CYP450 inhibition

Cytochrome P450 (CYP450) Inhibition Studies

Cytochrome P450 (CYP450) inhibition has been implicated in a majority of reported clinically relevant DDIs. CYP450 enzymes are grouped into 18 families based on their sequence similarity. Only a few of these enzymes, belonging to the 1, 2, and 3 CYP-families, are responsible for the metabolism of the majority of drugs and other xenobiotics. Therefore, regulatory agencies globally recommend in vitro evaluation of an investigational drug’s potential to inhibit the following specific CYP450 enzymes[1]

  • CYP1A2
  • CYP2B6
  • CYP2C8
  • CYP2C9
  • CYP2C19
  • CYP2D6
  • CYP3A4/5

Types of Inhibition

P450 enzymes can be inhibited either via direct inhibition or time-dependent inhibition (TDI)[2]):

  • Direct inhibition involves rapid association and dissociation of an inhibitor drug and the enzyme, and may be competitive, non-competitive, or uncompetitive. A list of reversible inhibitors includes α-naphthoflavone, sertraline, montelukast, sulfaphenazole, nootkatone, quinidine and, ketoconazole.
  • Time-dependent inhibition, on the other hand, is defined as an interaction where there is an increase in the extent of inhibition when the inhibitor is incubated with the enzyme before addition of the substrate. Time dependent inhibitors include furafylline, ticlopidine, phenelzine, tienilic acid, and paroxetine.

For TDI there are two major mechanisms by which the phenomenon is observed. The most important and common one being TDI by mechanism-based inhibition (MBI) and the rarer and less impactful TDI by reversible inhibition via a metabolite.

  • TDI by MBI is the result of irreversible covalent binding or quasi-irreversible noncovalent tight binding of a chemically reactive intermediate to the enzyme that catalyzes its formation, resulting in loss of enzyme function. The consequences of TDI via the mechanism of irreversible inhibition are often more concerning then TDI due to a reversible inhibition, because the CYP activity gets fully inactivated when the mechanism is irreversible and can only be rescued via de novo synthesis of the enzyme, a process that naturally takes time. TDI can display a delayed onset due to the time-dependence and can persist even after the inhibitor has been eliminated. Many time-dependent inhibitors of various human P450 identified in in vitro studies have been shown to cause DDI.[3]
  • TDI by reversible inhibition via a metabolite occurs, as implied, when the extent of inhibition is increased due to the reversible inhibition of CYP activity by a metabolite generated in situ.

Given the meaningful difference between the types of inhibition and their possible clinical impact, it is important to study inhibition of CYP450 enzymes in an assay setup that allows us to understand whether the compound is a reversible or a time-dependent inhibitor, and in case of a time-dependent inhibitor, can also help understanding if MBI or reversible TDI occurs.

Did you know…?
TDI by MBI, as explained in the main text, occurs usually when a drug is a mechanism-based inhibitor of the affected enzymes(s). It can however also happen that a metabolite of a drug is causing a mechanism-based inhibition on a different enzyme. One such example is gemfibrozil, of which the glucuronide (formed by UDP-glucuronosyltransferases (UGTs)) is a MBI of CYP2C9.

Assay description and technical considerations

Specific in vitro methods to evaluate compounds as a direct or time dependent inhibitor of CYP450 enzymes have been described. The most important part of these protocols is preincubation of the test compound with metabolic enzymes, in the presence and absence of NADPH, the cofactor needed for substrate transformation, before the probe substrate is added to the incubation. In case a compound is a mechanism-based inhibitor, preincubation with enzymes in the presence of NADPH increases the inhibitory potential compared to preincubation without NADPH, due to the inhibiting effect of the formed reactive intermediates. If a third, 0-minute preincubation treatment group is applied, the comparison of inhibitory potential of this treatment group and preincubation with NADPH can identify slowly associating inhibitors, or inhibitors which are transformed during preincubation without NADPH (for example, α-naphthoflavone).

Inhibition of CYP450 activity is most frequently examined in human liver microsomal preparations using specific probe substrates for all major metabolizing CYPs. Cocktail approaches, also known as ‘N-in-One’ assays, have been developed to test for inhibition of several P450 isoforms simultaneously.[4] Known selective time-dependent and non-time dependent inhibitors at one concentration are commonly included as positive controls for inhibition.

A test compound is typically studied at least over a 1000-fold concentration range. The compound is pre-incubated with human liver microsomes (HLM) for 30 minutes with and without NADPH. Following pre-incubation, NADPH and the CYP-specific substrate or substrate cocktail (for ‘N-in-One’ type assay) are added to initiate the reaction, and then a second incubation takes place. Optionally, a parallel incubation of the compound without preincubation can be added. Wells are assayed for metabolites specific to single CYP enzymes, as outlined in Table 1. According to FDA recommendations, applying the substrates at approximate Km concentrations allows the use of half of measured IC50 values an estimate of Ki based on the Cheng-Prusoff equation[5]:

K i = I C 50 ( 1 + S K M )

Did you know…?
Numerous physiochemical and biological factors have an impact on in vitro drug-drug interaction determinations. First of all, the choice of biological matrix determines the applicability and the limits of the system, while each has their drawbacks and advantages. Recombinant CYP enzymes are the most common choice, due to their specificity and relatively easy handling. Microsomes contain a more complete complement of hepatic drug metabolizing enzymes when compared to recombinant enzymes and thus is a more physiologically relevant system. Hepatocytes represent a higher level of complexity and therefore should be the most representative of in vivo processes. However, using a more complex model also means additional sources of variation, such as drug accumulation inside the cells, transporter activity, choice of preparation protocols. Furthermore, hepatocytes are more costly and their supply is limited.


