Transport of compound through Caco-2 cell monolayer is used for measuring the rate of membrane transport, expressed as apparent (Papp) permeability. Resulting Papp values from both transport directions (apical to basolateral side and basolateral to apical side) are used for calculation of efflux ratio. In addition, the inclusion of the P-gp inhibitor could identify whether membrane transport is mediated by P-glycoprotein.
Therefore, Caco-2 permeability is considered to be a golden standard for in vitro prediction of permeability and for estimation of the potential for different efflux/uptake transporters (most importantly P-glycoprotein, P-gp) to transport the compound of interest.
Caco-2 cells are used after adequet culturin in multi-well Transwell plates. Test and reference compounds are prepared in Hanks’ Balanced Salt Solution containing 25 mM HEPES (pH 7.4) and added to either the apical or basolateral chambers of the Transwell plate assembly at a concentration. Lucifer Yellow is added to the donor buffer in all wells to assess integrity of the cell layers by monitoring Lucifer Yellow permeation. As Lucifer Yellow (LY) cannot freely permeate lipophilic barriers, a high degree of LY transport indicates poor integrity of the cell layer. After incubation at 37°C, aliquots are taken from both apical (A) and basal (B) chambers and added to analytical internal standard in a 96-well plate. Concentrations of compound in the samples are measured by high performance liquid-chromatography/mass spectroscopy (LC-MS/MS). Apparent permeability (Papp) values are calculated from the relationship: Papp = compound conc × acceptor final Vacceptor / (compound conc × donor initial Vdonor) / Tinc × Vdonor / surface area × 60 × 10 -6 cm/s. Where V = chamber volume and Tinc = incubation time. The Efflux ratios, as an indication of active efflux from the apical cell surface, are calculated using the ratio of Papp B>A/ Papp A>B.
Deliverable – Caco-2 permeability report includes
- Papp(A>B) and Papp(B>A)
- Efflux ratio
Test Compound Requirements
- Molecular mass (exact mass) of the test compound and its salt form
- MSDS or handling and storage information