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Caco-2 Permeability Assay
About Caco-2 permeability assays
The Caco-2 permeability assay is an established in vitro model for evaluating intestinal drug absorption. Derived from human colon carcinoma cells, Caco-2 monolayers mimic the epithelial barrier of the small intestine. These cells form tight junctions, develop microvilli, and express key transporters, including P-glycoprotein (P-gp), BCRP, and MRP2.
This assay enables the prediction of oral bioavailability and identifies potential efflux mechanisms early in drug development. It supports compound ranking, transporter profiling, and regulatory risk mitigation.
Key benefits
- Physiologically relevant barrier model: Mimics intestinal epithelial structure and transporter expression
- Efflux assessment: Detects directional transport and identifies involvement of P-gp or BCRP
- Standardised, reliable outputs: Delivers Papp, efflux ratio, and membrane integrity assessment
- Flexible protocol: Available as unidirectional or bidirectional transport assay
- Robust quantitation: LC-MS/MS readout supported by validated internal standards and UV/Vis lucifer yellow control
Protocol
Unidirectional transport assay
Test compound is added to apical well (gentle agitation) and transport determined by measuring concentration in basolateral well at 2 h (AB).
Bidirectional transport assay
At the same time as AB incubation, a parallel incubation is also performed where test compound is added to the basolateral well and then measured in the apical well (BA) at 2hr. The efflux ratio is then calculated by comparing the rates of transport in both directions (BA / AB) to determine if test compound is an efflux transporter substrate.
P-gp or BCRP involvement
If efflux has been predetermined, then an additional study may be performed using Pgp or BCRP specific inhibitors that are co-incubated with test compound to establish if these transporters are involved.
The final solvent concentration is 0.1%DMSO.
For all assays after the 2 h incubation, 200 μL aliquots are removed from all apical and basolateral wells and transferred to a new deep well plate to halt the transport.
- Aliquots are removed from the deep well plate and added to internal standard in a separate plate ready for LC-MSMS analysis.
- A second set of aliquots from the deep well plate representing the basolateral wells are removed and transferred to a separate plate ready for UV/vis absorbance to measure lucifer yellow.
Compound requirements
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Test systems
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Assay format
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Controls
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Quantitation
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Deliverables
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Data analysis & results
The response ratio is compared with the standard curve to determine the concentrations of the receiver and donor wells. The % recovery is determined to indicate the level of non-specific binding of the test compound, by comparing the concentrations with the initial concentration of the donor well.
The apparent permeability Papp is calculated according to:
Where V = receiver volume (cm3)
A = transwell membrane surface area (cm2)
Co = initial donor well concentration
dC/dt = rate of conce
Conclusions
The Caco-2 permeability assay provides critical insight into intestinal drug absorption and efflux transporter interactions. By replicating physiological conditions, this assay helps de-risk clinical development and supports bioavailability optimisation. Results guide early decisions in candidate selection, formulation strategy, and regulatory planning.