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Early prediction of drug metabolism and pharmacokinetic properties during drug discovery and development can help simplify the selection of lead compounds that are most likely to be useful drugs. In addition, due to time constraints and the large number of compounds produced through combinatorial chemistry and high-throughput screening, the demand for in vitro has increased dramatically. An analytical method that can be used to qualitatively and quantitatively evaluate biological systems that affect the pharmacokinetics of drug candidates. However, intestinal absorption is a strong barrier, which limits the oral bioavailability of many potential new drugs. In order to meet the needs of in vitro testing of the intestinal permeability of potential new drugs, a variety of methods have been developed as part of the drug development process. Among these methods, Caco-2 cell monolayer analysis has become one of the standard in vitro tools.

Although the etiology of these diseases is not yet known, it is now generally accepted that the intestinal barrier plays an important role in health and disease. Studying intestinal permeability is essential for the development of treatments aimed at alleviating and avoiding the severe symptoms associated with the destruction of the intestinal barrier. From a drug discovery perspective, pills are needed to enable researchers to screen thousands of candidate drug models. The scalability of conventional membrane insert-based systems at this level is limited, and usually cannot meet the requirements of 3D cell models, which is an aspect required to improve physiological relevance. Although you may have the ideal cell type for simulating intestinal permeability, having the potential to incorporate 3D models or other cell types would be very beneficial.

Caco-2 cells are a human colonic epithelial cancer cell line used as a model for human intestinal absorption of drugs and other compounds. When cultured as a monolayer of cells, Caco-2 cells differentiate to form tight junctions between cells, thereby becoming a model of paracellular movement of the compound across the monolayer. In addition, Caco-2 cells express transport proteins, efflux proteins and phase II binding enzymes to simulate a variety of cross-cell pathways and the metabolic transformation of the test substance. In many ways, the Caco-2 cell monolayer mimics the human intestinal epithelium. One of the functional differences between normal cells and Caco-2 cells is the lack of cytochrome P450 isoenzymes, especially the expression of CYP3A4, which is usually expressed at high levels in the intestine. However, treatment with vitamin D3 can induce Caco-2 cells to express higher levels of CYP3A4.

Generally, Caco-2 cells are cultured in a monolayer on a semipermeable plastic carrier that can be fitted into the wells of a multi-well culture plate. The test compound is then added to the top or basolateral side of the monolayer. After various times of incubation, aliquots of the buffer solution are placed in opposite chambers to determine the concentration of the test compound and calculate the permeability of each compound (called the apparent permeability coefficient).

Although radiolabeled compounds were used in the initial Caco-2 cell monolayer assay, most laboratories have replaced radioactivity by using liquid chromatography-mass spectrometry (LC-MS) and LC-tandem mass spectrometry (LC-MS-MS) Labeled compound. Mass spectrometry not only eliminates the need for radiolabeled compounds, but also allows multiple compounds to be measured simultaneously. The measurement of multiple compounds in each assay reduces the number of incubations that need to be performed, thereby increasing the throughput of the experiment.

In addition, LC-MS and LC-MS-MS add a new dimension to Caco-2 analysis by promoting the study of compound metabolism by Caco-2 cells. The measurement of multiple compounds in each assay reduces the number of incubations that need to be performed, thereby increasing the throughput of the experiment. In addition, LC-MS and LC-MS-MS add a new dimension to Caco-2 analysis by promoting the study of compound metabolism by Caco-2 cells. The measurement of multiple compounds in each assay reduces the number of incubations that need to be performed, thereby increasing the throughput of the experiment. In addition, LC-MS and LC-MS-MS add a new dimension to Caco-2 analysis by promoting the study of compound metabolism by Caco-2 cells.

In vitro ADME analysis methods come in many forms to adapt to different stages of the drug development process, thereby enabling the suppliers to provide flexible methods for ADME services. Creative Bioarray provides standard procedures for each of the detection methods listed below, experienced research team can also help design customized optimal strategies and protocols to meet any drug discovery needs.

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