Superior Efficacy of Liposomal Amphotericin B with Prolonged Circulation in Blood in the Treatment of Severe Candidiasis in Leukopenic Mice

In leukopenic mice with severe systemic candidiasis, single-dose treatment (5 mg of amphotericin B [AMB]/kg of body weight) with long-circulating polyethylene glycol-coated AMB liposomes (PEG-AMB-LIP) resulted in zero mortality and a significant reduction in the number of viable Candida albicans in the kidney, whereas 70% mortality was seen in mice treated with five daily doses of AmBisome (5 mg of AMB/kg · day). When the first of five daily doses of AmBisome was combined with a single low dose of Fungizone (0.1 mg of AMB/kg), the efficacy was equal to that of PEG-AMB-LIP.     

Comparative study on the efficacy of AmBisome and Fungizone in a mouse model of pulmonary aspergillosis

OBJECTIVES: The aim of this study was to evaluate the efficacy and tissue concentration of AmBisome and Fungizone in murine pulmonary aspergillosis, and to investigate the localization of AmBisome at the infection site. METHODS: Mice were infected intratracheally with Aspergillus fumigatus. A single dose of each of the antifungals was administered intravenously 4 h after infection. The efficacy of the antifungal treatment was assessed by the pulmonary fungal burden at 20 h post-treatment and the survival time over 1 month. The pulmonary amphotericin B (AMB) concentration was measured until 48 h after administration. The distribution of AmBisome in the lung was evaluated using rhodamine-labelled AmBisome and an anti-AMB antibody. RESULTS: AmBisome at a dose of > or =1 mg/kg significantly prolonged the survival time of infected mice compared with the control group. At the maximum tolerated dose, 10 mg/kg AmBisome exhibited greater efficacy than 1 mg/kg Fungizone in terms of increasing survival and reducing the fungal burden. The pulmonary AMB concentration of 10 mg/kg AmBisome was higher than that of 1 mg/kg Fungizone. Tissue distribution analysis showed that AmBisome was localized at the infection site in the lung, and this might explain the potent in vivo efficacy in this infection model. CONCLUSIONS: AmBisome is localized at the infection site in the lung and consequently may fully exhibit its in vivo activity. The efficacy of AmBisome is superior to that of Fungizone against pulmonary aspergillosis.     

Efficacy of NS-718, a Novel Lipid Nanosphere-Encapsulated Amphotericin B, against Cryptococcus neoformans

In vitro and in vivo efficacies of NS-718, a lipid nanosphere-encapsulated amphotericin B (AMPH-B), have been studied. Of the tested AMPH-B formulations, NS-718 had the lowest MIC for Cryptococcus neoformans. In a murine model, low-dose therapy (0.8 mg/kg of body weight) with NS-718 showed higher efficacy than that with AmBisome. High-dose therapy (2.0 mg/kg) with NS-718 was much more effective than those with Fungizone and AmBisome. In mice treated with a high dose of NS-718, only a few yeast cells had grown in lung by 7 days after inoculation. A pharmacokinetic study showed higher concentrations of AMPH-B in lung following administration of NS-718 than after administration of AmBisome. Our results indicated that NS-718, a new AMPH-B formulation, is a promising antifungal agent for treatment of pulmonary cryptococcosis and could be the most effective antifungal agent against C. neoformans infections.     

Antileishmanial Activity,Uptake, and Biodistribution of an Amphotericin B and Poly(α-Glutamic Acid) Complex

A noncovalent, water-soluble complex of amphotericin B (AMB) and poly(α-glutamic acid) (PGA), with AMB loadings ranging from 25 to 55% (wt/wt) using PGA with a molecular weight range of 50,000 to 70,000, was prepared as a potential new treatment for visceral leishmaniasis (VL). The AMB-PGA complex was shown to be as active as Fungizone (AMB deoxycholate) against intracellular Leishmania donovani amastigotes in differentiated THP-1 cells. The in vitro uptake of the AMB-PGA complex by differentiated THP-1 cells was similar to that of Fungizone and higher than that of AmBisome (liposomal AMB). The AMB-PGA complex also displayed a dose-response profile similar to that of AmBisome in vivo in BALB/c mice against L. donovani, with 50% effective doses (ED₅₀s) of 0.24 ± 0.03 mg/kg of body weight for the AMB-PGA complex and 0.24 ± 0.06 mg/kg for AmBisome. A biodistribution study with mice indicated that the AMB-PGA complex cleared more rapidly from plasma than AmBisome, with a comparable low level of distribution to the kidneys.     

