Tissue Fungal Burden Research Articles

IL-6 deficiency accelerates cerebral cryptococcosis and alters glial cell responses

Cryptococcus neoformans (Cn) is an opportunistic encapsulated fungal pathogen that causes life-threatening meningoencephalitis in immunosuppressed individuals. Since IL-6 is important for blood-brain barrier support and its deficiency has been shown to facilitate Cn brain invasion, we investigated the impact of IL-6 on systemic Cn infection in vivo, focusing on central nervous system (CNS) colonization and glial responses, specifically microglia and astrocytes. IL-6 knock-out (IL-6−/−) mice showed faster mortality than C57BL/6 (Wild-type) and IL-6−/− supplemented with recombinant IL-6 (rIL-6; 40 pg/g/day) mice. Despite showing early lung inflammation but no major histological differences in pulmonary cryptococcosis progression among the experimental groups, IL-6−/− mice had significantly higher blood and brain tissue fungal burden at 7-days post infection. Exposure of cryptococci to rIL-6 in vitro increased capsule growth. In addition, IL-6−/− brains were characterized by an increased dystrophic microglia number during Cn infection, which are associated with neurodegeneration and senescence. In contrast, the brains of IL-6-producing or -supplemented mice displayed high numbers of activated and phagocytic microglia, which are related to a stronger anti-cryptococcal response or tissue repair. Likewise, culture of rIL-6 with microglia-like cells promoted high fungal phagocytosis and killing, whereas IL-6 silencing in microglia decreased fungal phagocytosis. Lastly, astrogliosis was high and moderate in infected brains removed from Wild-type and IL-6−/− supplemented with rIL-6 animals, respectively, while minimal astrogliosis was observed in IL-6−/− tissue, highlighting the potential of astrocytes in containing and combating cryptococcal infection. Our findings suggest a critical role for IL-6 in Cn CNS dissemination, neurocryptococcosis development, and host defense.

Efficacy of an oral lipid nanocrystal formulation of amphotericin B (MAT2203) in the neutropenic mouse model of pulmonary mucormycosis.

Invasive mucormycosis (IM) is associated with high mortality and morbidity. MAT2203 is an orally administered lipid nanocrystal formulation of amphotericin B, which has been shown to be safe and effective against other fungal infections. We sought to compare the efficacy of MAT2203 to liposomal amphotericin B (LAMB) treatment in a neutropenic mouse model of IM due to Rhizopus arrhizus var. delemar or Mucor circinelloides f. jenssenii DI15-131. In R. arrhizus var. delemar-infected mice, 15 mg/kg of MAT2203 qd was as effective as 10 mg/kg of LAMB in prolonging median survival time vs placebo (13.5 and 16.5 days for MAT2203 and LAMB, respectively, vs 9 days for placebo) and enhancing overall survival vs placebo-treated mice (40% and 45% for MAT2203 and LAMB, respectively, vs 0% for placebo). A higher dose of 45 mg/kg of MAT2203 was not well tolerated by mice and showed no benefit over placebo. Similar results were obtained with mice infected with M. circinelloides. Furthermore, while both MAT2203 and LAMB treatment resulted in a significant reduction of ~1.0-2.0log and ~2.0-2.5log in Rhizopus delemar or M. circinelloides lung and brain burden vs placebo mice, respectively, LAMB significantly reduced tissue fungal burden in mice infected with R. delemar vs tissues of mice treated with MAT2203. These results support continued investigation and development of MAT2203 as a novel and oral formulation of amphotericin for the treatment of mucormycosis.

A ketogenic diet enhances fluconazole efficacy in murine models of systemic fungal infection.

