Amphotericin B (AMB) is a polyene antibiotic with broad spectrum antifungal activity, but its clinical toxicities and poor solubility limit the wide application of AMB in clinical practice. Recently, new drug-loaded nanoparticles (NPs) - diblock copolymer D-α-tocopheryl polyethylene glycol 1000 succinate-b-poly(ε-caprolactone-ran-glycolide) (PLGA-TPGS) - have received special attention for their reduced toxicity, and increased effectiveness of drug has also been reported. This study aimed to develop AMB-loaded PLGA-TPGS nanoparticles (AMB-NPs) and evaluate their antifungal effects in vitro and in vivo.

Cryptococcus neoformans is an encapsulated yeast responsible for approximately a quarter of a million deaths worldwide annually despite therapy, and upwards of 11% of HIV/AIDS-related deaths, rivaling the impact of tuberculosis and malaria. However, the most effective antifungal agent, amphotericin B, requires intravenous delivery and has significant renal and hematopoietic toxicity, making it difficult to utilize, especially in resource-limited settings. The present studies describe a new nanoparticle crystal encapsulated formulation of amphotericin B known as encochleated amphotericin B (CAmB) that seeks to provide an oral formulation that is low in toxicity and cost. Using a 3-day delayed model of murine cryptococcal meningoencephalitis and a large inoculum of a highly virulent strain of serotype A C. neoformans, CAmB, in combination with flucytosine, was found to have efficacy equivalent to parental amphotericin B deoxycholate with flucytosine and superior to oral fluconazole without untoward toxicity. Transport of fluorescent CAmB particles to brain as well as significant brain levels of amphotericin drug was demonstrated in treated mice, and immunological profiles were similar to those of mice treated with conventional amphotericin B. Additional toxicity studies using a standardized rat model showed negligible toxicity after a 28-day treatment schedule. These studies thus offer the potential for an efficacious oral formulation of a known fungicidal drug against intrathecal cryptococcal disease.IMPORTANCECryptococcus neoformans is a significant global fungal pathogen that kills an estimated quarter of a million HIV-infected individuals yearly and has poor outcomes despite therapy. The most effective therapy, amphotericin B, is highly effective in killing the fungus but is available only in highly toxic, intravenous formulations that are unavailable in most of the developing world, where cryptococcal disease in most prevalent. For example, in Ethiopia, reliance on the orally available antifungal fluconazole results in high mortality, even when initiated as preemptive therapy at the time of HIV diagnosis. Thus, alternative agents could result in significant saving of lives. Toward this end, the present work describes the development of a new formulation of amphotericin B (CAmB) that encapsulates the drug as a crystal lipid nanoparticle that facilitates oral absorption and prevents toxicity. Successful oral absorption of the drug was demonstrated in a mouse model that, in combination with the antifungal flucytosine, provided efficacy equal to a parental preparation of amphotericin B plus flucytosine. These studies demonstrate the potential for CAmB in combination with flucytosine to provide an effective oral formulation of a well-known, potent fungicidal drug combination.

The rise of drug-resistant fungal pathogens urges for the development of new tools for the discovery of novel antifungal compounds. Polyene antibiotics are potent agents against fungal infections in humans and animals. They inhibit the growth of fungal cells by binding to sterols in the cytoplasmic membrane that subsequently causes pore formation and eventually results in cell death. Many polyenes are produced by Streptomycetes and released into the soil environment, where they can then target fungal hyphae. While not antibacterial, these compounds could nevertheless be also perceived by bacteria sharing the same habitat and serve as signaling molecules. We therefore addressed the question of how polyenes such as amphotericin B are perceived by the soil bacterium, Bacillus subtilis. Global transcriptional profiling identified a very narrow and specific response, primarily resulting in strong upregulation of the lnrLMN operon, encoding an ABC transporter previously associated with linearmycin resistance. Its strong and specific induction prompted a detailed analysis of the lnrL promoter element and its regulation. We demonstrate that the amphotericin response strictly depends on the two-component system LnrJK and that the target of LnrK-dependent gene regulation, the lnrLMN operon, negatively affects LnrJK-dependent signal transduction. Based on this knowledge, we developed a novel whole-cell biosensor, based on a P lnrL -lux fusion reporter construct in a lnrLMN deletion mutant background. This highly sensitive and dynamic biosensor is ready to be applied for the discovery or characterization of novel amphotericin-like polyenes, hopefully helping to increase the repertoire of antimycotic and antiparasitic polyenes available to treat human and animal infections.

