Reduced toxicity of amphotericin B methyl ester (AME) vs. amphotericin B and fungizone in tissue culture.

The comparative toxicities of amphotericin B methyl ester (AME), the parent antibiotic amphotericin B (AB), and the deoxycholate solubilized complex of AB, Fungizone (FZ), toward five cell lines has been determined as measured by early membrane damage (51Cr release), 24 hr survival, 72 hr viability, and growth rate. Cells used were of turtle (TH-1), marsupial (PT K2), human MA 160), rabbit (RK-13) and hamster (BHK-21) origin. AME: (a) caused less membrane damage at 1 hr than AB or FZ; (b) was less toxic than AB or FZ as indicated by 24 hr cell survival and 72 hr cell viability; and (c) was required in higher levels than AB or FZ to reduce the growth rate of all five cell lines. Spectrophotometric analysis of residual polyene levels indicated that AME had good stability in tissue culture medium. Previous studies have indicated that AME has the same in vitro antifungal activity as the parent antibiotic AB (1, 2). These findings suggest that AME may prove to be superior to AB and FZ for use as an antifungal agent in tissue culture systems.     

The effect of fetal bovine serum on polyene macrolide antibiotic cytotoxicity and antifungal activity

Summary The relationships between fetal bovine serum (FBS) concentration and polyene macrolide antibiotic cytotoxicity to animal cells and to fungi were evaluated. The toxicity of amphotericin B (AB) and its derivative, amphotericin B methyl ester (AME), toward KB cells was found to be directly related to fetal bovine serum concentration. At higher FBS levels, increased concentrations of AB and AME were required to reduce 72-hr KB viable cell numbers to 50% of control values. Similarly, polyene macrolide antibiotic levels required to inhibit the growth ofSaccharomyces cerevisiae to 50% of controls, and for obtaining minimum fungicidal concentrations (MFC), were greater when higher levels of FBS were used. In addition, AME was less toxic than AB toward KB cells grown in media containing 2, 5, 10, 15 or 20% FBS, whereas the antifungal activities of AB and AME were similar. AME was also capable of eliminatingCandida albicans, Saccharomyces cerevisiae, Aspergillus niger orFusarium moniliforme from KB cultures at antibiotic levels which exhibited less cell toxicity than did the concentrations of AB required for a similar response. These findings indicate that AME may be a potentially useful antifungal antibiotic for tissue culture systems. Portions of this paper were presented at the 25th Annual Meeting of the Tissue Culture Association at Miami, Florida, 1974. This investigation was supported in part by contract NIH 69-2161, NIH grant no. AI-02095 and NIH training grant no. GM 507 from the National Institute of General Medical Sciences.     

Effect of nystatin,amphotericin B and amphotericin B methyl ester on Saccharomyces cerevisiae with different lipid composition

Saccharomyces cerevisiae was cultured under anaerobiosis in semi-complete medium to which either palmitoleic or oleic acid was added. Cells were grown at 20 °C or 30 °C. The levels of total lipids, total sterols, and phospholipids were higher in cells grown at 20 °C than at 30 °C. The effects of nystatin (NYS), amphotericin B (AMB), and amphotericin B methyl ester (AME) were evaluated by determining cell viability and liberation of intracellular compounds. The loss of cell viability is higher in the first 30 minutes of incubation with the drugs and is the same regardless of the type of cells obtained. Low molecular weight compounds and ions such as K⁺ are liberated a few minutes after incubation with the drugs whereas proteins and substances absorbing at 260 nm are liberated later. Phosphate liberation comes after K⁺ and before compounds of higher molecular weights.     