Comparison of In Vitro Antifungal Activities of Free and Liposome-Encapsulated Nystatin with Those of Four Amphotericin B Formulations

The in vitro activity of a multilamellar liposomal formulation of nystatin (Nyotran) was compared with those of free nystatin and four pharmaceutical preparations of amphotericin B. MICs for 200 isolates of two Aspergillus spp., seven Candida spp., and Cryptococcus neoformans were determined by a broth microdilution adaptation of the method recommended by the National Committee for Clinical Laboratory Standards. Minimum lethal concentrations (MLCs) of the six antifungal preparations were also determined. Both nystatin formulations possessed fungistatic and fungicidal activities against the 10 species tested. Liposomal nystatin appeared to be as active as free nystatin, with MICs and MLCs that were similar to, or lower than, those of the latter. Neither formulation of nystatin was as active as amphotericin B deoxycholate (Fungizone) or amphotericin B lipid complex (Abelcet), but both were more effective than liposomal amphotericin B (AmBisome). Our results suggest that further evaluation of liposomal nystatin is justified.     

Single-dose AmBisome (Liposomal amphotericin B) as prophylaxis for murine systemic candidiasis and histoplasmosis

AmBisome is a liposomal formulation of amphotericin B that has broad-spectrum antifungal activity and greatly reduced toxicity compared to the parent drug. In this study, amphotericin B deoxycholate (Fungizone) (1 mg/kg) and AmBisome (1 to 20 mg/kg) were tested as single-dose prophylactic agents in both immunocompetent and immunosuppressed C57BL/6 mice challenged with either Candida albicans or Histoplasma capsulatum. Prophylactic efficacy was based on survival and fungal burden in the target organ (kidneys or spleen). At 9 to 10 days after histoplasma challenge, 80 to 90% of both immunocompetent and immunosuppressed mice in the control and Fungizone groups had died. All AmBisome-treated mice survived, although in the AmBisome groups given 1 mg/kg, the mice became moribund by day 10 to 12. No spleen CFU were detected in the histoplasma-challenged mice given 10 or 20 mg of AmBisome per kg. By 23 to 24 days after histoplasma challenge, fungal growth and/or death had occurred in all immunosuppressed mice except for four mice receiving 20 mg of AmBisome per kg. There were still no detectable fungi in the spleens of immunocompetent mice given 10 or 20 mg of AmBisome per kg. In the C. albicans experiment at 7 days postchallenge, all animals in both untreated and treated groups were alive with culture-positive kidneys. The kidney fungal burdens in AmBisome groups given 5 to 20 mg/kg were at least 1 log unit lower than those in the Fungizone group and significantly lower than those in the untreated control group (P < 0.05). There was a trend toward decreasing fungal growth in the kidneys as the dose of AmBisome was increased. In conclusion, these results show that a single high dose of AmBisome (5 to 20 mg/kg) had prophylactic efficacy in immunocompetent and immunosuppressed murine H. capsulatum and C. albicans models.     

Comparative studies on the efficacy of AmBisome and Fungizone in a mouse model of disseminated aspergillosis

OBJECTIVES: The efficacy of intravenous injections of a liposomal formulation of amphotericin B (AmBisome) and amphotericin B deoxycholate (Fungizone) was evaluated in immunocompetent and temporarily leucopenic mouse models of disseminated aspergillosis using seven isolates of Aspergillus. METHODS: Mice were infected with the organisms via tail veins. At 4 h after infection, antifungals were administered intravenously. For 30 days the number of mice surviving was recorded. RESULTS: AmBisome at 1 mg/kg or higher significantly prolonged the survival time of mice infected with five out of seven isolates of Aspergillus compared with the control group. There was no difference in in vivo activity between AmBisome and Fungizone at 1 mg/kg in six isolates of Aspergillus. At the maximum tolerated dose of antifungals, however, AmBisome (10 mg/kg) showed greater efficacy than Fungizone (1 mg/kg). CONCLUSIONS: These results suggest that the overall protective activity of AmBisome against disseminated aspergillosis is superior to that of Fungizone.     

Enhanced antifungal efficacy in experimental invasive pulmonary aspergillosis by combination of AmBisome with Fungizone as assessed by several parameters of antifungal response

In common with a proportion of patients with invasive pulmonary aspergillosis (IPA), the efficacy of AmBisome treatment regimens in our rat model remains suboptimal. To investigate whether this might be the result of initially low antifungal activity of amphotericin B at the site of infection when administered in the liposomal form, Fungizone was added to AmBisome at the start of treatment. Groups of granulocytopenic rats with left-sided IPA received 10 day treatment regimens with either AmBisome 10 mg/kg/day (n = 25) or AmBisome 10 mg/kg/day combined with a single dose of Fungizone 1 mg/kg at day 1 (n = 27). Parameters of treatment response included survival, serum galactomannan (GM), size and quality of pulmonary macroscopic lesions, lung weight, viable fungal counts (cfu) and chitin content of the infected lung, and extra-pulmonary disseminated fungal infection. In a separate experiment the significance of early start of treatment to obtain therapeutic efficacy was investigated. Compared with untreated controls, both treatment regimens showed a significant increase in survival and change in parameters of fungal infection except left lung cfu. The combination treatment showed a significant increase in survival compared with AmBisome monotherapy (P = 0.02) and a significant decrease in left lung chitin content (P = 0.03). Differences in circulating GM concentrations between the two treatment regimes approached significance (P = 0.06). Delay in the start of treatment from 16 to 24 h after fungal inoculation resulted in a significant decrease in therapeutic efficacy (P = 0.02). It is concluded that the efficacy of AmBisome therapy can be enhanced by the addition of Fungizone at the start of treatment. This is probably a result of active amphotericin B being immediately available in the lung at the start of treatment.     