Single, high-dose liposomal amphotericin B (LAmB; AmBisome, Gilead Sciences) has demonstrated non-inferiority to amphotericin B deoxycholate in combination with other antifungals for averting all-cause mortality from HIV-associated cryptococcal meningitis. There are limited data on the pharmacokinetics (PK) of AmBisome. The aim of this study was to describe population PK of AmBisome and conduct a meta-analysis of the available studies to suggest the optimal dosing for cryptococcal meningoencephalitis.

Immunomodulatory drugs (IMiDs) present one example of immunomodulatory agents that improve cancer immunotherapy. Based on the cytotoxic activity of natural killer (NK) cells against cancer cells, a high throughput screening method for the identification of novel immunomodulatory molecules with the potential to stimulate NK cell cytotoxicity against cancer cells was designed and tested using an approved drug library. Among the primary hit compounds, the anti-fungal drug amphotericin B (AMP-B) increased the cytotoxicity of NK cell line and human primary NK cells in a direct manner. The increase in NK cell activity was related to increased formation of NK-target cell conjugates and the subsequent granule polarization toward target cells. The results of the present study indicate that AMP-B could serve a dual function as an anti-fungal and immunomodulatory drug.

candidiasis is the primary fungal infection encountered in patients undergoing prolonged hospitalization, and the fourth leading cause of nosocomial bloodstream infections. One of the most important Candida spp. virulence factors is the ability to form biofilms, which are extremely refractory to antimicrobial therapy and very difficult to treat with the traditional antifungal therapies. It is known that the prophylaxis or treatment of a systemic candidiasis are recurrently taken without considering the possibility of a Candida spp. biofilm-related infections. Therefore, it is important to assess the effectiveness of the available drugs and which formulations have the best performance in these specific infections.

The present study was designed to develop a thermoreversible gel of Pluronic (P407) loaded amphotericin B (AmB-gel) for the dermal and vaginal treatment of candidiasis. P407 was used as a copolymer to exploit potential advantages related to increasing drug concentration in the tissue layer in order to provide a local effect. Parameters including internal structure, swelling, porosity, and short-term stability were determined. In addition, drug release profile and ex vivo skin and vaginal permeation studies were carried out. Antifungal efficacy was evaluated against strains of Candida spp. and atomic force microscopy (AFM) supported the results. The tolerance of AmB-gel was studied by evaluating biomechanical properties of skin and determining the irritation level in scarified rabbit skin supported by histological analysis. Results confirmed the development of a thermoreversible AmB-gel with high porosity exhibiting Newtonian behavior at 4 °C and pseudoplasticity at 32 °C as well as optimal stability for at least 90 days. The Amb-gel provided a sustained drug release following a Boltzmann sigmoidal model. Non permeation was observed in skin and vaginal mucosa, showing a high retained amount of AmB of 960.0 and 737.3 µg/g/cm2, respectively. In vitro antifungal efficacy showed that AmB-gel was more effective than Free-AmB in inhibiting strains of Candida spp. and these results were corroborated by AFM. Finally, tolerance studies showed that its application did not induce skin irritation nor alter its biophysical properties. Together, these results confirmed that AmB-gel could be proposed as a promising candidate for the clinical status in the treatment of skin and vaginal candidiasis.

