EA253

ExpressPlus P-gp Substrate Assessment in Caco-2 Cells (+/- Inhibitor)

This assay is used to determine the interaction of a test compound with P-gp using Caco-2 cell monolayers with and without a P-gp inhibitor.

Required from Customer

  • Either a minimum of 300 µL of test compound at 10 mM in DMSO, or 5 mg of powder
  • Exact molecular mass of test compound and its salt form
  • Solubility of test compound in Hanks’ buffer
  • MSDS or handling and storage information, e.g., light sensitive, store at -20°C, etc.

Deliverables

  • The percent recovery of the test compound from Transwell® wells containing cell monolayers
  • The apparent permeability (Papp) in both directions with and without inhibitor
  • The efflux ratio (Papp B to A)/(Papp A to B) with and without inhibitor
  • P-gp substrate classification:
    • Positive when efflux ratio ≥3.0 in the absence of inhibitor and relative efflux ratio ≥2.0
    • Negative when efflux ratio ≥3.0 in the absence of inhibitor and relative efflux ratio <2.0, or when efflux ratio <3.0 in the absence and presence of inhibitor
    • Requires further study when the effux ratio ≥3.0 in the absence and presence of inhibitor

Substrate

  • Test compound at 5 µM in HBSSg with maximum DMSO concentration not greater than 1%

Assay System

  • Confluent monolayers of Caco-2 cells, 21 to 28 days old

Assay Conditions

  • Receiver well with 1% BSA in modified Hanks buffer (HBSSg)
  • Apical and basolateral side at pH 7.4 ± 0.2
  • Assay run in duplicate (N=2)
  • Single test compound concentration at 5 µM
  • Dose apical side for A to B assessment and basolateral side for B to A assessment
  • Sample both donor and receiver sides at 120 minutes
  • Determine the concentrations of test compound using a generic LC-MS/MS method with a minimum 6-point calibration curve

Assay QC

  • The quality of the cell monolayer batch is verified using control compounds before the monolayers are used
  • The quality of each monolayer used in the assay is verified by a TEER measurement and by calculating the Papp for a control compound

Notes

  1. The results from this assay are sent to the customer in the ExpressPlus report format, which may include graphical representations of data and comparison with historical data for reference compounds.
  2. Monolayer integrity will be verified by a pre-experimental TEER.
  3. This screening assay does not provide definitive information about the P-gp compound interactions or the apparent Km.
  4. Assay conditions with the inhibitor include:
    • A 30 minute pre-incubation with Valspodar
  5. Assay conditions without the inhibitor include:
    • A 30 minute pre-incubation with buffer

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