Figure. 1 Molecular structure of (A) imatinib, (B) sunitinib
and (C) gefitinib.
Materials and methods
Materials and chemicals
Rivaroxaban, sunitinib and NADPH were purchased from Shanghai Yuanye
Bio-Technology Co., Ltd. Imatinib and gefitinib were obtained from
Sigma-Aldrich (Missouri, USA). cDNA-expressed recombinant human CYP3A4
was purchased from Cypex Ltd (Dundee, UK). Pooled human liver microsomes
(HLM) and recombinant human CYP2J2 were purchased from
BioreclamationIVT
(Maryland, USA). The pooled HLM and recombined P450 isoforms were stored
at –80 ℃ before experimentation. PBS was prepared from dipotassium
bisulphate and potassium dihydrogen sulphate in the appropriate
proportions and stored at 4 ℃ until use. All analytical reagent grade
and HPLC grade solvents were from Tedia, Inc. (Ohio, USA).
High-performance liquid chromatography (HPLC) was performed using an
Agilent MSD/MS system controller, two 1260 series pumps, a 1200 series
autosampler and a 1200 series variable wavelength detector. Other
instrumentation included a constant temperature vibrating mixer
(Hangzhou ALLSHENG Instrument Co. Ltd, China), scroll machine,
centrifuge and elite ODS-BP analysis column (4.6 × 200 mm, 5 μm).
Analytical methods
The inhibitory effect of the TKIs on the metabolism of rivaroxaban in
recombined P450 isoforms and pooled human liver microsomes were compared
by quantifiably detecting the production of the major metabolite using
HPLC. The mobile phase consisted of 60% methanol (A pump) and 40% pure
water with 0.2% formic acid (B pump) with isocratic elution. Detection
conditions were as follows: column temperature, 30 °C; autosampler
injection volume, 20 μL; flow rate, 0.5 mL·min–1;
and, detector wavelength, 240 nm. The amount of major rivaroxaban
metabolite (M1) was determined from the rivaroxaban standard curve, for
which the correlation coefficient was > 0.999.
Enzyme inhibition assays
The total volume of the incubation system with HLM was 200 μL, which
contained rivaroxaban (200 μM), HLM (0.3 mg·mL–1),
TKIs, NADPH (10 mM final) and PBS. The total volume of the incubation
system with recombined P450 isoforms was 100 μL, which contained
rivaroxaban (400 μM), recombined enzymes (CYP2J2 0.4
mg·mL–1, CYP3A4 0.6 mg·ml–1),
TKIs, NADPH (10 mM final) and PBS. 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), as reported in our previous study (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). Briefly, rivaroxaban, TKIs, enzymes and PBS were pre-incubated at
37 ℃ for 5 min, following which NADPH was added to initiate the
reaction. After 60 min, the reaction was terminated by adding an equal
volume of ice-cold methanol. The samples were centrifuged at 2000 x g
for 15 minutes at 4 ℃. Aliquots of the supernatants were stored at –20
°C until analysis by HPLC.
Reversible inhibition of
CYP3A4 and CYP2J2 by TIKs
Rivaroxaban was used as the probe substrate for the determination of
reversible inhibition kinetics values. The incubation system with CYP3A4
included rivaroxaban (0–400 μM), potential inhibitor (imatinib: 0–10
μM; gefitinib: 0–10 μM; sunitinib: 0–20 μM), NADPH and PBS. The
incubation system with CYP2J2 included rivaroxaban (0–100 μM),
potential inhibitor (imatinib, gefitinib and sunitinib), NADPH and PBS.
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).
IC50shift assay
The 30 min pre-incubation of TKIs with NADPH and CYP2J2 preceded the
normal incubation, following which the IC50 values
(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.
