Drug absorption generally occurs either through passive transcellular or paracellular diffusion, active carrier transport, or active efflux mechanisms. Several methods have been developed to aid in the understanding of the absorption of new lead compounds. The most common ones use an immortalized cell line (e.g., Caco-2, Madin-Darby carine kidney (MDCK)) to mimic the intestinal epithelium. These in vitro models provide more predictive permeability information than artificial membrane systems (i.e., PAMPA and permeability assays described above) based on the cells' ability to promote (active transport) or resist (efflux) transport. Various in vitro methods listed in the U.S. FDA guidelines, acceptable to evaluate the permeability of a drug substance, include monolayers of suitable epithelial cells, and one such epithelial cell line that has been widely used as a model system of intestinal permeability is the Caco-2 cell line.
The kinetics of intestinal drug absorption, permeation enhancement, chemical moiety structure-permeability relationships, dissolution testing, in vitro-in vivo correlation (IVIVC), BE, and the development of novel polymeric materials are closely associated with the concept of Caco-2. Since most drugs are known to absorb via the intestine without using cellular pumps, passive permeability models came in the limelight. In a typical Caco-2 experiment, a monolayer of cells is grown on a filter separating two stacked micro-well plates. The permeability of drugs through the cells is determined after the introduction of a drug on one side of the filter. The entire process is automated, and when used in conjunction with chromatography and/or mass spectroscopy detection, it enables any drug's permeability to be determined. The method requires careful sample analysis to calculate permeability correctly. Limitations of the Caco-2 experiments are 21 days for preparing a stable monolayer, stringent storage conditions; however, tight junction formation prior to use is the better choice. The villus in the small intestine contains more than one cell type, the Caco-2 cell line does not produce the mucus as observed in the small intestine, and no P450 metabolizing enzyme activity has been found in the Caco-2 cell line. Test compound solubility may pose a problem in the Caco-2 assays because of the assay conditions. Finally, Caco-2 cells also contain endogenous transporter and efflux systems, the later of which works against the permeability process and can complicate data interpretation for some drugs.
The Caco-2 cell line is heterogeneous and was derived from a human colorectal adenocarcinoma. Caco-2 cells are used as in vitro permeability models to predict human intestinal absorption because they exhibit many features of absorptive intestinal cells. This includes their ability to spontaneously differentiate into polarized enterocytes that express high levels of brush border hydrolases and form well-developed junctional complexes. Consequently, it becomes possible to determine whether passage is transcellular or paracellular based on a compound's transport rate. Caco-2 cells also express a variety of transport systems including dipeptide transporters and P-gps. Due to these features, drug permeability in Caco-2 cells correlates well with human oral absorption, making Caco-2 an ideal in vitro permeability model. Additional information can be gained on metabolism and potential drug-drug interactions as the drug undergoes transcellular diffusion through the Caco-2 transport model.
Although accurate and well researched, the Caco-2 cell model requires a high investment of time and resources. Depending on the number of factors, including initial seeding density, culturing conditions, and passage number, Caco-2 cells can take as much as 20 days to reach confluence and achieve full differentiation. During this 20-day period, they require manual or automated exchange of media as frequently as every other day. The transport assays consume valuable drug compounds and normally require expensive, posttransport sample analyses (e.g., LC-MS). Therefore, the use of the Caco-2 transport model in a high-throughput laboratory setting is only possible if the platform is robust, automation compatible, reproducible, and provides high-quality data that correlate well with established methodologies.
The Millipore MultiScreen Caco-2 assay system is a reliable 96-well platform for predicting human oral absorption of drug compounds (using Caco-2 cells or other cell lines whose drug transport properties have been well characterized). The MultiScreen system format is automation compatible and is designed to offer more cost-effective, higher throughput screening of drugs than a 24-well system. The MultiScreen Caco-2 assay system exhibits good uniformity of cell growth and drug permeability across all 96 wells and low variability between production lots. The plate design supports the use of lower volumes of expensive media and reduced amounts of the test compounds. Using the MultiScreen Caco-2 assay system, standard drug compounds are successfully categorized as either "high" or "low" permeable, as defined
by the FDA, and the permeability data correlate well with established human absorption values. The components of Caco-2 Assay system are shown in Figure 3.
Components with single-well feeder plate and 96-well transport analysis plate (Courtesy of Millipore Corporation).
The apparent permeability (Papp) in cm/sec can be calculated for Caco-2 drug transport assays using the following equation:
app VArea time^ V [druginitial,donoj where Va is the volume (mL) in the acceptor well, "Area" is the surface area of the membrane (0.11 cm2 for MultiScreen Caco-2 plate and 0.3 cm2 for the 24-well plate), and "time" is the total transport time in seconds. For radio-labeled drug transport experiments, the count per minute (CPM) units obtained from the Trilux Multiwell Plate Scintillation Counter are used directly for the drug acceptor and initial concentrations such that the formula becomes
app VArea time; ^CPM^ donor
Historically, it has been shown that a sigmoidal relationship exists between drug absorption rates as measured with the in vitro Caco-2 model and human absorption.
% human absorption = 100 X exp (a + b X Papp)/[ 1 + exp (a + b X Papp)] (4)
The Caco-2 cells are heterogeneous and their properties in final culture may differ based on the selection pressures of a particular laboratory. Direct comparison of compound permeability rates between laboratories is not possible unless the same Caco-2 cells and conditions are used. Therefore, transport rates and permeability classification ranges of specific drugs are expected to vary between reported studies. Most important is the ability to successfully classify compounds as low-, medium-, or high-permeable drugs and produce transport results that correlate to established human absorption values.
Several modifications of the Caco-2 cell model have been tested; for example, CYP3A4-transfected Caco-2 cells are also used to define the biochemical absorption barriers. Oral BA and intestinal drug absorption can be significantly limited by metabolizing enzymes and efflux transporters in the gut. The most prevalent oxidative drug-metabolizing enzyme present in the intestine is CYP3A4. Currently, more than 50% of the drugs on the market metabolized by P450 enzymes are metabolized by CYP3A4. Oral absorption of CYP3A4 substrates can also be limited by the multidrug resistance transporter P-gp, because there is extensive substrate overlap between these two proteins. P-gp is an ATP-dependent transporter on the apical plasma membrane of enterocytes, which functions to limit the entry of the drugs into the cell. There is significant interaction between CYP3A4 and P-gp in the intestine can serve. Although Caco-2 cells express a variety of uptake and efflux transporters found in the human intestine, a major drawback to the use of Caco-2 cells is that they lack CYP3A4. As such, no data regarding the importance of intestinal metabolism on limiting drug absorption can be obtained from normal Caco-2 cells. Caco-2 cells pretreated with 1,25-dihydroxyvitamin-D3 (vitamin D3) express higher levels of CYP3A4 when compared with Caco-2 but still underestimate the amount of CYP3A4 in the human intestine.
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