In vitro and in vivo antifungal activities of liposomal amphotericin B,and amphotericin B lipid complex

The in vitro and in vivo antifungal activities of liposomal amphotericin B (L-AMPH) and amphotericin B lipid complex (ABLC), which is composed of amphotericin B and the phospholipids dimyristoyl phosphatidylcholine and dimyristoyl phosphatidylglycerol, were compared with those of conventional amphotericin B (Fungizone®, AMPH). The acute intravenous toxicity was markedly lower in BALB/c mice; 50% lethal doses (LD_50s) were 2.75 mg/kg in AMPH, 32.9 mg/kg in L-AMPH and >75 mg/kg in ABLC. In vitro antifungal activities againstCandida albicans, C. parapsilosis, C. tropicalis, C. glabrata, andC. krusei were evaluated by the agar plate dilution method. The activities were unchanged againstC. albicans, but MICs increased more than four fold in 18 of the 20 strains other thanC. albicans in L-AMPH and in 9 of the 20 in ABLC. L-AMPH and ABLC were as efficacious as AMPH in the treatment of mice infected withC. albicans, and at a dose of 0.5 and 1.0 mg/kg of body weight, ABLC was more efficacious on survival. A ten-times larger dose (10 mg/kg) of L-AMPH and ABLC was administered to mice with 100% survival, suggesting improved tolerability as compared to amphotericin B.     

Selection of mouse x hamster hybrids using hat medium and a polyene antibiotic

Summary In the present study we tested the feasibility of utilizing a structurally modified polyene antibiotic, amphotericin B methyl ester (AME), as a half-selection agent for isolating somatic cell hybrids. By using HAT medium supplemented with AME we have isolated interspecific mouse-hamster hybrids from mixed cultures of mouse (TK⁻C1ID or HPRT⁻ A9) and hamster (BHK/C 13) cells fused with Sendai virus, lysolecithin or polyethylene glycol. Hybrid cells proliferated and clones were isolated after 2 to 3 weeks growth in three changes of HAT-AME medium and subsequent growth in HAT medium alone. In contrast, genetically deficient parental C1 1D or A9 cells and AME-sensitive BHK/C 13 cells were killed using a similar growth protocol. The described technique is simple, efficient and permits one to use a cell line without a genetic defect in combination with a genetically deficient cell type in hybrid formation. This investigation was supported in part by Contract NIH 69-2161, NIH Grant No. AI-2095 and NIH Training Grant No. GM 507 from the National Institute of General Medical Sciences.     

Inhibition of HIV-1 replication by amphotericin B methyl ester: selection for resistant variants

Membrane cholesterol plays an important role in human immunodeficiency virus type 1 (HIV-1) particle production and infectivity. Here, we have investigated the target and mechanism of action of a cholesterol-binding compound, the polyene antifungal antibiotic amphotericin B methyl ester (AME). We found that AME potently inhibited the replication of a highly divergent panel of HIV-1 isolates in various T-cell lines and primary cells irrespective of clade or target cell tropism. The defects in HIV-1 replication caused by AME were due to profoundly impaired viral infectivity as well as a defect in viral particle production. To elucidate further the mechanism of action of AME, we selected for and characterized AME-resistant HIV-1 variants. Mutations responsible for AME resistance mapped to a highly conserved and functionally important endocytosis motif in the cytoplasmic tail of the transmembrane glycoprotein gp41. Interestingly, truncation of the gp41 cytoplasmic tail in the context of either HIV-1 or rhesus macaque simian immunodeficiency virus also conferred resistance to AME. The infectivity of HIV-1 virions bearing murine leukemia virus or vesicular stomatitis virus glycoproteins was unaffected by AME. Our data define the target and mechanism of action of AME and provide support for the concept that cholesterol-binding compounds should be pursued as antiretroviral drugs to disrupt HIV-1 replication.     

Comparative efficacy of fluconazole and amphotericin B in the parenteral treatment of experimental paracoccidioidomycosis in the rat

