CYP TDI/MBI – kinact/KI

When a drug candidate exhibits TDI and/or MBI, it is important to determine the kinetic parameters of inhibition constant, KI, and inactivation rate constant, kinact. KI and kinact, with the knowledge of the degradation rate of CYP proteins, are important in predicting the in vivo DDI effects.

Assay Methodology

To determine KI and kinact, the inhibitor is preincubated with HLM in the presence of NADPH for various time intervals at multiple concentrations. Upon adding the substrate, the remaining enzymatic activity is monitored by LC-MS/MS, and plotted as a function of preincubation time. The observed inactivation rate (kobs), determined from the slope of the plot, is analyzed over a range of inhibitor concentrations. Values of KI and kinact can be determined by nonlinear fit to the classic Michaelis-Menten enzyme kinetics.

Time-Dependent Inhibition – kinact/Ki: Standard Assay Conditions (Customizable)

CYP Isoforms
CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6 and CYP3A4 (other isoforms are available)

Pooled human liver microsomes

Testing Compound Concentration
0, 0.25xIC50, 0.5xIC50, 0.75xIC50, 1xIC50, 2.5xIC50, 5xIC50, 10xIC50

0, 5, 10, 20, 30, 60 min with 1 mM NADPH

Known time-dependent inhibitors

Detection Method
Specific metabolites by LC-MS/MS

KI, kinact, detailed Report

Turnaround Time
2-3 days

Compound requirements
2 mg solid