Invasive fungal infections are a significant public health concern, with mortality rates ranging from 20% to 85% despite current treatments. Therefore, we examined whether a ketogenic diet could serve as a successful treatment intervention in murine models of Cryptococcus neoformans and Candida albicans infection in combination with fluconazole-a low-cost, readily available antifungal therapy. The ketogenic diet is a high-fat, low-carbohydrate diet that promotes fatty acid oxidation as an alternative to glycolysis through the production of ketone bodies. In this series of experiments, mice fed a ketogenic diet prior to infection with C. neoformans and treated with fluconazole had a significant decrease in fungal burden in both the brain (mean 2.66 ± 0.289 log10 reduction) and lung (mean 1.72 ± 0.399 log10 reduction) compared to fluconazole treatment on a conventional diet. During C. albicans infection, kidney fungal burden of mice in the keto-fluconazole combination group was significantly decreased compared to fluconazole alone (2.37 ± 0.770 log10-reduction). Along with higher concentrations of fluconazole in the plasma and brain tissue, fluconazole efficacy was maximized at a significantly lower concentration on a keto diet compared to a conventional diet, indicating a dramatic effect on fluconazole pharmacodynamics. Our findings indicate that a ketogenic diet potentiates the effect of fluconazole at multiple body sites during both C. neoformans and C. albicans infection and could have practical and promising treatment implications.IMPORTANCEInvasive fungal infections cause over 2.5 million deaths per year around the world. Treatments for fungal infections are limited, and there is a significant need to develop strategies to enhance antifungal efficacy, combat antifungal resistance, and mitigate treatment side effects. We determined that a high-fat, low-carbohydrate ketogenic diet significantly potentiated the therapeutic effect of fluconazole, which resulted in a substantial decrease in tissue fungal burden of both C. neoformans and C. albicans in experimental animal models. We believe this work is the first of its kind to demonstrate that diet can dramatically influence the treatment of fungal infections. These results highlight a novel strategy of antifungal drug enhancement and emphasize the need for future investigation into dietary effects on antifungal drug activity.

Antifungal Effect of Vitamin D3 against Cryptococcus neoformans Coincides with Reduced Biofilm Formation, Compromised Cell Wall Integrity, and Increased Generation of Reactive Oxygen Species.

Cryptococcus neoformans is an invasive fungus that causes both acute and chronic infections, especially in immunocompromised patients. Owing to the increase in the prevalence of drug-resistant pathogenic fungi and the limitations of current treatment strategies, drug repositioning has become a feasible strategy to accelerate the development of new drugs. In this study, the minimum inhibitory concentration of vitamin D3 (VD3) against C. neoformans was found to be 0.4 mg/mL by broth microdilution assay. The antifungal activities of VD3 were further verified by solid dilution assays and "time-kill" curves. The results showed that VD3 reduced fungal cell adhesion and hydrophobicity and inhibited biofilm formation at various developmental stages, as confirmed by crystal violet staining and the 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide assay. Fluorescence staining of cellular components and a stress susceptibility assay indicated that VD3 compromised cell integrity. Reverse transcription quantitative PCR demonstrated that VD3 treatment upregulated the expression of fungal genes related to cell wall synthesis (i.e., CDA3, CHS3, FKS1, and AGS1). Moreover, VD3 enhanced cell membrane permeability and caused the accumulation of intracellular reactive oxygen species. Finally, VD3 significantly reduced the tissue fungal burden and prolonged the survival of Galleria mellonella larvae infected with C. neoformans. These results showed that VD3 could exert significant antifungal activities both in vitro and in vivo, demonstrating its potential application in the treatment of cryptococcal infections.

Nikkomycin Z for the treatment of experimental sporotrichosis caused by Sporothrix brasiliensis.

Sporotrichosis caused by Sporothrix brasiliensis is a global emergent infectious disease. Due to the scarcity of therapeutic options for fungal diseases, new antifungals are urgently needed. Nikkomycin Z (NikZ) is a future option as an agent against dimorphic fungi. We evaluated NikZ monotherapy and in combination with itraconazole (ITZ; the conventional therapy) in the treatment of experimental sporotrichosis caused by S. brasiliensis in a murine model. Animals were subcutaneously infected, and treated orally for 30 days. The study groups were as follows control (untreated), ITZ group (50 mg/kg/day), and three groups treated with NikZ, two by monotherapy (200 or 400 mg/kg/day), and one combining NikZ (400 mg/kg/day) and ITZ. Efficacy of treatments was evaluated via body weight gain, mortality and fungal burden in tissues. Efficacy was noted in all treatment groups, and the group receiving the drug combination showed even better results than those with monotherapy. Our study shows for the first time the high potential of NikZ to be used in the treatment of sporotrichosis caused by S. brasiliensis.

Antifungal Activity and Potential Action Mechanism of Allicin against Trichosporon asahii.

