Evaluating Drug Interactions with Cytochrome P450 (CYP450) Enzymes

Metabolism represents the major route of clearance for 75% of drugs on the market. Understanding a drug's metabolism is key to minimizing risks and supporting the development process. The rate at which a drug is metabolized can have significant implications on the level and frequency of dosing regimens required to maintain efficacious concentrations in vivo. Our in vitro metabolism assays are optimized to identify the route, rate, and extent of a drug’s metabolism using the appropriate experimental systems.

The majority of drugs are metabolized by enzymes belonging to the cytochrome P450 families 1, 2, and 3, that mainly mediate oxidative reactions. CYP450 reaction phenotyping can identify whether a drug is metabolized through these enzymes as well as which isoenzymes are responsible.

In addition to interactions of the drug as substrate, inhibition of CYP450 enzymes can have significant implications on metabolism of the drug of interest as well as any co-administered medicine. Understanding the extent and nature of this inhibition is critical to the derisking process during drug discovery and development. Some drugs may also upregulate the expression levels of CYP450s, which again, may impact the pharmacokinetic profiles of circulating drugs. Therefore, investigation of interactions of drug candidates with the most relevant set of CYP450s is required by international drug-drug interaction (DDI) guidelines. Our page on Drug-drug Interaction Studies for Regulatory Submission summarizes the in vitro DDI data that needs to be generated.

CYP450 enzyme interactions are routinely investigated in vitro, and such assays typically include using human recombinant enzymes, liver microsomes (LM), liver S9 fractions, and/or hepatocytes.

Whilst the focus is typically on human in vitro systems, the availability of these models for preclinical species (mouse, rat, dog, monkey, etc.) also allows for the study of species differences, which may consequently improve in vivo animal study design by identifying preclinical species likely to cover the full range of human metabolites. Using the correct model reduces animal use and also improves safety and risk predictions for humans based on in vivo preclinical studies.

Regulatory requirements for CYP450-mediated DDI

Metabolism-based drug-drug interactions (DDI) via inhibition or induction may pose significant safety concerns. This is exemplified by the fact that several drugs have been withdrawn from the market or their use has been restricted due to significant impact of the DDI on drug exposure relative to the associated therapeutic window.

Drug-drug interactions occur when multiple drugs are co-administered and the CYP450 inhibition or induction potential of one drug (precipitant) affects the metabolic clearance of another drug (object). Potential inhibition of drug metabolizing enzymes by the co-administered drug may lead to a significant decrease in clearance and a correspondingly large increase in exposure, resulting in an increased risk of adverse effects.

It is therefore crucial to assess the potential of an investigational drug to cause (inhibitor or inducer, precipitant) or be affected (substrate, object) by such DDIs. Consequently, regulatory agencies such as the ICH[1], the US FDA[2], the EMA[3], or the Japanese PMDA[4] require that potential drug interaction risks be investigated before in-patient clinical trials are conducted.

The potential for drug-drug interactions is therefore usually assessed during the hit-to-lead up to the development stages before clinical trials.

Specific requirements for CYP450 phenotyping, CYP450 inhibition, and CYP450 induction are described below.

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CYP450 Phenotyping

The aim of CYP450 phenotyping studies is to assess whether an investigational drug is metabolized by these enzymes and identify the metabolic pathways.

CYP450 enzymes are involved in the metabolic clearance of a large range of drugs, with major contributions from CYP3A4/5 (37% of drugs) followed by CYP2C9 (17%), CYP2D6 (15%), CYP2C19 (10%), CYP1A2 (9%), CYP2C8 (6%), and CYP2B6 (4%). These CYP450 enzymes mainly mediate oxidative reactions. Beyond the CYP450 enzymes, other oxidative enzymes (e.g. aldehyde oxidase, dehydrogenases, xanthine oxidoreductase) or enzymes mediating hydrolysis, reduction, and conjugation can also play a role in metabolic clearance of drugs.

The metabolic clearance of a drug depends on the activity and abundance of the relevant drug-metabolizing enzymes. Changes in the clearance rate of metabolism of a drug may move systemic exposure outside of the therapeutic window, thereby leading to a loss of efficacy or introduction of toxicity. Both activity and expression of these enzymes can be affected by genetic variance, environmental factors and/or drug-drug interactions (DDI).

Drugs metabolized by the same enzyme may affect each other’s clearance rate, leading to altered pharmacokinetics and potential changes in efficacy and effect. Regulatory Drug-Drug Interaction (DDI) assessment guidelines [1-4] call for the identification of the enzymes which contribute to ≥ 25% of drug elimination, in case of considerable metabolic clearance (based on metabolic stability and mass balance studies).

