Interaction between amorolfine and voriconazole against Fusarium species

doi:10.21203/rs.3.rs-53804/v1

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Interaction between amorolfine and voriconazole against Fusarium species

https://doi.org/10.21203/rs.3.rs-53804/v1

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Background: Fusarium species are environmentally ubiquitous fungi capable of causing diverse superficial, locally invasive, or disseminated infections, making them important pathogens. Compared with antibacterial drugs, the types of antifungal drugs are still limited, and the adverse reactions are significant. Therefore, novel treatments against Fusarium spp. are an urgent need.

Results: Here we investigated the interaction of amorolfine combined with voriconazole on Fusarium spp. Our study demonstrated that amorolfine in combination with voriconazole could inhibited the Fusarium spp. significantly. Galleria mellonella was also used as a model to show the interaction of the two-drug combination in vivo against Fusarium spp.; larval survival rates were significantly higher after treatment with the amorolfine-voriconazole combination compared to the monotherapy group.

Conclusions: This study is the first to demonstrate that voriconazole combined with amorolfine has a synergistic effect against Fusarium spp. infection and may be an effective method for antifungal therapy.

General Microbiology

Applied & Industrial Microbiology

Fusarium species

Antifungal

Amorolfine

Voriconazole

Synergy

Fusarium species are environmentally ubiquitous fungi [1], and they are capable of inducing a range of diverse superficial, locally invasive, or disseminated infections [2–21]. Fusariosis primarily occurs in humans as a consequence of inhaling or contacting materials that contain Fusarium conidia. Subsequently, when a suitable environment is provided, conidia germinate and develop into filamentous structures capable of invading local tissues [21]. There has been no international consensus on the prescriptive treatment of Fusarium spp. infecion. The therapeutic effects of all kinds of antifungal drugs against Fusarium spp. were not significant. Currently, voriconazole or liposomal amphotericin B are the two primary drugs used to treat invasive fusariosis, and they are the most effective drugs for Fusarium infections [22–24]. However, patient outcomes are often poor and primarily depend on a restoration of immune function in affected hosts, with neutropenia in particular being associated with poor outcomes [25]. Additionally, it has been known that patients treated with voriconazole or liposomal amphotericin B are prone to abnormal renal function and other adverse reactions. Compared with antibacterial drugs, the types of antifungal drugs are still lmited, and the adverse reactions are significant. As few novel antifungal drugs are available or in development, combination-based therapeutic approaches instead represent the most promising approach to treat serious fungal infections.

Amorolfine is a derivative of morpholine and a new kind of broad-spectrum antifungal agent that that has been used to treat fungal infections in humans, such as dermatophytes, yeast, and a few condition pathogenic fungi [26].

Herein, we first demonstrated the in vitro antifungal activity of amorolfine in combination with voriconazole on Fusarium spp. by the checkerboard microdilution method. Moreover, we also investigated the in vivo therapeutic effects of the two-drug combination against Fusarium spp. infection in the Galleria mellonella model by survival analysis.

MIC of amorolfine and voriconazole against Fusarium spp.

For all of the strains, the minimal inhibitory concentration (MIC) ranges for individual agents when used to treat Fusarium isolates were 4 to 16 µg/mL for amorolfine, and 2 to 8 µg/ml for voriconazole (Table 1). A combination of amorolfine with voriconazole showed synergistic antifungal effects against 11 (73.3%) strains of Fusarium spp. and the effective MIC ranges of amorolfine and voriconazole were primarily in the 1–2 µg/ml and 0.5–2 µg/ml ranges, respectively. These effects were also indicated in vitro by the FICI: the FICIs for most F. solani (Jzfs1, Jzfs2, Jzfs3, Jzfs4, Jzfs6, Jzfs7, Jzfs8, Jzfs10) and F.oxysporum (Jzfo2, Jzfo3, Jzfo5) strains were all ≤ 0.5 (Table 1); nevertheless, the FICIs were > 0.5 for some of Fusarium strains including Jzfs5, Jzfs9, Jzfo1and Jzfo4 strains, confirming that the two-drug combination has no interaction on these strains.