Trichosporon asahii is an emerging opportunistic pathogen that causes potentially fatal disseminated trichosporonosis. The global prevalence of coronavirus disease 2019 (COVID-19) poses an increasing fungal infection burden caused by T. asahii. Allicin is the main biologically active component with broad-spectrum antimicrobial activity in garlic. In this study, we performed an in-depth analysis of the antifungal characteristics of allicin against T. asahii based on physiological, cytological, and transcriptomic assessments. In vitro, allicin inhibited the growth of T. asahii planktonic cells and biofilm cells significantly. In vivo, allicin improved the mean survival time of mice with systemic trichosporonosis and reduced tissue fungal burden. Electron microscopy observations clearly demonstrated damage to T. asahii cell morphology and ultrastructure caused by allicin. Furthermore, allicin increased intracellular reactive oxygen species (ROS) accumulation, leading to oxidative stress damage in T. asahii cells. Transcriptome analysis showed that allicin treatment disturbed the biosynthesis of cell membrane and cell wall, glucose catabolism, and oxidative stress. The overexpression of multiple antioxidant enzymes and transporters may also place an additional burden on cells, causing them to collapse. Our findings shed new light on the potential of allicin as an alternative treatment strategy for trichosporonosis. IMPORTANCE Systemic infection caused by T. asahii has recently been recognized as an important cause of mortality in hospitalized COVID-19 patients. Invasive trichosporonosis remains a significant challenge for clinicians, due to the limited therapeutic options. The present work suggests that allicin holds great potential as a therapeutic candidate for T. asahii infection. Allicin demonstrated potent in vitro antifungal activity and potential in vivo protective effects. In addition, transcriptome sequencing provided valuable insights into the antifungal effects of allicin.

LB1580. Efficacy Assessment of MAT2203 Encochleated Oral Formulation of Amphotericin B, in the Neutropenic Mouse Model of Pulmonary Mucormycosis

Abstract Background Invasive mucormycosis (IM) is associated with high mortality and morbidity. MAT2203 is an encochleated oral formulation of amphotericin B which has been shown to be safe and effective against murine aspergillosis and murine cryptococcal meningoencephalitis. We sought to compare the efficacy of MAT2203 to liposomal amphotericin B (LAMB) treatment in a neutropenic mouse model of IM. Methods ICR mice were immunosuppressed with cyclophosphamide and cortisone acetate on days -2, -3 and +8, relative to infection with intratracheally instilled Rhizopus delemar 99-880 or M. circinelloides f. jenssenii DI15-131. Treatment with placebo (diluent control), oral MAT2203 (5 to 45 mg/kg, given qd or bid for 7 days) or LAMB (10 mg/kg, iv, qd for 4 days), began 16 h post infection. Survival (n=10-20/group from 1/2 experiments) through Day +21 and tissue fungal burden of lungs or brain (n=10/group) euthanized on Day +4 post infection (conidial equivalents using qPCR) served as a primary and secondary endpoint, respectively. Results For Rhizopus delemar infection, doses of MAT2203 5,15 mg/kg qd or 7.5 mg/kg bid, significantly prolonged median survival time (MST) and enhanced overall survival vs. placebo-treated mice (MST of 9 Days and 0% survival for placebo-treated mice vs. 13 Days, and 20-40% for MAT2203 doses, P< 0.05 by Log-Rank). Importantly, MAT2203 treatments were as effective as LAMB (MST of 16 days and overall survival of 45%). For mice infected with M. circinelloides, MAT2203 at 15 mg/kg, qd significantly prolonged MST and enhanced overall survival vs. placebo-treated mice (MST of 5 Days and 0% survival, for placebo-treated mice vs. 13.5 Days and 50% survival for MAT2203, P< 0.05). In both infection models MAT2203 treatment of 15 mg/kg or LAMB resulted in significant ∼1.0-1.5 log reduction and ∼2.0-2.2 log reduction in lung and brain fungal burden vs. placebo, respectively (Wilcoxon Rank Sum). Conclusion MAT2203 demonstrated in vivo efficacy in treating R. delemar or M. circinelloides pulmonary infection in immunosuppressed mice, which was equivalent to the LAMB current standard of care. Continued investigation and development of MAT2203 as a novel, and oral formulation of amphotericin antifungal agent against mucormycosis is warranted. Disclosures Theresa Matkovits, PhD, Matinas BioPharma: Employee Jenel Cobb, PhD, Matinas BioPharma: Employee Raphael J. Mannino, PhD, Matinas BioPharma: Employee Ashraf S. Ibrahim, PhD, Matinas BioPharma: Advisor/Consultant|Matinas BioPharma: Grant/Research Support|SFunga: Grant/Research Support.

Recent Antifungal Pipeline Developments against Candida auris: A Systematic Review.

