P-gp Inhibition – 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 100 µL of test article at 10 mM in DMSO or 2 mg of powder
- Molecular weight of the test article and its salt form
- MSDS or handling and storage information, e.g., light sensitive, store at -20°C, etc.
- Cell batch QC results
- The percent recovery of digoxin 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 efflux ratio ≥ 2.0 in the absence of inhibitor and corrected efflux ratio reduced by ≥ 50% in the presence of inhibitor
- Negative: Digoxin efflux ratio ≥ 2.0 in the absence of inhibitor and corrected efflux ratio reduced by <50% in the presence of inhibitor
- Test article and digoxin each at 10 µM in HBSSg with maximum DMSO concentration ≤ 0.8%
- Confluent monolayers of Caco-2 (clone C2BBe1) cells, 21-28 days old
- 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 for each direction (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
- 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
- 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.
- 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.
- Assay conditions with the inhibitor include:
- A 30 minute pre-incubation with test article
- Assay conditions without the inhibitor includes
- A 30 minute pre-incubation with buffer
- Digoxin can be run in the presence and absence of Valspodar as an optional positive control
- Valspodar dosed at 1 µM