Combination of Amphotericin B and Flucytosine against Neurotropic Species of Melanized Fungi Causing Primary Cerebral Phaeohyphomycosis

ABSTRACTPrimary central nervous system phaeohyphomycosis is a fatal fungal infection due mainly to the neurotropic melanized fungiCladophialophora bantiana,Rhinocladiella mackenziei, andExophiala dermatitidis.Despite the combination of surgery with antifungal treatment, the prognosis continues to be poor, with mortality rates ranging from 50 to 70%. Therefore, a search for a more-appropriate therapeutic approach is urgently needed. Ourin vitrostudies showed that with the combination of amphotericin B and flucytosine against these species, the median fractional inhibitory concentration (FIC) indices for strains ranged from 0.25 to 0.38, indicating synergy. By use of Bliss independence analysis, a significant degree of synergy was confirmed for all strains, with the sum ΔE ranging from 90.2 to 698.61%. No antagonism was observed. These results indicate that amphotericin B, in combination with flucytosine, may have a role in the treatment of primary cerebral infections caused by melanized fungi belonging to the orderChaetothyriales. Furtherin vivostudies and clinical investigations to elucidate and confirm these observations are warranted.

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Surface-Engineered Dendrimeric Nanoconjugates for Macrophage-Targeted Delivery of Amphotericin B: Formulation Development andIn VitroandIn VivoEvaluation

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.04213-14 ◽

2015 ◽

Vol 59 (5) ◽

pp. 2479-2487 ◽

Cited By ~ 40

Author(s):

Keerti Jain ◽

Ashwni Kumar Verma ◽

Prabhat Ranjan Mishra ◽

Narendra Kumar Jain

Keyword(s):

Drug Release ◽

Amphotericin B ◽

Human Erythrocytes ◽

Antileishmanial Activity ◽

Cell Uptake ◽

Antiparasitic Activity ◽

Content Type ◽

Drug Localization

ABSTRACTThe present study aimed to develop an optimized dendrimeric delivery system for amphotericin B (AmB). Fifth-generation (5.0G) poly(propylene imine) (PPI) dendrimers were synthesized, conjugated with mannose, and characterized by use of various analytical techniques, including Fourier transform infrared spectroscopy (FTIR),1H nuclear magnetic resonance (1H-NMR) spectroscopic analysis, and atomic force microscopy (AFM). Mannose-conjugated 5.0G PPI (MPPI) dendrimers were loaded with AmB and evaluated for drug loading efficiency,in vitrodrug release profile, stability, hemolytic toxicity to human erythrocytes, cytotoxicity to and cell uptake by J774A.1 macrophage cells, antiparasitic activity against intracellularLeishmania donovaniamastigotes,in vivopharmacokinetic and biodistribution profiles, drug localization index, toxicity, and antileishmanial activity. AFM showed the nanometric size of the MPPI dendrimers, with a nearly globular architecture. The conjugate showed a good entrapment efficiency for AmB, along with pH-sensitive drug release. Highly significant reductions in toxicity toward human erythrocytes and macrophage cells, without compromising the antiparasitic activity of AmB, were observed. The dendrimeric formulation of AmB showed a significant enhancement of the parasiticidal activity of AmB toward intramacrophagicL. donovaniamastigotes. In thein vitrocell uptake studies, the formulation showed selectivity toward macrophages, with significant intracellular uptake. Further pharmacokinetic and organ distribution studies elucidated the controlled delivery behavior of the formulation. The drug localization index was found to increase significantly in macrophage-rich organs.In vivostudies showed a biocompatible behavior of MPPIA, with negligible toxicity even at higher doses, and promising antileishmanial activity. From the results, we concluded that surface-engineered dendrimers may serve as optimized delivery vehicles for AmB with enhanced activity and low or negligible toxicity.

