CYP and UGT Reaction Phenotyping Assay

Reaction phenotyping (identifying which drug-metabolizing enzymes contribute to a candidate's clearance) has always been an early foundational step in preclinical ADME and DDI risk assessment. The primary goals are to determine the fraction metabolized (fm) by individual enzymes, characterize enzyme kinetics, and flag enzymes that could mediate clinically relevant drug–drug interactions. These data feed quantitative DDI predictions and guide the need and design of clinical DDI studies.

In recent years, the scope of reactions typically explored during reaction phenotyping has been extended beyond classical CYP450 isoforms to also focus on phase II conjugation enzymes (particularly UGTs) as well as non-CYP pathways (CES, AOX, FMOs, SULTs, NATs, etc). The recognition of UGT- and non-CYP–mediated metabolism has increased due to their potential impact on drug exposure and interaction risk for specific chemotypes.

Why reaction phenotyping is necessary?

• The FDA and ICH M12 final guidance now clearly support in vitro enzyme phenotyping for drug–drug interaction assessments and to determine when follow-up clinical studies are needed.
• Identification of enzymes contributing to ≥ 25% of metabolic clearance (fm ≥ 25%) is a common threshold used as a trigger for more detailed DDI evaluation or clinical interaction studies.
• Regulators expect robust, well-documented in vitro approaches (recombinant enzymes, human liver microsomes/hepatocytes, inhibitor cocktails, and orthogonal methods) to support mechanistic conclusions.

Creative Bioarray's Reaction Phenotyping Assay

Brief Workflow

Step 1: Metabolite Generation

Add the test compound to human hepatocytes, liver microsomes (HLMs) or recombinant CYP/UGT enzymes for in vitro incubation

Step 2: Reaction Optimization

Optimize the incubation reaction conditions (pH, temperature, cofactor concentration, etc.).

Step 3: Sample Analysis

The incubated samples are then extracted and analyzed by high-resolution LC-MS/MS, providing sensitive detection, structural elucidation and quantification of the primary and secondary metabolites.

Step 4: Enzyme Identification

A range of selective chemical inhibitors are used to probe the contribution of specific enzymes to the metabolism of the parent drug.

Step 5: Data Interpretation & Reporting

The results are analyzed to determine which enzymes are responsible for the formation of each major metabolite, followed by a comprehensive report detailing the findings.

Figure 1. Flowchart for typical reaction phenotyping evaluation of a lead drug candidate.

Our Capabilities:

• Cytochrome P450 (CYP) Enzymes: CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP3A4/5
• UDP-glucuronosyltransferase (UGT) Enzymes: UGT1A1, UGT1A3, UGT1A4, UGT1A6, UGT1A9, UGT2B7, UGT2B15
• Other Non-CYP Enzymes: Such as Flavin-containing Monooxygenases (FMO), Aldehyde Oxidase (AO), Xanthine Oxidase (XO), Monoamine Oxidase (MAO), Carboxylesterase (CES)

Protocol

Reaction phenotyping is part of our broad drug metabolism and pharmacokinetics (DMPK) services. Other DMPK services include Cytochrome P450 Induction Assay, Cytochrome P450 Inhibition Assay, UGT Induction, and UGT Inhibition. These services provide a complete picture of your compound's interaction with the major metabolic pathways, crucial for a robust DDI risk assessment.

Key Features

Comprehensive enzyme coverage

full CYP + broad UGT panel and optional non-CYP pathways for compounds with atypical metabolism.

Experienced DMPK team

senior scientists with long track records in reaction phenotyping, metabolite identification, and regulatory interactions.

Flexible reporting

clear interpretative reports targeted for discovery, preclinical packages, or regulatory submissions.

FAQ

Reference

1. Chen Y, Fretland AJ, et al. Utility of intersystem extrapolation factors in early reaction phenotyping and the quantitative extrapolation of human liver microsomal intrinsic clearance using recombinant cytochromes P450. Drug Metab Dispos. 2011 Mar; 39 (3):373-82.