Metabolic Stability – Liver S9 Fractions
This assay is used to determine the percent remaining of a test compound incubated with liver S9 fraction (mouse, rat, dog, monkey, or human) in the presence of cofactors.
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 controls
- Calculated in vitro intrinsic clearance of test compound
- Test compound at 1 µM dissolved in DMSO, methanol, or acetonitrile
- Pooled liver S9 fraction (≥3 donors), with 1 mM NADPH, UDPGA, PAPS and GSH
- 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)
- Incubate test compound (1 µM) at 37°C in buffer containing 1.0 mg/mL S9 protein
- Initiate the reaction by adding cofactors, sampling at 0, 10, 20, 30, and 60 minutes
- Incubate positive controls (testosterone and 7-hydroxycoumarin) in parallel, sampling at 0, 10, 30, and 60 minutes
- The control compounds, testosterone and 7-hydroxycoumarin, are run in parallel to verify the enzymatic activity of the S9 fraction
- After the final time point, fluorimetry is used to confirm the addition of NADPH to the reaction mixture
- T1/2 of control compounds 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 S9 incubations without cofactors.
- 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/P (mL/min·mg protein), where k is the elimination rate constant (min-1) and P is the protein concentration (mg/mL).