This assay uses BCRP-transfected MDCK cells to assess whether the test compound is a potential substrate or inhibitor of the efflux transporter BCRP

Catalog numbers: EA234

Required from Customer

  • 50 mg of test compound powder
  • Molecular weight of the test compound and its salt form
  • Relevant solubility of test compound
  • The MSDS and handling and storage information, e.g., light sensitive, store at -20ºC, etc.

Deliverables

  • Determination of substrate classification: positive when the net flux ratio of the efflux ratios in BCRP-transfected cells vs. MDCK control cells >2, followed by a ≥ 50% decrease in the corrected efflux ratio in the presence of inhibitor.
  • IC50 of the test compound

Substrate

Study I:

  • Test compound at three concentrations in modified Hanks’ buffer with <0.8% DMSO

Study II:

  • BCRP probe substrate, cladribine, at 10 µM
  • Test compound at seven concentrations in modified Hanks’ buffer with <0.8% DMSO

Assay System

BCRP-transfected MDCK cells and MDCK control cells cultured in Transwell® plates

Assay Conditions

Pre-study work is done to establish the calibration range and method reproducibility, and to evaluate chemical stability, solubility, non-specific binding, and tolerability

Study I: BCRP substrate assessment

  • Measure bidirectional permeability of test compound in BCRP-transfected and MDCK control cells
  • Three treatments:
    • Test compound at 3 concentrations in BCRP-MDCK cells
    • Test compound at 3 concentrations in MDCK control cells
    • Test compound at 1 concentration in BCRP-MDCK cells, co-dosed inhibitor
  • Perform assay in triplicate (N=3 per treatment)
  • Sampling time points:
    • Donor: 5 and 120 min
    • Receiver: 60 and 120 min
  • Analyze dosing solution, donor, and receiver samples using LC-MS/MS to determine test compound concentration

Study II: BCRP inhibitor assessment

  • Measure efflux of cladribine in BCRP-transfected MDCK with and without test compound
  • Test compound at six concentrations
  • Negative control: vehicle only
  • Positive control: Ko143
  • One cell line: BCRP-transfected MDCK
  • Eight treatments as follows:
    • BCRP-transfected with cladribine + test compound (6 concentrations)
    • Positive control: BCRP-transfected with cladribine + Ko143 (1 concentration)
    • Negative control: BCRP-transfected with cladribine + vehicle only
  • Perform assay in duplicate (N=2 per treatment)
  • Sampling time points:
    • Donor: 5 and 120 min
    • Receiver: 120 min
  • Analyze dosing solution, donor, and receiver samples using LC-MS/MS to determine test compound (dosing solution only) and cladribine concentration

Batch QC

  • Each batch of BCRP-MDCK and MDCK cell monolayers will be certified using internally established criteria
  • Inhibition of probe substrate efflux by a known inhibitor of BCRP

Options

  • The customer may be asked to specify:
    • The concentrations of the test compound
  • The customer can request:
    • A single concentration inhibition assessment prior to IC50 determination
    • A unidirectional assay format for the IC50 determination