Table 1

MIC and FICI results with combination of AMO and VOR against *Fusarium* spp.
	Strains	MICs (µg/ml)
	Strains	AMO	VOR	AMO/VOR	FICI	IN
F. Solani
	Jzfs1	4	4	1/0.5	0.375	SYN
	Jzfs2	8	4	1/0.5	0.25	SYN
	Jzfs3	4	2	1/0.5	0.5	SYN
	Jzfs4	8	4	2/1	0.5	SYN
	Jzfs5	4	2	2/0.5	0.75	NI
	Jzfs6	16	8	1/1	0.1875	SYN
	Jzfs7	8	4	1/0.5	0.25	SYN
	Jzfs8	8	2	1/0.5	0.375	SYN
	Jzfs9	8	2	1/1	0.625	NI
	Jzfs10	16	8	2/2	0.375	SYN
F. oxysporum
	Jzfo1	8	2	1/1	0.625	NI
	Jzfo2	16	4	1/0.5	0.1875	SYN
	Jzfo3	8	4	2/0.5	0.375	SYN
	Jzfo4	8	2	2/1	0.75	NI
	Jzfo5	8	2	1/0.5	0.375	SYN

AMO Amorolfine; VOR Voriconazole; MICs Minimal inhibitory concentrations; FICI, fractional inhibitory concentration index; NI no interaction; IN interaction; SYN synergism; NI no interaction

Efficacy of voriconazole and amorolfine in Fusarium-infected G. mellonella

We investigated the in vivo antifungal activity of voriconazole (100 µg/mL), amorolfine (100 µg/mL), or the combination thereof using G. mellonella infected with Fusarium isolate (jzfs1). Our study illustrated that after 2 days of infection, larvae treated with the two-drug combination had the highest survival rate(68%) compared to monotherapy (voriconazole, 51%; amorolfine, 43%) and conidial alone groups (28%) (P < 0.05). At 4 days after infection, larvae treated with the combination group exhibited survival rates that were 3-fold higher than those of the conidial alone group. At 6 days after infection, G. mellonella with monotherapy exhibited lower survival rate (voriconazole, 15%; amorolfine, 12%) than G. mellonella with combination therapy (30%) with 3% of G. mellonella in the conidial group (P < 0.05). However, larvae treated with amorolfine alone exhibited slightly decreased survival rates compared to larvae treated with voriconazole alone (Fig. 1).

Histopathology Study

At 2 days of infection, we conducted histopathologic staining of G. mellonella larvae infected with F. solani ( jzfs1) and treated with different drugs. Histopathologic staining revealed the differences in the presence and abundunce of fungi and cytoplasmic staining. We found that plenty of mycelia and spores existed in infected larvae. In different groups, the number of spores decreased significantly in the group treated with a combination of voriconazole (100 µg/mL) and amorolfine (100 µg/mL) (Fig. 2d). In addition, the number of yeast cells in the voriconazole alone group (Fig. 2c) was lower than that in the amorolfine alone group (Fig. 2b). Mean infected area size in the combination group was smaller relative to the voriconazole group. The conidial (treatment with the conidia alone) group (Fig. 2a) had higher infection levels than other assessed groups.

Fusariosis can be treated effectively using voriconazole or amphotericin B monotherapeutic approaches[21, 27]. However,the above two treatments have major side effects and higher costs. Therapeutic approaches, such as the application of novel compounds or existing drugs in combination with azoles, may represent an efficacious approach for fungal infections, as they may enhance antifungal efficacy, enable a wider antifungal spectrum, and reduce side effects. Previous studies have demonstrated that licofelone can synergistically function with fluconazole when used to treat conidia of fluconazole- resistant C. albicans [28]. And pyrvinium pamoate was also a synergist of azoles against Exophilal dermatitidis [29]. Kathrin H et al. [30]reported that the combination of voriconazole and micafungin was synergistic in vitro against F.solani. Amorolfine, as broad- spectrum antifungal agent, has been found to have antifungal activity against dermatophytes, yeast, and a few condition pathogenic fungi [26]. We then evaluated the interaction between voriconazole and amorolfine against Fusarium spp. In vitro, amorolfine alone has a moderate effect on Fusarium spp.: the MICs of amorolfine alone against Fusarium spp. were 4, 8 and 16 µg/mL. In addition, amorolfine can synergistically function with voriconazole; the MIC of voriconazole fell from 8, 4,2 to 2,1 and 0.5 µg/mL when combined with amorolfine (1,2 µg/mL), whereas the combination had little impact on the F. solani (jzfs 5,9)and F.oxysporum strains (jzfo 1,4) (Table 1). Therefore, it is reasonable to speculate that the combination of amorolfine and voriconazole could improve the susceptibility of Fusarium spp. by decreasing the MICs of the above two drugs. Based on FICIs, we found that voriconazole could synergistically work with amorolfine against Fusarium spp. (Table 1).

