CYP Reaction Phenotyping Using Supersomes™
This assay is used to identify the CYP(s) involved in the metabolism of a test compound, through the use of CYP-specific Supersomes.
Required from Customer
- The test compound in powder form—a minimum of 10 mg
- Exact molecular mass of the test compound and its salt form
- Relevant solubility of test compound
- The MSDS or handling and storage information, e.g., light sensitive, store at -20ºC, etc.
- In vitro intrinsic clearance of test compound determined for each CYP, scaled to the relative abundance of each isoform
- Rank order of CYP isoform(s) responsible for test compound’s metabolism
- Substrate Test compound at a single concentration (based on Km; if not available, 1 µM will be used) dissolved in aqueous buffer, acetonitrile (≤1% final), methanol (≤1% final), or DMSO (≤0.1% final)
- Individual CYP-specific Supersomes with NADPH
- Non-transfected control with NADPH run in parallel
- LC-MS/MS is used to determine the peak area response ratio (peak area corresponding to the test compound divided by that of an analytical internal standard), without running a standard curve
- This assay is run with a single incubation (N=1) per treatment
- Incubate test compound at 37°C in the presence of CYP-specific Supersomes and non-transfected control
- Sample the reaction mixture at 0, 5, 10, 15 and 30 minutes
- Use fluorimetry to confirm the addition of NADPH to the reaction mixture
|CYP Specific Substrates as Positive Controls|
|CYP Isoform||Probe Substrate||Metabolite|
- The second time point may be selected based on the half-life of the test compound in human liver microsomes.
- CYP-specific substrates as positive controls:
- An optional complete analytical method development includes:
- Optimization of MS: Initial conditions will be established by infusing a solution containing the test compound into an appropriate mass spectrometer. The goals in MS optimization are to maximize detector response for the test compound and determine the approximate lower limit of quantitation (LLOQ) of the test compound.
- Optimization of HPLC: HPLC parameters will be optimized using an appropriate selection of LC column(s) and mobile phase. The goal in HPLC optimization is to develop appropriate chromatographic run-times while maintaining adequate levels of sensitivity.
- Optimization of Sample Preparation: Various means of sample preparation e.g. direct injection, dilution with appropriate solution, protein precipitation, solid phase extraction etc., will be evaluated for their efficiency in extracting the test compound from the biological matrix. The goal in extraction optimization is to achieve the required lower limit of quantitation (LLOQ) while alleviating matrix suppression effects.
- An optional validation is performed using calibration standards and quality control (QC) samples containing the test compound at known concentrations. In order for the analytical method to be considered acceptable the results from at least one method validation run must meet the following criteria:
- The standard curve must include a minimum of five standards with their concentration back calculated to ±15% (±20% at the LLOQ) of their theoretical concentration when the correlation coefficient of the regression model is ≥ 0.990.
- The calculated concentration of at least two-thirds of all QC samples and at least one-third of the QC replicates must fall with ±15% of their respective theoretical concentration.
- The intra-assay coefficient of variation (CV) of the replicate determinations of the low, medium and high QC samples must not exceed ±15%, and the average concentration for each set of QC replicates as calculated using the standard curve must be within ±15% of their theoretical concentration.
- The lowest standard is accepted as the lower limit of quantitation if the typical response at this concentration is at least 5 times greater than any interference in the blank matrix at the retention time of the analyte(s).
- The response ratio of any interfering peak(s) in the matrix blanks at the retention time of the analyte(s) must be <20% of the response to the LLOQ standard.
- The customer will be asked to specify: • the concentration(s) of the test compound • the CYP(s) to be screened
- The customer can request: • additional replicates • additional or alternative time points • the use of a standard curve • that a positive control be run performed in parallel for each CYP