Patients with severe and complicated paracoccidioidomycosis are treated with amphotericin B by the intravenous route. Fluconazole is active in vitro against Paracoccidioides brasiliensis and can also be administered intravenously, but few clinical or experimental data are available about its action against the infection caused by this fungus. In the present study, the efficacy of fluconazole andamphotericin B was assessed comparatively in rats inoculated parenterally with P. brasiliensis. The treatment was performed 3 times a week for 4 weeks starting one week after infection. Fluconazole administered intraperitoneally (14 mg/kg bodyweight/dose) was more effective (P > 0.001)than amphotericin B (2 mg/kg body weight/dose) in reducing the number of colony forming units in the lungs and spleen. When administered intravenously at the dose of 3 mg/kg body weight, fluconazole was as effective as amphotericin B (0.8 mg/kg body weight) in reducing the pulmonary fungal burden. Under these conditions, the rats treated with fluconazole had a smaller number of colony forming units than untreated animals (P > 0.001), but amphotericin B was more effective than fluconazole in reducing spleen infection (P > 0.005). Except for this result obtained with a low dose, fluconazole showed an antifungal action equal to or higher than that of amphotericin B. The activity of fluconazole at doses equivalent to those used for human treatment suggests that this antifungal agent may be an alternative to amphotericin B for the early intravenous treatment of patients with paracoccidioidomycosis. This revised version was published online in June 2006 with corrections to the Cover Date.     

Antifungal pharmacodynamic characteristics of amphotericin B against Trichosporon asahii, using time-kill methodology

We determined the MIC of amphotericin B against 45 Trichosporon asahii isolates from various clinical and environmental sources, and used in vitro time-kill methods to characterize the relationship between amphotericin B concentrations and MIC for four representative T. asahii isolates. Amphotericin B had concentration-dependent antifungal activity. MICs ranged from 0.5 to 16 microg/ml, and most T. asahii isolates (76%, 34/45) were inhibited at safely achievable amphotericin B serum concentrations (< or = 2 microg/ml). However, 40% (18/45) of isolates were not killed at these concentrations (MFCs from 1.0 to 32 microg/ml). At concentrations > or = 2 x MIC, amphotericin B exhibited fungicidal activity (< 99.9% reduction in CFU) over a 12-hr time-period; the maximal effect was achieved at > or =4 x MIC. Susceptibility testing confirmed the resistance of T. asahii to amphotericin B, and in vitro pharmacodynamic results also suggest that amphotericin B is not suitable therapy for T. asahii infection.     

In Vitro Amphotericin B Effects on Growth,Viability and Dimorphism of Paracoccidioides brasiliensis: Reversal of the Treatment

The in vitro effects of amphotericin B deoxycholate suspension (fungizone) on Paracoccidioides brasiliensis growth, cell viability and transformation were investigated. We also analyzed the protein synthesis patterns of both cellular forms, yeast and mycelium in the presence of AmB. This drug, at 30 μg/ml, highly inhibited yeast growth, which could be recovered depending on treatment time, where the most effective reversion was observed after 6 hr of incubation. The yeast cell viability, that had been partially affected by the drug, could also be efficiently recovered after AmB was removed. The effect of AmB on the cellular dimorphism process showed a strong reduction in the mycelium to yeast transformation (80% inhibition compared to the control without the drug). On the other hand, the transformation from yeast to mycelium in the presence of AmB was 50% affected, relative to the control. In contrast to the growth and cell viability experiments, the reversion effects on dimorphism were partial when the drug was removed, even with only 6 hr treatment. The two-dimensional gels of ³⁵S-labeled proteins revealed a strong reduction in the three species of 80, 71 and 56 kDa in yeast and mycelium when treated with AmB.     

Enhancing the fungicidal activity of amphotericin B via vacuole disruption by benzyl isothiocyanate,a cruciferous plant constituent

Amphotericin B (AmB), a typical polyene macrolide antifungal agent, is widely used to treat systemic mycoses. In the present study, we show that the fungicidal activity of AmB was enhanced by benzyl isothiocyanate (BITC), a cruciferous plant-derived compound, in the budding yeast, Saccharomyces cerevisiae. In addition to forming a molecular complex with ergosterol present in fungal cell membranes to form K⁺-permeable ion channels, AmB has been recognized to mediate vacuolar membrane disruption resulting in lethal effects. BITC showed no effect on AmB-induced plasma membrane permeability; however, it amplified AmB-induced vacuolar membrane disruption in S. cerevisiae. Furthermore, the BITC-enhanced fungicidal effects of AmB significantly decreased cell viability due to the disruption of vacuoles in the pathogenic fungus Candida albicans. The application of the combinatorial antifungal effect of AmB and BITC may aid in dose reduction of AmB in clinical antifungal therapy and consequently decrease side effects in patients. These results also have significant implications for the development of vacuole-targeting chemotherapy against fungal infections.     