Fungicidal Mechanisms of Cathelicidins LL-37 and CATH-2 Revealed by Live-Cell Imaging

Antifungal mechanisms of action of two cathelicidins, chicken CATH-2 and human LL-37, were studied and compared with the mode of action of the salivary peptide histatin 5 (Hst5). Candida albicans was used as a model organism for fungal pathogens. Analysis by live-cell imaging showed that the peptides kill C. albicans rapidly. CATH-2 is the most active peptide and kills C. albicans within 5 min. Both cathelicidins induce cell membrane permeabilization and simultaneous vacuolar expansion. Minimal fungicidal concentrations (MFC) are in the same order of magnitude for all three peptides, but the mechanisms of antifungal activity are very different. The activity of cathelicidins is independent of the energy status of the fungal cell, unlike Hst5 activity. Live-cell imaging using fluorescently labeled peptides showed that both CATH-2 and LL-37 quickly localize to the C. albicans cell membrane, while Hst5 was mainly directed to the fungal vacuole. Small amounts of cathelicidins internalize at sub-MFCs, suggesting that intracellular activities of the peptide could contribute to the antifungal activity. Analysis by flow cytometry indicated that CATH-2 significantly decreases C. albicans cell size. Finally, electron microscopy showed that CATH-2 affects the integrity of the cell membrane and nuclear envelope. It is concluded that the general mechanisms of action of both cathelicidins are partially similar (but very different from that of Hst5). CATH-2 has unique features and possesses antifungal potential superior to that of LL-37.     

Activity of a Heat-Induced Reformulation of Amphotericin B Deoxycholate (Fungizone) against Leishmania donovani

The heat treatment of amphotericin B deoxycholate (Fungizone), which was previously shown to induce superaggregation and decrease the toxicity of the drug to mammalian cells, increased its activity against Leishmania donovani in BALB/c mice, whereas it reduced its toxicity. Heat treatment preserved the activity of Fungizone against L. donovani HU3-infected mouse peritoneal macrophages.     

High-Throughput Screening of a Collection of Known Pharmacologically Active Small Compounds for Identification of Candida albicans Biofilm Inhibitors

Candida albicans is the most common etiologic agent of systemic fungal infections with unacceptably high mortality rates. The existing arsenal of antifungal drugs is very limited and is particularly ineffective against C. albicans biofilms. To address the unmet need for novel antifungals, particularly those active against biofilms, we have screened a small molecule library consisting of 1,200 off-patent drugs already approved by the Food and Drug Administration (FDA), the Prestwick Chemical Library, to identify inhibitors of C. albicans biofilm formation. According to their pharmacological applications that are currently known, we classified these bioactive compounds as antifungal drugs, as antimicrobials/antiseptics, or as miscellaneous drugs, which we considered to be drugs with no previously characterized antifungal activity. Using a 96-well microtiter plate-based high-content screening assay, we identified 38 pharmacologically active agents that inhibit C. albicans biofilm formation. These drugs were subsequently tested for their potency and efficacy against preformed biofilms, and we identified three drugs with novel antifungal activity. Thus, repurposing FDA-approved drugs opens up a valuable new avenue for identification and potentially rapid development of antifungal agents, which are urgently needed.     

Antifungal Activity of 3′-Deoxyadenosine (Cordycepin)

The antifungal activity of the nucleoside analog 3′-deoxyadenosine (cordycepin) was studied in a murine model of invasive candidiasis. When protected from deamination by either deoxycoformycin or coformycin, both of which are adenosine deaminase inhibitors, cordycepin exhibited potent antifungal efficacy, as demonstrated by prolongation of survival and a decrease in CFU in the kidneys of mice treated with cordycepin plus an adenosine deaminase inhibitor. The antifungal effect was seen with three different Candida isolates: Candida albicans 64, a relatively fluconazole-resistant clinical isolate of C. albicans (MIC, 16 μg/ml), and the fluconazole-resistant Candida krusei. Cordycepin and related compounds may provide another avenue for the discovery of clinically useful antifungal drugs.     

