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
• MSDS or handling and storage information, e.g., light sensitive, store at -20°C, etc.

Deliverables

• Percent recovery of test compound from Transwell^® wells containing Caco-2 cell monolayers
• Apparent permeability (P_app) of test compound in the apical-to-basolateral (A to B) direction in the presence and absence of a PepT1 inhibitor
• PeptT1 interaction potential of test compound classified as either positive or negative

Substrate

• Test compound at 5 µM in modified Hanks’ buffer (HBSSg) with final DMSO concentration <0.8%

Assay System

• Confluent monolayers of PepT1-induced Caco-2 cells, 21-28 days old, in Transwell dual-chamber plates

Assay Conditions

• Receiver well with 1% BSA in modified Hank’s buffer (HBSSg)
• Pre-incubate monolayers with assay buffer (apical pH 5.0, basolateral pH 7.4), with or without GlySar, for 30 minutes prior to initiation of the assay
• Dose duplicate monolayers (N=2) with test compound on the apical side only
• Dose two other monolayers (N=2) with test compound and a PepT1 inhibitor, GlySar, on the apical side only
• Sample both apical and basolateral sides at 120 minutes
• Determine the concentration of test compound using a generic LC-MS/MS method with a minimum 4-point calibration curve

Assay QC

• Verify the quality of the cell monolayer batch using control compounds before the monolayers are released for use
• Verify the quality of each monolayer by pre-experiment TEER and P_app of a co-dosed control compound

Notes

1. 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.

Options

1. The customer can request:
   • that a positive control (valacyclovir) be run in parallel