Cytochrome P450 time dependent inhibition (IC50 shift) assay
Understand the potential drug-drug interaction liabilities of your compounds by using our cytochrome P450 (CYP450) time dependent inhibition assays for a range of isoforms.
Our range of cytochrome P450 time dependent inhibition services form part of our portfolio of in vitro ADME screening services. Cyprotex deliver consistent, high quality data with cost-efficiency that comes from a highly automated approach.
Time dependent inhibition of cytochrome P450 (CYP450) enzymes
Inhibition of cytochrome P450 enzymes is one of the most common mechanisms resulting in clinically relevant drug-drug interactions. This inhibitory effect can either be a reversible or irreversible (time dependent) interaction.
Time dependent inhibition (TDI) of cytochrome P450 is of particular concern as typically de novo synthesis of the enzyme is required in order to restore activity. The consequences of TDI can be termination of drug development, drug withdrawal or serious restrictions of use.
Cyprotex’s IC50 shift assay determines the IC50 (inhibitor concentration which results in 50% inhibition of activity) following a pre-incubation in the absence and presence of NADPH. This assay enables discrimination between compounds which cause reversible, irreversible, or both reversible and irreversible inhibition.
The IC50 shift method has the capability for simultaneous detection of both reversible inhibitors and time-dependent inhibitors, plus the added benefit for potentially predicting drug-drug interactions.
1Grimm SW, Einolf HJ, Hall SD, He K, Lim H-K, John Ling K-H, Lu C, Nomeir AA, Seibert E, Skordos KW, Tonn GR, Horn RV, Wang RW, Wong YN, Yang TJ and Obach RS. (2009) Drug Metab Dispos37; 1355-1370
Cytochrome P450 time dependent inhibition (IC50 shift) assay protocol
CYP Isoforms Available
CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6 and CYP3A4
Data from Cyprotex's Cytochrome P450 Time Dependent Inhibition (IC50 Shift) assay
A number of known time dependent inhibitors were screened in the IC50 shift assay in triplicate on the plate on three separate occasions. Inhibitors which were known to be solely reversible inhibitors were screened alongside the time dependent inhibitors as negative controls. The results show a high level of consistency over a range of inhibition values.
Figure 1 IC50 shift data for phenacetin O-deethylation inhibition by furafylline, a time dependent inhibitor. Furafylline was pre-incubated with human liver microsomes in the presence and absence of NADPH prior to the addition of the CYP1A2 substrate, phenacetin. The 17 fold shift in IC50 value (IC50 (30 Minus) = 10.1 µM; reversible inhibition component, IC50 (30 Plus) = 0.586 µM; time-dependent effect) when NADPH is included in the pre-incubation indicates that furafylline is a time dependent inhibitor.
Figure 2 Graph illustrates the fold shift in IC50 for tienilic acid (a reversible and time dependent inhibitor) and sulfaphenazole (a reversible inhibitor). Data are mean ± standard deviation of triplicate incubations for three separate experiments.It is recommended that compounds which exhibit an IC50 shift of ≥ 1.5 with a 30 min pre-incubation are classified as time dependent inhibitors1. Tienilic acid, a known reversible and time dependent inhibitor, gives a mean fold shift of 6.8, whereas sulfaphenazole, which is solely a reversible inhibitor, gives a mean fold shift of 0.84.
Figure 3 Graph illustrating mean fold shift in IC50 of midazolam 1-hydroxylation by 4 known CYP3A4 time dependent inhibitors. The data illustrate the mean fold shift in IC50 over 3 experiments with the error bars representing the triplicate incubations on each run of the assay.
Questions and answers on cytochrome P450 time dependent inhibition (IC50 shift)
Please provide an overview of the Cytochrome P450 Time Dependent Inhibition; IC50 Shift Assay.
The Cytochrome P450 Time Dependent Inhibition IC50 Shift assay is able to identify both reversible and time-dependent inhibitors. The IC50 shift assay determines the IC50 value (concentration which produces 50% inhibition) of test compound under three different experimental conditions; 0 min pre-incubation, 30 min pre-incubation minus NADPH and 30 min pre-incubation plus NADPH. Following the pre-incubation with/without NADPH, isoform-specific substrates are added to measure the residual enzyme activity without a dilution step, running the incubation under linear conditions. If the compound is a time-dependent inhibitor a shift to the left (increase in potency) will occur between the 30 min pre-incubation minus NADPH and 30 min pre-incubation plus NADPH. The ratio of these two values gives the IC50 shift.
Figure 4 Example IC50 shift profile
Why is it important to evaluate time dependent inhibition?
Cytochromes P450 (CYP) are a family of enzymes which play a major role in the metabolism of drugs. Inhibition of CYP enzymes is one of the most common causes of drug-drug interactions. The mechanism of inhibition can be reversible, quasi-irreversible or irreversible.
The consequences of irreversible inhibition are considered to be more serious than reversible inhibition because the inactivated enzyme must be re-synthesized before activity is restored. In addition, the irreversible inactivation usually implies the formation of a covalent bond between the metabolite and the enzyme, which can lead to hapten formation and can in some cases trigger an autoimmune response. For these reasons it is important to study the mechanism of the CYP inhibition of new potential drugs as early as possible during the drug discovery process.