Efficacy of SPK-843, a novel polyene antifungal, in comparison with amphotericin B, liposomal amphotericin B, and micafungin against murine pulmonary aspergillosis

SPK-843, a new polyene antifungal, exhibited dose-dependent efficacy on murine pulmonary aspergillosis models. SPK-843 doses of higher than 1.0 mg/kg of body weight exhibited no renal toxicities and a tendency toward better survival prolongation than the estimated maximum tolerated doses of amphotericin B (Fungizone) (1.0 mg/kg) and liposomal amphotericin B (AmBisome) (8.0 mg/kg).     

Comparative efficacies of four amphotericin B formulations--Fungizone, amphotec (Amphocil), AmBisome, and Abelcet--against systemic murine aspergillosis

We compared various amphotericin B formulations (no treatment or 0.8 mg of Fungizone [conventional deoxycholate amphotericin B] per kg of body weight, or 0.8, 4, or 8 mg of Amphocil, AmBisome, or Abelcet per kg of body weight) for treatment of systemic murine aspergillosis. In two studies, all formulations prolonged survival, with the results for AmBisome nearly equivalent to those for Fungizone; Amphocil and Abelcet were less effective or equivalent depending on the severity of infection. No survivors were cured in both kidneys and brain, but each formulation showed efficacy, especially in the kidneys. Although higher doses could be given, no lipid-based formulation showed consistent superiority over Fungizone or over each other.     

Decreased Accumulation or Increased Isoleucyl-tRNA Synthetase Activity Confers Resistance to the Cyclic β-Amino Acid BAY 10-8888 in Candida albicans and Candida tropicalis

BAY 10-8888, a cyclic β-amino acid, exerts its antifungal activity by inhibition of isoleucyl-tRNA synthetase activity after accumulation to a millimolar concentration inside the cell. We have selected and characterized BAY 10-8888-resistant Candida albicans mutants. Reduced BAY 10-8888 accumulation as well as increased isoleucyl-tRNA synthetase activity was observed in these mutants. Some of the mutants were cross-resistant to cispentacin, a structurally related β-amino acid, while sensitivities to 5-fluorocytosine and fluconazole remained unchanged in all mutants. All except two in vitro-resistant mutants were pathogenic in a murine candidiasis model, and BAY 10-8888 failed to cure the infection. Furthermore, we have characterized BAY 10-8888 transport and isoleucyl-tRNA synthetase activity in several Candida tropicalis strains which showed MICs higher than those of other Candida strains. An analysis of the C. tropicalis strains revealed that intracellular concentrations of BAY 10-8888 were in the millimolar range, comparable to those for C. albicans. However, these isolates expressed isoleucyl-tRNA synthetase activities about fourfold higher than those for C. albicans. To test the possibility of resistance modeling, we determined the correlations between the intracellular concentration of BAY 10-8888, the specific activity of isoleucyl-tRNA synthetase, the number of free, i.e., noninhibited, isoleucyl-tRNA synthetase molecules/cell, and growth, assuming a linear relation. We found significant correlations between growth and the intracellular concentration of BAY 10-8888 and between growth and the number of free isoleucyl-tRNA synthetase molecules/cell, but not between growth and the specific activity of isoleucyl-tRNA synthetase.     

Paradoxical Growth of Candida albicans in the Presence of Caspofungin Is Associated with Multiple Cell Wall Rearrangements and Decreased Virulence

In the last decade, echinocandins have emerged as an important family of antifungal drugs because of their fungicidal activity against Candida spp. Echinocandins inhibit the enzyme β-1,3-d-glucan synthase, encoded by the FKS genes, and resistance to echinocandins is associated with mutations in this gene. In addition, echinocandin exposure can produce paradoxical growth, defined as the ability to grow at high antifungal concentrations but not at intermediate concentrations. In this work, we have demonstrated that paradoxical growth of Candida albicans in the presence of caspofungin is not due to antifungal degradation or instability. Media with high caspofungin concentrations recovered from wells where C. albicans showed paradoxical growth inhibited the growth of a Candida krusei reference strain. Cells exhibiting paradoxical growth at high caspofungin concentrations showed morphological changes such as enlarged size, abnormal septa, and absence of filamentation. Chitin content increased from the MIC to high caspofungin concentrations. Despite the high chitin levels, around 23% of cells died after treatment with caspofungin, indicating that chitin is required but not sufficient to protect the cells from the fungicidal effect of caspofungin. Moreover, we found that after paradoxical growth, β-1,3-glucan was exposed at the cell wall surface. Cells grown at high caspofungin concentrations had decreased virulence in the invertebrate host Galleria mellonella. Cells grown at high caspofungin concentrations also induced a proinflammatory response in murine macrophages compared to control cells. Our work highlights important aspects about fungal adaptation to caspofungin, and although this adaptation is associated with reduced virulence, the clinical implications remain to be elucidated.