Comparative in vitro studies on liposomal formulations of amphotericin B and its derivative,N-methyl-N-D-fructosyl amphotericin B methyl ester (MFAME)

N-Methyl-N-D-fructosyl amphotericin B methyl ester (MFAME) is a semisynthetic derivative of the antifungal antibiotic amphotericin B (AMB). In contrast to the parent antibiotic, the derivative is characterised by low toxicity to mammalian cells and good solubility in water of its salts. Comparative studies on biological properties of free MFAME, AMB and their liposomal formulations were performed. To obtain liposomal forms, the antibiotics were incorporated into small unilamellar vesicles composed of dimyristoyl phosphatidylcholine (DMPC) and DMPC:cholesterol or ergosterol, 8:2 molar ratio. The effectivity of the liposomal and free forms of AMB and MFAME were compared by determination of fungistatic and fungicidal activity against Candida albicans ATCC 10261, potassium release from erythrocytes, and haemolysis. The results obtained indicate that in contrast to AMB, incorporation of MFAME into liposomes did not further improve its selective toxicity. Studies on the antagonistic effect of ergosterol and cholesterol on the antifungal activity of the antibiotics indicated that sterol interference was definitely less pronounced in the case of MFAME than in the case of AMB.     

Treatment of systemic candidiasis in a neutropenic murine model using immunoglobulin G bearing liposomal amphotericin B

Efficacy of immunoglobulin G (IgG) bearing liposomal amphotericin B (LAMB-IgG), liposomal amphotericin B without IgG (LAMB) or free amphotericin B (fAMB/Fungizone) was investigated in the treatment of systemic candidiasis in a neutropenic mouse model. Treatment with a single dose (0.6 or 0.9 mg amphotericin B per kg body weight) of LAMB-IgG resulted in a significant increase in the survival rate of neutropenic mice infected with 3×10⁵ cfu ofCandida albicans compared to untreated controls, mice injected with IgG, or liposome alone. Survival was also better in neutropenic mice treated with LAMB-IgG than in neutropenic mice treated with the same dose of LAMB or fAMB. Moreover, 65% of all mice survived the infection after treatment with a single dose of 0.6 mg AMB of the LAMB-IgG formulation. Quantitative culture counts of organs showed that both fAMB and LABM-IgG formulations even at a dose of 0.3 mg AMB/kg, clearedC. albicans from the spleens, livers, and lungs but not from the kidneys. However, a decreasd number ofC. albicans cells was recovered from the kidneys of mice that survived the infection. Results of the study suggest that LAMB-IgG is more effective than LAMB or fAMB in the therapy of disseminated candidiasis in neutropenic mice.     

The relationship between adjuvant and mitogenic effects of amphotericin methyl ester

The antifungal polyene amphotericin B (AmB) and its methyl ester derivative (AME) both show potent murine immunostimulant as well as B-cell activating effects. Under certain experimental conditions, AME is a much more potent polyclonal B-cell activator (PBA) than AmB. Notable features of the murine B-cell stimulation induced by AME include: (i) High concentrations of AME (50-100 microgram/ml) are required and even at this level exhibit little or no spleen cell toxicity. (ii) Several lines of evidence suggest that the B-cell activating properties of AME are not involved in the cellular mechanism of adjuvant activity in vivo. (iii) There is a strong correlation between the magnitude of the in vitro PBA effects and the in vivo adjuvant effects of AME in a survey of different mouse strains. This evidence suggests that there is genetic control of the murine lymphoid cell-stimulatory effects of AME and that a small number of genes determines the responsive phenotype.     