Table 1 - Substrates and inhibitors for CYP inhibition assays

Enzyme Substrate Metabolite PC; Reversible inhibitor PC; TDI inhibitor
CYP1A2 Phenacetin Acetaminophen Alpha-naphtoflavone Furafylline
CYP2B6 Bupropion 4-OH Bupropion Voriconazole Deprenyl
CYP2C8 Amodiaquin N-desethylamodiaquine Quercetin Phenelzine
CYP2C9 Diclofenac 4´-OH diclofenac Sµlphaphenazole Tienilic acid
CYP2C19 S-Mephenytoin 4’-OH-mephenytoin Nootkatone (S)-fluoxetine
CYP2D6 Dextromethorphan Dextrorphan Quinidine Paroxetine
CYP3A4 Testosterone 6β-OH testosterone Ketoconazole Troleandomycin
Midazolam 3-hydroxylation Ketoconazole Verapamil

Figure 1 – Inhibition of CYP2C9-mediated diclofenac-4’-hydroxilation by tienilic acid. Sulfaphenazole was pre-incubated with human liver microsomes (0.1 mg/ml) at 37 °C with or without NADPH for 30 minutes, followed by 10 minutes incubation by addition of diclofenac (5 µM) and NADPH (1 mM).

Determination of TDI parameters

To estimate the potential DDI risk due to time-dependent inhibition, the apparent inactivation constant (KI) and the maximal inactivation rate constant (Kinact) are determined using multiple concentrations and pre-incubation times. [6]

The assay starts with pre-incubating the test article at multiple concentrations (plus a vehicle control) selected to achieve a scale from no inactivation to maximal inactivation, for different pre-incubation times (including 0 min) with human liver microsomes and NADPH. Following the pre-incubation, an aliquot of the pre-incubation is diluted 10-fold with buffer containing NADPH and the CYP isoform-specific substrate, and then a second incubation is conducted to allow formation of metabolites of the marker substrate. The dilution step and applying the substrates at concentrations 5-10-fold higher than the Km helps to minimize the rate of direct inhibition. The reaction is quenched, samples are centrifuged, and supernatant is collected for analyses.

Following expression of the obtained data of the LC/MS measurements as % remaining activity of the vehicle control, the natural logarithm (ln) % of the remaining CYP450 activities are plotted against the time of preincubation. Then Kinact and KI are calculated by a non-linear regression analysis of the negative slopes against inhibitor concentration according to the following equation:

k o b s   = k i n a c t   × [ I ] K I + [ I ]

where kobs represents the inactivation rate for a given concentration of the test compound in the preincubation mixture and is calculated from the negative slope of the natural logarithm of the % activity remaining after incubation of the inhibitor as a function of preincubation time, kinact is the maximal inactivation rate when [ I ] reaches infinity and KI is the inhibitor concentration for which kobs = 0.5 x kinact.


[1]        R. J. Riley and K. Grime, “Metabolic screening in vitro: metabolic stability, CYP inhibition and induction,” Drug Discov. Today Technol., vol. 1, no. 4, pp. 365–372, Dec. 2004, doi: 10.1016/j.ddtec.2004.10.008.

[2]        R. Evers et al., “Critical Review of Preclinical Approaches to Investigate Cytochrome P450–Mediated Therapeutic Protein Drug-Drug Interactions and Recommendations for Best Practices: A White Paper,” Drug Metab. Dispos., vol. 41, no. 9, pp. 1598–1609, Sep. 2013, doi: 10.1124/dmd.113.052225.

[3]        R. J. Riley, K. Grime, and R. Weaver, “Time-dependent CYP inhibition,” Expert Opin. Drug Metab. Toxicol., vol. 3, no. 1, pp. 51–66, Feb. 2007, doi: 10.1517/17425255.3.1.51.

[4]        D. Spaggiari, L. Geiser, Y. Daali, and S. Rudaz, “A cocktail approach for assessing the in vitro activity of human cytochrome P450s: An overview of current methodologies,” J. Pharm. Biomed. Anal., vol. 101, pp. 221–237, Dec. 2014, doi: 10.1016/j.jpba.2014.03.018.

[5]        R. J. Riley and C. E. Wilson, “Cytochrome P450 time-dependent inhibition and induction: advances in assays, risk analysis and modelling,” Expert Opin. Drug Metab. Toxicol., vol. 11, no. 4, pp. 557–572, Apr. 2015, doi: 10.1517/17425255.2015.1013095.

[6]        R. S. Obach, R. L. Walsky, and K. Venkatakrishnan, “Mechanism-Based Inactivation of Human Cytochrome P450 Enzymes and the Prediction of Drug-Drug Interactions,” Drug Metab. Dispos., vol. 35, no. 2, pp. 246–255, Feb. 2007, doi: 10.1124/dmd.106.012633.