Sodium alginate (SA) was oxidized using periodate and amphotericin B (AmB) was conjugated via imine and amine linkages to the oxidized alginate. Oxidization drastically reduced the molecular weight (MW) of the alginate. The conjugates were highly water-soluble to the extent of 1000mg/mL making them useful for therapeutic applications. SA-AmB conjugates derived from 20 and 50% oxidized alginate were non-toxic to HEK 293T and RAW 264.7 cell line at 100μg/mL and was also non-hemolytic to human blood at 100μg/mL. In vitro release of AmB into phosphate buffer from the imine conjugates was negligible with less than 0.2% of the drug released in 48h. Capping of residual aldehyde handles using 2-ethanolamine or glycine resulted in increased release of the drug in vitro. Injectable gels of gelatin crosslinked with oxidized alginate incorporating the SA-AmB conjugates as well as AmB were also fabricated and drug release was examined. In vitro release from the gel discs showed that AmB was released to the extent of 15-20% in 2days. The SA-AmB conjugates showed potent anti-fungal activity against C. albicans, C. neoformans and C. parapsilosis. The injectable gels seem to have potential for prolonged release of AmB when implanted.

Cryptococcus neoformans is one of the leading causes of invasive fungal infections worldwide. Cryptococcal meningoencephalitis is the main challenge of antifungal therapy due to high morbidity and mortality rates, especially in low- and middle-income countries. This can be partly attributed to the lack of specific diagnosis difficulty accessing treatment, antifungal resistance and antifungal toxicity.

Antibiotics play an important role in human health. Most antibiotics are derived from microbial secondary metabolites. Amphotericin is a polyene macrolide antibiotic synthesized by Streptomyces nodosus. S. nodosus ZJB2016050 with high-yield amphotericin B (AmB) was obtained by traditional mutagenesis using S. nodosus ATCC14899 as the original strain. The differences in the characterization of the two strains were found in color, mycelium morphology, and AmB yield. Subsequent comparative transcriptome explained the yield differences between the two strains. Pathways including the carbohydrate metabolic pathway and the secondary product synthesis pathway were targeted. The upregulation of glucokinase, phosphoglycerate mutase, and pyruvate dehydrogenase accelerates the consumption of glucose and has great effects on the accumulation of precursors. One of the competitive secondary metabolites of the polyketone synthetase (PKS) II type sapromomycin analog synthesis gene cluster was downregulated, which competes for malonyl-CoA. Five PKS modules (except for the first module amphA) of the amphotericin synthetic gene cluster in the high-yielding strain were downregulated, which resulted in the total amphotericin A (AmA) and AmB of S. nodosus ZJB2016050 being less than that of the wild-type S. nodosus ATCC14899. Combined with gene differential expression in the pentose phosphate pathway and the reaction mechanism of the ER5 domain, the reason that S. nodosus ZJB2016050 preferred to synthesize AmB was probably related to intracellular reduction.

Disseminated aspergillosis is responsible for a high mortality rate, despite the use of antifungal drugs. Adjuvant therapies are urgently needed to improve the outcome. The aim of this study was to demonstrate that the cytoprotective effect of erythropoietin (EPO) combined with amphotericin B can reduce the mortality rate in a murine model of disseminated aspergillosis. After infection with Aspergillus fumigatus, neutropenic mice were randomized to receive vehicle or 7.5 mg/kg liposomal amphotericin B (LAmB) or 7.5 mg/kg LAmB combined with 1000 IU/kg EPO (16 mice per group). Aspergillus galactomannan and organ cultures were performed to evaluate fungal burden at day 5. Cumulative long-term survival was analyzed at day 12 post-infection according to the Kaplan-Meier method. At day 5, fungal burden was similar between non-treated and treated groups. At day 12, mortality rates were 75, 62.5, and 31% in control group, LAmB group, and EPO/LAmB group, respectively. We observed a significant decrease in mortality using EPO/LAmB combination compared to control group (p < 0.01). LAmB single treatment did not improve the survival rate compared to control group (p = 0.155). Our results provide the first evidence that EPO improved the outcome of mice presenting with disseminated aspergillosis when combined with amphotericin B.

