Enzyme phenotyping, also known as reaction phenotyping, identifies the drug-metabolizing enzymes responsible for the metabolism of a drug candidate. These studies determine whether a compound is a substrate of specific enzymes, such as cytochrome P450 (CYP) enzymes, UDP-glucuronosyltransferases (UGTs), or other drug-metabolizing enzymes. Understanding which enzymes contribute to drug clearance is an important part of drug discovery and development, particularly when assessing the potential for drug-drug interactions (DDIs).
Enzyme phenotyping helps predict whether co-administered drugs could alter the exposure of a drug candidate by inhibiting or inducing the enzymes responsible for its metabolism. Such changes in drug exposure can potentially reduce efficacy or increase the risk of adverse effects. This makes reaction phenotyping particularly relevant for compounds intended for patients receiving multiple medications, including populations where polypharmacy is common.
Beyond DDI assessment, enzyme phenotyping can help explain interindividual differences in drug exposure. Genetic polymorphisms in drug-metabolizing enzymes can affect metabolic capacity and contribute to variability in pharmacokinetics and treatment response. By identifying the enzymes involved in drug metabolism early on, researchers can better understand metabolic pathways, assess DDI and pharmacogenetic risks, and support more informed decisions throughout the drug development process.
Cytochrome P450 (CYP) enzymes are most studied enzymes due to their major role in drug metabolism. Screening assays can be used to study contribution of these enzymes to metabolism of a new molecule early in discovery phase to flag possible DDI-risks early and help in compound selection and optimization. More comprehensive enzyme phenotyping is a key component of DDI risk assessment and supporting IND, and current recommendations are outlined in the ICH M12 guideline. According to ICH M12, if in vitro data indicate that 25% or more of a drug candidate’s total elimination is mediated by a single enzyme, further investigation is generally warranted, including clinical drug-drug interaction studies. Enzyme phenotyping studies are typically required for small molecules, whereas oligonucleotides, siRNAs, and peptides are generally outside the scope of ICH M12. For antibody-drug conjugates (ADCs), enzyme phenotyping may be needed for the small-molecule payload component of the conjugate.
When oxidative metabolism represents an important clearance pathway, enzyme phenotyping typically begins with in vitro CYP reaction phenotyping to determine the relative contribution of individual cytochrome P450 (CYP) enzymes to drug metabolism. These studies are commonly performed using human liver microsomes (HLM) in combination with CYP-selective chemical inhibitors and/or recombinant human CYP enzymes expressing individual isoforms. Depending on the study design, parent compound depletion and/or metabolite formation is measured to determine which CYP enzymes contribute to the metabolism of the drug candidate and, where possible, to quantify their relative contribution. Metabolite identification can be performed alongside reaction phenotyping, but identifying the major metabolites first is often advantageous. Knowing which metabolites are formed can help define the most relevant metabolic pathways and guide the selection of the most informative phenotyping approach. When recombinant CYP enzymes are used, differences in enzyme abundance, catalytic activity, and expression systems compared with human liver need to be considered when interpreting the results. These differences can be addressed using relative activity factors (RAFs) or by scaling recombinant enzyme data according to human liver CYP abundance. Such approaches help translate the activity observed in recombinant systems into a more physiologically relevant estimate of the contribution of individual CYP enzymes to drug clearance.
Recent research suggests that CYP3A4 contribution can be overestimated when reaction phenotyping is performed using human liver microsomes (HLM) or recombinant CYP enzymes. In contrast, human hepatocyte-based assays may provide a more physiologically relevant assessment of CYP-mediated metabolism and can, in some cases, better reflect observations from in vivo studies. For this reason, suspension human hepatocytes combined with selective CYP inhibitors can be used as an alternative or complementary approach to conventional HLM and recombinant CYP phenotyping. This strategy can be particularly valuable when results from different experimental systems are inconsistent or when there is uncertainty about the contribution of CYP3A4. Incorporating hepatocyte-based phenotyping can therefore provide additional confidence in the metabolic pathways identified and support a more robust understanding of the compound’s in vivo clearance.
