| Objective:This study was conducted to investigate the pharmacodynamic and pharmacokinetic characteristics at 6 dosing times after oral administrations of sunitinib and explore its possible mechanism via the expression of circadian genes and metabolic enzymes in ACHN xenograft-bearing nude mice.Methods:1.The ACHN xenograft-bearing nude mice were acclimated under strict 12 h light/12 h dark cycles for at least 2 weeks before the begain of the study.The mice model was established by inoculating by 0.1m L of 3.0 × 106 viable ACHN cells on the flank of the Balb/c nude mice.2.In pharmacological study,all ACHN xenograft-bearing nude mice were randomly grouped into six circadian groups(4:00,8:00,12:00,16:00,20:00,24:00).Each circadian group was randomly divided into sunitinib administration group and model group.3.Sunitinib administration group was gavaged single dose of sunitinib-malate(16.25mg·kg-1,0.2m L)at the corresponding circadian time(4:00,8:00,12:00,16:00,20:00,24:00),at the same time,model group was gavaged the same volume fraction of citrate buffer.Gavage administration lasted for 21 consecutive days.Tumor size was measured with caliper every 3 days and the tumor growth curves were drawn.on day 21 after gavage administration,all of mice were sacrificed for stripping the solid tumors and weighting at the corresponding circadian time.Antitumor rate was calculated.Solid tumors were collected for making pathological sections.4.In pharmacological study,all ACHN xenograft-bearing nude mice were randomly grouped into 6 circadian groups(4:00,8:00,12:00,16:00,20:00,24:00).Each circadian group was randomly divided into sunitinib administration group and model group.The sunitinib administration group was randomly divided into 10 blood groups.An additional group of tumour non-bearing nude mice served as negative control.5.Sunitinib-malate was given to sunitinib administration group at a dose of16.25mg·kg-1(0.2m L)at the corresponding circadian time(4:00,8:00,12:00,16:00,20:00,24:00),blood samples of 0.5m L were obtained from the ACHN xenograft-bearing nude mice by eye ectraction before dose(0h)and 15 min,30min,1h,3h,5h,7h,9h,14 h,19h,24 h after gavage administration,the plasma was stored at-80 ? after centrifugation.The livers were taken from 6 circadian groups and frozen under-80?conditions.High-performance liquid chromatography coupled with tandem mass spectrometry(HPLC/MS/MS)was applied to plasma samples to dected the plasma concentions of sunitinib and of SU12662,the pharmacokinetic parameters were calculated by Win Nonlin 6.3.6.The relative expression of cytochrome P450 enzyme 3a11(cyp3a11),cyp3a13,brain and muscle Arnt-like protein-1(Bmal-1),circadian locomotor output cycles kaput(Clock),D site-binding protein(Dbp)were quantificated with Quantificational Real Time-PCR(q RT-PCR).Results:1.Chronopharmacokinetics of sunitinib in ACHN xenograft-bearing nude mice had significant circadian rhythms.Compared with model groups,tumor growth speed and tumor weight of sunitinib administration groups significantly decreased.Tumor growth increased most rapidly and the tumor inhibition rate was the lowest at 16:00 sunitinib administration group while the tumor growth were significantly suppressed at8:00,12:00 sunitinib administration group and the tumor inhibition rate was high(P<0.05).The tumor inhibition at 8:00 sunitinib administration group was the hightest.The results of pathological sections showed that the tumor cells in the 16:00 sunitinib administration group were more closely aligned and the border was clear,the tumor necrosis and inflammatory infiltration were mild compared with 8:00 and 12:00groups,in which the tumor cells were necrotic,inflammatory infiltration and severe rupture of bleeding were serious.2.Chronopharmacokinetics of sunitinib in ACHN xenograft-bearing nude mice had significant circadian rhythms.The AUC0-24 h and MRT0-24 h of 16:00,20:00 sunitinib administration groups were lower and the CL/F was higher.8:00,12:00 sunitinib administration groups had higher AUC0-24 h,MRT0-24 h and lower CL/F compared with16:00 sunitinib administration groups(P<0.05),which was consistent with the results of pharmacological studies.3.The relative expression of cyp3a11,cyp3a13,Bmal-1,Clock,Dbp had significant circadian rhythms.The expression of cyp3a11 and cyp3a11 in the model group were rising from 12:00 and peaked at 16:00.The expression of cyp3a13 and Clock was increasing from 8:00 and in the range of 20:00 to 24:00 reached the fastest growth.DBP was highly expressed at 16:00 and 24:00.The total expression of Bmal-1 and cyp3a11 was the highest in the 16:00 sunitinib administration group.The highest total expression of Clock,Dbp and cyp3a13 were found in 24:00 sunitinib administration group,The total expression of Clock and cyp3a13 were higher in 20:00 sunitinib administration group too.The expression trend of the circadian genes were matched with the expression trend of metabolic enzyme genes.Conclusion:Circadian rhythmicity chronopharmacology characteristics were observed in ACHN xenograft-bearing nude mice.Tumor growth was more suppressed and the tumor inhibitory rate was higher in the early of light phase,at the same time,the concentration of sunitinib and of SU12662 were higher compared with other circadian time.The mechanism is likely ralated to the expression of metabolic enzymes were regulated by circadian genes. |
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