The Caco-2 permeability assay is an FDA required assay for companies applying for an Investigational New Drug (IND) and delivers key information on the readiness of a compound for oral dosing.

When run with IONTOX the Caco-2 permeability assay is overseen by a Diplomat of the American Board of Toxicology, run in replicants of 3 to 4, and follows the internationally recognized guidance documents on Good In Vitro Method Practices (GIVMP) and Good Cell Culture Practices (GCCP).

In the human intestine, absorption is controlled by a barrier comprised of a single polarized layer of epithelial cells that line the intestinal wall. The Caco-2 human colon carcinoma cell line mimics this epithelial barrier in culture by forming a well-differentiated monolayer of cells. The Caco-2 monolayer contains an apical brush border, forms of tight junctions, and contains many of the brush border enzymes and transport proteins that mediate the active uptake or efflux of drugs and chemicals in the intestine. For these reasons, the Caco-2 permeability assay has become the gold standard for in vitro prediction of intestinal drug permeability and absorption.

In this assay, Caco-2 cells are cultured on transwell membranes (0.4 um) for approximately 3 weeks in order to establish a fully differentiated monolayer. The test compound and controls are applied to the apical surface and the rate of transport across the barrier over time is measured. Control compounds for low (ranitidine) and high (pindolol) permeability are always included in order to monitor assay performance. These data are then used to determine the apparent permeability coefficient (P_app; units are cm/s), which is calculated using the following equation: P_app = (dQ/dt) (1/(AC₀)) where dQ/dt is the steady-state flux (mmol/second), A is the surface area of the filter (cm²) and C₀ is the initial concentration in the donor chamber (mM). The mass balance (experimental recovery) is also calculated.

Additional assessments of the test compound can be made as well. These include the efflux ratio (measured by assessing the basal to apical P_app as well as the standard apical to basal P_app). This information can also determine whether the test compound is actively transported by P-glycoprotein (P-gp).