Cytochrome P450 time dependent inhibition (IC₅₀ shift) assay

Cytochrome P450 time dependent inhibition (IC₅₀ shift) assay protocol

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 (IC_{50 (30 Minus)} = 10.1 µM; reversible inhibition component, IC_{50 (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 K_I and k_inact 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× K_m 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 K_I and k_inact determination assay should be determined. This ascertains the time-dependent inhibition kinetic constants, where k_inact is the maximal rate of enzyme inactivation at a saturating concentration of inhibitor and K_I 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 (AUC_i/AUC).⁵

¹ Grimm SW et al. (2009) Drug Metab Dispos 37; 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 J 273(pt3); 533-539
⁴ Berry LM et al. (2008) Drug Metab Lett 2; 51-59
⁵ Burt HJ et al. (2010) Xenobiotica 40(5); 331-343