The alarming spread and impact of multidrug-resistant Candida auris infections alongside the limited therapeutic options have prompted the development of new antifungals. These promising agents are currently in different stages of development, offering novel dosing regimens and mechanisms of action. A systematic search in MEDLINE, EMBASE, Web of Science, and Scopus up to 27 June 2022 was conducted to find relevant articles reporting data of in vitro activity and in vivo efficacy of investigational antifungals against C. auris. These included new additions to existing antifungal classes (rezafungin and opelconazole), first-in-class drugs such as ibrexafungerp, manogepix/fosmanogepix, olorofim and tetrazoles (quilseconazole, oteseconazole and VT-1598), as well as other innovative agents like ATI-2307, MGCD290 and VL-2397. From 592 articles retrieved in the primary search, 27 met the eligibility criteria. The most studied agent was manogepix/fosmanogepix (overall MIC90: 0.03 mg/L), followed by ibrexafungerp (overall MIC90: 1 mg/L) and rezafungin (overall MIC mode: 0.25 mg/L), while VT-1598 and ATI-2307 were the least explored drugs against C. auris. All these compounds demonstrated significant improvements in survival and reduction in tissue fungal burden on neutropenic animal models of candidemia due to C. auris. Continual efforts towards the discovery of new treatments against this multidrug-resistant fungus are essential.

Assessment of Biofilm Formation by Candida albicans Strains Isolated from Hemocultures and Their Role in Pathogenesis in the Zebrafish Model.

Candida albicans, an opportunistic pathogen, has the ability to form biofilms in the host or within medical devices in the body. Biofilms have been associated with disseminated/invasive disease with increased severity of infection by disrupting the host immune response and prolonging antifungal treatment. In this study, the in vivo virulence of three strains with different biofilm formation strengths, that is, non-, weak-, and strong biofilm formers, was evaluated using the zebrafish model. The survival assay and fungal tissue burden were measured. Biofilm-related gene expressions were also investigated. The survival of zebrafish, inoculated with strong biofilms forming C. albicans,, was significantly shorter than strains without biofilms forming C. albicans. However, there were no statistical differences in the burden of viable colonogenic cell number between the groups of the three strains tested. We observed that the stronger the biofilm formation, the higher up-regulation of biofilm-associated genes. The biofilm-forming strain (140 and 57), injected into zebrafish larvae, possessed a higher level of expression of genes associated with adhesion, attachment, filamentation, and cell proliferation, including eap1, als3, hwp1, bcr1, and mkc1 at 8 h. The results suggested that, despite the difference in genetic background, biofilm formation is an important virulence factor for the pathogenesis of C. albicans. However, the association between biofilm formation strength and in vivo virulence is controversial and needs to be further studied.

The Combination Treatment of Fosmanogepix and Liposomal Amphotericin B Is Superior to Monotherapy in Treating Experimental Invasive Mold Infections.

ABSTRACTInvasive pulmonary aspergillosis (IPA), invasive mucormycosis (IM), and invasive fusariosis (IF) are associated with high mortality and morbidity. Fosmanogepix (FMGX) is a first-in-class antifungal in clinical development with demonstrated broad-spectrum activity in animal models of infections. We sought to evaluate the benefit of combination therapy of FMGX plus liposomal amphotericin B (L-AMB) in severe delayed-treatment models of murine IPA, IM, and IF. While FMGX was equally as effective as L-AMB in prolonging the survival of mice infected with IPA, IM, or IF, combination therapy was superior to monotherapy in all three models. These findings were validated by greater reductions in the tissue fungal burdens (determined by quantitative PCR) of target organs in all three models versus the burdens in infected vehicle-treated (placebo) or monotherapy-treated mice. In general, histopathological examination of target organs corroborated the findings for fungal tissue burdens among all treatment arms. Our results show that treatment with the combination of FMGX plus L-AMB demonstrated high survival rates and fungal burden reductions in severe animal models of invasive mold infections, at drug exposures in mice similar to those achieved clinically. These encouraging results warrant further investigation of the FMGX–plus–L-AMB combination treatment for severely ill patients with IPA, IM, and IF.

Candida auris Bloodstream Infection Induces Upregulation of the PD-1/PD-L1 Immune Checkpoint Pathway in an Immunocompetent Mouse Model.