Amphotericin B (AmB) is a highly effective antimicrobial, with broad antimycotic and antiparasitic effect. However, AmB poor water-solubilisation and aggregation tendency limits its use for topical applications. We studied the capacity of nanostructures formed by alkyl esters of L-ascorbic acid (ASCn) to solubilise AmB and tested the relationship between the prevalence of the monomeric form of AmB and its effectiveness as antimicrobial agent.

Leishmaniasis is a major health problem in many parts of the world, caused by various species of Leishmania. Amastigotes are the clinically relevant form of the parasite in the human host and reside in the parasitophorous vacuole within macrophages. Polymer-drug conjugates have been used for lysosomotropic drug delivery and have already shown potential in anticancer and antileishmanial chemotherapy. We synthesised N-(2-hydroxypropyl)methacrylamide-amphotericin B (HPMA-AmB) copolymer conjugates in which the AmB was attached to the polymer through a degradable GlyPheLeuGly linker. Antileishmanial activity was assessed in vitro against intracellular amastigotes in host macrophages [murine peritoneal exudate macrophages (PEMs), murine bone marrow-derived macrophages (BMMs) and differentiated THP-1 cells]. The most potent copolymers had 50% effective concentration (EC(50)) values of 0.03 microg/mL AmB equivalent against Leishmania donovani amastigotes in PEMs and BMMs and an EC(50) of 0.57 microg/mL AmB equivalent against L. donovani in THP-1 cells. This activity was comparable with free AmB (EC(50)=0.03-0.07 microg/mL against L. donovani in PEMs and BMMs and 0.24-0.42 microg/mL against amastigotes in THP-1 cells) and Fungizone (EC(50)=0.04-0.07 microg/mL against amastigotes in PEMs). Conjugates also showed potent in vivo activity with ca. 50% inhibition of parasite burden at 1mg/kg body weight.

Invasive infections caused by Trichosporon spp. have increased considerably in recent years, especially in neutropenic and critically ill patients using catheters and antibiotics. The genus presents limited sensitivity to different antifungal agents, but triazoles are the first choice for treatment. Here, we investigated the biofilm production and antifungal susceptibility to triazoles and amphotericin B of 54 Trichosporon spp. isolates obtained from blood samples (19), urine (20) and superficial mycosis (15). All isolates and 7 reference strains were identified by sequence analysis and phylogenetic inferences of the IGS1 region of the rDNA. Biofilms were grown on 96-well plates and quantitation was performed using crystal violet staining, complemented with Scanning Electron Microscopy (SEM). Susceptibility tests for fluconazole, itraconazole, voriconazole and amphotericin B were processed using the microdilution broth method (CLSI) for planktonic cells and XTT reduction assay for biofilm-forming cells. Our results showed that T. asahii was the most frequent species identified (66.7%), followed by T. faecale (11.1%), T. asteroides (9.3%), T. inkin (7.4%), T. dermatis (3.7%) and one T. coremiiforme (1.8%). We identified 4 genotypes within T. asahii isolates (G1, G3, G4 and G5) and 2 genotypes within T. faecale (G1 and G3). All species exhibited high adhesion and biofilm formation capabilities, mainly T. inkin, T. asteroides and T. faecale. Microscopy images of high biofilm-producing isolates showed that T. asahii presented mainly hyphae and arthroconidia, whereas T. asteroides exhibited mainly short arthroconidia and few filaments. Voriconazole exhibited the best in vitro activity against all species tested. Biofilm-forming cells of isolates and reference strains were highly resistant to all antifungals tested. We concluded that levels of biofilm formation by Trichosporon spp. were similar or even greater than those described for the Candida genus. Biofilm-forming cells were at least 1,000 times more resistant to antifungals than planktonic cells, especially to voriconazole.

