Developing CES Reaction Phenotyping Assay:
Insights from our APA 2025 Poster Presentation.

Reaction phenotyping is a cornerstone of drug metabolism studies, routinely applied to cytochrome P450 (CYP) and UDP-glucuronosyltransferase (UGT) enzymes, to identify the main enzymes responsible for the metabolism of a new drug. Carboxylesterases (CES), particularly CES1b, CES1c, and CES2, play a critical role in the biotransformation of ester- and amide-containing compounds. Their involvement is essential for the metabolic fate of such drug molecules, especially when CYP-mediated metabolism is minimal or absent.

Our work, presented at APA 2025, focuses on developing an in vitro CES reaction phenotyping using p-nitrophenyl acetate (p-NPA) as a universal substrate. In this study, we compared different CES substrates, optimized the experimental conditions and developed an LC-MS method for simultaneous detection of p-NPA and its metabolites.

Key Highlights:

• The incubation concentrations of CES substrate (p-NPA )and the protein concentrations of various supersomes were optimized.
• p-NPA showed measurable depletion and metabolite formation in CES1b, CES1c, and CES2 supersomes.
• Half-lives of p-NPA in CES isoforms were significantly shorter than in buffer controls, confirming enzymatic activity.

We employed p-NPA as a universal substrate to optimize and establish a CES reaction phenotyping assay, which supports the metabolic evaluation and drug-drug interaction assessment of ester-containing compounds in early-stage drug development. These findings support the inclusion of CES phenotyping in early drug development, particularly for ester-containing compounds, and aid in improving in vitro–in vivo extrapolation (IVIVE) models.

Download the poster below and contact us if you would like to discuss in more detail.