Kinetic study
Given
the obvious metabolic activity displayed by
CYP2D6, CYP2J2, CYP3A4 and CYP4F3
for rivaroxaban, kinetic studies were conducted to estimate the
catalytic capability of HLM (Figure 4A) and these four recombinant CYPs
(Figure 4B) in rivaroxaban hydroxylation. All of these reactions obeyed
Michaelis-Menten kinetics, as depicted by their respective linear
Eadie-Hofstee plots. As shown in Figure 4B, there was a wide range in M1
formation rates with the four recombinant CYPs, from 29.42 to 753.9
pmol·min−1·mg protein−1. TheV max value of CYP2J2 was 753.9
pmol·min−1·mg protein−1, which was
12.6−25.6-fold higher than that of the other CYPs (Table 1).
Additionally, the apparent K m value of CYP2J2 was
the lowest among these four recombinant CYPs, which further gave rise to
the highest intrinsic clearance value of CYP2J2
(V max/K m = 38.92
μL·min−1·mg protein−1). The
intrinsic clearance values indicated that CYP2J2 dominated in the
metabolism of rivaroxaban, being approximately 39- to 100-fold higher
for CYP2J2 than for the other CYPs. Detailed kinetic parameters are
shown in Table 1.
CYP-specific
inhibitory study
To further estimate the contributions of the P450 isoforms in
rivaroxaban hydroxylation, six specific inhibitors were used. As shown
in Figure 5, significant inhibition was observed in the groups with
CYP3A inhibitor ketoconazole and CYP2J2 inhibitor danazol, reducing
enzyme activity to 56.6% and 58.9% of control activity, respectively.
In addition, CYP2C9-specific inhibitor sulfaphenazole slightly inhibited
the formation of M1, remaining about 74.6% of control. However,
inhibition by other inhibitors including quinidine, montelukast and
8-methoxypsoralen was not significant, with the resulting activity
remaining at > 90% that of the control.
Molecular
docking simulation
Molecular docking simulation was used to elucidate the binding
conformations of the interaction between rivaroxaban and CYP2J2. The
distance between the O atom of rivaroxaban and the haem iron atom of
CYP22 was 3.9 Å (Figure 6A). The optimal orientation of rivaroxaban in
CYP2J2 and the corresponding 2D ligand interaction diagram are shown
in Figure 6. As shown in Figure 6B,
the benzene and thiophene rings of rivaroxaban interacted with ALA311
and PHE310, respectively, via π-alkyl bonds. In addition, alkyl
interactions between the methyl group of rivaroxaban and ILE487, VAL380
and ILE376 were apparent. The TotalScore, ChemScore and CScore for
rivaroxaban docking to CYP2J2 were 5.35, −27.398 and 4, respectively.
Taken together, these results showed a marked binding affinity of
rivaroxaban for CYP2J2.