DDIM vs. TDI in CYP Enzyme Inhibition Studies: Can Results from a CYP Direct Inhibition Assay be Used to Predict Time-Dependent Inhibition Risk?

In early drug development, the evaluation of cytochrome P450 (CYP) inhibition is a critical component of drug-drug interaction assessment, which is further categorized into direct inhibition (often referred to as reversible inhibition) and time-dependent inhibition (TDI). Both studies measure changes in enzyme activity by comparing the formation of a specific metabolite from its probe substrate in the presence and absence of a test compound. Due to this similarity, some researchers assume a sequential or causal relationship between the results of the two studies. For example, a compound that shows a significant inhibition in a direct inhibition (DDIM) study (i.e., those with low IC₅₀ values) is sometimes presumed to exhibit comparable effects in a TDI study. This assumption overlooks key differences between DDIM and TDI, including their mechanisms, experimental designs, and evaluation criteria. Sole reliance on direct inhibition data to determine TDI testing necessity is scientifically unsupported. The reasons for this include the following:  

Fundamentally Different Objectives of CYP Inhibition Assays

• Direct Inhibition Assay: This assay aims to evaluate the direct inhibitory effects of the test compound on CYP enzymes.

• Time-Dependent Inhibition Assay: This assay is designed to assess whether the test compound exhibits time-dependent inhibition against CYP enzymes.

Experimental Design and Evaluation Criteria Are Independent

• Direct Inhibition Assay: This assay measures the immediate effect of series concentrations of a test compound on CYP enzyme activity. The half-maximal inhibitory concentration (IC₅₀) is then calculated to evaluate the compound’s direct inhibition potential.

• Time-Dependent Inhibition (TDI) Assay: This assay uses a pre-incubation step. It compares the shift in IC₅₀ values after pre-incubating the compound with or without nicotinamide adenine dinucleotide phosphate (NADPH). The difference in IC₅₀ curves—known as the IC₅₀ shift—is used to determine whether the compound exhibits time-dependent inhibition of the enzyme.

Because the two assays differ fundamentally in both experimental design and evaluation criteria, their core parameters (IC₅₀ for direct inhibition and IC₅₀ shift for TDI) are not directly correlated. Consequently, results from a direct inhibition assay cannot be used to reliably predict or inform the risk of time-dependent inhibition. This will be elaborated in the following sections.

What Are Reversible Inhibition and Time-Dependent Inhibition?

Drug-drug interactions (DDIs) are a common concern in clinical practice and can affect both the efficacy and safety of pharmacotherapy. A key cause for DDIs is the inhibition of cytochrome P450 enzymes (CYPs), which play a central role in the metabolic clearance of many drugs. Inhibition of CYPs can be mechanistically categorized into reversible and irreversible or quasi-irreversible time-dependent inhibition. Drug interactions resulting from time-dependent inhibitors can be particularly insidious because enzyme function is not restored after the withdrawal of the drug and must be regenerated de novo. According to the latest ICH M12 guideline on drug interaction studies, it is recommended to conduct in vitro assessments for both reversible inhibition and time-dependent inhibition for key metabolic enzymes such as CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, and CYP3A ^[1]. The primary distinctions between these two types of inhibition are outlined below.

Fundamental Differences in Mechanism of Action

Reversible inhibition arises from the rapid and dynamic binding and dissociation between the compound and the metabolic enzyme. There are four types of reversible inhibition: competitive, non-competitive, uncompetitive, and mixed inhibition. For all types of reversible inhibition, enzyme function is restored after dissociation of the inhibitor from the active or allosteric site ^[2]. As a result, the duration of reversible inhibition in vivo mainly depends on the elimination half-life of the inhibitor. In in vitro studies, the risk of reversible inhibition is typically assessed by determining the half-maximal inhibitory concentration (IC₅₀ value) through the DDIM assay or by calculating the inhibition constant (K_i) through mechanistic enzyme kinetic assays.

There are commonly two different mechanisms leading to time-dependent inhibition of CYPs.

Irreversible inhibition (also referred to as "suicide inhibition"): The inhibitor is metabolized by the enzyme to generate reactive metabolites, which react with the P450 to form covalent adducts of the heme or apoprotein, resulting in enzyme inactivation.

Quasi-irreversible inhibition: The reactive intermediates generated during inhibitor metabolism tightly bind to the heme prosthetic site to form stable metabolite-intermediate complexes whose off-rate is so low that it leads to functional inactivation of the enzyme ^[3,4].

Inactivation of CYP enzymes by compounds can lead to long-lasting inhibition effects, and new enzyme formation is necessary to restore activity. 

