Unidirectional Permeability – Caco-2 Cell Monolayers

This assay is used to determine the permeability of a test article through Caco-2 cell monolayers in the apical-to-basolateral direction.

Required from Sponsor

• Either a minimum of 100 µL of test article at 10 mM in DMSO or 2 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.

Deliverables

• Cell batch QC results
• The percent recovery of the test article from the assay wells containing cell monolayers
• The apparent permeability (P_app) of the test article
• The absorption potential of a test article classified as either low or high using the following criteria for the apical-to-basolateral (A→B) direction:
  • Low: P_app <1.0 x 10-6 cm/s
  • High: P_app ≥1.0 x 10-6 cm/s

Substrate

• Test article at 5 µM in HBSSg with maximum DMSO concentration ≤ 0.8%

Assay System

• Confluent monolayers of Caco-2 (clone C2BBe1) cells, 21 to 28 days old

Assay Conditions

• Unidirectional permeability of the test article in Caco-2 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 on the apical side only
• 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

Assay QC

• The quality of the monolayer batch is verified using control articles before the monolayers are released for use
• The quality of each monolayer used in the assay is verified by calculating the P_app for the control article lucifer yellow, dosed post-experimentally

Notes

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 assay broadly classifies the P_app of a test article

3. For reproducible results, the solubility of the test article must be greater than 5 µM in pH 7.4 HBSSg.

Required from Sponsor

• Either a minimum of 100 µL of test article at 10 mM in DMSO or 2 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.

Deliverables

• Cell batch QC results
• The percent recovery of the test article from the assay wells containing Caco-2 cell monolayers
• The apparent permeability (Papp) in both directions
• The absorption potential of a test article classified as either low or high using the following criteria for the apical-to-basolateral (A→B) direction:
  • Low: Papp <1.0 x 10-6 cm/s
  • High: Papp ≥1.0 x 10-6 cm/s
• The efflux ratio (Papp B→A)/(Papp A→B)
  • The efflux is classified as significant when: Efflux ≥2.0 and Papp (B→A) ≥ 1.0 x 10-6 cm/s

Substrate

• Test article at 5 µM in HBSSg with maximum DMSO concentration ≤ 0.8%

Assay System

• Confluent monolayers of Caco-2 (clone C2BBe1) cells, 21 to 28 days old

Assay Conditions

• Bidirectional permeability of the test article in Caco-2 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 for each direction (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

Assay QC

• 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

Notes

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. For reproducible results the solubility of the test article must be greater than 5 µM in HBSSg.

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 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.

Deliverables

• 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

Substrate

• Test article at 5 µM in HBSSg with maximum DMSO concentration ≤ 0.8%

Assay System

• Confluent monolayers of MDR1-MDCK cells, 7 to 12 days old

Assay Conditions

• 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 for each direction (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

Assay QC

• 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

Notes

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.