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Why is it important to investigate 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.2
The draft FDA guidance for drug interactions (2012)3 and the draft EMA guidelines (2010)8 recommend evaluating time dependent inhibition for investigational drugs.
Please provide an overview of the Cytochrome P450 Time Dependent Inhibition: kinact/KI.
The Cytochrome P450 Time Dependent Inhibition: kinact/KI assay determines kinetic constants of time dependent inhibition. The 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. The experimental conditions are determined from the results of previously performed assays such as P450 reversible inhibition and time-dependent inhibition: single point or IC50 shift. Compounds are evaluated by pre-incubating a range of 5 test compound concentrations (plus a vehicle control) selected so to achieve a scale from no inactivation to maximal inactivation, for 7 differing pre-incubation times (including 0 min) with human liver microsomes and NADPH. Following the pre-incubation, an aliquot of the pre-incubation is diluted with buffer containing a specific cytochrome P450 probe substrate (at 5×Km concentration) and NADPH for a specific incubation time. Following least-squares regression analysis to determine the negative slope of the logarithm of the % remaining activity (corrected for negative control) versus pre-incubation time, non-linear regression analysis of the negative slopes against inhibitor concentration enables kinact and KI to be calculated (Figure 3).
Figure 3
The inactivation plots of CYP3A4 by diltiazem:
(Above) Plot of natural logarithm of the corrected % activity remaining against pre-incubation time.
(Below) Non-linear regression analysis of the negative slopes against inhibitor concentration.
Each data point represents the mean of n=2.
What information is needed prior to performing a kinact/KI determination?
The kinact/KI determination assay is a later stage assessment of inhibitor kinetics. The data analysis and utility of the data from this assay is completely dependent upon the choice of inhibitor concentrations and pre-incubation times. Ideally the combination of both of these variables should be so that a range of inactivation is achieved from maximal inactivation to no inactivation. This can be difficult to achieve if the compound undergoes considerable reversible inhibition during the substrate incubation. Therefore it is important to have knowledge of the potency of reversible inhibition beforehand so suitable conditions can be determined e.g. a greater than 1:10 dilution to dilute the test compound before substrate incubation, or increased pre-incubation time compared to incubation time. An indication of the amount of time-dependent inhibition expected is also required, from either a single point or IC50 shift assay. This provides an indication of the highest inhibitor concentration required to give maximal inhibition.
How are the results from the kinact/KI assay interpreted?
The kinact and KI values determined can be used to predict the risk of drug-drug interactions. The draft FDA guidance for drug interactions (Feb 2012)3 recommend determining kinact and KI if any time dependent loss of initial product formation rate is observed in preliminary in vitro pre-incubation studies. An estimated R value of greater than 1.1 where R= (Kobs + Kdeg)/Kdeg and Kobs = kinact x [I]/(KI +[I]) is considered positive and thus may warrant further in vivo investigations. [I] represents the maximal total (free and bound) systemic inhibitor concentration in plasma. For CYP3A4 inhibitors that are orally dosed, [I] should also be estimated by [I] = Igut = molar dose/250mL and if the alternate R is greater than 11 then further in vivo investigations may be warranted. Mechanistic and dynamic models may also be used to help make the decision on whether to proceed to in vivo studies.
Within the draft EMA guidelines (2010)8, a multiple dose in vivo study is recommended if greater than or equal to 30% inhibition is observed. The suggested approach is to calculate the fold reduction in clearance using the following equation;
Where kdeg is the degradation constant of the enzyme, kinact is the maximum inactivation constant and [I] is the concentration of inhibitor.
How can you further analyse the type of mechanism involved in the time dependent inhibition?
Mechanistic studies can be used to derive whether the inhibition observed is mechanism (metabolism)-based or time-dependent, i.e. forms a metabolite that covalently binds to amino acids or quasi-irreversibly coordinates to the heme group. Mechanism dependent inhibition can be confirmed as irreversible inhibition by undergoing dialysis or ultra-filtration where activity will not be restored if true mechanism dependent inhibition is observed. Formation of metabolite-inhibitor complexes can be measured by an increase in absorbance at 455nm compared to standard absorbance at 450nm of the ferrous-carbon monoxide complex. Further experiments using potassium ferricyanide should reverse the formation of the metabolite-inhibitor complex.2
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