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EA258

P-gp Inhibition – MDR1-MDCK

This MDR assay is used to screen for inhibition of P-gp by a test compound, by measuring its effect on the bidirectional permeability of a P-gp substrate through MDR1-MDCK cell monolayers.

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 and chemical stability of the test compound in Hanks’ buffer
  • MSDS or handling and storage information, e.g., light sensitive, store at -20°C, etc.

Deliverables

  • Apparent permeability (Papp) of digoxin in both directions, in the presence and absence of test compound
  • Efflux ratio [(Papp B to A)/(Papp A to B)] of digoxin in the presence and absence of test compound
  • Classification of test compound as a likely inhibitor or non-inhibitor of P-gp at the concentration tested

Substrate

  • Test compound and digoxin each at 10 µM in modified Hanks’ buffer (HBSSg) with final DMSO concentration < 0.8%

Assay System

  • Confluent monolayers of MDR1-MDCK cells, 7-14 days old, in Transwell® dual-chamber plates, with apical and basolateral pH 7.4

Assay Conditions

  • Dose duplicate monolayers (N=2) on apical side for A to B digoxin permeability
  • Dose duplicate monolayers (N=2) on basolateral side for B to A digoxin permeability
  • Repeat above steps on monolayers pretreated and co-dosed with test compound on both apical and basolateral sides
  • Sample both apical and basolateral sides at 120 minutes
  • Determine the concentration of digoxin using a generic LC-MS/MS method with a minimum 4-point calibration curve

Assay QC

  • Verify the quality of the cell monolayer batch using control compounds before the monolayers are released for use
  • Verify the quality of each monolayer used in an assay by a pre-experiment TEER measurement and by calculating the Papp for the co-dosed control compound, Lucifer yellow

Notes

  1. The results from this assay are provided to the customer in the ExpressPlus report format, which may include graphical representations of data and comparison with historical data for reference compounds.
  2. The solubility of the test compound in Hanks’ buffer containing < 0.8% DMSO must be greater than the test concentration. If the solubility of the test compound is unavailable, Absorption Systems can conduct a solubility assessment at an additional charge.

Options

  1. The customer can request:
    • that a positive control be performed in parallel (additional fees apply)