Enzyme inhibition assays
The concentration of HLM, CYP2J2 and CYP3A4 was 0.3, 0.4 and 0.6
mg·mL–1. The selection of the rivaroxaban
concentration depended on the Km values of the kinetic
studies (22.81, 19.37 and 46.98 μM for HLM, CYP2J2 and CYP3A4,
respectively) (Zhao et al., 2021). The concentration of CYP3A4 (0.6
mg·mL–1) was selected to correspond with the lowest
detected concentration of M1. The detailed method can be found in our
previous publication (Zhao et al., 2021).
Reversible inhibition of
CYP3A4 and CYP2J2 by TIKs
The concentration range of rivaroxaban in incubation of CYP2J2 and
CYP3A4 was 0–100 μM and 0–400 μM respectively. The
inhibition
constant (Ki ) was determined using various
concentrations of inhibitors and rivaroxaban. Kiwas calculated by three inhibition mode formulae (competitive,
non-competitive and mixed-mode) using Prism v.6.0 (GraphPad, San Diego,
CA, USA). Detailed information on the fitting formulae and related
parameters can be found in our previous publication (Li, Cao, He, Ge,
Guo & Wu, 2018).
IC50 shift
assay
The 30 min pre-incubation of TKIs with NADPH and CYP2J2 or CYP3A4
preceded the normal incubation, following which the IC50values (IC50 shift) were re-determined. These
IC50 shift values were compared with the
IC50 values that were determined without the 30 min
pre-incubation, with a more than 1.5-fold decrease considered to be
evidence of time-dependent inactivation. Other reaction conditions were
as mentioned above.