Reaction Phenotyping Assay - Creative Bioarray

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Drug metabolism is largely mediated by phase I enzymes, including the cytochrome P450 (CYP) superfamily, and phase II enzymes, such as UDP-glucuronosyltransferases (UGTs). A large number of these drug-metabolizing enzymes are genetically polymorphic, leading to interindividual variation in drug exposure, efficacy, and safety among patients. In addition, drug–drug interactions (DDIs) can occur through the inhibition or induction of these enzymes by co-administered compounds, leading to therapeutic failure or toxicity. As a result, regulatory agencies, such as the FDA and EMA, have recommended conducting reaction phenotyping studies during drug development to identify the enzymes responsible for the metabolism of new chemical entities.

By integrating systems such as recombinant enzymes, human liver microsomes, and hepatocytes, reaction phenotyping provides a clear picture of metabolic pathways, ensuring better decision-making during drug development and a stronger foundation for regulatory submission.

Our Reaction Phenotyping Assay Service

Creative Bioarray's Reaction Phenotyping Assay is designed to systematically identify and quantify the contribution of individual CYP and UGT enzymes to the metabolism of your compound.

General Workflow:

Selection of test system (recombinant CYPs/UGTs, HLM, or hepatocytes).
Incubation with your compound under optimized assay conditions.
Application of selective enzyme inhibitors to confirm contribution.
Sample processing and quantitative analysis using LC-MS/MS.
Data interpretation and report generation.

Protocol:

Enzymes	Recombinant CYP isoforms (1A2, 2B6, 2C8, 2C9, 2C19, 2D6, 3A4) and UGT isoforms (1A1, 1A3, 1A4, 1A6, 1A9, 2B7, 2B15, 2B17).
Model Systems	Recombinant enzymes, human liver microsomes (HLM), or hepatocytes
Analytical Method	LC-MS/MS
Incubation Time	5–60 minutes depending on compound stability.
Test Concentrations	at least three concentrations
Replicates	n = 3
Deliverables	Detailed study report with enzyme contribution estimates, kinetic parameters (Km, Vmax), and regulatory-ready data visualization.

Key Features

Regulatory Compliance: Our protocols are designed in accordance with FDA and EMA guidelines for in vitro DDI studies, ensuring your data meets regulatory standards.
High-Quality Reagents: We employ only validated, high-purity enzyme systems and selective inhibitors to ensure your data is accurate and dependable.
Experienced Scientists: Our team of DMPK scientists have a wealth of experience and technical expertise in the design and conduct of highly complex metabolic studies.
Customizable Assays: We can tailor the assay to your specific compound, including the selection of enzymes and assay conditions, to meet the unique needs of your project.

FAQ

What is the difference between enzyme inhibition and reaction phenotyping assays?

In vitro enzyme inhibition studies assess the ability of your drug to inhibit enzymes that are important for the clearance of other drugs (predicting your drug as a DDI perpetrator), while reaction phenotyping studies determine which enzymes are responsible for your drug clearance (predicting your drug as a DDI victim). These assays should be run in parallel.

Can assays be customized for specific compounds?

Yes, we tailor enzyme selection, concentrations, and matrices according to your program needs.

What is the typical amount of test compound required for the study?

The amount of test compound necessary for the study can vary depending on the solubility of your compound and the design of the study. As a rule of thumb, we generally request at least 10mg of the test compound in order to ensure we have enough material for all incubations (as well as repeat analyses if necessary).

Should I choose recombinant enzymes, human liver microsomes (HLM), or hepatocytes for my study?

The selection of in vitro systems depends on the objective of the study and on the stage of drug development. Recombinant enzymes provide a means of directly assessing individual isoforms in isolation, HLM are a pooled system that reflect the overall hepatic metabolism and hepatocytes provide the most physiologically relevant model (covering both Phase I and Phase II metabolism). Please contact us to discuss your needs and the best approach for your particular program.

How can reaction phenotyping data be integrated with other DDI studies?

Reaction phenotyping studies define the enzymes responsible for the clearance of your drug, and in vitro inhibition and induction studies then determine whether or not your compound is an inhibitor or inducer of these same enzymes. Both datasets are required to predict the potential for clinical DDIs with your drug (PBPK models), and these in vitro findings will also be required to satisfy FDA/EMA regulatory requirements. Sponsors can use phenotyping data in conjunction with inhibition/induction results to estimate the probability that their drug will be both a victim of (affected by co-medication) and a perpetrator of DDIs (affecting co-medication).

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