EA222-B

ExpressPlus P-gp Inhibition Assessment in Caco-2 Cells

This assay is used to screen for inhibition of P-gp by a test article, by measuring its effect on the bidirectional permeability of a P-gp substrate through Caco-2 cell monolayers.

Required from Sponsor

  •  Either a minimum of 300 µL of test article at 10 mM in DMSO, or 5 mg of powder
  •  Molecular mass (exact mass) of the test article and its salt form
  •  MSDS or handling and storage information, e.g., light sensitive, store at -20°C, etc.

Deliverables

  •  Cell batch QC results
  •  The percent recovery of the test article from the assay wells containing Caco-2 monolayers
  •  The apparent permeability (Papp) of digoxin in both directions, in the presence and absence of inhibitor
  •  The efflux ratio [(Papp B to A)/(Papp A to B)] of digoxin in the presence and absence of inhibitor
  •  P-gp substrate classification:
    •  Positive: Digoxin corrected efflux ratio ≥ 1.0 in the absence of inhibitor and corrected efflux ratio reduced by ≥ 50% in the presence of inhibitor
    •  Negative: Digoxin corrected efflux ratio ≥ 1.0 in the absence of inhibitor and corrected efflux ratio reduced by <50% in the presence of inhibitor
    • Negative: Digoxin corrected efflux ratio < 1.0 in both the absence and presence of inhibitor

Substrate

  •  Test article and digoxin each at 10 µM in HBSSg with maximum DMSO concentration ≤ 0.8%

Assay System

  •  Confluent monolayers of Caco-2 (clone C2BBe1) cells, 21-28 days old

Assay Conditions

  •  Bidirectional permeability of Digoxin in Caco-2 cells in the presence and absence of inhibitor
  •  Transport buffer: HBSSg, pH 7.4 ± 0.2
  •  Apical and basolateral side at pH 7.4
  •  Dose two cell monolayers (n=2), in the presence and absence of inhibitor
    •  Dose apical side for (A→B) assessment
    •  Dose basolateral side for (B→A) assessment
  •  Sample from both apical and basolateral sides at 120 minutes
  •  Determine the concentrations of digoxin using a generic LC-MS/MS method with a minimum 6-point calibration curve

Assay QC

  •  The quality of the monolayer batch is verified using control compounds before the monolayers are released for use
  •  The quality of each monolayer used in the assay is verified by calculating the Papp for the control article, lucifer yellow, dosed post-experimentally

Notes

1. The results from this assay are sent to the sponsor in the ExpressPlus report format, which may include graphical representations of data and comparison with historical data for reference articles.

2. The solubility of the test article in Hanks’ buffer containing ≤ 0.8% DMSO must be greater than the test concentration. If the solubility of the test article is unavailable, Absorption Systems can conduct a solubility assessment at an additional charge.

3. Assay conditions with the inhibitor include:

  •  A 30 minute pre-incubation with test article

4. Assay conditions without the inhibitor include:

  •  A 30 minute pre-incubation with buffer

5. Digoxin can be run in the presence and absence of Valspodar as an optional positive control

  •  Valspodar dosed at 1 µM

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