P-gp Inhibition assay (Caco-2 cells)

Aim: To determine the inhibition potential of test compound for P-glycoprotein (P-gp), an efflux 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 (Breast Cancer Resistance Protein Multidrug Transporter). Efflux transporters are involved in drug absorption and disposition. Inhibition of transporter-mediated efflux may affect the efficacy and safety of potential concomitant drugs in a clinical setting.

Inhibition transport assays are conducted both in the absorption (apical-to-basolateral) and efflux (basolateral-to-apical) directions. Probe substrate is added to either the apical or basolateral sides of the monolayer in the absence and presence of test compound. Probe substrate concentrations are quantified by UPLC-MS/MS using a calibration curve.

Compound requirement: 5 mg

Turnaround time: 4 weeks

Experimental design:

  • Test compound concentrations: 3 (variable on request and compound solubility)
  • 24-well plate
  • Number of replicates: 2
  • DMSO concentration: 1%
  • Incubation time: 120 min
  • Probe substrate: colchicine
  • Positive control: verapamil
  • Monolayer integrity marker: lucifer yellow
  • Analytical method: UPLC-MS/MS with calibration curves

Delivered results:

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

C1, C2, C3: test compound concentrations

COL: Colchicine (probe substrate)

V: Verapamil (inhibitor)