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.

Deliverables

• In vitro intrinsic clearance of test compound determined for each CYP isoform
• Rank order of CYP isoform(s) responsible for test
• compound’s metabolism

Substrate

• Test compound at a single concentration (based on K_m; if not available, 1µM will be used) dissolved in aqueous buffer, acetonitrile (≤1% final), methanol (≤1% final), or DMSO (≤0.1% final)

Assay System

• Pooled, mixed-donor, human liver microsomes (minimum 50 donors) with and without NADPH
• CYP-specific chemical inhibitor(s)
• 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

Assay Conditions

• This assay is run with a single incubation (N=1) per treatment
• Incubate test compound at 37°C in the presence of human liver microsomes (0.5 mg protein/mL) and:
  • No NADPH (negative control)
  • NADPH (no-inhibitor control)
  • NADPH + a CYP-specific inhibitor (single concentration)
• Sample the reaction mixture at 0, 10, 20, 30 and 60 minutes

Batch QC

• Batch certification of human liver microsomes with CYP-specific probe substrates and CYP-specific chemical inhibitors
• Use fluorimetry to confirm the addition of NADPH to the reaction mixture

Options

1. The customer will be asked to specify: • the concentration(s) of the test compound • the CYP(s) to be screened
2. The customer can request: • additional replicates • additional or alternative time points • the use of a standard curve • that a positive control be run for each CYP