For metabolic enzyme reaction phenotyping, usually two different but complementary experimental approaches are applied. The "chemical inhibition" approach uses inhibitors specific to each CYP450 enzyme to assess changes in the test compound's metabolism in human liver microsomes or hepatocytes. If inhibition of each tested CYP450 enzyme (CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2D6, and CYP3A4/3A5) using a known selective inhibitor increases the recovery of the unchanged test compound, the enzyme in question is identified to be involved the test compounds metabolism. The "recombinant enzyme" approach looks at the depletion of the test compound in the presence of recombinant CYP450 enzymes, where the direct effect of each selected enzyme can be observed individually on test compound metabolism. Using output from both experiments, the CYP450 enzymes responsible for catalyzing test compound metabolism can be identified.

  • Technical specifications
    • Human liver microsomes, hepatocytes, or recombinant enzymes
    • CYP450-selective chemical inhibitors to determine which enzyme(s) are involved in metabolism
    • Metabolite profiling and/or metabolite identification
    • Investigative or regulatory-compliant study setup

CYP450 Inhibition

CYP450 inhibition has been implicated in the majority of reported clinically relevant DDIs. Our cytochrome CYP450 inhibition assays are designed to elucidate the potential drug-drug interaction liabilities of your compounds and their metabolites for a range of CYP450 isoforms.

CYP450 enzymes can be inhibited either via direct inhibition or time-dependent inhibition (TDI):

  • Direct inhibition involves rapid association and dissociation of an inhibitor drug and the enzyme, and may be competitive, non-competitive, or uncompetitive.
  • Time-dependent inhibition, on the other hand, is defined as an interaction where there is an increase in the extent of inhibition when the inhibitor is incubated with the enzyme before addition of the substrate. The most important and common type of TDI is mechanism-based inhibition (MBI) where the formation of reactive metabolites irreversibly inhibits the metabolizing enzyme. TDI can also be observed where the metabolite itself is a reversible inhibitor or there is a slow covalent binding process of the parent compound to the enzyme.

Given the meaningful difference between the types of inhibition and their possible clinical impact, it is important to study inhibition of CYP450 enzymes in an assay setup which identifies and separates direct and time-dependent inhibitors, and in case of a time-dependent inhibitor, the mechanism underlying this effect.

Inhibition of CYP450 activity is most frequently examined in human liver microsomal preparations using specific probe substrates for all major metabolizing CYP450s. High throughput screen formats are available for initial CYP450 inhibition assessment. Known selective time-dependent and non-time dependent inhibitors at one concentration are included as positive controls for inhibition.

A CYP450 inhibition study usually begins with the assessment of the direct and time-dependent inhibition potential of a compound. In the case of direct inhibition observed in a screening format, a more definitive assessment of the inhibitor constant Ki and the mechanism of this inhibition can be assessed (competitive, non-competitive, or uncompetitive). In the case of a shift between preincubated and non-preincubated IC50 values (indicative of TDI), further experiments may be performed to investigate the related drug-drug interaction risk. This typically includes the determination of KI and kinact using the appropriate selection of multiple test compound concentrations and pre-incubation times.

  • Technical specifications
    • Human liver microsomes, hepatocytes, or recombinant enzymes
    • Major CYP450 isozymes: 1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6, 3A4, and 2E1
    • Reversible, metabolic, and time-dependent inhibition (IC50 shift)
    • Initial screening assessments (% inhibition) or definitive determinations (IC50, Ki)
    • Kinact/KI determinations
    • Determine formation of MIC and/or covalent binding
    • Activity of CYP450 enzymes measured via LC-MS/MS analysis
    • Drug-drug interaction experiments with known co-medications
    • Investigative or regulatory-compliant study setup

CYP450 Induction

CYP450 induction-mediated interactions are one of the major concerns in clinical practice, as an ever-increasing number of patients undergo multidrug therapy. Due to the relatively broad substrate specificity of CYP450 enzymes, metabolic routes of elimination for many substrate drugs can be altered when these enzymes are induced by concomitant xenobiotic administration/exposure.

Induction of a CYP450 enzyme may significantly enhance the clearance of substrate drugs, which could lead to a reduction in their therapeutic effect. CYP450 induction may also create an undesirable increase in formation of reactive metabolites, leading to increased incidence of metabolite-induced toxicity.

The most common mechanism of CYP450 enzyme induction is receptor-mediated transcriptional gene activation. At the molecular level, CYP450 induction is initiated by the binding of an endogenous or exogenous ligand to one of the nuclear receptors/transcription factors:

  • Aryl hydrocarbon Receptor (AhR)
  • Constitutive Androstane Receptor (CAR)
  • Pregnane X Receptor (PXR)

PXR primarily induces the transcription of the CYP3A and CYP2C families, CAR induces the CYP2B family and AhR induces the CYP1A family.

