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).
(1)
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.
(2)
Here, A is the time-dependent inactivation of each P450 isoform that was observed in the liver, as described by Eq. 3.
(3)(4)
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).