ABSTRACTCandida auris is a globally spreading yeast pathogen causing bloodstream infections with high mortality in critically ill patients. The inherent antifungal drug resistance of most C. auris isolates and threat of multidrug-resistant strains create a need for adjunct immunotherapeutic strategies. While C. albicans candidemia was shown to induce immune paralysis and activation of inhibitory immune checkpoints, in vivo data on host responses to C. auris bloodstream infection are lacking as is an immunocompetent murine infection model to study the immunopathology and immunotherapy of C. auris sepsis. Therefore, herein, we developed an immunocompetent C. auris sepsis model by intravenously infecting C57BL/6 mice with 1.5 × 108 to 8 × 108 yeast cells of aggregate-forming (AR-0384) and nonaggregative (AR-0381) C. auris reference isolates. Both isolates caused reproducible, inoculum-dependent increasing morbidity, mortality, and fungal burden in kidney tissue. Notably, morbidity and mortality outcomes were partially decoupled from fungal burden, suggesting a role of additional modulators of disease severity such as host immune responses. Flow cytometric analyses of splenic immune cells revealed significant upregulation of the programmed cell death protein 1 (PD-1) on T cells and its ligand PD-L1 on macrophages from mice infected with C. auris AR-0384 compared to uninfected mice. PD-L1 expression on macrophages from AR-0384-infected mice strongly correlated with fungal tissue burden (Spearman’s rank correlation coefficient [ρ] = 0.95). Altogether, our findings suggest that C. auris sepsis promotes a suppressive immune phenotype through PD-1/PD-L1 induction, supporting further exploration of PD-1/PD-L1 blockade as an immunotherapeutic strategy to mitigate C. auris candidiasis.IMPORTANCE Health authorities consider Candida auris to be one of the most serious emerging nosocomial pathogens due to its transmissibility, resistance to disinfection procedures, and frequent antifungal drug resistance. The frequency of multidrug-resistant C. auris isolates necessitates the development of novel therapeutic platforms, including immunotherapy. However, in vivo data on host interactions with C. auris are scarce, compounded by the lack of reliable immunocompetent mammalian models of C. auris candidemia. Herein, we describe a C. auris sepsis model in immunocompetent C57BL/6 mice and demonstrate reproducible and inoculum-dependent acute infection with both aggregate-forming and nonaggregative reference isolates from different clades. Furthermore, we show that C. auris sepsis induces upregulation of the PD-1/PD-L1 immune checkpoint pathway in infected mice, raising the potential of a therapeutic benefit of immune checkpoint blockade. Our immunocompetent model of C. auris sepsis could provide a facile preclinical platform to thoroughly investigate immune checkpoint blockade and combination therapy with antifungals.

Ambisome shows potent anti-Candida auris in vitro and in vivo activity

Candida auris is an emerging multidrug resistant Candida species that has been reported in many parts of the world for causing severe illness in hospitalized patients especially bloodstream infections. The challenge with this type of yeast is its resistance to commonly used antifungal drugs, thus identifying antifungals that are effective against this species is critical. In this study, we determined the in vitro activity of Ambisome against 35 clinical isolates of C. auris using minimum inhibitory concentration (MIC) assay as well as the efficacy of Ambisome compared to Amphotericin B and fluconazole using a C. auris murine disseminated model. Antifungal activity of C. auris against Ambisome and comparators was assessed using amicrodilution method performed according to the Clinical and Laboratory Standard Institute (CLSI) M27-A4 methodology. Mice were immunocompromised and challenged with 3 x 107 C. auris blastopores in 0.1 ml of normal saline (via the tail vein). Treatment efficacy was assessed by determining reductions in mortality as well as decrease in tissue fungal burden (CFUs). Ambisome showed lower MIC50 and MIC90 values (1 and 2 μg/mL, respectively) than the comparators tested. Significant efficacy was observed in the Ambisome 7.5 mg/kg -treated group (100% and 90% survival by day 7- and 14-days post inoculation, respectively). Additionally, Ambisome and fluconazole treated groups showed significant reduction in CFUs in the kidneys (P- values of 0.028 and 0.022, respectively) compared to the untreated group. Our data shows that Ambisome shows significant antifungal activity against C. auris in vitro as well as in vivo.

Caspofungin Inhibits Mixed Biofilms of Candida albicans and Methicillin-Resistant Staphylococcus aureus and Displays Effectiveness in Coinfected Galleria mellonella Larvae.

