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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-synthesised 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 metabolising 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.3
The draft FDA guidance for drug interactions (February 2012)1 recommend evaluating time dependent inhibition for investigational drugs.
Please provide an overview of the Cytochrome P450 Time Dependent Inhibition: Single Point Assay.
In the Cytochrome P450 Time Dependent Inhibition: Single Point assay a single concentration of the test compound is pre-incubated with NADPH and human liver microsomes at a 10 fold higher concentration than the final incubation concentration for 30 minutes. If the test compound is a time dependent inhibitor then it will be activated in the presence of the NADPH and bind to the active site of the enzyme. Following the pre incubation stage, an aliquot of the pre-incubation is diluted 10 fold with buffer containing the CYP isoform-specific substrate and then a second incubation takes place. By diluting the test compound 10 fold for the final incubation with the marker substrate, any potential reversible inhibition is minimised. The potential for reversible inhibition is also reduced by using a high concentration for the CYP isoform-specific substrate (typically 5 × Km). Several control incubations are included in addition to the test well where the pre-incubation is performed in the absence of NADPH and absence of test compound. Details of all incubations are illustrated in Figure 4.
Figure 4
Overview of the time dependent inhibition protocol
1. Pre-incubation 1: Test Cmpd + NADPH. This pre-incubation consists of test compound, NADPH and 10 fold the concentration of microsomes. If the test compound is a time dependent inhibitor then it should be activated and bind to the active site of the CYP isoform being screened.
2. Pre-incubation 2: Test Cmpd – NADPH. This pre-incubation consists of test compound, buffer and 10 fold the concentration of microsomes. As no NADPH is present in the pre-incubation, no metabolic activation of the test compound by CYP isoforms should occur. Therefore, time dependent (or mechanism based) inhibition should not occur during the pre-incubation.
3. Pre-incubation 3: Vehicle + NADPH. This pre-incubation is one of the vehicle solvent incubations where the test compound is absent. It consists of vehicle solvent, NADPH and 10 fold the concentration of microsomes. As the test compound is absent, no inhibition of the CYP-specific isoform should occur during the pre-incubation. Any loss of activity observed in this incubation could possibly be due to CYP futile cycling.
4. Pre-incubation 4: Vehicle – NADPH. This pre-incubation is one of the vehicle solvent incubations where the test compound is absent and the NADPH is replaced by buffer. It consists of vehicle solvent, buffer and 10 fold the concentration of microsomes. As the test compound and NADPH are absent, no inhibition of CYP should occur in the pre-incubation.
The percentage inhibition observed following pre-incubation is calculated for the test compound using Equation 1 below:
R is the response of the metabolite measured in the sample from the incubation.
This equation is adapted from Atkinson et al., 20052 and accounts for any loss of activity which may occur in the solvent vehicle incubations in the presence of NADPH.
How do I interpret the data from the single point time dependent inhibition assay?
Using the time dependent inhibition assay, it is possible to distinguish between reversible and irreversible inhibition. Figure 5 illustrates the effect of a reversible (ketoconazole), time dependent (troleandomycin) and mixed reversible and time dependent (mibefradil) inhibitor on the formation of 1-hydroxymidazolam in the time dependent inhibition assay. For reversible inhibitors, it is possible to observe inhibition when NADPH is absent from the pre-incubation as the inhibition occurs in the second incubation stage. However, this is unlikely to be picked up if the compound is a weak reversible inhibitor due to the 10 fold dilution step and the probe substrate concentration in the incubation being well above Km. For compounds which are only time dependent inhibitors, inhibition will be observed only when NADPH is present in the pre-incubations. A mixed inhibitor which exhibits both reversible and time dependent inhibition will exhibit inhibitory potential in both the absence and presence of NADPH in the pre-incubation. However, the compound will inhibit to a greater extent when NADPH is present in the pre-incubation. The results from the time dependent studies can be used in conjunction with the reversible inhibition results to identify the nature of inhibition.
Potent irreversible inhibition is typically considered unfavourable and may preclude the development of a compound. However, the way in which the data is interpreted is project specific. For example, time dependent inhibition may be irrelevant if the concentration of inhibitor in the in vitro studies is much higher than the anticipated concentration in vivo. Typically, for the time dependent inhibition assay, the test compound is pre-incubated at the highest concentration investigated in the reversible inhibition assay. If the compound is identified to be a time dependent inhibitor then more detailed studies can be performed to calculate the KI and kinact to further characterise the extent of inhibition.
