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.
The Caco-2 cell transport model is the first drug screening model used by foreign pharmaceutical companies and laboratories to simulate intestinal absorption. It has been widely used in the evaluation of drug absorption in the small intestine and the study of transport mechanisms. The function is similar to human small intestinal epithelial cells, containing enzymes related to the brush edge epithelium of the small intestine. Different from the reverse differentiation of normal mature small intestinal epithelial cells during in vitro cultivation, Caco-2 cells can spontaneously differentiate into intestinal epithelial cells after being cultured on a porous and permeable polycarbonate membrane for 3 weeks, forming a dense cell monolayer , Which can then be used as an intestinal transit model (as shown in the figure).
The Caco-2 cell model culture and verification method is simple, and it is more convenient and economical than in vivo animal experiments.
Caco-2 cell culture
1) Using 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 Cultivate Caco-2 cells;
2) Change the culture medium every other day. When the confluence of the cells reaches 80%, the cells are passaged. Aspirate the cell culture solution, carefully wash it twice with HBSS free of Ca2+ and Mg2+ ions, and add 0.25% trypsin-0.02% EDTA mixed digestion solution for digestion;
3) Inoculate an appropriate amount of cells into a 75 cm2 culture flask to continue culturing or plating.
Construction and verification of Caco-2 cell model
1) Collect Caco-2 cells in 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 medium on the Basolateral side of the Transwell plate (polycarbonate membrane, 0.4 µm, 1.12 cm2) (Figure 2), and 0.5 mL of cell suspension on the Apical side;
3) Put it in a cell culture box and change the culture medium every two days, and change the medium every day after one week of culture;
4) Continue to cultivate until 21 days;
5) Use the Millicell ERS resistance meter (Millipore) to measure the transepithelial cell resistance (greater than 500 Ω•cm2) to determine the compactness and integrity of the cell monolayer.
Let’s take fluorescently labeled drugs as an example, and observe the drug transport across the membrane with the help of a laser confocal microscope.
1) Take out the Transwell plate for resistance measurement, and select cell wells that meet the transport conditions (resistance greater than 500 Ω•cm2). Wash with pre-heated (37℃) Hank’s buffer for 2~3 times, and incubate at 37℃ for 20~30 min. Aspirate and discard the washing solution;
2) Take 0.5 mL of fluorescently labeled drug solution and add it to the Apical side as the supply solution, while adding 1.5 mL of blank Hank’s buffer to the Basolateral side as the receiving solution;
3) Transit at 37℃ for 2 h;
4) After the transfer is completed, remove the fluorescent solution in the well, and carefully wash 3 times with HBSS to wash off the fluorescent dye that has not entered the cells to complete the transfer; add an appropriate amount of 4% paraformaldehyde solution to the well, and fix it at room temperature for 20 minutes;
5) After the fixation is completed, wash 3 times with HBSS, add an appropriate amount of 10 μg/mL Hoechst 33258, stain the nucleus at room temperature for 1 h; after washing 3 times with HBSS, add 2 drops of anti-fluorescence quencher, and carefully cut the monolayer of cells to In a glass-bottomed cuvette, perform Z-axis cross-section under a laser confocal microscope to observe the transmembrane process of fluorescently labeled drugs.
It can be seen from the image taken by the laser confocal microscope that the transmembrane transport rate of the green fluorescent drug A and the red fluorescent drug B in the Caco-2 cell model is different, and the transmembrane transport rate of the drug A is slower than that of the drug B. The difference in transmembrane transport rate is closely related to the physical and chemical properties of the drug itself.
In addition to the use of fluorescently labeled drugs for transmembrane transport research, non-fluorescent labeled drugs can also be used for transmembrane research. At this time, other detection methods (such as high performance liquid chromatography) are needed to determine the transmembrane transport capacity of drugs. .
As a rapid screening tool for drug absorption research, the Caco-2 cell model 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 research.