How does the study design to determine the Ki and the kinetic parameters of inactivation, kinact and KI, differ from that used to determine IC50?
For determination of Ki, multiple concentrations of the test article between 0.1x and 2x the estimated IC50 value are incubated with up to six concentrations of isoform-specific probe substrate. For determination of the kinact and KI values, concentrations of test article equivalent to those used to calculate IC50 are incubated with human liver microsomes (10x excess) for up to five non-zero time points prior to dilution into secondary incubations containing isoform-specific probe substrate.