Figure 5
Inhibition of 1-hydroxymidazolam by (a) ketoconazole (b) troleandomycin and (c) mibefradil
What is the difference between irreversible and quasi-irreversible inhibition?
Irreversible inhibition occurs when a drug is activated to form a covalently modified enzyme-drug complex. Quasi-irreversible inhibition occurs when the test compound is metabolised by CYP to form a stable complex with the haem of the CYP. For both irreversible and quasi-irreversible inhibition, synthesis of new enzyme is required before normal CYP activity is restored. For the time dependent inhibition method, both irreversible and quasi-irreversible inhibition is detected, but cannot be distinguished between.
Why is CYP3A4 inhibition often considered to be the interaction of most concern?
Consistent with its highest abundance in humans CYP3A4 is responsible for the metabolism of about 60% of currently known drugs. However, this unusual low substrate specificity also makes CYP3A4 susceptible to reversible or mechanism-based inhibition by a variety of drugs. Clinically important time dependent CYP3A4 inhibitors include antibacterials (e.g. clarithromycin, erythromycin and isoniazid), anticancer agents (e.g. tamoxifen and irinotecan), anti-HIV agents (e.g. ritonavir and delavirdine), antihypertensives (e.g. dihydralazine, verapamil and diltiazem), sex steroids and their receptor modulators (e.g. gestodene and raloxifene), and several herbal constituents (e.g. bergamottin and glabridin). Drugs inactivating CYP3A4 often possess several common moieties such as a tertiary amine function, furan ring, and acetylene function4.
Compared with reversible inhibition of CYP3A4, mechanism-based inhibition of CYP3A4 more frequently causes pharmacokinetic-pharmacodynamic drug-drug interactions, as the inactivated CYP3A4 has to be replaced by newly synthesised CYP3A4 protein. The resultant drug interactions may lead to adverse drug reactions, and even fatality.
Why do we not use the equation below from Obach et al., 20075?
Concerns have been raised about how compounds that exhibit both reversible and irreversible inhibition may be overlooked when using Equation 2 from Obach et al., 20075.
Equation 2
where R is the response of the metabolite measured in the sample from the incubation.
In Grime et al., 20096 it is noted that the methods used in Obach et al., 20075 had the potential to contaminate irreversible CYP inhibition with reversible inhibition and as such, the equation reported by Obach et al., does not account for reversible inhibition. This means that for compounds that cause reversible CYP inhibition, in addition to irreversible inhibition, the percent irreversible inhibition is vastly underestimated. An example of such a compound is illustrated in Figure 6. In this experiment, performed in duplicate, the mean values for % reversible inhibition given by both equations are as follows:
Figure 6
Example data from an experiment carried out in duplicate where the mean inhibition values calculated from equations 1 and 2 vary significantly.
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Examples of Calculated Inhibition |
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Equation 1 |
44.5% |
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Equation 2 |
5.80% |
How can I further characterize any inhibition observed?
If time dependent inhibition is observed, an IC50 shift assay may be performed. The IC50 shift assay determines the IC50 value of test compound (concentration which produces 50% inhibition) under three different experimental conditions; 0 min pre-incubation, 30 min pre-incubation minus NADPH and 30 min pre-incubation plus NADPH. 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. 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.7
If an IC50 shift is observed, further work could be completed to establish the mechanism of inhibition and kinetics of inactivation using a KI and kinact determination assay. 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).8
Why and when would you run a single point time dependent inhibition assay compared with a reversible inhibition and IC50 shift assay?
The single point time dependent inhibition study can be run in parallel with the standard reversible CYP inhibition assay. It is advisable however, to run a standard reversible CYP inhibition assay prior to running a time dependent inhibition study. This is because compounds that cause potent reversible inhibition may need to be run at a lower concentration in the time dependent inhibition assay to minimise the reversible inhibition that it likely to occur in the probe substrate incubation. These two assays are considered early stage ADME assays designed for larger sets of compounds. The IC50 shift assay however is typically run at a later stage as it delivers a greater amount of information than the single point assay which can be further used to plan kinetic studies to predict in vivo risk of drug-drug interactions.
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