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.