ABSTRACTCandida albicans and Staphylococcus aureus are pathogens commonly isolated from bloodstream infections worldwide. While coinfection by both pathogens is associated with mixed biofilms and more severe clinical manifestations, due to the combined expression of virulence and resistance factors, effective treatments remain a challenge. In this study, we evaluated the activity of echinocandins, especially caspofungin, against mixed biofilms of C. albicans and methicillin-resistant (MRSA) or methicillin-susceptible S. aureus (MSSA) and their effectiveness in vivo using the Galleria mellonella coinfection model. Although caspofungin (CAS) and micafungin (MFG) inhibited the mixed biofilm formation, with CAS exhibiting inhibitory activity at lower concentrations, only CAS was active against preformed mixed biofilms. CAS significantly decreased the total biomass of mixed biofilms at concentrations of ≥2 μg/ml, whereas the microbial viability was reduced at high concentrations (32 to 128 μg/ml), leading to fungus and bacterium cell wall disruption and fungal cell enlargement. Notably, CAS (20 or 50 mg/kg of body weight) treatment led to an increased survival and improved outcomes of G. mellonella larvae coinfected with C. albicans and MRSA, since a significant reduction of fungal and bacterial burden in larval tissues was achieved with induction of granuloma formation. Our results reveal that CAS can be a therapeutic option for the treatment of mixed infections caused by C. albicans and S. aureus, supporting additional investigation.IMPORTANCE Infections by microorganisms resistant to antimicrobials is a major challenge that leads to high morbidity and mortality rates and increased time and cost with hospitalization. It was estimated that 27 to 56% of bloodstream infections by C. albicans are polymicrobial, with S. aureus being one of the microorganisms commonly coisolated worldwide. About 80% of infections are associated with biofilms by single or mixed species that can be formed on invasive medical devices, e.g., catheter, and are considered a dissemination source. The increased resistance to antimicrobials in bacterial and fungal cells when they are in biofilms is the most medically relevant behavior that frequently results in therapeutic failure. Although there are several studies evaluating treatments for polymicrobial infections associated or not with biofilms, there is still no consensus on an effective antimicrobial therapy to combat the coinfection by bacteria and fungi.

Design, Synthesis, and In Vitro and In Vivo Evaluation of Novel Fluconazole-Based Compounds with Promising Antifungal Activities.

Demand has arisen for developing new azole antifungal agents with the growth of the resistant rate of infective fungal species to current azole antifungals in recent years. Accordingly, the present study reports the synthesis of novel fluconazole (FLC) analogues bearing urea functionality that led to discovering new azole agents with promising antifungal activities. In particular, compounds 8b and 8c displayed broad-spectrum activity and superior in vitro antifungal capabilities compared to the standard drug FLC against sensitive and resistant Candida albicans (C. albicans). The highly active compounds 8b and 8c had potent antibiofilm properties against FLC-resistant C. albicans species. Additionally, these compounds exhibited very low toxicity for three mammalian cell lines and human red blood cells. Time-kill studies revealed that our synthesized compounds displayed a fungicidal mechanism toward fungal growth. Furthermore, a density functional theory (DFT) calculation, additional docking, and independent gradient model (IGM) studies were performed to analyze their structure–activity relationship (SAR) and to assess the molecular interactions in the related target protein. Finally, in vivo results represented a significant reduction in the tissue fungal burden and improvements in the survival rate in a mice model of systemic candidiasis along with in vitro and in silico studies, demonstrating the therapeutic efficiency of compounds 8b and 8c as novel leads for candidiasis drug discovery.

Targeted Propolis-Loaded Poly (Butyl) Cyanoacrylate Nanoparticles: An Alternative Drug Delivery Tool for the Treatment of Cryptococcal Meningitis.