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In Vitro Antifungal Combination of Flucytosine with Amphotericin B, Voriconazole, or Micafungin against Candida auris Shows No Antagonism

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01393-19 ◽

2019 ◽

Vol 63 (12) ◽

Cited By ~ 8

Author(s):

A. L. Bidaud ◽

F. Botterel ◽

A. Chowdhary ◽

E. Dannaoui

Keyword(s):

Amphotericin B ◽

Inhibitory Concentration ◽

Geometric Mean ◽

Multidrug Resistant ◽

Candida Auris ◽

Content Type ◽

Hospital Acquired Infections ◽

Resistant Mutants ◽

Hospital Acquired

ABSTRACT Candida auris is an emerging, multidrug-resistant pathogen responsible for invasive hospital-acquired infections. Flucytosine is an effective anti-Candida species drug, but which cannot be used as a monotherapy because of the risk of development of resistant mutants during treatment. It is, therefore, noteworthy to test possible combinations with flucytosine that may have a synergistic interaction. In this study, we determined the in vitro interaction between flucytosine and amphotericin B, micafungin, or voriconazole. These combinations have been tested against 15 C. auris isolates. The MIC ranges (geometric mean [Gmean]) of flucytosine, amphotericin B, micafungin, and voriconazole were 0.125 to 1 μg/ml (0.42 μg/ml), 0.25 to 1 μg/ml (0.66 μg/ml), 0.125 to 0.5 μg/ml (0.3 μg/ml), and 0.03 to 4 μg/ml (1.05 μg/ml), respectively. When tested in combination, indifferent interactions were mostly observed with fractional inhibitory concentration index values from 0.5 to 1, 0.31 to 1.01, and 0.5 to 1.06 for the combinations of flucytosine with amphotericin B, micafungin, and voriconazole, respectively. A synergy was observed for the strain CBS 10913 from Japan. No antagonism was observed for any combination. The combination of flucytosine with amphotericin B or micafungin may be relevant for the treatment of C. auris infections.

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New Triazole NT-a9 Has Potent Antifungal Efficacy against Cryptococcus neoformans In Vitro and In Vivo

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01628-19 ◽

2019 ◽

Vol 64 (2) ◽

Cited By ~ 1

Author(s):

Ren-Yi Lu ◽

Ting-Jun-Hong Ni ◽

Jing Wu ◽

Lan Yan ◽

Quan-Zhen Lv ◽

...

Keyword(s):

Cryptococcus Neoformans ◽

Amphotericin B ◽

Pathogenic Fungi ◽

Control Group ◽

Survival Times ◽

Content Type ◽

Fungal Burden ◽

Wide Range

ABSTRACT In the past decades, the incidence of cryptococcosis has increased dramatically, which poses a new threat to human health. However, only a few drugs are available for the treatment of cryptococcosis. Here, we described a leading compound, NT-a9, an analogue of isavuconazole, that showed strong antifungal activities in vitro and in vivo. NT-a9 showed a wide range of activities against several pathogenic fungi in vitro, including Cryptococcus neoformans, Cryptococcus gattii, Candida albicans, Candida krusei, Candida tropicalis, Candida glabrata, and Candida parapsilosis, with MICs ranging from 0.002 to 1 μg/ml. In particular, NT-a9 exhibited excellent efficacy against C. neoformans, with a MIC as low as 0.002 μg/ml. NT-a9 treatment resulted in changes in the sterol contents in C. neoformans, similarly to fluconazole. In addition, NT-a9 possessed relatively low cytotoxicity and a high selectivity index. The in vivo efficacy of NT-a9 was assessed using a murine disseminated-cryptococcosis model. Mice were infected intravenously with 1.8 × 106 CFU of C. neoformans strain H99. In the survival study, NT-a9 significantly prolonged the survival times of mice compared with the survival times of the control group or the isavuconazole-, fluconazole-, or amphotericin B-treated groups. Of note, 4 and 8 mg/kg of body weight of NT-a9 rescued all the mice, with a survival rate of 100%. In the fungal-burden study, NT-a9 also significantly reduced the fungal burdens in brains and lungs, while fluconazole and amphotericin B only reduced the fungal burden in lungs. Taken together, these data suggested that NT-a9 is a promising antifungal candidate for the treatment of cryptococcosis infection.

