EA204

ExpressPlus P-gp Substrate Assessment in MDR1-MDCK Cells (+/- Inhibitor)

This assay is used to determine the interaction of a test article with P-gp using MDR1- MDCK cell monolayers with and without a P-gp inhibitor

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 MDR1-MDCK monolayers
  •  The apparent permeability (Papp) of the test article in both directions in the presence and absence of inhibitor
  •  The efflux ratio in the presence and absence of inhibitor
  •  The brain penetration potential of a test article classified as either low, moderate, or high using the following criteria
    • Low: Papp A→B < 3.0 x 10-6 cm/s
    •  Low: Papp A→B ≥ 3.0 x 10-6 cm/s, Efflux ≥ 10
    •  Moderate: Papp A→B ≥ 3.0 x 10-6 cm/s, 10 > Efflux ≥ 3.0
    •  High: Papp A→B ≥ 3.0 x 10-6 cm/s, Efflux < 3.0

 

  •  P-gp substrate classification:
    • Positive: Efflux ratio ≥2.0 in the absence of inhibitor and corrected efflux ratio reduced by ≥50% in the presence of inhibitor
    •  Negative: Efflux ratio ≥2.0 in the absence of inhibitor and corrected efflux ratio reduced by <50% in the presence of inhibitor
    •  Negative: Efflux ratio < 2.0 in both the absence and presence of inhibitor

Substrate

  •  Test article at 5 µM in HBSSg with maximum DMSO concentration ≤ 0.8%

Assay System

  •  Confluent monolayers of MDR1-MDCK cells, 7 to 12 days old

Assay Conditions

  • Bidirectional permeability of the test article in MDR1-MDCK cells in the presence and absence of inhibitor
  • Transport buffer: HBSSg, pH 7.4 ± 0.2
  • Receiver well containing 1% BSA
  • 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 test article 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. This screening assay does not provide definitive information about the P-gp article interactions or the apparent Km.

3. Assay conditions with the inhibitor include:

  •  A 30 minute pre-incubation with Valspodar

4. Assay conditions without the inhibitor include:

  •  A 30 minute pre-incubation with buffer

5. Digoxin can be run as an optional positive control

  •  Dosed at 10 µM
  •  BSA is not included in the receiver chamber

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