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.