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In Vitro Evaluation of Radiolabeled Amphotericin B for Molecular Imaging of Mold Infections

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.02377-19 ◽

2020 ◽

Vol 64 (7) ◽

Cited By ~ 1

Author(s):

Lukas Page ◽

Andrew J. Ullmann ◽

Fabian Schadt ◽

Sebastian Wurster ◽

Samuel Samnick

Keyword(s):

Molecular Imaging ◽

Amphotericin B ◽

Invasive Pulmonary Aspergillosis ◽

Nuclear Imaging ◽

Molecular Probes ◽

Content Type ◽

Diagnostic Potential ◽

Gallium 68

ABSTRACT Invasive pulmonary aspergillosis and mucormycosis are life-threatening complications in immunocompromised patients. A rapid diagnosis followed by early antifungal treatment is essential for patient survival. Given the limited spectrum of biomarkers for invasive mold infections, recent studies have proposed the use of radiolabeled siderophores or antibodies as molecular probes to increase the specificity of radiological findings by nuclear imaging modalities. While holding enormous diagnostic potential, most of the currently available molecular probes are tailored to the detection of Aspergillus species, and their cost-intensive and sophisticated implementation restricts their accessibility at less specialized centers. In order to develop cost-efficient and broadly applicable tracers for pulmonary mold infections, this study established streamlined and high-yielding protocols to radiolabel amphotericin B (AMB) with the gamma emitter technetium-99m (99mTc-AMB) and the positron emitter gallium-68 (68Ga-AMB). The radiochemical purity of the resulting tracers consistently exceeded 99%, and both probes displayed excellent stability in human serum (>98% after 60 to 240 min at 37°C). The uptake kinetics by representative mold pathogens were assessed in an in vitro Transwell assay using infected endothelial cell layers. Both tracers accumulated intensively and specifically in Transwell inserts infected with Aspergillus fumigatus, Rhizopus arrhizus, and other clinically relevant mold pathogens compared with their accumulation in uninfected inserts and inserts infected with bacterial controls. Inoculum-dependent enrichment was confirmed by gamma counting and autoradiographic imaging. Taken together, this pilot in vitro study proposes 99mTc-AMB and 68Ga-AMB to be facile, stable, and specific probes, meriting further preclinical in vivo evaluation of radiolabeled amphotericin B for molecular imaging in invasive mycoses.

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New Insight into Amphotericin B Resistance in Aspergillus terreus

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01283-12 ◽

2013 ◽

Vol 57 (4) ◽

pp. 1583-1588 ◽

Cited By ~ 40

Author(s):

Gerhard Blum ◽

Caroline Hörtnagl ◽

Emina Jukic ◽

Thomas Erbeznik ◽

Thomas Pümpel ◽

...

Keyword(s):

Amphotericin B ◽

Molecular Basis ◽

Aspergillus Terreus ◽

Minor Role ◽

Content Type ◽

Ergosterol Content ◽

Disseminated Aspergillosis ◽

A Minor

ABSTRACTAmphotericin B (AMB) is the predominant antifungal drug, but the mechanism of resistance is not well understood. We compared thein vivovirulence of an AMB-resistantAspergillus terreus(ATR) isolate with that of an AMB-susceptibleA. terreusisolate (ATS) using a murine model for disseminated aspergillosis. Furthermore, we analyzed the molecular basis of intrinsic AMB resistancein vitroby comparing the ergosterol content, cell-associated AMB levels, AMB-induced intracellular efflux, and prooxidant effects between ATR and ATS. Infection of immunosuppressed mice with ATS or ATR showed that the ATS strain was more lethal than the ATR strain. However, AMB treatment improved the outcome in ATS-infected mice while having no positive effect on the animals infected with ATR. Thein vitrodata demonstrated that ergosterol content is not the molecular basis for AMB resistance. ATR absorbed less AMB, discharged more intracellular compounds, and had better protection against oxidative damage than the susceptible strain. Our experiments showed that ergosterol content plays a minor role in intrinsic AMB resistance and is not directly associated with intracellular cell-associated AMB content. AMB might exert its antifungal activity by oxidative injury rather than by an increase in membrane permeation.

