Caco-2 Intestinal Drug Transporter Models for More Predictive Drug Absorption

Why use Caco-2 cells?

Drug-drug interactions are an important area of research as they can affect pharmacokinetics and safety or efficacy of drugs taken concomitantly. Guidance documents from the U.S. Food and Drug Administration (FDA) and the European Medicines Agency (EMA) state the need to identify the potential of an investigational drug to be a substrate or inhibitor of clinically relevant transporters. Caco-2 cells are ideal for this purpose as they are human-derived, express multiple transporters and grow in a homogeneous monolayer that forms tight junctions. The C2BBe1 cells, a subclone of Caco-2 cells (ATCC CRL-2101) utilized in this model are adenocarcinoma, epithelial cells from a human caucasian male (aged 72 years).

Figure 1. Transporter knockout(s) in each CompoZr^® Transporter Knockout Cell Line. The total loss of MDR1, BCRP and/or MRP2 function in each respective cell line enables explicit identification of transporter/substrate interactions without the use of chemical inhibitors.

What are Caco-2 Drug Transporter Models?

To aid in the investigation of specific transporters, our scientists have engineered single and double transporter knockout (KO) cell lines using CompoZr^® zinc finger nuclease (ZFN) technology in the Caco-2 intestinal cell line. ZFNs bind DNA at a sequence-specific location and create a double strand break. The break is repaired by non-homologous end joining resulting in frame-shift modifications at the desired locus. Stable cell lines were established from single cell KO clones. The KO’s target ATP-binding cassette (ABC) transmembrane proteins, transporters known to play a critical role in the development of multidrug resistance. Our double transporter KO, MDR1/BCRP, MDR1/MRP2, and BCRP/MRP2, Caco-2 models effectively create a single transporter expression model. Unlike common single transporter expressing models in MDCKII or HEK293 cell lines, the Caco-2 double transporter KO cell lines allow more precise investigation of single transporter drug interactions in a more physiologically relevant, human cell model.

Advantages of Caco-2 Drug Transporter Models

• Model of intestinal permeability: Express multiple drug transporters and commonly used to predict human intestinal drug permeability and to investigate drug efflux
• Genome editing for precision, efficiency, and specificity: Transporter KO Caco-2 cell lines made using CompoZr^® ZFN
• Highly predictive: Transporter KO cell lines eliminate the ambiguity from nonspecific substrates and inhibitors
• Meets regulatory guidelines: The drug transporters targeted include those referenced in the regulatory guidance for investigating drug-drug interactions (DDI) from the US Food and Drug Administration (FDA), the European Medicines Agency (EMA), and the Japanese Ministry of Health, Labour, and Welfare (MHLW): MDR1 (P-gp), BCRP, MRP2; and those of emerging clinical importance: MRP1, MRP3, MRP4, MRP5, MRP6, and MRP7

Figure 2. Caco-2 cells in culture. Caco-2 cells at low density (first panel) and high density (second panel).

Analysis of Cimetidine with Knockout Cell Lines

Cimetidine is an example of a crossover substrate that has an efflux ratio of approximately six in the parental (control) cell line. The efflux ratio is partially decreased in both the MDR1 and BCRP single KO cells but not the MRP2 KO cells. This result suggests that cimetidine is a dual substrate for MDR1 and BCRP, which is confirmed in the MDR1/BCRP double-KO cell line where the efflux ratio returns to unity (~ 1). The complete inhibition of cimetidine efflux in the double KO cells clearly demonstrates that cimetidine is a substrate for both MDR1 and BCRP (Figure 3).

Figure 3. Cimetidine transport. Values are the mean +/- standard deviation, n ≥3 assays of triplicates

Available Caco-2 Models

To aid in the investigation of drug absorption across the human intestinal epithelium, we have generated transporter knockout (KO) Caco-2 cell lines using CompoZr^® ZFN technology.

Assay-ready Vials

• Enables direct seeding to the transwell plate from the assay-ready vials
• Eliminates time-consuming sub-culturing procedures