Caco-2 Permeability Assay
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 has become one of the standard in vitro methods.

CaCO-2 cells are derived from human colon cancer, which 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 cell-based 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 required 10 mM DMSO 100µL or 2 mg
Compound Test Concentration 10µM
Cell Culture 21 days
Replicates N=2
Incubation Conditions Incubation in CO2 incubator at 37ºC for 90 min
Positive control compound digoxin (or other compounds)
Integrity of the monolayer TEER>200 Ω•cm2
Analysis method LC-MS/MS
Data delivery Papp, Efflux Ratio, %Recovery
Data calculation Papp = 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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