Although the terms time dependent inhibition (TDI) and mechanism based inhibition (MBI) are often used inter-changeably, there is a distinct difference scientifically. Time dependent inhibition is defined as an interaction where there is an enhanced inhibition if the test compound is pre-incubated with the metabolizing system prior to addition of the substrate. Mechanism based inhibition specifically refers to a subset of time dependent inhibition which defines inactivation of the enzyme by a chemically reactive metabolite.1
The FDA guidance for drug interactions (January 2020)2 recommend evaluating time dependent inhibition for investigational drugs.
How do I interpret the data from the IC50 shift assay?
Using the IC50 shift assay, it is possible to distinguish between reversible and irreversible inhibition. For solely reversible inhibitors addition of a 30 min pre-incubation gives no change in the IC50. Consequently all three experimental conditions will give the same IC50. Alternatively for compounds which are only time dependent inhibitors, an IC50 will be calculated only when NADPH is present in the pre-incubation and no effect is observed in the 0 min pre-incubation or 30 min pre-incubation minus NADPH. Test compounds which exhibit both reversible and time dependent inhibition will give a lower IC50 with a 30 min pre-incubation in the presence of NADPH compared to a 30 min pre-incubation minus NADPH and 0 min pre-incubation and thus an IC50 shift can be calculated as shown in Figure 4.
Very occasionally a shift may be observed between the 0 min pre-incubation and 30 min pre-incubation minus NADPH. This indicates potential non-NADPH mediated metabolism of one test compound into a more potent inhibitor species although this is rarely observed with CYP450 enzymes.3
A fold shift of greater than 1.5 is considered to be a significant shift and the compound is classed as a time dependent inhibitor.4 Inhibition potency must always be considered in the context of expected in vivo concentrations of the test compound. Although the criteria for acceptance are project and isoform-specific, potent inhibition is considered unfavorable and may preclude the development of a compound. Further work should then be completed to evaluate the mechanism of time-dependent inhibition or determine KI and kinact values to predict the risk of drug-drug interactions in vivo.5
Why and when would you run an IC50 shift assay compared with single point TDI and reversible inhibition screens?
Reversible and single point TDI assays are considered early stage ADME assays and are designed for larger sets of compounds, whereas the IC50 shift assay is run at a later stage to give more detail on leading compounds. It is advisable to run a reversible inhibition assay prior to running an IC50 shift assay. Although this provides the same information as a 0 min pre-incubation it enables an appropriate concentration range for the assay to be pitched so to achieve the most accurate IC50 shift calculation. Useful information can also be achieved from running a single point TDI assay prior to the IC50 shift assay, as it gives an indication of how much of a time-dependent element of inhibition the compound undergoes.
The IC50 shift screen gives a greater amount of information than the single point assay which can be further used to plan kinetic studies so to predict in vivo risk of drug-drug interactions.
Why is there no dilution step between the pre-incubation and incubation steps?
A dilution step is an additional factor in the assay conditions which can skew the behavior of the inhibitor and complicate data processing. Pre-incubating the test compound at 10x higher microsomal protein concentration than that in the incubation step introduces the possibility for higher protein binding, reducing the amount of free compound available for inhibition. This can give an under-prediction of potency. Additionally, differences can be observed in data processing depending on whether the inhibitor concentration in the ‘pre-incubation’ is taken, compared with the ‘incubation’ i.e. diluted 1 in 10. Thus could give a difference in potency of 10×. The effect is more pronounced upon compounds which have a reversible inhibition element compared with those which are solely time-dependent inhibitors.
Consequently an IC50 shift assay which does not have a dilution step gives the most accurate representation of a 30 min pre-incubation, providing unambiguous information for analyzing compounds for further work. For further information, our poster title ‘Comparison of the effect of assay conditions on interpretation of time-dependent inhibition of cytochrome P450 enzymes’ is available to download here.
Why are different assay conditions used for the single point time dependent inhibition assay and IC50 shift assay?
The single point TDI assay is very much an early ADME screen designed for a higher number of compounds where it is important to get a snapshot of the amount of time-dependent inhibition at a top concentration relative to expected therapeutic concentrations. Therefore by completing a 1 in 10 dilution step between the pre-incubation and incubation this reduces the potential for reversible inhibition to occur. This dilution is not sufficient for all compounds to remove the reversible inhibition element, especially for potent inhibitors, and thus the % inhibition results are processed accounting for all inhibition. Using a substrate concentration that is 5× Km also helps to decrease the reversible inhibition component so an accurate measure of TDI can be obtained.
Can I further characterize the time-dependent inhibition determined?
If an IC50 shift is observed, a KI and kinact determination assay should be determined. This ascertains the time-dependent inhibition kinetic constants, where kinact is the maximal rate of enzyme inactivation at a saturating concentration of inhibitor and KI is the concentration of inhibitor which gives half the maximal rate of inactivation. Alongside in vivo data, this can be used to predict the risk of drug-drug interactions (AUCi/AUC).5
1 Grimm SW et al. (2009) Drug Metab Dispos37; 1355-1370 2 FDA Guidance for Industry – In Vitro Drug Interaction Studies - Cytochrome P450 Enzyme- and Transporter-Mediated Drug Interactions (January 2020) 3 Wright JN et al. (1991) Biochem J273(pt3); 533-539 4 Berry LM et al. (2008) Drug Metab Lett2; 51-59 5 Burt HJ et al. (2010) Xenobiotica40(5); 331-343
Learn more about drug metabolism in our popular Everything you need to know about ADME guide.