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