Amphotericin B (AMB) is commonly used to treat life-threatening systemic fungal infections. AMB formulations that are more efficient and less nephrotoxic are currently unmet needs. In the current study, new ZnO-PEGylated AMB (ZnO-AMB-PEG) nanoparticles (NPs) were synthesized and their antifungal effects on the Candida spp. were investigated. The size and zeta potential values of AMB-PEG and ZnO-AMB-PEG NPs were 216.2 ± 26.9 to 662.3 ± 24.7 nm and -11.8 ± 2.02 to -14.2 ± 0.94 mV, respectively. The FTIR, XRD, and EDX spectra indicated that the PEG-enclosed AMB was capped by ZnO, and SEM images revealed the ZnO distribution on the surface NPs. In comparison to ZnO-AMB NPs and free AMB against C.albicans and C.neoformans, ZnO-AMB-PEG NPs significantly reduced the MIC and MFC. After a week of single and multiple dosage, the toxicity was investigated utilizing in vitro blood hemolysis, in vivo nephrotoxicity, and hepatic functions. ZnO-AMB-PEG significantly lowered WBC count and hematocrit concentrations when compared to AMB and ZnO-AMB. RBC count and hemoglobulin content, on the other hand, were unaltered. ZnO-AMB-PEG considerably lowered creatinine and blood urea nitrogen (BUN) levels when compared to AMB and ZnO-AMB. The difference in liver function indicators was determined to be minor by all formulae. These findings imply that ZnO-AMB-PEG could be utilized in the clinic with little nephrotoxicity, although more research is needed to determine the formulation's in vivo efficacy.

Amphotericin B (AmB) is a polyene antifungal drug and is reported to be one of a few reagents having therapeutic effects on prion diseases, that is, a delay in the appearance of clinical signs and prolongation of the survival time in an animal model. In prion diseases, glial cells have been suggested to play important roles; however, the therapeutic mechanism of AmB on prion diseases remains elusive. We have previously reported that AmB changed the expression of neurotrophic factors in microglia and astrocytes (Motoyoshi et al., 2008, Neurochem. Int. 52, 1290-1296; Motoyoshi-Yamashiro et al., 2013, ibid. 63, 93-100). These results suggested that neurotrophic factors derived from glial cells might be involved in the therapeutic mechanism of AmB. In the present study, we examined immunohistochemically the effects of AmB on the expression of neurotrophic factors in the rat brain. We found that direct injection of AmB into the striatum significantly enhanced the expression of glial cell line-derived neurotrophic factor protein. Amphotericin B also increased the expressions of CD11b and glial fibrillary acidic protein, markers of microglia and astrocytes, respectively. Moreover, expressions of the two neurotrophic factors by AmB were co-localized with the expression of CD11b or glial fibrillary acidic protein. These results suggest that AmB in vivo might also activate glial cells and induce the production of neurotrophic factors protecting neurons in prion diseases.

The purpose of our research was the development of Amphotericin B-loaded in situ gelling nanofibers for the treatment of keratomycosis. Different formulation strategies were applied to increase the drug load of the sparingly water-soluble Amphotericin B in electrospun Gellan Gum/Pullulan fibers. These include bile salt addition, encapsulation in poly(lactic-co-glycolic acid) (PLGA) nanoparticles and formation of a polymeric Amphotericin B polyelectrolyte complex. The Amphotericin B polyelectrolyte complex (AmpB-Eu L) performed best and was very effective against the fungal strain Issatchenkia orientalis in vitro. The complex was characterized in detail by attenuated total reflection infrared spectroscopy, X-ray powder diffraction, and differential scanning calorimetry. A heat induced stress test was carried out to ensure the stability of the polyelectrolyte complex. To gain information about the cellular tolerance of the developed polyelectrolyte complex a new, innovative multilayered-stratified human cornea cell model was used for determination of the cellular toxicity in vitro. For a safe therapy, the applied ophthalmic drug delivery system has to be sterile. Sterilization by electron irradiation caused not degradation of pure Amphotericin B and also for the bile salt complex. Furthermore, the developed Amphotericin B polyelectrolyte complex was not degraded by the irradiation process. In conclusion, a new polyelectrolyte Amphotericin B complex has been found which retains the antifungal activity of the drug with sufficient stability against irradiation-sterilization induced drug degradation. Furthermore, in comparison with the conventional used eye drop formulation, the new AmpB-complex loaded nanofibers were less toxic to cornea cells in vitro. Electrospinning of the Amphotericin B polyelectrolyte complex with Gellan Gum/ Pullulan leads to the formation of nanofibers with in situ gelling properties, which is a new and promising option for the treatment of keratomycosis.

