Intestinal Transporter Assays

The intestinal transporter assay provides a means to assess whether a test compound is a potential substrate or inhibitor of specific drug transporters. We provide intestinal transporter assays for small molecule formulations such as pharmaceuticals, industrial chemicals and consumer products.

Our intestinal transporter assays use genetically modified versions of the C2BBe1 cell line, a subclone of the original Caco-2 cell line created with our proprietary CompoZr® zinc finger nuclease (ZFN) technology.

Of particular interest to groups involved in drug development are the ABC efflux transporters, including P-glycoprotein (P-gp, MDR1, ABCB1), multidrug resistance-associated protein 2 (MRP2, ABCC2) and breast cancer resistance protein (BCRP, ABCG2). If a test compound undergoes active efflux, the next step involves determining the identity of the transporter(s) involved. Transporter interactions are currently assessed using small molecule inhibitors, although known lack of specificity among inhibitors complicates the interpretation of results.

To address this limitation, we developed a series of single and double transporter knockout (KO) cell lines. When used with the parental line, results from these knockout cell lines can be used to unambiguously identify drug-transporter interactions without the use of chemical inhibitors. The double knockout cell lines can be used to verify test compounds that are dual substrates or to allow the study of a single efflux transporter in the absence of the other efflux transporters.

Substrate Assessment
Here the identification of a test compound as a substrate for a given transporter can be assessed by comparing the rate of transport (efflux ratio) in the wild type and the various single and double transporter KO lines.

Inhibition Assessment
Here the ability of a test compound to inhibit a given transporter is assessed by using a model substrate in which the rate of transport (efflux ratio) is measured in both the presence and absence of the test compound. Additionally, inhibition studies can be performed in Sigma-Aldrich’s double knockout cell lines to study a test compound’s potential inhibition of the remaining single efflux transporter in the absence of the other efflux transporters.

 Intestinal Transporter Assay Protocol

Test System Caco-2 cell line, wild type
Caco-2 cell line, MDR1 KO
Caco-2 cell line, BCRP KO
Caco-2 cell line, MRP1 KO
Caco-2 cell line, MRP2 KO
Caco-2 cell line, MRP3 KO
Caco-2 cell line, MRP4 KO
Caco-2 cell line, MRP5 KO
Caco-2 cell line, MRP7 KO
Caco-2 cell line, MDR1/BCRP KO
Caco-2 cell line, MDR1/MRP2 KO
Caco-2 cell line, BCRP/MRP2 KO
Test Compound Concentration 10 µM (or custom)
Passage Number 40-60
Cell Culture 21 days in 24-well Transwell® plates
Number of Replicates 3
Incubation Time 2 hours at 37°C
Compound Requirements 100 µL of 10 mM DMSO solution
Integrity Marker Lucifer yellow
Control Compounds Atenolol (low passive permeability)
Metoprolol (high passive permeability)
Digoxin (substrate)
Estrone 3-Sulfate [E3S] (substrate)
5(6)-Carboxy-2’,7’-dichlorofluorescein diacetate
[CDCF-DA] (substrate)
Or custom
Analysis Method LC-MS/MS quantification of test substance