In this study, we describe a nano-carrier system for propolis that is able to cross an in vitro model of the blood-brain barrier (BBB) and effectively reduce the virulence of Cryptococcus neoformans in animal models. Antimicrobial properties of propolis have been widely studied. However, propolis applications are limited by its low water solubility and poor bioavailability. Therefore, we recently formulated novel poly (n-butyl cyanoacrylate) nanoparticles (PBCA-NP) containing propolis. PBCA-NP are biocompatible, biodegradable and have been shown to effectively cross the BBB using apolipoprotein E (ApoE) as a ligand. Prepared nanoparticles were characterized for particle size, zeta potential, propolis entrapment efficiency and in vitro release. Additionally, the PBCA-NP were functionalized with polysorbate 80, which then specifically adsorbs ApoE. Using an in vitro BBB model of human brain microvascular endothelial cells hCMEC/D3, it was shown that fluorescence labelled ApoE-functionalized PBCA-NP were internalized by the cells and translocated across the cell monolayer. Propolis-loaded PBCA-NP had in vitro, antifungal activity against C. neoformans, which causes meningitis. To utilize the invertebrate model, Galleria mellonella larvae were infected with C. neoformans and treated with propolis-loaded PBCA-NP. The larvae exhibited normal behavior in toxicity testing, and treatment with propolis-loaded PBCA-NP increased survival in the C. neoformans-infected larvae group. In addition, following cryptococcal infection and then 7 days of treatment, the tissue fungal burden of mice treated with propolis-loaded PBCA-NP was significantly lower than control groups. Therefore, our ApoE-functionalized propolis-loaded PBCA-NP can be deemed as a potential targeted nanoparticle in the therapeutic treatment of cerebral cryptococcosis.

Combination treatment of liposomal amphotericin B and isavuconazole is synergistic in treating experimental mucormycosis.

ObjectivesLiposomal 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.MethodsNeutropenic mice were intratracheally infected with either Rhizopus delemar or Mucor circinelloides. Treatment with L-AMB, isavuconazonium sulphate, or a combination of both started 8 h post-infection and continued through to Day +4. Placebo mice received vehicle control. Survival to Day +21 and tissue fungal burden (by conidial equivalent using quantitative PCR) on Day +4, served as primary and secondary endpoints, respectively.ResultsFor mice infected with R. delemar, L-AMB and isavuconazonium sulphate equally prolonged median survival time and enhanced survival versus placebo (an overall survival of 50% for either drug alone, versus 5% for placebo). Importantly, combination treatment resulted in an overall survival of 80%. Both antifungal drugs reduced tissue fungal burden of lungs and brain by ∼1.0–2.0 log versus placebo-treated mice. Treatment with combination therapy resulted in 2.0–3.5 log reduction in fungal burden of either organ versus placebo and 1.0 log reduction versus either drug alone. Similar treatment outcomes were obtained using mice infected with M. circinelloides.ConclusionsThe L-AMB/isavuconazonium sulphate combination demonstrated greater activity versus monotherapy in immunosuppressed mice infected with either of the two most common causes of mucormycosis. These studies warrant further investigation of L-AMB/isavuconazonium sulphate combination therapy as an optimal therapy of human mucormycosis.

Liposomal Ellagic Acid Alleviates Cyclophosphamide-Induced Toxicity and Eliminates the Systemic Cryptococcus neoformans Infection in Leukopenic Mice.

Cryptococcus neoformans infections rose sharply due to rapid increase in the numbers of immunocompromised individuals in recent years. Treatment of Cryptococcosis in immunocompromised persons is largely very challenging and hopeless. Hence, this study aimed to determine the activity of ellagic acid (EA) in the treatment of C. neoformans in cyclophosphamide injected leukopenic mice. A liposomal formulation of ellagic acid (Lip-EA) was prepared and characterized, and its antifungal activity was assessed in comparison to fluconazole (FLZ). The efficacy of the drug treatment was tested by assessing survival rate, fungal burden, and histological analysis in lung tissues. The safety of the drug formulations was tested by investigating hepatic, renal function, and antioxidant levels. The results of the present work demonstrated that Lip-EA, not FLZ, effectively eliminated C. neoformans infection in the leukopenic mice. Mice treated with Lip-EA (40 mg/kg) showed 70% survival rate and highly reduced fungal burden in their lung tissues, whereas the mice treated with FLZ (40 mg/kg) had 20% survival rate and greater fungal load in their lungs. Noteworthy, Lip-EA treatment alleviated cyclophosphamide-induced toxicity and restored hepatic and renal function parameters. Moreover, Lip-EA treatment restored the levels of superoxide dismutase and reduced glutathione and catalase in the lung tissues. The effect of FLZ or EA or Lip-EA against C. neoformans infection was assessed by the histological analysis of lung tissues. Lip-EA effectively reduced influx of inflammatory cells, thickening of alveolar walls, congestion, and hemorrhage. The findings of the present study suggest that Lip-EA may prove to be a promising therapeutic formulation against C. neoformans in immunocompromised persons.

Evaluation of Sex Differences in Murine Diabetic Ketoacidosis and Neutropenic Models of Invasive Mucormycosis.

