Stability with Metabolite Detection – Phase I Metabolites and Glucuronidation Liver Microsomes
This assay is used to determine the metabolic stability of a test compound incubated with human, rat, dog, primate or mouse liver microsomes and to detect expected Phase I and Phase II metabolites.
Required from Sponsor
- Test article: 20 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.
- Table of % remaining of test compound at each time point
- Calculated t1/2 of test compound and control
- Calculated in vitro intrinsic clearance of test compound
- Detection of expected Phase I metabolites:
- Retention time, peak area and number of isobars detected for up to three major expected biotransformations per species
- Detection of expected Phase II metabolites (glucuronidation)
- Results of positive control (7-HC)
- Indication if glucuronide conjugates were detected, including retention time and signal intensity of the conjugates
- Test compound at 1 µM in liver microsomal reaction mixture
- Human liver microsomes: 0.5 mg/mL (mixed gender, ≥ 10 donors)
- Mouse, rat, dog, or monkey liver microsomes: 0.5 mg/mL (male, ≥ 3 donors)
- Reaction mixture:
- Potassium phosphate buffer, pH 7.4: 100 mM
- Magnesium Chloride: 5 mM
- Phase I Metabolism: NADPH 1mM
- Phase II Metabolism: UDPGA 1mM
- Positive controls:
- Phase I Metabolism: Testosterone
- Phase II Metabolism: 7-HC
- The assay is run with a single incubation (N=1)
- Stability and Phase I/Phase II Metabolism
- Incubate test compound (1 µM) at 37°C in buffer containing 0.5 mg/mL microsomal protein
- Incubate solvent control in parallel, sampling at 60 minutes
- Initiate the reaction by adding cofactors (NADPH & UDPGA), sampling at 0, 10, 20, 30, and 60 minutes
- Incubate positive control (5 µM testosterone) in parallel, sampling at 0, 10, 20, 30, and 60 minutes
- Incubate positive control (100 µM 7-HC) in parallel, sampling at 60 minutes
- The control compounds, testosterone and 7-HC, are run in parallel assays to verify the activity of the CYPs
- After the final time point, fluorimetry is used to confirm the addition of cofactors to the reaction mixture
- T1/2 of controls 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 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).