Caco-2 Permeability Assay

Caco-2 permeability assay protocol

Data from Cyprotex's Caco-2 Permeability assay

For the validation, a set of compounds were screened through Cyprotex's Caco-2 Permeability assay over 3 separate experiments. Data generated were reproducible over a range of permeabilities.

The bidirectional assay is able to correctly distinguish between those compounds which are reported to undergo active efflux and those which are not.

Figure 1
The graphs illustrate the consistency of Cyprotex's Caco-2 Permeability data over 3 separate experiments for the apical to basolateral assay.

These data illustrate the high level of reproducibility provided by this assay for a set of compounds with a range of permeabilities.

Figure 2
Graph illustrates the efflux ratio of a set of 21 compounds generated by Cyprotex's Caco-2 Permeability assay.

Cyprotex's bi-directional Caco-2 Permeability assay can identify and quantify levels of active efflux. Screening compounds in both the A to B and B to A direction provides a ratio of B-A/A-B (efflux ratio). When a compound has an efflux ratio of greater than 2, it suggests that the compound may be subject to active efflux.

Questions and answers on Caco-2 permeability

Please provide an overview of Cyprotex's Caco-2 Permeability assay.

Caco-2 cells are widely used as an in vitro model for predicting human drug absorption. The Caco-2 cell line is derived from a human colorectal carcinoma, and when cultured, the cells spontaneously differentiate into monolayers of polarized enterocytes.

The cells are seeded on multiwell-insert plates and form a confluent monolayer over 20 days prior to the experiment. On day 20, the test compound is added to the apical side of the membrane and the flux of the compound across the monolayer is monitored over a 2 hour time period. To study drug efflux, it is also necessary to investigate transport of the compound from the basolateral compartment to the apical compartment.

The permeability coefficient (P_app) is calculated from the following equation:

Where dQ/dt is the rate of permeation of the drug across the cells, C₀ is the donor concentration at time zero and A is the area of the cell monolayer.

How do I interpret the data from the Caco-2 permeability assay?

There are several ways in which data from the Caco-2 permeability assay can be used. Firstly, the compounds can be ranked by their P_app values. Two reference compounds, atenolol (passive paracellular transport) and propranolol (passive transcellular transport) are screened alongside the test compounds. Atenolol and propranolol have known human absorption of 50% and 90% respectively^2,3, and can be used as markers for ranking test compounds. Secondly, data can be used in conjunction with other in vitro parameters to predict the oral pharmacokinetics of a compound in vivo using PBPK prediction. Thirdly, the Caco-2 data can be used to predict blood brain barrier (BBB) permeability (Figure 3).

Figure 3
Correlation between Caco-2 derived P_active ((P_appB-A - P_appA-B)/2) and in situ BBB permeation rate, logPS (permeability-surface area product). Drugs known to permeate the BBB (caffeine, imipramine, progesterone) have logPS >-2.0 (green shaded area), whilst drugs known to not cross the BBB (aldosterone, hydrocortisone, cimetidine) have a logPS ≤-3.5 (pink shaded area).

A regression line is shown which shows a clear correlation between P_active and logPS for compounds which are not subject to significant levels of efflux activity in the Caco-2 cells i.e., for compounds with a P_active of < 5 × 10 ^-6 cm/s. Using this correlation, Cyprotex's Caco-2 assay can be used to evaluate BBB potential for compounds with P_active < 5 × 10 ^-6 cm/s.

What is the relationship between Caco-2 permeability and human intestinal absorption?

The relationship between Caco-2 permeability (using pH7.4 HBSS buffers in both the apical and basolateral compartments) and human intestinal absorption is displayed in Figure 4. This correlation is typical of those observed in the literature for these two parameters⁴. It is important to note that this plot is influenced by the accuracy of the intestinal absorption data. The intestinal absorption values used in this plot are taken from Zhao et al. 2001². In this paper the human intestinal absorption has been extracted from various sources and varies considerably in quality. Several compounds are also known to exhibit dose-dependent absorption (shown by error bars on the graph).

Figure 4
Relationship between Caco-2 permeability and % human intestinal absorption.

What are the differences between the PAMPA and the Caco-2 permeability assay?

The Caco-2 permeability screen is considered to be more representative of human absorption in vivo than PAMPA (parallel artificial membrane permeability assay). PAMPA solely provides a measure of passive diffusion whereas the Caco-2 model provides better prediction of the human absorption for compounds which display active uptake or efflux or pass through the membrane via the paracellular route. The information from both assays used in conjunction can be valuable in identifying the root cause for poor absorption.

How do I measure drug efflux?

A bi-directional Caco-2 permeability assay is performed where the transport of the compound is measured in the apical to basolateral direction as well as the basolateral to apical direction. The result is typically reported as an efflux ratio i.e. P_app(B-A)/P_app(A-B). If the efflux ratio is greater than two then this indicates drug efflux is occurring. Talinolol, a known P-gp substrate, is screened as a control compound to confirm that the cells are expressing functional efflux transporter proteins.

How do you know if the cells have formed a confluent monolayer?

Transepithelial electrical resistance (TEER) measurement is used to determine tight-junction formation between cells. In addition, lucifer yellow, a membrane integrity marker, is co-incubated with the test compound at the start of the experiment. If the P_app of lucifer yellow exceeds 1.0 × 10 ^-6 cm/s in one well, but the derived P_app result for the test compound or control compound in that well is qualitatively similar to that determined in the remaining replicate well(s) (within the lucifer yellow threshold) then, based upon the scientific judgement of the responsible scientist, the cell monolayer may be considered acceptable. If this is not the case, then the result from the affected monolayer is excluded and an n=1 result is reported, or the compound may be re-tested. If both replicates are affected then the compound is re-screened. If both lucifer yellow P_app values fail for the same compound on two separate occasions then it is assumed that the compound exhibits either cytotoxic effects against the Caco‑2 cells or inherent fluorescence.

How and why is the % recovery calculated?

The % recovery can be useful in interpreting the Caco-2 data. If the recovery is very low, this may indicate problems with poor solubility, binding of the compound to the plate, metabolism by the Caco-2 cells or accumulation of the compound in the cell monolayer.

¹ Wang Z et al. (2000) Determination of in vitro permeability of drug candidates through a caco-2 cell monolayer by liquid chromatography/tandem mass spectrometry. J Mass Spectrom. 35(1); 71-6
² Zhao YH et al. (2001) Evaluation of human intestinal absorption data and subsequent derivation of a quantitative structure-activity relationship (QSAR) with the Abraham descriptors. J Pharmaceut Sci 90; 749-784
³ Yazdanian M et al. (1998) Correlating partitioning and caco-2 cell permeability of structurally diverse small molecular weight compounds. Pharmaceut Res 15; 1490-1494
⁴ Kansy M et al. (2001) Pharmacokinetic optimisation in drug research Ed. Testa et al; 447-464