In in vitro experiments, it typically determines the IC₅₀ shift, the pseudo-first-order rate constant (k_obs), or the inactivation kinetic parameters (specifically, the maximum inactivation rate constant (k_inact) and the inhibitor concentration at 50% of the maximum inactivation rate (K_I)) to evaluate the time-dependent inhibitory effect of the test compound on metabolic enzymes.

Fundamental Differences in Experimental Setup and Evaluation

The DDIM assay (Figure 1) employs a one-step incubation approach, in which the probe substrate and various concentrations of the test compound are co-incubated in the metabolic enzyme system. Enzyme activity is assessed by measuring the amount of metabolites generated from the probe substrate. The rate of metabolite formation at each inhibitor concentration is compared to a control incubation with no inhibitor. The data (percent inhibition vs. log [Inhibitor]) are fitted to a sigmoidal dose-response curve to determine the IC₅₀ value. The smaller the IC₅₀ value, the lower the concentration of the inhibitor required to reduce the activity of an enzyme by 50%.

The TDI assay (Figure 1, non-dilution method) is designed to determine if pre-incubation and enzymatic turnover increase the potency of an inhibitor. After a 10-minute warm-up, two groups of reactions were conducted. For group 1, the Inhibitor and enzyme are pre-incubated for a set period of time (30 minutes at 37°C) in the absence of the NADPH-regenerating system. After a 30-minute pre-incubation, the substrate with the NADPH-regenerating system was added to initiate the reactions. For group 2, the Inhibitor and enzyme are pre-incubated for a set period of time (30 minutes at 37°C) in the presence of the NADPH-regenerating system. After a 30-minute pre-incubation, the substrate was added to initiate the reactions. The pre-incubation with the NADPH-regenerating system step provides favorable conditions for the test compound to be metabolized into reactive metabolites/reactive intermediates. In this setup, the pre-incubation group without the NADPH-regenerating system serves as the control. The inhibition against CYP enzymes exhibiting at least 1.5-fold IC₅₀ shift between 30-min preincubation without NADPH and 30-min preincubation with NADPH is classified as a time-dependent inhibition.

Figure 1. DDIM and TDI experimental workflows

When comparing the evaluation metrics of the two assays, the DDIM assay provided a single IC₅₀ value that reflects the compound’s direct inhibitory effect on the metabolic enzyme. In contrast, the TDI assay assesses time-dependent inhibition by calculating the IC₅₀ shift value.

Therefore, even if a compound shows a low IC₅₀ value in the direct inhibition assay, one can not conclude that it also exhibits time-dependent inhibition.

Case Study on CYP450 Inhibitors

Several commonly used commercially available CYP direct inhibitors produced low IC₅₀ values in the DDIM assay, yet none demonstrated time-dependent inhibition against their respective metabolic enzymes (see Table 1).

Table 1. IC₅₀ and IC₅₀ shift values for common commercially available CYP enzyme direct inhibitors

Note: Values in the table are retained to three significant figures.

As shown in Fig. 2, montelukast, exhibiting an IC₅₀ value of 0.0412 μM without pre-incubation, did not exhibit significant shifting in its inhibition curves obtained with NADPH in pre-incubation and without NADPH in pre-incubation, respectively. Its IC₅₀ shift was only 0.627 and far below the cutoff value, indicating that montelukast does not exhibit a time-dependent inhibitory effect on CYP2C8.

Figure 2. Inhibition of CYP2C8 Activity (Amodiaquine N-Deethylase Reaction) in Human Liver Microsomes Treated with Montelukast

Conversely, if a test compound is confirmed to be a time-dependent inhibitor, it does not mean that it will have a low IC₅₀ value in the direct inhibition assay. As shown in Figure 3, phenelzine exhibits a time-dependent inhibitory effect on CYP2C8 in the TDI assay, but its IC₅₀ value in the direct inhibition assay is greater than 100 μM.

Figure 3. Inhibition of CYP2C8 Activity (Amodiaquine N-Deethylase Reaction) in Human Liver Microsomes Treated with Phenelzine

Therefore, in vitro direct inhibition (DDIM) and time-dependent inhibition (TDI) assays represent two distinct and independent systems for evaluating enzyme inhibition. Each is designed to assess a different inhibitory mechanism—direct inhibition versus time-dependent inhibition of metabolic enzymes. Because these assays differ in their underlying principles, measurement parameters, and data interpretation criteria, there is no inherent correlation that allows one set of results to be used to predict the other. In other words, the need for a TDI assay cannot be determined based solely on direct inhibition data. To fully understand a compound’s inhibitory risk toward metabolic enzymes, both assays must be conducted and interpreted independently under their respective experimental conditions.

Authors: Xi Liu, Hongying Ma, Lifang Jiang, Genfu Chen

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