In Vitro And In Vivo Efficacy Of A Synergistic Combination Of Itraconazole And Verapamil Against Aspergillus Fumigatus

Background and PurposeInvasive aspergillosis is one of the most important life-threatening fungal infections and mainly affects immunocompromised hospitalized patients,such as patients with hematological malignancies,AIDS and solid organ or hematopoietic stemcell transplant(HSCT)recipients.Currently,there are only three categories of antifungals for aspergillosis treatment:polyenes,azoles and echinocandins.Itraconazole and voriconazole are the first-line recommended options for aspergillosis.Long-term administration of azoles to susceptible populations for prevention or treatment results in natural selection of resistant isolates.Many researchers have searched for alternative drug targets or tried to develop safe and broad-spectrum antifungals.However,development of novel antifungal agents is very tough,costly and time-consuming.Thus,mining existing agents that can enhance the efficacy of antifungal drugs is a promising approach to improve the drug susceptibility of A.fumigatus.Previous studies have demonstrated that calcium signaling pathway is requred in the regulatory mechanism for fungal stress adaption under azole environments in fungi.The putative plasma membrane Ca2+influx system of A.fumigatus is the CchA-MidA complex protein,which is homologous to mammalian Ltype voltage-gated calcium channels.The CchA-MidA complex mediates a rapid influx of calcium ions and leads to transient increases in intracellular calcium concentrations.Calcium channel blockers are clinical commonly used class Ⅳ antiarrhythmia agents,which target the L-type voltage-gated calcium channels in mammalian cells.They are possible candidates to interfere with Ca2+ entry in fungi.However,little is known about whether clinical calcium channel blockers could enhance the in vitro and in vivo antifungal activity of azoles against Aspergilli.In this study,after a preliminary screening of different calcium channel blockers and antifungals,we showed that verapamil displayed a synergistic effect with itraconazole against an A.fumigatus strain in vitro and in vivo.The purpose of this study was to study the synergistic effect of verapamil and itraconazole in vitro and in vivo,and to explore its possible mechanism.MethodsThe effects of different calcium channel blockers combined with antifungal drugs on the colony phenotype of A.fumigatus were compared.The effects of verapamil combined with itraconazole on colony phenotypes of 17 itraconazole sensitive strains(ITC-S)and itraconazole resistant strains(ITC-R)were compared.The combined effect of the two drugs on A.fumigatus was evaluated by microliquid-based dilution method and chessboard method.The intracellular calcium level was analyzed by Aequorin(jellyfish luminescent protein)-labeled strain.The ergosterol content was analyzed by HPLC.Flow cytometry was used to detect the intracellular retention of drug pump activity indicator Rh 6G.And in vivo bio luminescence imaging was used to further test the combined effect of the two drugs in the mouse model of invasive pulmonary aspergillosis.Results1.In vitro studies:1)Verapamil was superior to nifedipine and diltiazem in inhibiting the diameter of A.fumigatus.The inhibitory effect of verapamil combined with itraconazolev was superior to that of verapamil combined with voriconazole and amphotericin B on A.fumigatus colony diameter.Verapamil and itraconazole also affected the colony diameter and sporulation of Aspergillus flavus,Aspergillus terrestris and Aspergillus Niger.2)Verapamil had no antifungal activity against A.fumigatus,but the combination of verapamil and itraconazole had synergistic effect on all ITC-S strains from clinical sources,but no obvious regularity was found in ITC-R strain.2.Mechanism research:1)Itraconazole stimulation resulted in an instaneous increase of intracellular calcium concentration.However,after pretreatment with verapamil or calcium chelator EGTA,the peak value of this transient response was significantly inhibited.2)Verapamil had no effect on the absorption of Rh 6G,but significantly inhibited the efflux of Rh 6G,which led to the retention of intracellular Rh 6G3)Verapamil alone had no effect on ergosterol biosynthesis.Itraconazole led to a slight decrease in ergosterol content.However,the drug combination significantly decreased the content of ergosterol.3.Animal experimentsThe survival rate and lung fluorescence of mice treated with verapamil alone were similar to those of the untreated group.All of the mice in this two groups died whthin 6 days after infection,and there was no significant difference in the pathological sections of the lungs between the two groups.A large number of Aspergillus hyphae invaded into the bronchial cavity.The survival rate of the group with itraconazole monotherapy was 30%,the amount of fluorescence in the lung was significantly weaker than that in the untreated group,and the pulmonary pathology showed less mycelial infiltration and inflammatory reaction.The survival rate of mice in the combined group was 50%,which was higher than that in the group with itraconazole monotherapy.The amount of lung fluorescence in the combined group was lower than that in the itraconazole group,and the lung pathology was basically normal lung tissue morphology.ConclusionThis study confirmed that verapamil combined with itraconazole could enhance the antifungal effect of A.fumigatus,elucidated the possible mechanism of the combination of verapamil and itraconazole,and for the first time proved the therapeutic effect of the combination of verapamil and itraconazole in mouse model by using in vivo bio luminescence imaging.It provides theoretical support for calcium blockers to enhance antifungal therapy.