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Investigation of the Efficacy of Micafungin in the Treatment of Histoplasmosis Using Two North American Strains of Histoplasma capsulatum

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01681-10 ◽

2011 ◽

Vol 55 (9) ◽

pp. 4447-4450 ◽

Cited By ~ 15

Author(s):

Chadi A. Hage ◽

Patricia Connolly ◽

Daniel Horan ◽

Michelle Durkin ◽

Melinda Smedema ◽

...

Keyword(s):

Amphotericin B ◽

North American ◽

Liposomal Amphotericin ◽

Histoplasma Capsulatum ◽

Liposomal Amphotericin B ◽

Content Type ◽

Yeast Form

ABSTRACTMicafungin alone and combined with liposomal amphotericin B was evaluated against two strains ofHistoplasma capsulatum. Micafungin was activein vitroagainst the mold but not the yeast form but was ineffectivein vivo. Micafungin appears to be ineffective in treatment of histoplasmosis.

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Comparison of antifungal efficacies of moxifloxacin, liposomal amphotericin B, and combination treatment in experimental Candida albicans endophthalmitis in rabbits

Canadian Journal of Microbiology ◽

10.1139/w09-112 ◽

2010 ◽

Vol 56 (1) ◽

pp. 1-7 ◽

Cited By ~ 16

Author(s):

Yudum Tiftikcioğlu Deren ◽

Şengül Özdek ◽

Ayşe Kalkanci ◽

Nalan Akyürek ◽

Berati Hasanreisoğlu

Keyword(s):

Candida Albicans ◽

Minimum Inhibitory Concentration ◽

Amphotericin B ◽

Combination Treatment ◽

Liposomal Amphotericin ◽

Inhibitory Concentration ◽

Liposomal Amphotericin B ◽

Control Group

The goal of this study was to compare in vitro and in vivo efficacy of moxifloxacin and liposomal amphotericin B (Amp-B) monotherapies and combination treatment against Candida albicans in an exogenous endophthalmitis model in rabbit eyes. Microplate dilution tests and checkerboard analysis were performed to detect in vitro efficacies. Endophthalmitis was induced by intravitreal injection of C. albicans in 40 rabbit eyes with simultaneous intravitreal drug injection according to prophylactic treatment groups. Group 1 (control group) received 0.1 mL of balanced salt solution, group 2 (moxi group) 100 µg moxifloxacin/0.1 mL, group 3 (Amp-B group) 10 µg liposomal Amp-B/0.1 mL, and group 4 (combi group) both 100 µg moxifloxacin/0.05 mL and 10 µg liposomal Amp-B/0.05 mL intravitreally. Clinical examination, quantitative analysis of microorganisms, and histopathologic examination were performed as in vivo studies. The minimum inhibitory concentration of liposomal Amp-B against C. albicans was found to be 1 µg/mL. Moxifloxacin showed no inhibition of in vitro C. albicans growth. The minimum inhibitory concentration values of liposomal Amp-B for C. albicans were reduced two- to eightfold with increasing concentrations of moxifloxacin in vitro. In vivo, there was no C. albicans growth in the combi group (zero of eight eyes), whereas three eyes (37.5%) showed growth in the Amp-B group. Vitreous inflammation, retinal detachment, focal retinal necrosis, and outer nuclear layer loss were found to be lower in the moxi group compared with the control group. Ganglion cell and inner nuclear layer loss was observed in all eyes (100%) in both the moxi and combi groups, whereas only in 25% (two of eight eyes) in the Amp-B group. Moxifloxacin strongly augments the efficacy of liposomal Amp-B against C. albicans in vitro, although it has no in vitro antifungal activity when used alone. It is interesting that we found a synergistic effect for in vitro tests but failed to demonstrate it in vivo. When 100 µg moxifloxacin/0.1 mL is given intravitreally, it has some toxic effects that are limited to the inner retinal layers.