Evaluation of antifungal pharmacodynamic characteristics of AmBisome against Candida albicans

A liposomal formulation of Amphotericin B (AmBisome), with small unilamellar vesicles containing amphotericin B, shows characteristic pharmacokinetics as liposomes, and in consequence, has different pharmacological activity and toxicity from amphotericin B deoxycholate (Fungizone). In this study, we evaluated the antifungal pharmacodynamic characteristics of AmBisome against Candida albicans using the in vitro time-kill method and murine systemic infection model. A time-kill study indicated that the in vitro fungicidal activities of AmBisome and Fungizone against C. albicans ATCC 90029 increased with increasing drug concentration. For in vivo experiments, leucopenic mice were infected intravenously with the isolate 4 hr prior to the start of therapy. The infected mice were treated for 24 hr with twelve dosing regimens of AmBisome administered at 8-, 12-, 24-hr dosing intervals. Correlation analysis between the fungal burden in the kidney after 24 hr of therapy and each pharmacokinetic/pharmacodynamic parameter showed that the peak level/MIC ratio was the best predictive parameter of the in vivo outcome of AmBisome. These results suggest that AmBisome, as well as Fungizone, has concentration-dependent antifungal activity. Furthermore, since AmBisome can safely achieve higher concentrations in serum than Fungizone, AmBisome is thought to have superior potency to Fungizone against fungal infections.     

Synergistic anticandidal activity of xanthorrhizol in combination with ketoconazole or amphotericin B

Candida species are responsible for the fourth most common nosocominal bloodstream infection. Xanthorrhizol, a sesquiterpene compound isolated from Curcuma xanthorrhiza Roxb. has been reported to have anticandidal activity. The aim of this study is to investigate the synergistic anticandidal effect of xanthorrhizol in combination with ketoconazole or amphotericin B against Candida albicans, Candida glabrata, Candida guilliermondii, Candida krusei, Candida parapsilosis , and Candida tropicalis . Mostly, xanthorrhizol in combination with ketoconazole or amphotericin B exhibited the synergistic anticandidal effects against all species of Candida tested. In combination with xanthorrhizol, the concentration of ketoconazole or amphotericin B for inhibiting the growth of the tested Candida species could be reduced by ≥50%. Time–kill curves showed that 1/2 minimum inhibitory concentration (MIC) dose of xanthorrhizol, amphotericin B, or ketoconazole alone against each of the six Candida species did not inhibit the growth of all Candida species tested. However, 1/2 MIC dose of xanthorrhizol in combination with 1/2 MIC dose of ketoconazole or 1/2 MIC dose of amphotericin B exhibited growth inhibition of all Candida species tested and reduced viable cells by several logs within 4 h. These results support the potential use of xanthorrhizol as an anticandidal agent, and it can be used complementarily with other conventional antifungal agents.     

Optimizing efficacy of amphotericin B through nanomodification

Fungal infections and leishmaniasis are an important cause of morbidity and mortality in immunocompromised patients. The macrolide polyene antibiotic amphotericin B (AmB) has long been recognized as a powerful fungicidal and leishmanicidal drug. A conventional intravenous dosage form of AmB, AmB- deoxycholate (Fungizone or D-AmB), is the most effective clinically available for treating fungal and parasitic (leishmaniasis) infections. However, the clinical efficacy of AmB is limited by its adverse effects mainly nephrotoxicity. Efforts to lower the toxicity are based on synthesis of AmB analogues such as AmB esters or preparation of AmB-lipid associations in the forms of liposomal AmB (L-AmB or AmBisome), AmB lipid complex (Abelcet or ABLC), AmB colloidal dispersion (Amphocil or ABCD), and intralipid AmB. These newer formulations are substantially more expensive, but allow patients to receive higher doses for longer periods of time with decreased renal toxicity than conventional AmB. Modifications of liposomal surface in order to avoid RES uptake, thus increased targetability has been attempted. Emulsomes and other nanoparticles are special carrier systems for intracellular localization in macrophage rich organs like liver and spleen. Injectable nano-carriers have important potential applications as in site-specific drug delivery.     

