In Vitro Interaction And Mechanism Research Of Amiodarone Combined With Azole Antifungal Agents

In the past decades,with the rapid development of medicine technology,the widespread application of antibiotics,the extensive use of immunosuppressant,hormone,antitumor chemotherapy and cytotoxic drugs,the prevalence of conditioned pathogen—especially fungi infections have increased significantly,Candiada albicans has the highest isolating rate among them.Given no promptly cure,fungi infection would progress quickly with a high mortality rate.Meanwhile,following by the frequent administration of antifungals,resistant rate is increasing continuously,as a result of selected pressure of chemotherapeutics,which made fungi infection more difficult to treat.Now,drugs combinations method has become one of the hottest and most effective strategies to solve this problem.Antiarrhythmic drug amiodarone(AMD)is the first line medication in treating atrial fibrillation,and the choice drug for patients with pre-excitation syndrome and refractoriness arrhythmia.Recent literatures reported amiodarone had a novel,broad fungicidal activity,that can acted synergistic with low dosage of fluconazole.Static and dynamic antifungal activity of AMD and azoles acted alone or in combinations were fully examined;mechanisms of sensitization of amiodarone on azoles against resistant Candida albicans were futher studied;and the cellular morphology changes after drugs actions were observed as well.â… Static antifungal activity of amiodarone combined with azolesReferring to the revised CLSI M27-A2 document,susceptibility testing was performed by amiodarone within 0.5～256μg/ml,fluconazole within 0.25～256μg/ml,itraconazole and voriconazole within 0.008～64μg/ml.CA5,CA8,CA12,CA14 and CA129 were tested to be susceptible,while CA10,CA15,CA16,CA135 and CA137 resistant.We also found that amiodarone had nearly no antifungal acitivity on all strains.Static combined antifungal effect testing was carried according to checkerboard method,concentrations ranged 0.125～8μg/ml for amiodarone,0.125～128μg/ml for fluconazole and 0.008～8μg/ml for itraconazole and voriconazole.FICI,RSM and AE methods were employed for overall assessment.The combinations of amiodarone and azoles were synergistic against resistant isolates with a average FICI of 0.010,a average ICαand its 95%confidence interval(CI)exceed zero,a averageΣSYN of 1250%andΣANT of -18.7%.While The combinations of amiodarone and azoles displayed indifferent against susceptible strains with a average FICI of 1.84,a average ICa exceed zero but its 95%CI containing,a averageΣSYN of 59%andΣANT of -138.5%.After comparising all three methods,non-parameter method was better for assessing the combined effect of antifungals and non-antifungal agents.â…¡Dynamic antifungal activity of amiodarone combined with azolesTime-kill method had been applied to test the dynamic activity of amiodarone combined with azoles against CA10 and CA14.Concentrations of amiodarone (4μg/ml),fluconazole(8μg/ml),itraconazole(0.5μg/ml)and voriconazole(0.5μg/ml) were screened.At determined time point(0,6,12,24,48h),viable count and XTT assay were used to obtain the number of cells.After analysis of log₁₀CFU/ml at 48h, weak antifungal activity of amiodarone was confirmed;synergism of amiodarone combined with azoles against resistant strain were confirmed by a≥2 decrement in log₁₀CFU/ml compared to the corresponding azole;indifference of amiodarone combined with azoles against sensitive strain were confirmed by a≤1 decrement in log₁₀CFU/ml compared to the corresponding azole.Finally,we demonstrated that viable count and XTT assay had an excellent coincidence,with r=0.9622.â…¢Extracellular calcium modulation and drug effectPlate streaking was occupied to research the influences of calcium modulators on the combined effect of amiodarone and azoles against CA10,CA137,CA8,CA14. After streaking the fungi suspension onto YPD agar medium with certain concentrations of drugs and calcium modulators,plates were incubated at 35℃for 48h.Results displayed that amiodarone strikingly reinforced the effect of azoles against resistant strains,and slightly enhanced the effect of azoles against sensitive strains.BayK8644 had no influence on drugs combinations;low concentration of calcium can promote fungi growth,while high concentration of calcium,EGTA and EDTA can inhibit fungi growth,and strengthened the antifungal actions.Addition of benidipin largly augments the combination activity against resistant fungi and slightly affects the combination activity against sensitive isolates.â…£Relationship between sensitization of amiodarone and intracellular calciumFluo-3/AM is a trans-membrane fluorescent indicator,which can immediately give out fluorescence after bounding to Ca²⁺,and reflect the square pulse changes of intracellular calcium.Flow cytometry was introduced to test the effect of drugs combinations on intracellular calcium of CA10(resistant).CA10 was incubated in YPD liquid medium until log phase growth,then washed by D-Hanks buffer and resuspended to 10⁷CFU/ml.Cells were loaded with isovolumic Fluo-3/AM at 37℃and 120rpm for 30min to a final concentration of 3μM.Results showed that an application of AMD resulted in a biphasic elevation of cytoplasmic calcium,which may relate to its weak antifungal activity;fluconazole has no effect on intracellular [Ca²⁺]i,but can reversed the biphasic elevation(phase 1 and 3)of cytoplasmic[Ca²⁺]i of "control" to monophasic elevation(phase 2);sensitization of AMD may due to the release of calcium from calcium store,and extremely raised intracellular calcium.â…¤Effect of amiodarone on intracellular azoles accumulationRhodamine 6G(R6G)can passive diffuse into fungi cells,and has the same substrate of CDR(Candida Drug Resistance)with fluconazole,itraconazole and voriconazole.Fluorometric method and flow cytometry were introduced to test the accumulation,uptake and effluxion of R6G(10μM)with or without amiodarone in CA10,CA135,CAB,CA14.Strains were incubated in YPD liquid medium until log phase growth.Samples of R6G accumulation were carried out in YPD liquid medium, R6G uptake was performed in PBS buffer and R6G effluxion was done in 5% glucose-containing PBS buffer.Results exhibited that sensitization mechanism of amiodarone is the increment of R6G accumulation,which is resulted from increased R6G uptake,not reduced R6G effluxion;and amiodarone had no obvious effect on R6G accumulation,uptake,effluxion in sensitive strains.â…¥Effect of drugs on cellular morphology of Candida albicansImmersion objective(×1000)of light microscope was used to overview the effects of amiodarone and azoles alone or in combinations against CA10 and CA14.There are no cellular morphology changes among drugs actions after 6h incubation,which demonstrated that most bacterium and tissue dyeing methods suitable for fungi cells, too.Strains after 24 and 48h incubation with drugs showed:cells of "growth control" have clear wall and nucleus,with pack of budding and filaments;cells of "amiodarone alone" have no clear wall and nucleus,with aniso-size of cell body;"azoles alone" has less cells than "growth control",with aniso-size of cell body and gloomy staining; cells of "drugs combinations" have no significant deviation with "azoles alone " in CA14,while in CA10 there has the least number of cells,with appearance of no staining or debris-liking cells.