Time-dependent inactivation of CYP3A4
To investigate the time-dependent inactivation of CYP3A4 by sunitinib,
seven gradient concentrations (0–5 μM) and six time points (0–20 min)
were used. It is worth noting that a higher substrate concentration than
its Michaelis-Menten constant is required to reduce reversible
inhibition. The data were then fitted to a linear regression model,
which reflected the linear relation between ‘ln remaining activity’ and
‘inactivation concentration’ (I ). The negative slope of this
linear relationship reflected the observed inactivation rates
(Kobs) value, which could be plotted against I to
allow the fitting of inactivation kinetic parametersK I and K inact to the
nonlinear least-squares regression based on Eq. 1. Using Prism v.6.0
(GraphPad, San Diego, CA, USA).
Molecular
docking simulations
The CYP2J2 crystal structure homology model was used to conduct
molecular docking simulations between TKIs and rivaroxaban in SYBYL
(X-1.1). The CYP2J2 model was constructed using the Clustal Omega
webserver (https://www.ebi.ac.uk/Tools/msa/clustalo/), as previously
described (Ning et al., 2019). The crystal structure of CYP3A4 (PDB:
4D7D) was from the crystal structures that bound to a known inhibitor.
The 3D structures of the TKIs were subjected to energy minimisation
using the default Tripos force field parameters, and the
Gasteiger-Hückel charges were calculated for each compound. The
Surflex-Dock mode was used to generate binding conformations of TKIs
with CYP2J2, from which the optimal conformations were determined by
their empirical functions ChemScore. The PyMOL Molecular Graphics System
v.16.1.0.15350 (DeLano Scientific LLC) was used to visualise the docking
results.
Quantitative prediction of DDI risk
Kinetic constants were included in the mechanistic static model to
explore reversible inhibition and time-dependent inactivation. This
static model was previously developed and refined by Fahmi et al.
(Fahmi, Maurer, Kish, Cardenas, Boldt & Nettleton, 2008) and
Isoherranen et al. (Isoherranen, Lutz, Chung, Hachad, Levy &
Ragueneau-Majlessi, 2012) to account for the inhibition of multiple P450
isoforms. In the present study, this model was designed to explore the
contributions of enzyme inhibition in the prediction of DDI risk. The
area under the curve ratio (AUCR) in the presence of a pharmacokinetic
DDI was used as the index, as described by Eq. 2.
Here, A is the time-dependent inactivation of each P450 isoform that was
observed in the liver, as described by Eq. 3.
Here, B is the reversible inhibition of each P450 isoform that was
observed in the liver, as described by Eq. 4. The degradation rates
(Kdeg) of CYP2J2 and CYP3A4 were 0.00026 and 0.00032
min–1, respectively (Cheong et al., 2017), where I
represented the in vivo concentration of inhibitors in healthy and solid
tumour patients. Additionally, the fraction of rivaroxaban metabolised
by CYP2J2 or CYP3A4 was input from our previous study (Zhao et al.,
2021), which were 0.95 for CYP2J2 and 0.025 for CYP3A4.
Results
Initial screening of
TKI inhibitory activity
The inhibitory effects of the three TKIs, imatinib, sunitinib and
gefitinib, on rivaroxaban metabolism with HLM, CYP3A4 and CYP2J2 were
initially investigated at three TKI concentrations (1, 10, and 100 μM).
Generally, imatinib and gefitinib showed strong inhibition of the
metabolism of rivaroxaban, but sunitinib only exerted inhibitory
activity in the incubation with HLM and CYP3A4.
As shown in Figure 2A, imatinib
had the most potent inhibitory effect on rivaroxaban metabolism with
HLM, inhibiting approximately 50% of catalytic activity at 1 μM.
Imatinib also showed the strongest inhibitory effect on CYP3A4-mediated
rivaroxaban metabolism, with undetectable formation of M1 in the
incubation with 100 μM imatinib (Figure 2B). Imatinib and gefitinib
showed similar inhibition at 1 and 10 μM towards the metabolism mediated
by CYP2J2 (Figure 2C). While sunitinib had minimal inhibitory effect on
the metabolism of rivaroxaban by CYP2J2, the residual enzyme activity
was more than 70% at 100 μM (Figure 2C).