An In Vitro And In Vivo Study On The Synergistic Effect And Main Mechanism Of Itraconazole Or Voriconazole Alone And In Combination With Tetrandrine Against Aspergillus Fumigatus

ObjectivesTo investigate the synergistic effect and main mechanism of itraconazole(ITR)or voriconazole(VRC) alone and in combination with tetrandrine(TET) against Aspergillus fumigatus.MethodsIn this study, we investigated the in vitro antifungal effects of ITR/VRC alone and in combination with TET against 23 clinical isolates of A. fumigatus using the microdilution method described in M38-A. Additionally, we investigated the in vitro antifungal effects of ITR/VRC alone and in combination with TET against 23 clinical isolates of A. fumigatus using the checkerboard microdilution method described in M38-A2, and the effect of the interactions was assessed by two non-parametric approaches: the fractional inhibitory concentration index(FICI) model and ΔE model(the difference between the predicted and measured fungal growth percentages).Furthermore, The dynamic antifungal effects of drug combinations against AF17,AF23 were assessed by using time-killing curves.The antifungal effects of TET with ITR/VRC against A. fumigatus were assessed in vivo using systemic infection of mice with A. fumigatus.After treatment, efflux pump activity and expression levels of MDR1, MDR2,MDR3, MDR4 and ATRF were determined by the efflux of rhodamine 6G(R6G) and RT-PCR, respectively.ResultsThe minimum inhibitory concentrations(MICs) of ITR alone and combined with TET against the A. fumigatus were 0.5 μg/m L ~32 μg/m L and 0.0625 μg/m L~ 4μg/m L, respectively(P < 0.05); The MICs of VRC alone and combined with TET against the A. fumigatus were 0.25 μg/m L ~ 2 μg/m L and 0.03125 μg/m L ~ 0.25μg/m L, respectively(P < 0.05).When ITR was combined with TET, ITR minimum inhibitory concentrations(MIC) were reduced from 0.125~32 mg/L to 0.0625~2 mg/L,and TET MICs were reduced from 256~512 mg/L to 8~64 mg/L. When VRC was combined with TET, VRC MICs were reduced from 0.125~2 mg/L to 0.03125~0.5mg/L, and TET MICs were reduced from 256~512 mg/L to 8~256 mg/L. Time-killing curves revealed that A. fumigatus viability was reduced after treatment with ITR/VRC combined with TET versus ITR/VRC alone.ITR/VRC combined with TET significantly prolonged mouse survival and reduced kidney and brain tissue burdens versus ITR/VRC alone(P < 0.05).Moreover, TET inhibited R6 G efflux of A. fumigatus, and ITR/VRC combined with TET down-regulated MDR2, MDR3, MDR4 and ATRF expression versus ITR/VRC alone(P < 0.05).ConclusionsThus, TET acts synergistically with ITR/VRC against A. fumigatus by inhibiting drug efflux via down-regulation of the efflux pump genes MDR2, MDR3, MDR4 and ATRF.