Efflux Transporter Substrate Identification assay (Caco-2 cells)
Aim: To determine if a test compound is substrate of an efflux transporter, either P-gp (P-glycoprotein) or BCRP (breast cancer resistance protein multidrug transporter), using the Caco-2 cell model.
Background information: Caco-2 cells are a human colon adenocarcinoma cell line which express efflux transporters, such as P-gp and BCRP. Efflux transporters are involved in drug absorption and disposition. As transporter substrates, drugs can be victims of drug-drug interactions in the clinical practice.
Transport assays are conducted both in the absorption (apical-to-basolateral) and efflux (basolateral-to-apical) directions. Test compound is added to either the apical or basolateral sides of the monolayer in the absence and presence of transporter reference inhibitor. Concentrations are quantified by UPLC-MS/MS using a calibration curve.
Compound requirement: 50 µl 10 mM in DMSO
Turnaround time: 4 weeks
- Test compound concentration: 10 µM
- 24-well plate
- Number of replicates: 2
- DMSO concentration: 1%
- Incubation time: 120 min
- Probe substrate: nitrofurantoin (BCRP) and colchicine (P-gp)
- Reference inhibitor: Ko143 (BCRP) and verapamil (P-gp)
- Monolayer integrity marker: lucifer yellow
- Analytical method: UPLC-MS/MS with calibration curves
- Absorption and efflux permeability (+/- transporter inhibitor)
- Absorption and efflux recovery (+/- transporter inhibitor)
- Efflux ratio of the probe substrate (+/- transporter inhibitor)