In recent years, drug discovery programs have increasingly prioritized metabolically stable compounds with low intrinsic clearance. While this can be desirable from a drug development perspective, it also makes the accurate determination of the fraction metabolized (Fm) more challenging. Conventional in vitro enzyme phenotyping approaches using human liver microsomes (HLM), recombinant CYP enzymes, or suspension human hepatocytes are generally most informative for compounds with moderate to high metabolic turnover. For low-turnover compounds, however, the limited formation of metabolites during standard incubation periods can make it difficult to reliably quantify the contribution of individual drug-metabolizing enzymes. To address this challenge, long-term hepatocyte-based models, such as hepatocyte co-cultures or hepatocytes embedded in an extracellular matrix such as Matrigel, can be used in combination with CYP-selective chemical inhibitors. These systems maintain hepatocyte functionality over extended incubation periods—up to 72 hours compared with the typical 1–2 hour incubation used in conventional assays—providing greater opportunity to detect low levels of metabolism. Mechanism-based inhibitors can also be used to achieve sustained inhibition of specific CYP enzymes throughout the extended incubation period. These approaches can therefore improve the sensitivity and reliability of reaction phenotyping for low-clearance compounds, helping to generate more robust estimates of enzyme contributions and Fm when conventional short-term assays provide insufficient turnover.
When CYP-mediated metabolism is not the primary metabolic pathway, reaction phenotyping may need to be extended to other phase I and phase II drug-metabolizing enzymes. The ICH M12 guideline recommends considering a broader range of enzymes where relevant, including less common CYP isoforms as well as flavin-containing monooxygenases (FMOs), monoamine oxidases (MAOs), aldehyde oxidase (AO), xanthine oxidase (XO), carboxylesterases (CES), and alcohol/aldehyde dehydrogenases (ADH/ALDH). Phase II pathways may also be important and can include uridine diphosphate-glucuronosyltransferases (UGTs), sulfotransferases (SULTs), and N-acetyltransferases (NATs). The contribution of these enzymes can typically be investigated using recombinant human enzymes, human liver subcellular fractions, and selective chemical inhibitors, depending on the metabolic pathway and study objective. Chemical structure, observed metabolites, and metabolic stability data can help prioritize the most plausible enzymes and establish an efficient, stepwise phenotyping strategy. Starting with the enzymes most likely to contribute can focus experimental efforts while providing a clearer understanding of the pathways responsible for the drug candidate’s metabolism and clearance.
Among phase II drug-metabolizing enzymes, UDP-glucuronosyltransferases (UGTs) are the most commonly included in an enzyme phenotyping strategy. As with CYP phenotyping, UGT reaction phenotyping is typically performed using recombinant human UGT enzymes to identify which isoforms contribute to the glucuronidation of a drug candidate. The ICH M12 guideline recommends considering 11 UGT isoforms for reaction phenotyping, although additional isoforms may be relevant depending on the compound’s chemical structure, metabolic profile, and observed metabolites. For hepatic UGTs, confirmatory studies using human liver microsomes (HLM) in combination with selective UGT inhibitors can provide complementary evidence for the contribution of individual isoforms. However, the limited availability and selectivity of suitable UGT inhibitors can make this approach challenging. An alternative is to apply relative activity factors (RAFs) to recombinant UGT data. RAF-based scaling can help translate activity measured with individual recombinant enzymes to the enzyme abundance and activity observed in human liver microsomes, providing a more quantitative estimate of the contribution of the major hepatic UGT isoforms to drug metabolism.
A robust enzyme phenotyping strategy combines metabolite identification, in vitro reaction phenotyping, and, where appropriate, hepatocyte-based follow-up studies to build a comprehensive understanding of the enzymes and metabolic pathways responsible for drug clearance. The most effective approach is not necessarily a single assay, but a stepwise strategy tailored to the compound’s metabolic profile, incorporating complementary experimental systems when needed to increase confidence in the results.
By identifying the enzymes that contribute to drug metabolism and estimating their relative contribution to clearance, enzyme phenotyping provides valuable insight into drug-drug interaction (DDI) risk, pharmacogenetic variability, and clinical pharmacokinetics. These data can support IND-enabling development and regulatory submissions, inform clinical pharmacology strategies, and ultimately help guide the development of safer and more predictable medicines.
If you would like to learn more about how to incorporate enzyme phenotyping into your drug development program, our experts are happy to help. Get in touch with us to discuss your specific needs and identify the most appropriate strategy for your compound.
References:
Bapiro, T.E., Martin, S., Blacker, T.S. et al. A mismatch in enzyme-redox partnerships underlies divergent cytochrome P450 activities between human hepatocytes and microsomes. Commun Biol 8, 1539 (2025). https://doi.org/10.1038/s42003-025-08903-1
International Council for Harmonisation. (2024). M12: Drug interaction studies
