Reaction phenotyping assay

Aim: Identification of the drug metabolizing enzymes involved in the metabolism of test compound using recombinant human enzymes.

Background information: During Drug Discovery, the prediction of the routes of elimination in humans is challenging. For compounds predicted to be eliminated by metabolism, the identification of the enzymes involved in the metabolite formation is an important step in the risk assessment of potential metabolism-based drug-drug interactions. Reaction phenotyping helps to assess compounds as potential victims of interactions with concomitant drugs in the clinical practice. In addition, certain cytochrome P450 ezymes are polymorphic and metabolism can vary considerably between individuals, which may require dose adjustment.

Reaction phenotyping is evaluated independently with several recombinant enzymes (CYP, FMO, MAO). After incubation of test compound, the enzymes involved in the formation of the metabolites observed are identified by UPLC-MS/MS.

Compound requirement: 100 µl of 10 mM DMSO solution

Turnaround time: 2 weeks

Experimental design:

  • Test compound concentration: 10 µM (variable on request)

  • Human recombinant enzymes: CYP1A2, CYP2B6,  CYP2C9, CYP2C19, CYP2D6 and CYP3A4, FMO1, FMO3, MAO-A and MAO-B

  • Number of replicates: 2 per time point

  • Final organic solvent: 1%

  • Time points: 0 and 60 min

  • Positive controls: known probe substrates

  • Stability in incubation media and human  liver microsomes run in parallel

  • Analytical method: UPLC-MS/MS

Delivered results:

  • -% test compound remaining relative to time 0 for each isoform

  • Enzymes involved in the metabolism

  • NADPH-dependence of metabolism

  • Enzymes responsible for the formation of detected metabolites