Criteria for Determining MBI

1. Inhibition of DME is time dependent.

2. Inhibition of DME is cofactor dependent (e.g., NADPH dependent in the case of P450 enzymes).

3. The presence of another substrate slows down (or removes) inactivation by competing with the MBI.

4. Inactivation is irreversible and dialysis does not retain its activity.

5. Inactivation takes places with no lag time.

6. Rate of inactivation is first order and saturable.

7. Trapping agents and reactive oxygen species (ROS) scavengers (catalase and superoxide dismutase) do not save the enzyme from inactivation. This means that MBIs do not form reactive metabolites or ROS that escape the enzyme's active site and inactivate the enzyme.

The partition ratio (r) is the ratio of kcat to kinact. The smaller this number the more effective the enzyme is inactivated by the MBI.

The inactivation process results in depletion of enzyme activity over time, which can be expressed by:

Et = enzyme concentration at time t E0 = initial enzyme concentration

5.4.2.1 Practical Tips

TDI studies are typically performed for the major CYP isoforms, or at least for CYP3A4. These assays are performed in a two-incubation process. The first incubation includes different inhibitor concentrations with enzymes in the presence or absence of cofactors. At different time points, aliquots are transferred to a new incubation that includes the probe substrate for the enzyme and, if necessary, cofactors (but no enzymes). This step is usually, but not always, a 10-20-fold dilution in order to minimize the competitive inhibition properties of the inhibitor.

One important factor to consider is the solubility of the inhibitors.

In the first step of the incubation, it is necessary to include these controls:

1. Known positive and negative controls for the enzymes

2. No inhibitor plus NADPH

3. Inhibitor but no NADPH

A nonlinear regression analysis is used to determine kinact and KI. The quality of the correlation can be checked graphically by plotting the log of the percent remaining activity (y-axis) versus incubation time (x-axis; starting from the first incubation) for each [I] (see Fig. 5.2a). The observed rate constant for inactivation (kobs) is the slope of the line for each [I], which should be first order with respect to time. Then, a plot of 1/kobs versus 1/[1] gives an x-intercept of 1/KI and ay-intercept of 1/kinact (see Fig. 5.2b).

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