Amphotericin B is increasingly used in treatment of leishmaniasis. Here, fourteen independent lines of Leishmania mexicana and one L. infantum line were selected for resistance to either amphotericin B or the related polyene antimicrobial, nystatin. Sterol profiling revealed that, in each resistant line, the predominant wild-type sterol, ergosta-5,7,24-trienol, was replaced by other sterol intermediates. Broadly, two different profiles emerged among the resistant lines. Whole genome sequencing then showed that these distinct profiles were due either to mutations in the sterol methyl transferase (C24SMT) gene locus or the sterol C5 desaturase (C5DS) gene. In three lines an additional deletion of the miltefosine transporter gene was found. Differences in sensitivity to amphotericin B were apparent, depending on whether cells were grown in HOMEM, supplemented with foetal bovine serum, or a serum free defined medium (DM). Metabolomic analysis after exposure to AmB showed that a large increase in glucose flux via the pentose phosphate pathway preceded cell death in cells sustained in HOMEM but not DM, indicating the oxidative stress was more significantly induced under HOMEM conditions. Several of the lines were tested for their ability to infect macrophages and replicate as amastigote forms, alongside their ability to establish infections in mice. While several AmB resistant lines showed reduced virulence, at least two lines displayed heightened virulence in mice whilst retaining their resistance phenotype, emphasising the risks of resistance emerging to this critical drug.

Amphotericin B (AMB) is used most commonly in severe systemic life-threatening fungal infections. There is currently an unmet need for an efficacious (AMB) formulation amenable to oral administration with better bioavailability and lower nephrotoxicity. Novel PEGylated polylactic-polyglycolic acid copolymer (PLGA-PEG) nanoparticles (NPs) formulations of AMB were therefore studied for their ability to kill Candida albicans (C. albicans). The antifungal activity of AMB formulations was assessed in C. albicans. Its bioavalability was investigated in nine groups of rats (n = 6). Toxicity was examined by an in vitro blood hemolysis assay, and in vivo nephrotoxicity after single and multiple dosing for a week by blood urea nitrogen (BUN) and plasma creatinine (PCr) measurements. The MIC of AMB loaded to PLGA-PEG NPs against C. albicans was reduced two to threefold compared with free AMB. Novel oral AMB delivery loaded to PLGA-PEG NPs was markedly systemically available compared to Fungizone® in rats. The addition of 2% of GA to the AMB formulation significantly (p < 0.05) improved the bioavailability from 1.5 to 10.5% and the relative bioavailability was > 790% that of Fungizone®. The novel AMB formulations showed minimal toxicity and better efficacy compared to Fungizone®. No nephrotoxicity in rats was detected after a week of multiple dosing of AMB NPs based on BUN and PCr, which remained at normal levels. An oral delivery system of AMB-loaded to PLGA-PEG NPs with better efficacy and minimal toxicity was formulated. The addition of glycyrrhizic acid (GA) to AMB NPs formulation resulted in a significant oral absorption and improved bioavailability in rats.

Liposomal amphotericin B (L-AMB) and isavuconazonium sulphate are commonly used antifungal drugs to treat mucormycosis. However, the efficacy of combination therapy of L-AMB/isavuconazonium sulphate versus monotherapy is unknown. We used an immunosuppressed mouse model of pulmonary mucormycosis to compare the efficacy of L-AMB/isavuconazonium sulphate versus either drug alone.