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

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

Substrate

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

Assay System

• Pooled hepatocytes (≥3 donors)
  • 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)
• Cell viability assessed by trypan blue exclusion prior to assay
• Incubate test compound (1 µM) at 37^oC in buffer containing 1.5 x 10⁶ hepatocytes/mL, sampling at 0, 15, 30, 60, and 120 minutes
• Incubate positive controls (testosterone and 7-hydroxycoumarin) in parallel, sampling at 0, 5, 15, 30, 60, and 120 minutes (testosterone) and 0, and 15 minutes (7-hydroxycoumarin)

Assay QC

• The control compounds, testosterone and 7-hydroxycoumarin, are run in parallel to verify the enzymatic activity of the hepatocytes
• T_1/2 of testosterone elimination and rate of formation of 7-hydroxycoumarin conjugates 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/D (mL/min·million cells), where k is the elimination rate constant (min^-1) and D is the cell density (cells/mL).