Cytochrome P450 (CYP450) Reaction Phenotyping Studies
Understanding the metabolic stability of a drug candidate is key to its further development, as it affects not only compound efficacy and target concentrations but also its clearance route and overall ADME properties. Metabolism is the process of biotransformation of both endo- and xenobiotics, including drugs. In fact, for 75% of all drugs, metabolism represents the major route of clearance, therefore compound metabolic stability is usually addressed early in the development process in vitro or using in vivo mass balance experiments. At this stage, however, identifying the exact metabolic processes involved in a drug’s or API’s degradation may not be necessary and if often completed at later stages of development.
In addition to its contribution to a drug’s overall ADME properties and clearance path, enzymatic metabolism can also be a mediator of drug-drug interactions. 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 (including FDA 2020 final and ICH M12 2022 draft guidelines) call for the identification of the enzymes that contribute to ≥ 25% of drug elimination, in case of considerable metabolic clearance (based on metabolic stability and mass balance studies).
The majority of drugs that undergo extensive metabolism are metabolized by the enzymes belonging to the cytochrome P450 families 1, 2 and 3, 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 are mainly mediating 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.
In vitro data on the investigational drug as a substrate of metabolic enzymes generally should be obtained before starting phase 1 (first-in-human) to evaluate metabolic stability and identify the potential main metabolic pathway(s) and enzyme(s) that metabolize the investigational drug (reaction phenotyping studies). In vitro enzymatic reaction phenotyping data is often used to identify and quantify elimination pathways of an investigational drug, together with e.g., in vivo pharmacokinetics, mass-balance study, pharmacogenetic data or available DDI data.
Assay description and technical considerations – metabolic stability
First, the test compound’s metabolism is assessed using human liver microsomes. In case there is no significant metabolic degradation of the compound is observed, reaction phenotyping may not be warranted. If the compound loss due to metabolism is NADPH-independent, its chemical degradation is not driven by CYP450 enzymes.
In such cases, other phase I and II metabolic enzymes may still be involved, these can often be predicted based on the type of metabolic reaction and change in molecular structure. Deliverables from such metabolic stability studies are the in vitro half-life (min) and the in vitro clearance (μL/min per mg protein or per million cells) of the compound. Functionality of CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2D6 and CYP3A4/3A5 is confirmed in each experiment using a set of probe substrate drugs specific to each respective enzyme as control.
In vitro metabolic clearance values are determined for the test compound and can be used to predict in vivo metabolic clearance values, and also potentially ranking multiple candidate compounds based on the results. At this stage, metabolite identification (MetID) may also be useful to drive further decisions.
Learn more about our Metabolic Stability assays here.
Figure 1. Drug metabolism assessment and CYP450 phenotyping workflow with assay types and decision points.
Assay description and technical considerations – CYP450 reaction phenotyping
For compounds where NADPH-dependent loss is observed in microsomes, CYP450 reaction phenotyping is necessary. As follow-up, two experiments are conducted to identify the responsible enzymes:
Figure 2. Specific metabolism of Diclofenac into its OH-Diclofenac metabolite by the CYP2C9 enzyme assessed using different assay systems. A) Recombinant CYP450 enzymes. Significant depletion of Diclofenac in the assay system is observed only when recombinant CYP2C9 was added. Data is presented as % remaining diclofenac upon incubation with each respective enzyme compared to a control where the empty vector is present, but no CYP450 enzyme is expressed. B) Enzyme inhibition using human liver microsomes (HLM). Upon addition of Tienic acid, a selective CYP2C9 inhibitor, Diclofenac recovery from human liver microsomes is increased, while inhibition of other CYP450 enzymes do not affect its metabolism significantly. Data is presented as % remaining diclofenac upon incubation with each respective enzyme inhibitor compared to a control where no NADPH was added.
Table 1. Summary of specific reference substrates and inhibitors applied for each respective CYP450 enzymes studies in the reaction phenotyping assay. All inhibitors selected show time-dependent CYP450 inhibition kinetics.
If upon inhibition of each tested CYP enzyme (CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2D6 and CYP3A4/3A5) individually using a known selective inhibitor increases the recovery of the unchanged test compound, the enzyme in question is identified to be involved in its metabolism. In each experiment, a known substrate of each respective enzyme is run in parallel with the test compound, in the presence of their specific inhibitor, to confirm enzyme functionality as well as inhibitor performance. Inhibition effect is provided as % recovery of the parent compound (control substrate or unknown TA) compared to a control condition where no NADPH is added and therefore no CYP450-mediated metabolism occurs (for selective probe substrates, quantification of their specific metabolite formation is also a possible readout). As many compounds are metabolized by multiple CYP450 enzymes to some degree, comparison to the no NADPH condition allows clearly and separately identifying the impact of inhibiting a single enzyme.
Using a recombinant CYP450 enzyme system, direct effect of each selected enzyme can be observed individually on test compound breakdown. Recombinant CYP450 enzymes are expressed using a bactosomal system which is used for the reaction phenotyping. Substrate depletion in each reaction is determined as % remaining parent compound compared to a control incubation using bactosomes containing the empty vector. Again, known reference probe substrates for each enzyme are tested in parallel with any unknown test compound as control for system functionality (with parent depletion or specific metabolite formation as potential readout).
Using output from both experiments, CYP450 enzymes mainly catalyzing test compound metabolism can be identified. If the drug is not found to undergo significant metabolism by these major CYP enzymes, other enzymes can be investigated.