BBB Penetration Potential Determined – MDR1-MDCK Cell Monolayers
This assay is used to determine the blood-brain barrier (BBB) penetration potential of a test article using MDR1-MDCK cell monolayers.
Required from Sponsor
- Either a minimum of 300 µL of test article at 10 mM in DMSO, or 5 mg of powder
- Molecular mass (exact mass) 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 the test article from the assay wells containing MDR1-MDCK cell monolayers
- The apparent permeability (Papp) in both directions
- The efflux ratio (Papp B→A)/(Papp A→B)
- The blood-brain barrier penetration potential classification:
- High when
- Papp A→B ≥ 3.0 x 10-6 cm/s, and Efflux <3.0
- Moderate when
- Papp A→B ≥ 3.0 x 10-6 cm/s, and 10 > Efflux ≥3.0
- Low when either
- Papp A→B ≥ 3.0 x 10-6 cm/s, and Efflux ≥10, or when
- Papp A→B < 3.0 x 10-6 cm/s
- High when
- Test article at 5 µM in HBSSg with maximum DMSO concentration ≤ 0.8%
- Confluent monolayers of MDR1-MDCK cells, 7 to 12 days old
- Bidirectional permeability of the test article in MDR1-MDCK cells Transport buffer: HBSSg, pH 7.4 ± 0.2
- Receiver well containing 1% BSA
- Apical and basolateral side at pH 7.4
- Dose two cell monolayers (n=2)
- 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 test article 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 a control article, lucifer yellow, dosed post-experimentally
1. 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.
2. This screening assay does not provide definitive information about P-gp interactions or apparent Km values.