CaCO-2 Permeability Assay

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A thorough understanding of intestinal epithelial transport is crucial for evaluating the potential for oral dosing of drug candidates. In vitro studies with CaCo-2 cell monolayers have proven to be a valuable tool for predicting human in vivo intestinal permeability.¹

Figure 1. Correlation of apparent permeability as measured at Apredica in the CaCO-2 permeability assay with reported human fraction absorbed (literature).

In vitro permeability across differentiated monolayers of CaCO-2 cells is measured to estimate human intestinal permeability.

CaCO-2 Cells as a Model of the Small Intestine for the Study of Drug Transport

Originally isolated from a colorectal carcinoma in the 1970s,² 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.^{3, 4} CaCO-2 cells grown on permeable filter supports form tight junctions and express transporters on the apical (e.g. P-gp^{5, 6}, MRP-2⁶, BCRP⁷) and basolateral (e.g. MRP-1⁶, PepT1^{8, 9}) surfaces, and drugs that are predicted to be bioavailable are not transported across the intestinal mucosa due to the activity of efflux transporters.^{10, 11} 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.¹² 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.¹³

Figure 2. Scheme 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. Compounds are applied to the apical (A) or basolateral (B) side of the monolayer and incubated for 2 h (Figure 2). The amount of compound 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.

Principle of the CaCO-2 Assay

CaCo-2 cells are grown to confluence and allowed to differentiate on filters. Test agent is added to one side of the monolayer, and permeability is assessed using HPLC or LC/MS.

The CaCO-2 Cells

CaCO-2 human colon adenocarcinoma cells are grown to confluence and differentiated for 3 weeks on filters.

CaCO-2 Assay Sample Requirements

20 µL of a 10 mM DMSO solution, based on a test concentration of 50 µM.

CaCO-2 Assay Modes

Screening: Test agent is incubated for 2 hr on either side of the monolayer (apical and basolateral), and the concentration of the test agent on both sides is measured by HPLC or LC/MS. High permeability predicts good human oral bioavailablity. High asymmetry index indicates possible PGP efflux.

Transporter Inhibition: Test agent is incubated for 2 hr on either side of the monolayer in the presence and absence of a transporter's inhibitor (such as PGP inhibitor verapamil), and thus interaction with transporters is estimated using the CaCO-2 model.

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.

Footnotes

1. Balimane, PV. AAPS J. 8:1 (2006); Wessel, MD. J Chem Inf Comput Sci 38:726 (1998)

2. Fogh J. et al. 1977. One hundred and twenty seven cultured human tumor cell lines producing tumors in nude mice. J Natl Cancer Inst 59:221-6.

3. 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.

4. Hidalgo IT et al. 1989. Characterization of the human colon carcinoma cell line (Caco-2) as a model system for intestinal epithelial permeability. Gastroenterology 96:736-49.

5. Takano M et al. 1998. Interaction with P-glycoprotein and transport of erythromycin, midazolam and ketoconazole in Caco-2 cells. Eur J Pharmacol 358:289-94.

6. Taipalensuu J et al. 2001. Correlation of gene expression of ten drug efflux proteins of the ATP-binding cassette transporter family in normal human jejunum and in human intestinal epithelial Caco-2 cell monolayers. J Pharmacol Exp Ther 299:164-170.

7. Xia CQ et al. 2005 Expression, localization, and functional characteristics of breast cancer resistance protein in Caco-2 cells. Drug Metab Dispos 33:637-43.

8. Brandsch M.1994. Expression and protein kinase C-dependent regulation of peptide/H+ co-transport system in the Caco-2 human colon carcinoma cell line. Biochem J 299:253-60.

9. Thwaites DT. 1993. Transepithelial glycylsarcosine transport in intestinal Caco-2 cells mediated by expression of H+-coupled carriers at both apical and basal membranes. J Biol Chem 268:7640-42.

10. Balimane PV et al. 2006. Current industrial practices of assessing permeability and P-glycoprotein interaction. The AAPS Journal 8: Article 1.

11. Braun A et al. 2000. Cell cultures as tools in biopharmacy. 2000. Eur J Pharm Sci 11:S51-S60.

12. Hubatsch I et al. Determination of drug permeability and prediction of drug absorption in Caco-2 monolayers. 2007. Nat Protoc 2:2111-9.

13. FDA Guidance for Industry. Drug interaction studies - study design, data analysis, and implications for cosing and labeling. September, 2006