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Amphotericin B- and Voriconazole-Echinocandin Combinations against Aspergillus spp.: Effect of Serum on Inhibitory and Fungicidal Interactions

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00597-13 ◽

2013 ◽

Vol 57 (10) ◽

pp. 4656-4663 ◽

Cited By ~ 21

Author(s):

Antigoni Elefanti ◽

Johan W. Mouton ◽

Paul E. Verweij ◽

Athanassios Tsakris ◽

Loukia Zerva ◽

...

Keyword(s):

Amphotericin B ◽

Colorimetric Method ◽

Antifungal Drugs ◽

Content Type ◽

Synergistic Interactions ◽

Inhibitory Activities ◽

Additive Interactions ◽

Species Specific

ABSTRACTAntifungal combination therapy with voriconazole or amphotericin B and an echinocandin is often employed as primary or salvage therapy for management particularly of refractory aspergillosis. The pharmacodynamic interactions of amphotericin B- and voriconazole-based combinations with the three echinocandins caspofungin, micafungin, and anidulafungin in the presence of serum were tested against 15Aspergillus fumigatuscomplex,A. flavuscomplex, andA. terreuscomplex isolates to assess both their growth-inhibitory and fungicidal activities. Thein vitroactivity of each drug alone and in combination at a 1:1 fixed concentration ratio was tested with a broth microdilution colorimetric method, and interactions were assessed by isobolographic analysis. Synergy was found for all amphotericin B- and voriconazole-based combinations, with amphotericin B-based combinations showing strong inhibitory synergistic interactions (interaction indices of 0.20 to 0.52) and with voriconazole-based combinations demonstrating strong fungicidal synergistic interactions (interaction indices of 0.10 to 0.29) (P< 0.001). Drug- and species-specific differences were found, with caspofungin and theA. fumigatuscomplex exhibiting the weakest synergistic interactions. In the presence of serum, the synergistic interactions were reduced in the order (from largest to smallest decrease) micafungin > anidulafungin > caspofungin, andA. flavuscomplex >A. fumigatuscomplex >A. terreuscomplex, resulting in additive interactions, particularly for inhibitory activities of amphotericin B-echinocandin combinations and fungicidal activities of voriconazole-echinocandin combinations. Drug- and species-specific differences were found in the presence of serum for inhibitory activities of antifungal drugs, with the lowest interaction indices being observed for amphotericin B-caspofungin (median, 0.77) and for theA. terreuscomplex (median, 0.56). The presentin vitrodata showed that serum had a major impact on synergistic interactions of amphotericin B-echinocandin and voriconazole-echinocandin combinations, resulting in additive interactions and explaining the indifferent outcomes usually observedin vivo.

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Estrogen Receptor Antagonists Are Anti-Cryptococcal Agents That Directly Bind EF Hand Proteins and Synergize with Fluconazole In Vivo

mBio ◽

10.1128/mbio.00765-13 ◽

2014 ◽

Vol 5 (1) ◽

Cited By ~ 58

Author(s):

Arielle Butts ◽

Kristy Koselny ◽

Yeissa Chabrier-Roselló ◽

Camile P. Semighini ◽

Jessica C. S. Brown ◽

...