According to the regulatory agencies, the CYP450 induction potential of a compound should be studied in vitro. Since the activation of PXR results in the co-induction of both CYP3A and CYP2C enzymes, a negative in vitro result for CYP3A induction eliminates the need for additional in vitro or in vivo induction studies with CYP3A and CYP2C enzymes. However, if CYP3A induction is observed and confirmed in a clinical study, the induction of CYP2C enzymes (CYP2C8, CYP2C9, and CYP2C19) should be studied either in vitro or in vivo.

CYP induction assays are conducted with plated or sandwich-cultured hepatocytes (SCH) using primary human hepatocytes, individually from at least three donors for a regulatory-compliant setup (screening formats also available for initial assessment using pooled hepatocytes). Readouts can be gene expression changes reported as fold-change in the mRNA levels of each enzyme, and/or as changes in each enzyme’s specific metabolic activity reported as fold-change of reference substrate depletion, except in the case of CYP2C19, where only activity readouts are considered reliable.

  • Technical specifications
    • Multiple human CYP450 enzymes: 1A2, 2B6, 3A4, 2C8, 2C9, and 2C19
    • Enzyme catalytic activity or mRNA levels using human cryopreserved hepatocytes
    • Gene expression of CYP450 isoforms measured via RT-qPCR
    • Catalytic activity of CYP450 isoforms measured via LC-MS/MS analysis
    • Emax and EC50 determination
    • Relative Induction Score (RIS) determination
    • screening format and regulatory studies available

CYP450 Assay Availability

Available SetupsScreeningMechanisticRegulatory
CYP Phenotyping ✓✓
CYP Inhibition✓✓✓
CYP Induction✓✓✓

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Frequently Asked Questions (FAQs) About Cytochrome P450 Drug Interaction Assays

  • Which is the preferred experimental approach to CYP450 Phenotyping?

    Historically, some regulatory guidelines, including the 2020 FDA DDI guidance, have required phenotyping data both from an enzyme inhibition and a recombinant system to be generated. More recently, the ICH M12 DDI guidelines offer the possibility to generate only one dataset, but without specifying the preferred approach. Our experience is that while HLM or hepatocyte phenotyping approaches can be more in vivo relevant than recombinant CYPs by mimicking in vivo isoform ratios, inhibition of an enzyme in a HLM or hepatocyte study may be compensated for by other enzymes present in the same system and can therefore mask its involvement in a compound’s metabolism. Such discrepancies are not uncommon, therefore generating both datasets is preferred to ensure no meaningful interactions are missed.

  • What is the preferred in vitro assay system for CYP450 inhibition studies?

    Numerous physiochemical and biological factors impact in vitro drug-drug interaction determinations. The choice of biological matrix determines the applicability and the limits of the system, as each has its drawbacks and advantages. Whilst recombinant CYP450 enzymes are specific and relatively easy to handle, human microsomes contain a more complete complement of hepatic drug metabolizing enzymes and may be considered more physiologically relevant. Hepatocytes represent a higher level of complexity, and therefore are considered to be the most representative of in vivo processes. However, using a more complex model also means additional sources of variation, such as drug accumulation inside the cells, non-CYP metabolism, transporter activity, and choice of preparation protocols. Furthermore, hepatocytes are more costly.

  • Can drug metabolites be Mechanism-Based Inhibitors (MBI) of other enzymes?

    TDI by MBI usually occurs when a drug is a mechanism-based inhibitor of the enzymes(s) for which it is a substrate. A metabolite may, however, inhibit a different enzyme for which it is not a substrate. One such example is gemfibrozil, where the glucuronide (formed by UDP-glucuronosyltransferases [UGTs]) is a MBI of CYP2C9 and CYP2C8.

  • Is mRNA fold-change data sufficient for CYP induction data submission?

    Induction potential is assessed via incubation of hepatocytes with a test article and is reported as the fold-change in CYP enzyme mRNA levels and/or the fold-change in enzyme activity, using selective probe substrates. Although both endpoints are accepted by regulatory authorities, it is mentioned that in case of activity studies, induction may be masked by concomitant inhibition. Therefore, in some cases, it may be useful to generate both read-outs for a complete study design. The induction of CYP2C19 can, however, only reliably be assessed in an enzyme –activity-based assay.

  • When do I need to generate CYP interaction data during drug development?

    Currently, according to the ICH M12 DDI guidance, in vitro CYP phenotyping data needs to be available when the investigational drug is first administered to patients. Inhibition and induction data are needed by the start of larger studies in patients. They also specify, however, that data generation timelines should be adapted specifically to each new drug and development project. Typically, however, it is beneficial to address potential DDI risk early, starting at the Hit-to-Lead phase of a drug discovery program, mitigating the risk later in the development.

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