title: Dynamic modeling of cytochrome P450 inhibition in vitro: impact of inhibitor depletion on IC₅₀ shift.
abstract: The impact of inhibitor depletion on the determination of shifted IC₅₀ (IC₅₀ determined after 30 minutes of preincubation with inhibitor) is examined. In addition, IC₅₀-shift data are analyzed using a mechanistic model that incorporates the processes of inhibitor depletion, as well as reversible and time-dependent inhibition. Anomalies such as a smaller-than-expected shift in IC₅₀ and even increases in IC₅₀ with preincubation were explained by the depletion of inhibitor during the preincubation. The IC₅₀-shift assay remains a viable approach to characterizing a wide range of reversible and time-dependent inhibitors. However, as with more traditional time-dependent inactivation methods, it is recommended that IC₅₀-shift experimental data be interpreted with some knowledge of the magnitude of inhibitor depletion. For the most realistic classification of time-dependent inhibitors using IC₅₀-shift methods, shifted IC₅₀ should be calculated using observed inhibitor concentrations at the end of the incubation rather than nominal inhibitor concentrations. Finally, a mechanistic model that includes key processes, such as competitive inhibition, enzyme inactivation, and inhibitor depletion, can be used to describe accurately the observed IC₅₀ and shifted IC₅₀ curves. For compounds showing an IC₅₀ fold shift >1.5 based on the observed inhibitor concentrations, reanalyzing the IC₅₀-shift data using the mechanistic model appeared to allow for reasonable estimation of Ki, KI, and kinact directly from the IC₅₀ shift experiments.
authors: Pharmacokinetics and Drug Metabolism, Amgen, Inc., 360 Binney St., Cambridge, MA 02142, USA. loren.berry@amgen.com
authors: Loren M
authors: LM
authors: Berry
authors: Zhiyang
authors: Z
authors: Zhao
authors: Min-Hwa Jasmine
authors: MH
authors: Lin
mesh_terms: D051544:Cytochrome P-450 CYP3A; D065692:Cytochrome P-450 CYP3A Inhibitors; D065607:Cytochrome P-450 Enzyme Inhibitors; D006801:Humans; D020128:Inhibitory Concentration 50; D008954:Models, Biological; D009529:Nicardipine / Q000494:pharmacology; D019258:Saquinavir / Q000494:pharmacology; D013997:Time Factors
chemical_list: D065692:Cytochrome P-450 CYP3A Inhibitors; D065607:Cytochrome P-450 Enzyme Inhibitors; D009529:Nicardipine; D051544:Cytochrome P-450 CYP3A; C510163:CYP3A4 protein, human; D019258:Saquinavir