| Backgroud and objective:It had been reported that the clinical incidence of combination of statins and triazole antifungal drugs was 1.5%.Most of these drugs are metabolized by liver metabolizing enzymes,so the drug-drug interaction may occur.Caution in combination is suggested in the instructions for both types of drugs,and it is recommended to adjust the dose of statins when used in combination.Hoewever,there 1s no basis for how to adjust the dose.The reason 1s that the mechanism of the interaction between the two types of drugs is not clear,and the reliable basis for dose adjustment can not be obtained.This reasearch will proceed from the point of view of bioavailability barrier which composed of liver drug metabolizing enzymes and efflux transporters,based on the interaction of voriconazole/fluconazole and atorvastatin,to discuss the mechanism of interaction between two kinds of drugs.Methods1.To develop a HPLC-MS/MS method for simultaneous determination of atorvastatin and voriconazole in rat plasma.Plasma samples were treated with sodium acetate acidification and methyl tert-butyl ether was used as the the extractant to extracte the analytes through liquid-liquid extraction method,then separated on a Thermo Hypersil Gold C18(2.1×100 mm5 1.9 μm)column within 6 min with gradient elution using acetonitrile and water(containing 0.1%formic acid)as the mobile phase;the mass spectrometry detection was achieved in selective reaction monitoring(SRM)mode under the positive ion scanning mode of heated electrospray ion source(H-ESI)and using transition mass of m/z559.2→440.2 for atorvastatin and m/z350→280 for vonconazole and m/z370.2→252 for internal standard as quantitative ion.2.The experimental rats were divided into 5 groups:①Normal saline was given intragastrically for 6 days,and on day 6,after 1 hour of saline infusion,atorvastatin was administered to the stomach;② Voriconazole was given intragastrically for 6 days,and on the day 6,after voriconazole was given for 1 hour,atorvastatin was given to the stomach;③Fluconazole was given intragastrically for 6 days and on the day 6,after fluconazole was given for 1 hour,atorvastatin was given to the stomach.Fasting for 12 hours before the last administration,drinking water freely,and collecting blood for 24 hours continuously after administration,to determine the concentration of atorvastatin in plasma based on HPLC-MS/MS,the pharmacokinetic parameters were calculated with the DAS2.0 pharmacokinetic software and the statistical analysis was performed;④Single gavage of voriconazole;⑤Single gavage of voriconazole and atorvastatin.Fasting for 12 hours before administration,drinking water freely,and collecting blood continuously for 24 hours after administration,to determine the concentration of voriconazole in plasma based on HPLC-MS/MS,the pharmacokinetic parameters were calculated with the DAS2.0 pharmacokinetic software and the statistical analysis was performed.3.The mixed rat liver microsomes were prepared by differential centrifugation method,and the vitro incubation system was established.The concentration of 6β-hydroxytestosterone,a metabolite of CYP3A4 substrate,testosterone,was measured by HPLC to explore the effects of voriconazole and fluconazole on the activity of hepatic drug metabolizing enzyme CYP3A4.The effects of voriconazole and fluconazole on the metabolism of atorvastatin were studied by detecting the concentration of atorvastatin in rat mierosomes by HPLC.4.The caco-2 cell model in vitro was constructed by measuring the transmembrane resistance value(TEER)and alkaline phosphatase activity of the cells.The effects of voriconazole and fluconazole on the transmembrane transport of atorvastatin were investigated in caco-2 cell model based on HPLC-MS/MS,measuring the concentration of atorvastatin in the intracellular and terminal soIlution of the receiving cell after transport.The effects of voriconazole and fluconazole on the activity of extrorporated transporter were measured by using fluorescence probe substrate combined with multifunctional enzymatic labeling instrument.Results1.The calibration curves were linear with the concentration of atorvastati n andvoriconazole ranged from 0.01-100 ng/mL(r=0.9957)and 0.025-100 ng/m L(r=00.9981),respectively.The intra-day and inter-day precision were all less than 13%,the recovery was between 66.50%and 82.67%,the plasma samples were stable under experimental conditions.2.Compared with the atorvastatin control group,the AUC0-24h of atorvastatin was increased by 109.9%(P<0.05)and 40.17%(P<0.05),CLz decreased by 49.85%(P<0.05)and 25.14%(P<0.05),respectively,after administration of voriconazole or fluconazole for 6 days.Time of maximum concentration was all delay.There was no significant difference in other pharmacokinetic parameters(P>0.05);Compared with the voriconazole control group,there was no significant difference in the pharmacokinetic parameters of voriconazole after a single oral administration of atorvastatin(P>0.05).3.Compared with the control group,the production of 6β-hydroxytestosterone in the voriconazole and fluconazole groups was significantly decreased,and the IC50 value of inhibition of testosterone metabolism was 49.96 and 231.6 μmol/L,respectively.Compared with the control group,the residual amount of atorvastatin in voriconazole and fluconazole groups was significantly increased,and its IC50 value was 45.94 and 106.9 μmol/L,respectively.4.Compared with the control group,voriconazole and:fluconazole had no significant effects on the uptake and transport of atorvastatin in caco-2 cells(p>0.05).In caco-2 cells,different concentrations of voriconazole and fluconazole did not significantly change the average fluorescence intensity of intracellular Rh123,CDFDA,Hoechst33342(p>0.05),which indicated that voriconazole and fluconazole have no significant effect on the efflux activity of P-gp,MRP-2 and BCRP in caco-2 cells(p<0.05).ConclusionsThe results showed that voriconazole and fluconazole could significantly increase the exposure and bioavailability of atorvastatin in rats,and the effect of voriconazole was stronger than fluconazole.Voriconazole and fluconazole may decrease the metabolism of atorvastatin in the liver and increase the bioavailability by inhibiting the activity of hepatic drug metabolizing enzyme CYP3A4.Atorvastatin had little effect on the pharmacokinetics of voriconazole in rats. |
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