Keyword(s):

Breast Cancer ◽

Estrogen Receptor ◽

Amphotericin B ◽

Receptor Antagonists ◽

Content Type ◽

New Therapies ◽

Ef Hand ◽

Estrogen Receptor Antagonists

ABSTRACT Cryptococcosis is an infectious disease of global significance for which new therapies are needed. Repurposing previously developed drugs for new indications can expedite the translation of new therapies from bench to beside. Here, we characterized the anti-cryptococcal activity and antifungal mechanism of estrogen receptor antagonists related to the breast cancer drugs tamoxifen and toremifene. Tamoxifen and toremifene are fungicidal and synergize with fluconazole and amphotericin B in vitro. In a mouse model of disseminated cryptococcosis, tamoxifen at concentrations achievable in humans combines with fluconazole to decrease brain burden by ~1 log10. In addition, these drugs inhibit the growth of Cryptococcus neoformans within macrophages, a niche not accessible by current antifungal drugs. Toremifene and tamoxifen directly bind to the essential EF hand protein calmodulin, as determined by thermal shift assays with purified C. neoformans calmodulin (Cam1), prevent Cam1 from binding to its well-characterized substrate calcineurin (Cna1), and block Cna1 activation. In whole cells, toremifene and tamoxifen block the calcineurin-dependent nuclear localization of the transcription factor Crz1. A large-scale chemical genetic screen with a library of C. neoformans deletion mutants identified a second EF hand-containing protein, which we have named calmodulin-like protein 1 (CNAG_05655), as a potential target, and further analysis showed that toremifene directly binds Cml1 and modulates its ability to bind and activate Cna1. Importantly, tamoxifen analogs (idoxifene and methylene-idoxifene) with increased calmodulin antagonism display improved anti-cryptococcal activity, indicating that calmodulin inhibition can be used to guide a systematic optimization of the anti-cryptococcal activity of the triphenylethylene scaffold. IMPORTANCE Worldwide, cryptococcosis affects approximately 1 million people annually and kills more HIV/AIDS patients per year than tuberculosis. The gold standard therapy for cryptococcosis is amphotericin B plus 5-flucytosine, but this regimen is not readily available in regions where resources are limited and where the burden of disease is highest. Herein, we show that molecules related to the breast cancer drug tamoxifen are fungicidal for Cryptococcus and display a number of pharmacological properties desirable for an anti-cryptococcal drug, including synergistic fungicidal activity with fluconazole in vitro and in vivo, oral bioavailability, and activity within macrophages. We have also demonstrated that this class of molecules targets calmodulin as part of their mechanism of action and that tamoxifen analogs with increased calmodulin antagonism have improved anti-cryptococcal activity. Taken together, these results indicate that tamoxifen is a pharmacologically attractive scaffold for the development of new anti-cryptococcal drugs and provide a mechanistic basis for its further optimization.

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Spiroindolone That Inhibits PfATPase4 Is a Potent, Cidal Inhibitor of Toxoplasma gondii TachyzoitesIn VitroandIn Vivo

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.02225-13 ◽

2013 ◽

Vol 58 (3) ◽

pp. 1789-1792 ◽

Cited By ~ 15

Author(s):

Ying Zhou ◽

Alina Fomovska ◽

Stephen Muench ◽

Bo-Shiun Lai ◽

Ernest Mui ◽

...

Keyword(s):

Body Weight ◽

Toxoplasma Gondii ◽

Inhibitory Concentration ◽

Parasite Burden ◽

Content Type ◽

Medicine Development ◽

Lead Candidate ◽

Oral Doses

ABSTRACTHere, we show that spiroindolone, an effective treatment for plasmodia, is also active againstToxoplasma gondiitachyzoites.In vitro, spiroindolone NITD609 is cidal for tachyzoites (50% inhibitory concentration [IC50], 1μM) and not toxic to human cells at ≥10μM. Two daily oral doses of 100 mg/kg of body weight reduced the parasite burden in mice by 90% (P= 0.002), measured 3 days after the last dose. This inhibition ofT. gondiitachyzoitesin vitroandin vivoindicates that spiroindolone is a promising lead candidate for further medicine development.

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