In the development of small molecule drugs, it is necessary to evaluate the bioavailability of the compound as early as possible, so as to lay the foundation for subsequent development. In addition to using experimental animals to evaluate bioavailability, various in vitro analysis methods have been developed to evaluate the ability of compounds to penetrate the intestinal wall. These in vitro experiments are not only lower in cost but also higher in throughput and shorter experimental time. Among them, Caco-2 monolayer cell permeability analysis assay has become one of the standards in vitro methods.

CaCO-2 cells are derived from human colon cancer and have the typical characteristics of intestinal epithelial cells, such as the formation of a polarized monolayer columnar epithelium, and densely differentiated microvilli on the top surface of the cells neatly arranged to form a striated border and intercellular connection.

The determination of Caco-2 cell permeability not only helps to determine intestinal permeability but also can identify specific transporters or efflux proteins and intestinal phase II drug-metabolizing enzymes. Therefore, this Caco-2 permeability assay provides valuable information for drug development in an effective and reproducible manner, which is not available in non-biological models, and even in many other biological models.

The Efflux Ratio can be calculated by measuring the transport from A-B and B-A in two different directions, so as to determine whether a molecule has active efflux.

Caco-2 Permeability Assay Protocol

Compound required10 mM DMSO 100µL or 2 mg
Compound Test Concentration10µM
Cell Culture21 days
Incubation ConditionsIncubation in CO2 incubator at 37ºC for 90 min
Positive control compounddigoxin (or other compounds)
Integrity of the monolayerTEER>200 Ω•cm2
Analysis methodLC-MS/MS
Data deliveryPapp, Efflux Ratio, %Recovery
Data calculationPapp = CRecx VRec/ (A x t x C0)
Efflux Ratio = Papp (B→A) /Papp (A→B)
Recovery = [(Vrec x Crec) + (Vd x Cd)] / (Vd x C0)

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