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

Experimental design:

  • 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

Delivered results:

  • Absorption and efflux permeability (+/- transporter inhibitor)
  • Absorption and efflux recovery (+/- transporter inhibitor)
  • Efflux ratio of the probe substrate (+/- transporter inhibitor)