| OBJECTIVE:The method of high performance liquid chromatography?HPLC?detecting ibrutinib and its metabolite PCI-45227 has been established,which to observe the effects of sodium cantharidate on the metabolism of ibrutinib and its metabolite PCI-45227 in rats.METHODS:?1?Agilent ZORBAX SB-C18 chromatographic column?4.6 mm×250 mm,5?m?was adopted.The chromatographic condition is water-acetonitrile-0.2%TFA system as mobile phase,flow rate of 1.0 mL·min-1,column temperature of35°C.The plasma was extracted by ethyl acetate and carbamazepine as internal standard.The probe drug was detected with 234 nm.Ibrutinib and its metabolite were detected at 258nm.?2?The 36 male SD rats were randomly divided into 4 groups?group A,group B,group C,group D?.Group A?probe drug control group?injecting physiological saline of 0.5 mL/kg dose by intraperitoneal.Group B?probe drug experimental group?injecting 0.5 mL/kg dose sodium cantharidate by intraperitoneal for 10 days.On the 11th day,group A and group B gavage administrating 5 mL/kg dose probe drugs at the same time.Group C?ibrutinib control group?injecting physiological saline of 0.5 mL/kg dose by intraperitoneal.Group D?ibrutinib experimental group?injecting 10mg/kg dose sodium cantharidate by intraperitoneal for 10 days.On the 11th day,group C and group D gavage administrating 5 mL/kg dose ibrutinib at the same time.The blood of group A and group B was collected at the different time after the probe drug was given.The blood of group C and group D was took to EP at different time after giving ibrutinib.The detecting method was used to determine the concentration of the probe drug,ibrutinib and metabolites in plasma.The main pharmacokinetic parameters were calculated by the DAS program.?3?Liver microsomal incubation system in vitro was established to detecting the concentration of probe drugs and ibrutinib for calculating Km,Vmaxax and IC500 to observe the activities of sodium cantharidate to CYP2D6,CYP3A4,and ibrutinib.Metoprolol and midazolam as the substrate,sodium cantharidate as the intervention medicine water-acetonitrile-0.2%TFA system as the mobile phase,carbamazepine as the internal standard.RESULTS:?1?The linear relationship of probe drug,ibrutinib and PCI-45227were good in the range of 102000 ng/mL.The standard curve of metoprolol was established as y=0.1247x+0.0040?r=0.999 4?,midazolam's standard curve was y=0.1122x+0.0149?r=0.999 3?,ibrutinib's standard curve was y=0.0597x+0.0039?r=0.999 7?,PCI-45227's standard curve was y=0.1493x+0.0028?r=0.999 5?.The RSD below 15%of intra and inter day precision for low,middle and high concentration.The stability of plasma samples were good at the conditions of room temperature for4h,4°C for 24h,the circulating freeze-thaw three times and freezing for 30 days.?2?The Cmaxax of metoprolol in group A and group B was?613.55±92.37?and?998.46±120.14?ng/mL,AUC?0-??was?4881.10±220.79?and?7745.15±1383.88?ng·h/mL.The Cmaxax of midazolam was?1111.98±86.88?and?1298.05+64.91?ng/m L,AUC?0-??was?6624.46±1680.99?and?8970.28±3219.13?ng·h/mL.The results showed that both Cmaxax and AUC?0-??of the probe drug were increased after injecting sodium cantharidate.Sodium cantharidate increased the exposure of the probe drug.It is suggested that sodium cantharidate inhibits the metabolism of probe drug.The Cmaxax of ibrutinib of group C and group D were?1019.43±74.88?and?1333.71±65.18?ng/mL,AUC?0-??was?10956.72±2491.09?and?13447.88±1038.442?ng·h/mL.The Cmaxax of PCI-45227 was?230.59±15.36?and?167.28±38.99?ng/mL,AUC?0-??was?3203.80±345.78?and?2443.95±680.71?ng·h/m L.The results showed that the Cmaxax and AUC?0-??of ibrutinib were increased after giving sodium cantharidate.The Cmaxax and AUC?0-??of PCI-45227 were reduced after giving sodium cantharidate.It provided that sodium cantharidate increased the exposure of ibrutinib and reduced the concentration of PCI-45227.It suggested that sodium cantharidate inhibited the metabolism of ibrutinib in rats.?3?The linear relationship of probe drug and ibrutinib in 102000ng/mL range were good in vitro liver microsomal incubation.The standard curve of metoprolol was y=0.0729x+0.0306?r=0.999 3?.Vmax=1.21nmol/min/mg pro,Km=35.74?mol·L-1,IC50=1.7477?g·ml-1.The standard curve of midazolam was y=0.0825x+0.0331?r=0.999 1?,Vmax=0.62nmol/min/mg pro,Km=20.04?mol·L-1,IC50=1.7789?g·ml-1.The standard curve of ibrutinib's was y=0.0842x+0.0212?r=0.9991?,Vmax=2.53nmol/min/mg pro,Km=36.46 mol·l-1,IC50=1.90157?g·ml-1.CONCLUSIONS:The study established the method of detecting metoprolol and midazolam at the same time in rats plasma,another method of detecting ibrutinib and PCI-45227 both are complete separation,high specificity,short processing time.The method is suitable for the research of pharmacokinetics and drug interaction.Sodium cantharidate inhibits the metabolism of probe drug and ibrutinib in vivo and increases the exposure of metoprolol,midazolam and ibrutinib.The liver microsomal incubating system was established in vitro,which was accurate,reliable and simple.The method suitable for the enzyme activity research of CYP2D6 and CYP3A4 in rat vitro.Sodium cantharidate inhibits the effect of CYP2D6 and CYP3A4 enzyme in rats. |
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