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.
- 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
- Test compound and digoxin each at 10 µM in modified Hanks’ buffer (HBSSg) with final DMSO concentration < 0.8%
- Confluent monolayers of MDR1-MDCK cells, 7-14 days old, in Transwell® dual-chamber plates, with apical and basolateral pH 7.4
- 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
- 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
- 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.
- 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.
- The customer can request:
- that a positive control be performed in parallel (additional fees apply)