To further verify the interaction between amorolfine and voriconazole in our study, we detected the combined antifungal effect of voriconazole-amorolfine against Fusarium spp. at different time points in vivo. For monotherapy, voriconazole or amorolfine was used. For combination therapy, voriconazole with an amorolfine formulation was administered. We note that, compared to the conidia alone group, monotherapy with voriconazole or amorolfine improved the survival of G.mellonella, and the survival rate of vo riconazole monotherapy-treated larva was slightly higher than that of amorolfine monotherapy-treated larvae, potentially because this improvement in survival may be attributed to the availability of antifungal agents, and the antifungal activity of voriconazole was stronger than that of amorolfine. In addition, combination therapy in G. mellonella was better than voriconazole or amorolfine monotherapy (P < 0.05), suggesting that amorolofine could potentiate the activity of voriconazole and showed favorable synergistic effect with voriconazole against Fusarium spp.

Furthermore, the fungal virulence and antifungal drug activity has recently been investigated in G. mellonella model on account of a similar immune response as mammals and other advantages including lack of ethical concerns, low cost, and easy manipulation[28, 31, 32]. As illustrated in our study, the survival rate of larvae in voriconazole- amorolfine combination group was significantly improved compared to the monotherapy groups (including voriconazole or amorolfine-treated group) (P < 0.05). Moreover, we observed larva after infection microscopically (Fig. 2). In the histological tissue of G. mellonella, our results showed that the combined group with high survival rate contained fewer Fusarium spp. than the conidia group and monotherapy group. This suggested that amorolfine can further potentiate the therapeutic effect of voriconazole, and voriconazole combined with amorolfine showed a synergistic effect against Fusarium spp. infection.

Taken together, what we have identified in this study suggest that amorolfine could be an effective synergist with voriconazole against Fusarium spp. and the combination of the two drugs may represent a viable therapeutic strategy for antifungal treatment to significantly relieve the clinical symptoms of Fusarium spp.-related diseases. However, to some extent, the fact that amorolfine has no injection and oral preparation limites the possible application of the drug to invasive system infections. With ongoing development in pharmaceutical manufacturing technologies, we can look forward to innovative formulations of amorolfine enabling their broader application. More studies on the in-depth mechanistic basis for the antifungal properties of amorolfine may also guide new antifungal strategies.

Strains and Media

In the present study, Fifteen clinical isolates were stored in Department of Dermatology, Zhongnan hospital of Wuhan University in China, including ten Fusarium solani strains and five Fusarium oxysporum strains. Aspergillus flavus strain (ATCC 204304) was used for quality control. Clinical isolates were collected from patients that had provided written informed consent, and the Research Ethical Committee of Zhongnan Hospital of Wuhan University approved the present study.The susceptibility assay preparation for Fusarium spp. were performed in accordence to Clinical and Laboratory Standards Institute document M38-A2 [33]. The Fumarium spp. conidia were obtained from cultures grown on Potato Dextrose Agar(PDA) solid medium for 2 weeks at 28^◦C. Conidia were collected by washing the surface of these plates using PBS, after which a hemocytometer was used to count them. In the current study, we first determined the minimal inhibitory concentration (MIC) of voriconazole and amorolfine in single use before performing a large number of experiments.The MIC was defined as the lowest concentration of the drug that inhibited fungal growth by 100% compared with the growth of the control.

MIC Determination by Broth Microdilution Assays

Herein, the impact of amorolfine alone and combined with voriconazole was investigated against a total of 15 strains of Fusarium isolates (10 F. solani strains and 5 F.oxysporum strains). Aspergillus flavus strain (ATCC 204304) was included for quality control. All Fusarium isolates were identified by microscopic morphology and internal transcribed spacer (ITS) ribosomal DNA (rDNA) sequencing[34]. For the checkerboard assays [33], dimethyl sulfoxide (DMSO) was used to prepare stocks of each antifungal agent (3,200 µg/ml). Working solutions were prepared using sterile RPMI-1640 medium to serially dilute voriconazole (0.25 to 16 µg/ml; Shanghai Selleck Co., Ltd., China) and amorolfine (0.5 to 32 µg/ml; Shanghai Selleck Co., Ltd., China). For broth microdilution checkerboard analyses, the M38-A2 technique was adapted as per protocols developed by the Clinical and Laboratory Standards Institute, with MICs corresponding to

the lowest concentrations that completely inhibited growth (100% inhibition)[33].

