2.6 Tissue distribution of ningetinib and M1 in mice.
A total of 18 healthy male ICR mice were randomly divided into six
groups, which were fasted for 12 h with free access to water and
received a single oral administration of ningetinib at 10 mg
kg-1 (suspended in 0.5% CMC-Na solution; the
ningetinib dose used here in mice was basically equivalent to the
clinical dosage in NSCLC patients when corrected for interspecies
differences according to the Meeh-Rubner formula.). Each group of three
mice was sacrificed via exsanguination from the abdominal aorta under
anaesthesia at 0, 2, 6, 12, 24 or 48 h after administration. The blood,
brain, heart, liver, spleen, lung, kidney, bladder, pancreas, skeletal
muscle (hind limb), testis, stomach wall, small intestine, large
intestine, abdominal fat, adrenal gland and thymus were rapidly
collected. The tissues were washed by saline to rinse out contents or
blood, weighed and homogenised in five volumes of acetonitrile-water
(1:1, v/v) after wiping with filter paper. Plasma was obtained by
centrifugation of blood at 11,000 g for 10 min. All samples were stored
at -20 °C until LC-MS/MS analysis.
2.7 Transport
stud ies.
The MDCKII-MDR1/BCRP/MRP2 cells were cultured in DMEM supplemented with
10% FBS, 2 mM L-glutamine, 100 U penicillin G, 100 μg
mL-1 streptomycin, and 1% minimum essential medium
nonessential amino acids at 37 °C in a humidified 5%
CO2 atmosphere. The cells were seeded at a density of 2
× 105 cells/cm2 on a polycarbonate
membrane filter membrane on Transwell inserts (Millipore, Billerica,
MA). The medium was routinely replaced with a fresh one every other day.
The MDCKII-MDR1/BCRP/MRP2 cell lines were grown for 4 days before the
bidirectional transport study.
For substrate assessment, the apical and basolateral chambers were
washed twice with prewarmed HBSS (pH 7.4), and then, the cells were
equilibrated for 30 min in the presence or absence of positive
inhibitors GF120918 (10 μM, P-gp inhibitor), KO143 (10 μM, BCRP
inhibitor) or TAK875 [100 μM, MRP2 inhibitor (Li, Zhong, Guo, Zhong,
& Chen, 2015)]. Equilibration HBSS was removed at the end of the
equilibrium. HBSS containing ningetinib (10 μM) or M1 (10 μM) was added
to the donor side (either the apical or basolateral chamber), and the
positive inhibitors were added to both chambers. The cells were
incubated for 90 min at 37 °C, at which time aliquots (150 μL) were
collected from the receiver chambers for analysis. The samples were
stored at -20 °C for LC-MS/MS analysis. Digoxin (P-gp positive
substrate), imatinib (BCRP positive substrate) and vincristine (MRP2
positive substrate) were used for validation of the above efflux system.
Given that the metabolite M1 was eliminated more slowly than the parent
drug in the plasma, the effects of ningetinib and gefitinib on the
efflux of M1 mediated by P-gp, BCRP and MRP2 were further evaluated.
Experimental procedures were the same as above, whereas the positive
inhibitors were replaced by ningetinib (0–100 μM) or gefitinib (0–100
μM). All samples were stored at -20 °C before LC-MS/MS analysis.