Required from Customer

• Either a minimum of 300 µL of test compound at 10 mM in DMSO, or 5 mg of powder
• Molecular mass (exact mass) of test compound and its salt form
• MSDS or handling and storage information, e.g., light sensitive, store at -20°C, etc.

Deliverables

• Table of % remaining of test compound at each time point
• Calculated t_1/2 of test compound and control
• Calculated in vitro intrinsic clearance of test compound

Substrate

• Test compound at 1 µM dissolved in DMSO, methanol, or acetonitrile

Assay System

• Pooled liver microsomes (≥3 donors), with 1 mM NADPH
  • Human: mixed gender
  • Rat, dog, primate or mouse: male only
• LC-MS/MS is used to determine the peak area response ratio (peak area corresponding to test compound or control divided by that of an analytical internal standard) without running a standard curve

Assay Conditions

• The assay is run with a single incubation (N=1)
• Incubate test compound (1 µM) at 37°C in buffer containing 0.5 mg/mL microsomal protein
• Initiate the reaction by adding cofactors, sampling at 0, 10, 20, 30, and 60 minutes
• Incubate positive control (5 µM testosterone) in parallel, sampling at 0, 10, 30, and 60 minutes

Assay QC

• The control compound, testosterone, is run in a parallel assay to verify the activity of the CYPs
• After the final time point, fluorimetry is used to confirm the addition of NADPH to the reaction mixture
• T_1/2 of control must meet internal acceptance criteria

Notes

1. The results from this assay are sent to the customer in the ExpressPlus report format, which may include graphical representations of data and comparison with historical data for reference compounds.
2. This assay does not provide stability information of test compound in liver microsomes without NADPH.
3. Percent remaining at each time point is calculated from the peak area response ratio of test compound (or control) divided by the peak area response ratio of the 0-minute sample.
4. In vitro intrinsic clearance (CL_{int in-vitro}) = k/P (mL/min·mg protein), where k is the elimination rate constant (min^-1) and P is the protein concentration (mg/mL).