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
Types of Inhibition
P450 enzymes can be inhibited either via direct inhibition or time-dependent inhibition (TDI)):
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.
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.
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. 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:
Table 1 - Substrates and inhibitors for CYP inhibition assays
|Enzyme||Substrate||Metabolite||PC; Reversible inhibitor||PC; TDI inhibitor|
|CYP2C9||Diclofenac||4´-OH diclofenac||Sµlphaphenazole||Tienilic acid|
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. 
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:
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.
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