Metabolic Stability – Cyropreserved Hepatocytes
This assay is used to determine the percent remaining of a test compound incubated with cryopreserved hepatocytes (mouse, rat, dog, monkey, or human).
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.)
- Table of % remaining of test compound at each time point
- Calculated t1/2 of test compound and testosterone
- Calculated in vitro intrinsic clearance of test compound
- Test compound at 1 µM dissolved in DMSO, methanol, or acetonitrile
- 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
- 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 37oC 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)
- The control compounds, testosterone and 7-hydroxycoumarin, are run in parallel to verify the enzymatic activity of the hepatocytes
- T1/2 of testosterone elimination and rate of formation of 7-hydroxycoumarin conjugates must meet internal acceptance criteria
- 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.
- This assay does not provide stability information of test compound in liver microsomes without NADPH.
- 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.
- In vitro intrinsic clearance (CLint 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).