Apredica provides fast Caco-2 assay turnaround. Because of the large volume of
Caco-2 assays we perform, we continuously produce 21-day Caco-2 cell cultures in anticipation of client needs
to ensure rapid turnaround. The following Caco-2 assays are available:
Monodirectional: High permeability predicts good human oral bioavailablity.
Bidirectional: High efflux ratio indicates possible P-gp or other transporter efflux.
Bi-directional (P-gp substrate determination): Test agent is incubated on either
side of the monolayer in the presence and absence of a known P-gp inhibitor.
Bi-directional (P-gp inhibitor determination): Test agent is incubated on either
side of the monolayer in the presence and absence of a known P-gp substrate.
Bi-directional (Other transporters):Please inquire
regarding the testing of your test agents to be substrates or inhibitors of other efflux
transporters (e.g. BCRP, MRP1, MRP2, etc.). You may also
request the poster
we presented at the 16th North American Regional ISSX Meeting
where we fully characterized activity of transporters in CaCo-2 cells.
Contact us to learn more about our Caco-2 assay service.
Caco-2 Customization
Customized variants of the Caco-2 assay are available to address special issues, such as:
Evaluating the contribution of formulation on uptake.
Permeability assessment when the test compound is toxic to the endothelium.
Permeability assessment when solubility issues require that the assay simulate gut fluid conditions.
Please inquire about how the Caco-2 assay can be customized to your special situation.
Caco-2 cells are used to understand two issues related to drug uptake:
Permeability: Crucial for evaluating the potential for oral dosing of
drug candidates. Recommended as a screen during lead optimization.
Efflux: Identifies whether the compound is a substrate of active transport
mechanisms. This is often part of the in vitrodrug-drug interactions
package for IND-enabling studies.
Principle of the Caco-2 Assay
Caco-2 human colon adenocarcinoma cells are grown to confluence and differentiated
for 3 weeks on filters. Test agent is added to one side of the monolayer, and permeability
is assessed by analyzing the concentration of the test agent on the other side of the monolayer using LC/MS/MS.
Background and Validation of the Caco-2 Model
Originally isolated from a colorectal carcinoma in the 1970s,1 Caco-2 cell monolayers spontaneously
differentiate to express morphological and functional characteristics of mature small-intestinal enterocytes.
The differentiated monolayers are polarized, with microvilli on the apical side, and express small intestinal
hydrolase activities, including sucrase-isomaltase, lactase, aminopeptidases, on the apical surface.2, 3
Caco-2 cells grown on permeable filter supports form tight junctions and express transporters on the apical
(e.g. P-gp4, 5, MRP-25,
BCRP6) and basolateral (e.g. MRP-15, PepT17, 8) surfaces, and drugs that are
predicted to be bioavailable are not transported across the intestinal mucosa due to the
activity of efflux transporters.9, 10 Permeability across Caco-2 cell monolayers is used to predict human
permeability of drug candidates, to perform in-depth mechanistic and absorption studies, to
study the effects of transporters on permeability, and transporter-mediated drug-drug interactions.
The Caco-2 permeability assay is considered to be the industry gold standard for in vitro prediction
of in vivo human intestinal permeability and bioavailability of orally administered drugs.11
Fig. 1 represents a validation study conducted at Apredica on a subset of marketed drugs with fraction absorbed, reported in the literature.
The FDA recommends that drug-drug interactions should be performed during drug development.12
In vitro studies with Caco-2 cell monolayers have proven to be a valuable
tool for predicting human in vivo intestinal permeability.13
Figure 1. Correlation of apparent permeability as measured at Apredica in the Caco-2 permeability assay with reported human fraction absorbed (literature).
Figure 2. Diagram of the Caco-2 permeability assay.
Permeability across differentiated monolayers of Caco-2 is measured on fully differentiated cells grown for
3 weeks on permeable filter supports to estimate human intestinal permeability.
The integrity of the monolayer is determined by measurement of TEER or by Lucifer Yellow permeability.
In a typical experiment, the test agent is applied to the apical (A; "gut" side)
or basolateral (B; "blood") side of the monolayer and incubated for 2 h (Figure 2).
The amount of test agent on each side is measured by HPLC or LC/MS/MS. Permeability
(Papp) is calculated in the apical to basolateral (A → B) and basolateral to
apical (B → A) directions:
where dQ/dt is the rate of permeation, C0 is the initial concentration of test agent, and A is the area of the monolayer.
Passively transported compounds show equal permeability in both directions. The role of transporters
is demonstrated by asymmetry in the amount of permeability. A high B → A vs. A → B ratio indicates
the possibility that the compound is an efflux transporter substrate. The transporter can be identified
by performing the permeability assay in the presence of a specific inhibitor on both sides of the monolayer.
Follow-Ons to Caco-2 Studies
In Vivo Pharmacokinetics: The compound is administered to rodents,
and plasma samples are analyzed at different times to determine the concentration of test agent.
Customization (changes in concentration, time points, etc) is easily possible.
Contact us to learn more about how Caco-2 studies can be used in your programs.
2. Pinto M et al. 1983. Enterocyte-like differentiation and polarization of the human colon carcinoma cell line Caco-2 in culture. Biol Cell 47:323-30.