug candidates. This creates a huge inconsistency in experimental procedures, which jeopardizes the

SEARCH FOR PHYSIOLOGICALLY RELEVANT MEDIA

The ultimate goal of screening drug candidates using the Caco-2 model during lead optimization is to determine the permeability coefficient that will be as predictive as possible for the intestinal absorption after oral intake. Therefore, much effort has been done to design an experimental setup that mimics the physiological conditions. pH adjustment, the simulation of in vivo intestinal fluids, and the creation of sink conditions may increase the biological relevance and predictive value of

ANALYTICALLY FRIENDLY MEDIA

When performing high-throughput Caco-2 experiments, it is obvious that the throughput of the analytical part will be of major importance, as a huge amount of samples is generated. As most of these screening experiments are performed at an early stage during drug development, analytical methods are not always completely well defined and validated. Besides, the analytical method should be sensitive, simple, and rapid. Different options have been proposed to minimize the analytical workload

WHICH WAY TO GO?

Although the Caco-2 cell culture model is commonly accepted and used for the screening of potential drug candidates to predict intestinal absorption, many shortcomings remain associated with this model, and should be taken into account when designing and performing Caco-2 experiments as well as during the interpretation of the data. From the different examples, it becomes clear that a multitude of approaches has been investigated to overcome the different issues encountered when assessing the

REFERENCES (88)

  • P. Artursson et al.

    Correlation between oral drug absorption in humans and apparent drug permeability coefficients in human intestinal epithelial (Caco-2) cells

    Biochem Biophys Res Commun

    (1991)
  • P. Artursson et al.

    Caco-2 monolayers in experimental and theortical predictions of drug transport

    Adv Drug Del Rev

    (2001)
  • C. Hilgendorf et al.

    Caco-2 versus Caco-2/HT29-MTX co-cultured cell lines: Permeabilities via diffusion, inside- and outside-directed carrier-mediated transport

    J Pharm Sci

    (2000)
  • P. Annaert et al.

    In vitro ex vivo and in situ intestinal absorption characteristics of the antiviral ester prodrug adefovir dipivoxil

    J Pharm Sci

    (2000)
  • A. Braun et al.

    Cell cultures as tools in biopharmacy

    Eur J Pharm Sci

    (2000)
  • F. Mathieu et al.

    Transepithelial transport of bepiridil in the human intestinal cell line, Caco-2, using two media, DMEMc and HBSS

    Int J Pharm

    (1999)
  • P. Augustijns et al.

    Evidence of a polarized efflux system in Caco-2 cells capable of modulating cyclosporin A transport

    Biochem Biophys Res Commun

    (1993)
  • G. Krishna et al.

    Permeability of lipophilic compounds in drug discovery using in-vitro human absorption model, Caco-2

    Int J Pharm

    (2001)
  • S. Deferme et al.

    Intestinal absorption characteristics of the low solubility thiocarboxanilide UC-781

    Int J Pharm

    (2002)
  • B.D. Rege et al.

    Effects of non-ionic surfactants on membrane transporters in Caco-2 cell monolayers

    Eur J Pharm Sci

    (2002)
  • S.D. Flanagan et al.

    Contributions of saturable active secretion, passive transcellular, and paracellular diffusion to the overall transport of furosemide across adenocarcinoma (Caco-2) cells

    J Pharm Sci

    (2002)
  • S. Yamashita et al.

    Optimized conditions for prediction of intestinal drug permeability using Caco-2 cells

    Eur J Pharm Sci

    (2000)
  • P. Saha et al.

    Effect of solubilizing excipients on permeation of poorly water-soluble compounds across Caco-2 cell monolayers

    Eur J Pharm Biopharm

    (2000)
  • B.J. Aungst

    Intestinal permeation enhancers

    J Pharm Sci

    (2000)
  • E.D. Hugger et al.

    A comparison of commonly used polyethoxylated pharmaceutial excipitents on their ability to inhibit P-glycoprotein activity in vitro

    J Pharm Sci

    (2002)
  • E.D. Hugger et al.

    Effects of poly(ethylene glycol) on efflux transporter activity in Caco-2 cell monolayers

    J Pharm Sci

    (2002)
  • B.D. Rege et al.

    Effects of common excipients on Caco-2 transport of low-permeability drugs

    J Pharm Sci

    (2001)
  • M.E. Taub et al.

    Optimized conditions for MDCK permeability and turbidimetric solubility studies using compounds representative of BCS classes I–IV

    Eur J Pharm Sci

    (2002)
  • R.J. Mountfield et al.

    Potential inhibitory effects of formulation ingredients on intestinal cytochrome P450

    Int J Pharm

    (2000)
  • C. Udata et al.

    Enhanced transport of a novel anti-HIV agent—Cosalane and its congeners across human intestinal epithelial (Caco-2) cell monolayers

    Int J Pharm

    (2003)
  • T.S. Wiedmann et al.

    Examination of the solubilizaiton of drugs by bile salt micelles

    J Pharm Sci

    (2002)
  • I.J. Hidalgo et al.

    Carrier-mediated transport and efflux mechanisms in Caco-2 cells

    Adv Drug Del Rev

    (1996)
  • T. Ogihara et al.

    Structural characterization of substrates for the anion exchange transporter in Caco-2 cells

    J Pharm Sci

    (1999)
  • G.M. Friedrichsen et al.

    Synthesis of analogs of L-valacyclovir and determination of their substrate activity for the oligopeptide transporter in Caco-2 cells

    Eur J Pharm Sci

    (2002)
  • E.K. Anderberg et al.

    Epithelial transport of drugs in cell culture. VII: Effects of pharamceutical surfactant excipients and bile acids on transepithelial permeability in monolayers of human intestinal epithelial (Caco-2) cells

    J Pharm Sci

    (1992)
  • C.M. Meaney et al.

    A comparison of the permeation enhancement potential of simple bile salt and mixed bile salt: Fatty acid micellar systems using the Caco-2 cell culture model

    Int J Pharm

    (2000)
  • F. Ingels et al.

    Simulated intestinal fluid as transport medium in the Caco-2 cell culture model

    Int J Pharm

    (2002)
  • P. Saha et al.

    Effect of bovine serum ablumin on drug permeability estimation across Caco-2 monolayers

    Eur J Pharm Biopharm

    (2002)
  • Y.C. Martin

    A practitioner's perspective of the role of quantitative structure–activity analysis in medicinal chemistry

    J Med Chem

    (1981)
  • K. Palm et al.

    Evaluation of dynamic polar molecular surface area as predictor of drug absorption: Comparison wtih other computational and experimental predictors

    J Med Chem

    (1998)
  • R.A. Conradi et al.

    The influence of peptide structure on transport across Caco-2 cells

    Pharm Res

    (1991)
  • P. Stenberg et al.

    Prediction of membrane permeability to peptides from calculated dynamic molecular surface properties

    Pharm Res

    (1999)
  • P. Stenberg et al.

    Prediction of the intestinal absorption of endothelin receptor antagonists using three theoretical methods of increasing complexity

    Pharm Res

    (1999)
  • L.L. Wright et al.

    Role of desolvatation energy in the nonfacilitated membrane permeability of didoxyribose analogs of thymidine

    Mol Pharmacol

    (1992)
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