Caco-2 Cell Model of ADME Model in Vitro

2021-10-12

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The new drug development stage includes 4 important stages: target determination, model establishment, lead compound discovery, and lead compound optimization. After the target is determined, a biological model such as an in vitro ADME model of a drug should be established, which is mainly used to study drug absorption, distribution, metabolism and drug toxicity, in order to screen and evaluate the activity of the compound to improve the success rate of drug development. “ADME” means “toxic pharmacokinetics”, which is very important for pre-clinical drug safety evaluation. Medicilon Biopharmaceuticals can provide high-quality data and rapid turnaround in drug safety evaluation to support the safety of various drugs Evaluation research.

With the continuous development of various types of pharmaceutical preparations today, oral preparations are favored for their convenience, safety and effectiveness. Many factors can affect the bioavailability of oral drugs. Therefore, improving drug absorption and increasing bioavailability have become pharmaceutical work. The subject that the author devoted himself to research. Since Caco-2 is derived from human colon adenocarcinoma cells, it can differentiate into intestinal cells during the culture process. Caco-2 cells are seeded on a matrix such as a carbonate porous membrane, and spontaneously formed polar and microscopic under culture conditions. Villi and tight junctions are similar to the single cell layer on the brush border side of small intestine epithelial cells. Therefore, Caco-2 cells can mimic small intestinal epithelial cells and are widely used in the study of physical and biochemical barriers during drug absorption.

Since Caco-2 is derived from human colon adenocarcinoma cells and can undergo enterocyte differentiation in culture, Caco-2 cells inoculated onto a matrix such as a polycarbonate porous membrane spontaneously form a polar, single-cell layer with microvilli and tight junctions similar to the differentiation characteristics of the brush border side of small intestinal epithelial cells under culture conditions. Therefore, Caco-2 cells can mimic small intestinal epithelial cells and are widely used for the study of physical and biochemical barriers during drug absorption.

Caco-2 is derived from human colon adenocarcinoma cells and can undergo enterocyte differentiation in culture. Caco-2 cells inoculated onto a matrix such as a polycarbonate porous membrane spontaneously form a polar, single-cell layer with microvilli and tight junctions similar to the differentiation characteristics of the brush border side of small intestinal epithelial cells under culture conditions. Therefore, Caco-2 cells can mimic small intestinal epithelial cells and are used for the study of physical and biochemical barriers during drug absorption widely.

The Caco-2 cell transport model is the first drug screening model that has been applied by foreign pharmaceutical companies as well as laboratories to mimic intestinal absorption and has been widely used in the evaluation of drug absorption in the small intestine and the study of transport mechanisms. Caco-2 cells are structurally and functionally similar to human small intestinal epithelial cells and contain enzyme systems associated with the small intestinal brush border epithelium. Unlike normal mature small intestinal epithelial cells that show reverse differentiation during in vitro cultivation, Caco-2 cells can spontaneously differentiate into intestinal epithelial cells after three weeks of culture on porous, permeable polycarbonate membranes, forming a dense cell monolayer that can then be used as an intestinal transit model (as shown in the figurec).

The Caco-2 cell model is simple to culture and validate and more convenient and economical than in vivo animal experiments. According to online reports, the flow of Caco-2 cell culture, construction, validation, and transport studies is shown below.

Caco-2 cell culture

1) Caco-2 cells were cultured in MEM complete culture medium (containing 10% fetal bovine serum, 1% non-essential amino acids, 1% sodium pyruvate, 1% glutamine and 1% penicillin-streptomycin) at 37°C and 5% CO₂.

2) Change the culture medium every other day. When the cells reached 80% confluence, cell passaging was performed. Aspirate the cell culture medium, wash it carefully twice with HBSS without Ca²⁺ and Mg²⁺ions, and add 0.25% trypsin-0.02% EDTA digestion mixture for digestion.

3) Take an appropriate amount of cells and inoculate them into 75 cm² culture flasks for further culture or plate laying operation.

Construction and validation of the Caco-2 cell model

1) Collect Caco-2 cells in the log phase and adjust the cell concentration to 2.0×10⁵ cells/mL with MEM complete culture medium.

2) Add 1.5 mL of MEM complete culture medium to the basolateral side of the Transwell plate (polycarbonate membrane, 0.4 µm, 1.12 cm²) (Figure 2) and 0.5 mL of the cell suspension to the apical side.

3) Place in a cell culture incubator and change the culture medium every two days after one week of incubation.

4) Continue culture until 21 days.

5) Determine the trans-epithelial cell resistance (>500 Ω-cm²) using a Millicell ERS resistivity meter (Millipore) to determine the denseness and integrity of the cell monolayer.

Transmigration studies

The following is an example of fluorescently labeled drugs to observe drug translocation across the membrane with laser confocal microscopy.

1) Remove the Transwell plate for resistance measurement and select cell wells that meet the conditions for transport (resistance >500 Ω-cm²). Wash 2~3 times with pre-warmed (37℃) Hank's buffer and incubate at 37℃ for 20~30 min, aspirate, and discard the wash.

2) Add 0.5 mL of fluorescently labeled drug solution to the top side (Apical side) as the supply solution and 1.5 mL of blank Hank's buffer to the basolateral side (Basolateral side) as the receiving solution, respectively.

3) Transfer at 37°C for two h.

4) After the transfer, remove the fluorescent solution from the wells and wash carefully with HBSS three times to wash away the fluorescent dye that has not entered the cells to end the transfer; add an appropriate amount of 4% paraformaldehyde solution to the wells and fix them for 20 min at room temperature.

5) After fixation, the wells were washed three times with HBSS, 10 μg/mL Hoechst 33258 was added, and the nuclei were stained at room temperature for one h.

After three times washing with HBSS, two drops of anti-fluorescence quencher were added, the monolayer cells were carefully cut off into a small glass-bottomed dish, and the transmembrane process of fluorescently labeled drugs was observed by Z-axis transaction under a laser confocal microscope.

As can be seen from the graphs taken by laser confocal microscopy, the transmembrane transport rates of drug A with green fluorescence and drug B with red fluorescence are different in the Caco-2 cell model, where the transmembrane transport rate of drug A is slower than that of drug B. The difference in transmembrane transport rate is closely related to the physicochemical properties of the drug itself.

the transmembrane transport rate of drug A is slower than that of drug B.webp

In addition to fluorescently labeled drugs for transmembrane transport studies, non-fluorescently labeled drugs can also be used for transmembrane studies, in which case the determination of transmembrane transport capacity of the drugs needs to be done by other assays (e.g., high-performance liquid chromatography).

The Caco-2 cell model is a rapid screening tool for drug absorption studies, which can provide comprehensive information on the absorption, distribution, metabolism, transport, and toxicity of drug molecules through the small intestinal mucosa at the cellular level, providing a basis for drug studies.

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