CYP Reaction Phenotyping

About the Assay

The CYP450 Reaction Phenotyping (RePh) assay determines which human CYP450 isoforms are responsible for metabolising a test compound using recombinant enzymes expressed in E.coli bactosomes. This mechanistic assay supports early DMPK decision‑making by identifying metabolic pathways, potential drug–drug interaction liabilities, and whether clearance occurs through multiple CYP450 enzymes. The assay evaluates metabolism across the major human isoforms—CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6 and CYP3A4/5—using substrate depletion as the measurement endpoint. By monitoring loss of parent drug over time, the assay quantifies intrinsic clearance (CL_int), elimination rate constant (k) and half‑life (t_½), generating a clear profile of isoform‑selective metabolism. RePh includes FDA, EMA and ICH‑recommended probe substrates as positive controls, ensuring regulatory relevance. Because the bactosome system contains isolated, individually expressed recombinant CYPs, it enables unambiguous assessment of isoform involvement without confounding secondary pathways. Combined with high‑sensitivity LC‑MS/MS quantification, the assay delivers robust, reproducible enzyme‑specific clearance data. This makes RePh an essential screen for identifying metabolic soft spots, prioritising chemistry optimisation strategies and informing human PK predictions. Overall, the assay provides high‑confidence attribution of CYP involvement in NCE metabolism and is a critical component of modern DMPK workflows.

Protocol Summary

The RePh workflow begins with preparation of recombinant CYP bactosomes, probe substrates and test compounds. Bactosomes are thawed, diluted and combined with buffer components and test articles to create isoform‑specific incubation mixtures. After an initial equilibration step, reactions are initiated by cofactor addition, allowing CYP‑mediated metabolism to proceed. Samples are removed at scheduled timepoints to capture parent‑drug depletion profiles. A parallel control incubation using an empty‑plasmid bactosome preparation is run for each compound, supporting correction for non‑specific compound loss. Reaction termination is achieved by addition of acetonitrile, followed by centrifugation and sampling of supernatant for LC‑MS/MS analysis. Chromatographic methods quantify remaining parent compound across all timepoints. The subsequent analysis step calculates intrinsic clearance and related parameters using first‑order depletion kinetics, with results summarised on a per‑isoform basis. Collectively, this protocol yields a high‑quality comparison of metabolic turnover across each CYP isoform, enabling mechanistic attribution of clearance pathways and early identification of potential drug–drug interaction risks.

Validation Results

Validation demonstrated that the RePh assay delivers reliable and reproducible intrinsic clearance measurements across all major CYP450 isoforms using recombinant bactosomes. Inter‑assay performance was assessed using eight FDA‑recommended probe substrates, each tested in four independent assay runs with six technical replicates per run. The resulting intrinsic clearance values showed strong reproducibility, with coefficients of variation that confirm robust assay precision. Intra‑assay consistency was likewise strong, with low variability across replicate measures. Freeze–thaw stability was evaluated to confirm whether bactosome preparations maintained activity after multiple freeze–thaw cycles. Most CYP isoforms retained acceptable performance, with intrinsic clearance values remaining consistent and coefficients of variation below 25%, confirming that the reagent system is stable for limited reuse. Correction using control bactosomes further increased confidence in the accuracy of depletion‑based assessments by accounting for background disappearance not attributable to CYP‑mediated turnover. The collective dataset confirms that RePh provides dependable, mechanistically meaningful CYP phenotyping results suitable for informing early DMPK decisions.