Interactions between amorolfine and voriconazole were assessed based upon the fractional inhibitory concentration index (FICI), which was determined as follows: FICI= (Ac/Aa) +(Bc/Ba), wherein Ac and Bc correspond to antifungal MICs for drugs in combination and Aa and Ba correspond to MICs for drugs A and B as single agents. A FICI of ≤ 0.5 represents synergistic, while a FICI of > 0.5 to ≤ 4 indicates a lack of any interaction (indifference), and a FICI of > 4 represents an antagonism interaction[35]. Analyses were conducted in triplicate.

Efficacy of Voriconazole and Amorolfine in G. mellonella infected with Fusarium strains

As previously described [28, 36, 37], Galleria mellonella larval were used and divided into six groups: untreated group, growth control group (10 µl saline only), conidial group, voriconazole (100 µg/mL)- treated group, amorolfine (100 µg/mL)-treated group, and voriconazole (100 µg/mL) with amorolfine (100 µg/mL)-treated group. And each experimental group contained 20 larvae (0.3– 0.4 g in weight). Fusarium isolate (jzfs1) conidia, obtained from PDA cultures grown at 28 °C for 1 week, had been collected via washing the agar surface using PBS and counted via hemocytometer (10⁷CFU/ml). Ten microlitres of conidia suspension was injected into Galleria mellonella of different groups except untreated group and growth control group (10 µl saline only). The infected larvae were stored at 37^◦C for 2 h and then treated with antifungal drugs (5 µl). Finally, the larvae was incubated 37^◦C and monitored for survival rate every 24 h for 6 days. And the experiment was repeated thrice.

Histological Study

To detect the Fusarium isolate in the tissue of G. mellonella, we collected larvae separately from different groups, including conidial alone group, voriconazole- treated group, amorolfine-treated group, and voriconazole with amorolfine-treated group, at day 2 after infection. According to previously described [28], the larvae infected with 1 × 10⁵ cells/larva F. solani (jzfs1) were processed for ultrathin tissue sections, which were subsequently stained with hematoxylin and eosin (HE) and Periodic Acid-Schiff (PAS).

Statistical Analysis

GraphPad Prism 5.0 was used to prepare Figures, while SPSS 16.0 was used for all statistical analysis. Data are given as means ± SEM, and were compared using ANOVAs. Experiments were repeated in triplicate, with P < 0.05 as the significance threshold.

F. solani: Fusarium solani; F.oxysporum:Fusarium oxysporum; AMO:Amorolfine; VOR:Voriconazole; MICs:Minimal inhibitory concentrations; FICI:Fractional inhibitory concentration index; NI:No interaction; IN:Interaction; SYN:Synergism; NI:No interaction

Ethics approval and consent to participate

Research Ethical Committee of Zhongnan Hospital of Wuhan University granted permission to access the clinical samples used. And ethical approval was given by the Research Ethical Committee of Zhongnan Hospital of Wuhan University. Written consent was given by the patients for the use of clinical strains.

Consent for publication

Not applicable.

Availability of data and materials

All data generated or analyzed during this study are included in this published article. The data used or analysed during the current study are available from the corresponding author on reasonable request.

Competing interests

The authors declare that they have no competing interests.

Funding

This work was supported by National Natural Science Foundation of China (Grant 81602771). Funding bodies did not have a role in study design, in the collection, analysis, and interpretation of data, or preparation of the manuscript.

Authors’ contributions

PL, QL contributed to the conception of the project, data analysis, interpretation of results, creation of figures, writing and editing the manuscript. JS performed sample collection and fungal culture. The authors have read and approve of the manuscript.

Acknowledgements

We are grateful to the staff of Dermatology of Zhongnan Hospital of Wuhan University, for assistance in field sampling and processing samples in the laboratory.

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Interaction between amorolfine and voriconazole against Fusarium species

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Version 1

Abstract

Figures

Background

Results

Histopathology Study

Discussion

Conclusion

Methods

Strains and Media

MIC Determination by Broth Microdilution Assays

Histological Study

Statistical Analysis

Abbreviations

Declarations

Ethics approval and consent to participate

Funding

Authors’ contributions

Acknowledgements

References

Status:

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