Cytochrome P450 K_i assay

Cytochrome P450 K_i assay protocol

Typical Test Compound Concentrations 0, 0.25xIC50, 0.5xIC50, 0.75xIC50, 1xIC50, 2.5xIC50, 5xIC50 CYP Isoforms CYP1A, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP3A4 Typical Substrate Concentrations 0.3xKm, 1xKm, 3xKm, 6xKm and 10xKm Number of Replicates n=2 Compound Requirements Dependent on IC50 Analysis Method LC-MS/MS (Fluorescent endpoint for CYP1A) Data Delivery Ki Standard error of Ki Identification of type of inhibition Written report

For reversible inhibition, a simple classification, based on [I]/Ki ratio (where [I] is the inhibitor concentration at steady state Cmax of the highest clinical dose), is commonly used to predict clinical drug-drug interactions2.

Data from the Cloe Select Cytochrome P450 K_i assay

Compounds are evaluated at up to 6 inhibitor concentrations and 5 substrate concentrations in duplicate. Regression analysis is used to identify the type of inhibition (competitive, non-competitive, uncompetitive or mixed).

To select the most appropriate inhibition model, the goodness of fit criteria comprises of visual inspection of the data, correlation of determination (R²) and corrected Akaike’s Information Criterion (AICc). For visual inspection, data are presented as a direct plot of response against substrate concentration, in addition to Eadie-Hofstee and Lineweaver-Burk plots.

Inhibition type	Rate equation
Competitive
Non-competitive
Uncompetitive
Mixed

Table 1

Rate equations for different inhibition model types.

Where v is the rate, V_max is the maximal rate, K_m is the affinity constant, [S] is the substrate concentration, [I] is the inhibitor concentration, K_i is the inhibition constant and α is the interaction parameter which determines the degree to which the binding
of inhibitor changes the affinity of the enzyme for the substrate.

 

In the Cloe Select CYP3A4 K_i assay, the type of inhibition of CYP3A4-mediated midazolam -1´-hydroxylation by ketoconazole in human liver microsomes was identified as being non-competitive in nature. The K_i of ketoconazole was determined to be 53.1 nM. Table 2 illustrates that data generated in the Cloe Select CYP3A4 K_i assay for ketoconazole are comparable with data reported in the literature.

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Questions and answers on Cytochrome P450 K_i determination

Cytochromes P450 are a family of enzymes which play a major role in the metabolism of drugs. Assessment of the potential of a compound to inhibit a specific cytochrome P450 enzyme is important as co-administration of compounds may result in one or both inhibiting the other’s metabolism. This may affect plasma levels in vivo and potentially lead to adverse drug reactions or toxicity. Determination of the inhibition constant (K_i) of a compound is the current recommended approach by the FDA for studying the clinical relevance of reversible cytochrome P450 inhibitors.

Please provide an overview of the Cloe Select Cytochrome P450 K_i assay.

Compounds are evaluated incubating up to 6 inhibitor concentrations, (spanning the IC₅₀ determined using the Cloe Screen Cytochrome P450 Inhibition assay), and 5 substrate concentrations, spanning the K_m, in duplicate. Determination of the K_i value of a test compound defines the inhibitory affinity of the compound for a particular enzyme and can be used to estimate the potential clinical significance of in vitro inhibition.

Figure 1

Eadie-Hofstee (competitive inhibition) analysis of the effect of quinidine on dextrorphan formation in human liver microsomes.

Each data point represents the mean ± SE (n=2). [I] = nM.

 

What is the difference between IC₅₀ and K­­_i?

K_i is an intrinsic inhibitor constant which defines the affinity with which an inhibitor binds to an enzyme.  The IC₅₀ of an enzyme is the concentration of inhibitor which produces 50% inhibition. Preliminary IC₅₀ determination provides a guide to the inhibitor concentrations to be used in a K_i determination assay. The IC₅₀ value of a test compound is dependent upon experimental conditions such as the substrate concentration, incubation time and protein concentration, and therefore may not be reproducible from lab to lab. K_i is characteristic for each particular inhibitor and enzyme, and is not dependent on any particular substrate used. The K_i value is a more reproducible and more reliable value to give an indication whether future in vivo work is required.

How is the best-fit model chosen?

Regression analysis is used to identify the type of inhibition (competitive, non-competitive, uncompetitive or mixed) and the most appropriate inhibition model selected based upon the goodness of fit criteria of a visual inspection of the data, correlation of determination (R²) and corrected Akaike’s Information Criterion (AICc). For visual inspection, data are presented as a direct plot of response against substrate concentration, in addition to Eadie-Hofstee and Lineweaver-Burk plots. 

What does alpha mean?

Alpha (α) is the interaction parameter applied to the mixed inhibition model, which determines the degree to which the binding of inhibitor changes the affinity of the enzyme for the substrate. For example, when α = 1, the inhibitor does not change the binding of the substrate to the enzyme, thus making the mixed inhibition model identical to the non-competitive model. When α > 1 this means binding of the inhibitor is preventing binding of the substrate, reducing the formation of an enzyme-substrate-inhibitor complex, thus pushing the model towards a competitive inhibition model. When α < 1, binding of inhibitor enhances binding of substrate, pushing the model towards an uncompetitive inhibition model.

How do I interpret the data and utilise the K_i value?

For reversible inhibition, a sample classification, based on the [I]/K_i ratio (where [I] is the inhibitor concentration at steady state C_max of the highest clinical dose), is commonly used to predict clinical drug-drug interactions. As per FDA draft guidelines, an estimated [I]/K_i ratio of greater than 0.1 is considered positive and thus would warrant further in vivo investigations.

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