Polyene macrolide antibiotic cytotoxicity and membrane permeability alterations I. Comparative effects of four classes of polyene macrolides on mammalian cells

The relationship between polyene macrolide-induced early membrane damage and cytotoxicity in B1 (hamster), B82 (mouse), and RAG (mouse) cells has been investigated. Filipin (FIL) induced the greatest immediate damage, as monitored by ⁵¹Cr release, followed by mediocidin (MED), amphotericin B-deoxycholate (Fungizone®) (FZ) and pimaricin (PIM). For long term effect, PIM was the least toxic followed by MED, FZ, and FIL as indicated by 24-hour survival, 72-hour viability, and growth rate of cells. In evaluating polyene macrolide-induced permeability alterations and cytotoxicity two types of interactions with mammalian cells were found: (1) cell toxicity at polyene macrolide levels not eliciting immediate membrane permeability changes; and (2) immediate membrane damage without long range toxicity.     

The effect of amphotericin b-deoxycholate on proliferation and protein synthesis in human skin fibroblast cultures

Summary Cultures of adult human skin fibroblasts were grown in the presence of the recommended antifungal dose (3 μg per ml) of amphotericin B-deoxycholate. A reduction in cell culture growth, measured as DNA content and protein content per culture, was observed. However, radioisotope incorporation into noncollagen protein and, to a lesser extent, collagen protein was enhanced. These effects were due to amphotericin B, not to deoxycholate. These observations were made under several growth conditions and indicate that cell proliferation or isotope-labeling studies in fibroblasts in the presence of amphotericin B-deoxycholate are susceptible to errors in interpretation. Supported by PHS Grants AM-02456, AM-15312 and AM-17047, by the Kroc Foundation, and by the American Diabetes Association, Washington Affiliate. Recipient of Research Career Development Award AM-47142 from NIAMDD, and to whom requests for reprints should be addressed.     

The use of amphotericin B to detect inhibitors of cellular cholesterol biosynthesis

Pores formed in the membranes of animal cells by complexes of sterols and the polyene antibiotic amphotericin B can efficiently kill the cells. Thus, in the absence of exogenous sources of cholesterol, inhibitors of enzymes in the cholesterol biosynthetic pathway render cells resistant to amphotericin B. Preincubation of Chinese hamster ovary cells with compactin or 25-hydroxycholesterol, inhibitors of the synthesis of the key intermediate mevalonate, protected cells from amphotericin B killing and this protection was reversed by the addition of exogenous mevalonate. The ability of compactin to confer amphotericin B resistance on normal cells was abolished when cells were provided exogenous cholesterol by the receptor-mediated endocytosis of low density lipoprotein. Low density lipoprotein receptor-defective Chinese hamster ovary cells were not subject to this low density lipoprotein-dependent amphotericin B killing. Exogenous mevalonate did not prevent 4,4,10 beta-trimethyl-trans-decal-3 beta-ol, an inhibitor of mevalonate conversion to sterols, from protecting cells from amphotericin B. A simple two-step protocol in which cells are preincubated (15-24 h) with potential inhibitors and then treated (3-6 h) with amphotericin B was devised to provide a sensitive method for detecting direct (e.g., competitive) and regulatory inhibitors of cholesterol biosynthesis. This protocol may prove useful in detecting potential antihypercholesterolemia drugs and is currently being used to isolate mutants in receptor-mediated endocytosis.     

Transient permeability induced by alkyl derivatives of amphotericin B in lipid membranes

Individual ionic channels were shown to be formed in the brain cholesterol containing phospholipid membranes by two-sided addition of the amphotericin B alkyl derivatives. At concentrations between 10⁻⁸ and 10⁻⁷ M, the resulting conductance appeared to be transient. Existence of different antibiotic assemblies was justified by the kinetic analysis of the membrane conductance decline following the antibiotic washing out. In order to account for the transient characteristics of the induced conductance, it was proposed that the antibiotic oligomers incorporate into the membrane from the aqueous phase, form channels aggregating with cholesterol, and then dissociate in the bilayer into non-active degraded oligomeric or monomeric forms.