About the Assay

The Caco-2 cell line is derived from human colorectal adenocarcinoma. During extended culture, Caco-2 cells undergo both morphological and functional differentiation, acquiring characteristics similar to small intestinal enterocytes with formation of polarized monolayers of columnar epithelial cells with apical microvilli, brush borders, and inter-cellular tight junctions, expression of key ATP binding cassette transporters, including MDR1 (P gp/ABCB1) and BCRP (ABCG2).

Drug transport is measured bi-directionally, apical-to-basolateral (AtoB) and basolateral-to-apical (BtoA), which allows for the rate of permeability to be determined and for the identification of efflux substrates.

As a human derived cell line, cultured monolayers of Caco-2 cells represent a useful and relevant system for bidirectional transport studies which enable the identification of passive and active transport processes and can help predict the oral absorption and CNS penetrance of new chemical entities. 

Protocol Summary

Sygnature Discovery use in-house cultured Caco-2 cells to assess compound permeability and efflux potential in hanging insert plate format.

Compound Selection

A diverse set of compounds were selected to span a range of intrinsic permeabilities and to include both efflux and non‑efflux substrates.

Validation Results

Figure 1. Apparent permeability coefficient (Papp) results for test compounds evaluated in Sygnature Discovery’s Caco-2 assay with HBSS pH 7.4/7.4 buffer. Each bar represents the mean from triplicate technical replicates from a single experiment (n=3). A & B are the apical to basolateral determinations of Papp. C & D are the basolateral to apical determinations of Papp. Data is shown on different axis for ease of comparison purposes only.

Figure 2. Efflux ratio calculated for test compounds evaluated in Sygnature Discovery’s Caco-2 assay with HBSS pH 7.4/7.4 buffer. Data was assayed in triplicate in 3 independent experiments (n=3).

Figure 3. Efflux ratio of MDR1 and BCRP probe substrates in the presence of know inhibitors. A Digoxin, B Prazosin, C Estrone-3-Sulfate (E3S) and D Quinidine.Model inhibitors were Verapamil 18 µM (MDR1), Elacridar 3.5 µM (MDR1 & BCRP) and Ko143 1.5 µM (BCRP). Results are presented as mean +/- SD.Figure 3. Efflux ratio of MDR1 and BCRP probe substrates in the presence of know inhibitors. A Digoxin, B Prazosin, C Estrone-3-Sulfate (E3S) and D Quinidine.Model inhibitors were Verapamil 18 µM (MDR1), Elacridar 3.5 µM (MDR1 & BCRP) and Ko143 1.5 µM (BCRP). Results are presented as mean +/- SD.

References

Hubatsch, I., Ragnarsson, E. & Artursson, P. Determination of drug permeability and prediction of drug absorption in Caco-2 monolayers. Nat Protoc 2, 2111–2119 (2007).

The International Transporter Consortium, (2010), Nat Rev 9; 215-236

Role of the Breast Cancer Resistance Protein (ABCG2) in Drug Transport, 2005, The AAPS Journal, 7 (1) Article 12

 In Vitro Drug Interaction Studies — Cytochrome P450 Enzyme- and Transporter-Mediated Drug Interactions: Guidance for Industry. FDA (CDER) 2020.