2.9 Instruments and sample preparation.
Quantitation analysis of ningetinib and M1 was conducted using an API 5500 triple quadrupole mass spectrometer coupled with an LC-30AD high-performance liquid chromatography system (Shimadzu, Kyoto, Japan). Data acquisition and processing was conducted using Analyst 1.6.3 software (AB Sciex, MA, US). Chromatographic separation was achieved on YMC-Triart C18 (50 mm × 2.0 mm i.d., 5 μm; YMC Karasuma-Gojo Bldg, Japan) at 40 °C. The mobile phase was a mixture of 5 mM ammonium acetate (A) and acetonitrile (B) at a flow of 0.6 mL min-1. The gradient conditions were as follows: 20% B for 0.5 min; a stepwise linear increase to 80% B at 1.5 min; 1.5–2 min, 80% B; a stepwise linear decrease to 20% B at 2.5 min; 2.5–3 min, 20% B. Multiple reaction monitoring (m/z 557.3 → 215.0 for ningetinib, m/z 563.4 → 215.3 for D6-ningetinib, m/z 543.1 → 271.1 for M1 and m/z 549.4 → 271.5 for D6-M1) was used in the positive electrospray ionisation mode with an ion spray voltage of 4500 V and a source temperature of 400 °C. The nebuliser gas, heater gas and curtain gas were set to 50, 50 and 20 psi, respectively. All the in vitro and in vivo samples were prepared by protein precipitation with acetonitrile.
Data analysis.
Experimental procedures and statistical analysis were double-blind designed. The data and statistical analyses comply with the recommendations on experimental design and analysis in pharmacology (Curtis et al., 2018).
In the pharmacokinetic study, WinNonlin software (version 6.1; Pharsight Corp., Cary, NC) was used to calculate the pharmacokinetic parameters in a noncompartmental model.
For the inhibition kinetics studies, Vmax and Km values were determined by the nonlinear regression curve fit using the Michaelis–Menten equation. Intrinsic clearance (CLint) was calculated as CLint = Vmax/Km. The apparent kinetic parameters for inhibitory activity (Ki) were first estimated by graphical methods such as Dixon (1953) and Cornish-Bowden (1974) methods, and were more accurately determined by nonlinear least square regression analysis on the basis of the best enzyme inhibition model using the Graphpad Prism (version 8.01; GraphPad Software Inc., La Jolla, CA). On the basis of Dixon (1953) and Cornish-Bowden (1974) methods, the linear regression lines obtained in our experiments were all intersected at one point. Thus, the inhibition data were well fitted by the mixed-type inhibition v = (Vmax[S])/ (Km(1 + [I]/Ki) + [S](1+[I]/ α Ki). The models tested included pure and partial competitive, noncompetitive, uncompetitive and mixed-type inhibitions.
In the transport experiments, the apparent permeability coefficients (Papp) and efflux ratio (ER) was calculated using the following formula:
Papp = CT × V/ (C0 × T × S)
ER = Papp, B to A / Papp, A to B
where CT = the concentration of the test compound on the receiver side, V = the loading volume on the receiver side, S = the surface area of the cell monolayer (0.33 cm2 in a 24-well plate), C0 = the initial concentration of the test compound on the donor side and T = incubation time. Papp, A to B and Papp, B to A represent the extent of permeation generated by the transport from the apical to basolateral sides and from the basolateral to apical sides, respectively.
In the transport inhibition study, the IC50 values were calculated by plotting the log value of the inhibitor concentration against the normalised response as follows: Y = 100/[1 + 10(X-Log(IC50)].
Statistical comparisons between two groups were evaluated using the unpaired student’s t-test in GraphPad Prism. P < 0.05 was considered significant.
Results
3.1Pharmacokinetic interaction of ningetinib with gefitinib in patients with NSCLC.
As shown in Fig 2 and Table 1, when ningetinib was given alone, the peak concentrations (Cmax) of ningetinib and M1 in plasma were comparable, and AUC0-24h value of M1 was approximately 1.7-fold that of ningetinib. Moreover, the time to reach the peak concentration (Tmax) of M1 was significantly longer than that of ningetinib, and the elimination rate of M1 was much slower than that of ningetinib. After co-administration with gefitinib, the Cmax and AUC0-24h of ningetinib were almost unchanged, whereas those values for M1 significantly dropped by more than 80% on the first and 28th day. The elimination behaviours of both ningetinib and M1 were not obviously changed. These data suggested a DDI between ningetinib and gefitinib, mostly likely through metabolic mechanism.