There is increased concern that the quality, generalizability and reproducibility of biomedical research can be influenced by the sex of animals used. We studied the differences between male and female mice in response to invasive pulmonary mucormycosis including susceptibility to infection, host immune reaction and responses to antifungal therapy. We used diabetic ketoacidotic (DKA) or neutropenic mice infected with either Rhizopus delemar or Mucor circinelloides. The only difference detected was that when DKA mice were infected with M. circinelloides, female mice were more resistant to infection than male mice (median survival time of 5 vs. 2 days for female and male mice, respectively). However, a 100% lethality was detected among infected animals of both sexes. Treatment with either liposomal amphotericin B (L-AMB) or posaconazole (POSA) protected mice from infection and eliminated the difference seen between infected but untreated female and male mice. Treatment with L-AMB consistently outperformed POSA in prolonging survival and reducing tissue fungal burden of DKA and neutropenic mice infected with R. delemar or M. circinelloides, in both mouse sexes. While little difference was detected in cytokine levels among both sexes, mucormycosis infection in the DKA mouse model induced more inflammatory cytokines/chemokines involved in neutrophil (CXCL1) and macrophage (CXCL2) recruitment vs. uninfected mice. As expected, this inflammatory response was reduced in the neutropenic mouse model. Our studies show that there are few differences between female and male DKA or neutropenic mice infected with mucormycosis with no effect on the outcome of treatment or host immune response.

745. Combination Treatment of Liposomal Amphotericin B and Isavuconazole is Synergistic in Treating Experimental Mucormycosis

BackgroundMucormycosis is a life-threatening infection that predominantly occurs in immunocompromised hosts. Liposomal amphotericin B (L-AMB) and isavuconazole (ISAV) are commonly used antifungal drugs to treat mucormycosis. However, the efficacy of combination therapy of L-AMB + ISAV compared to monotherapy is unknown. We used an immunosuppressed mouse model of pulmonary mucormycosis to compare the efficacy of L-AMB + ISAV vs. either drug alone.MethodsICR mice were immunosuppressed with cyclophosphamide (200 mg/kg) and cortisone acetate (500 mg/kg) on Days -2, +3, and +8 relative to intratracheal infection with 2.5 x 105 cells of Rhizopus delemar 99-880, or 2.5 x 106 cells of Mucor circinelloides. Treatment with L-AMB (10 mg/kg, given intravenously qd), ISAV (56 mg/kg, by oral gavage TID), or a combination of both started 8 h post-infection and continued through day +4. Placebo mice received vehicle control. Survival studies through day +21 and tissue fungal burden (by conidial equivalent [CE] using qPCR) on Day +4, served as primary and secondary endpoints.ResultsFor mice (n=20) infected with R. delemar, L-AMB and ISAV equally prolonged median survival time and enhanced survival vs. placebo (19 and 16 days for L-AMB and ISAV, respectively, and overall survival of 50% for either drug alone, vs. 9 days and 5% overall survival for placebo, P< 0.002 for either drug vs. placebo by Log Rank test). Importantly, combination treatment enhanced median survival time (>21 days) and resulted in an overall survival of 80% (P< 0.05 vs. all treatments). Both antifungal drugs reduced tissue fungal burden of mice (n=10) lungs and brain by ~1.0-2.0 log vs. placebo-treated mice (P< 0.02 by Wilcoxon Rank Sum). Consistent with the survival data, treatment with combination therapy resulted in 2.0-3.5 log reduction in fungal burden of either organ vs. placebo and 1.0 log reduction vs. either drug alone (P< 0.005). Similar results were obtained using mice infected with M. circinelloides.ConclusionL-AMB + ISAV demonstrate greater activity vs. monotherapy treatment in immunosuppressed mice infected with either of two common causes of mucormycosis. These studies warrant further investigation of LAmB + ISAV combination therapy as an optimal therapy of human mucormycosis.DisclosuresTherese Kitt, MD, Astellas Pharma (Employee) Ashraf S. Ibrahim, PhD, Astellas Pharma (Research Grant or Support)

149. Efficacy of Cochleated Amphotericin B (CAMB) in Mouse and Human Mucocutaneous Candidiasis

Background Candida albicans causes debilitating mucosal infections in patients with inherited susceptibility to chronic mucocutaneous candidiasis (CMC), often requiring long-term azole-based treatment. Due to increasing azole resistance, alternative treatments are desirable. Acquired resistance to amphotericin B (AMB) is rare but AMB use is limited by parenteral administration and nephrotoxicity. Cochleated AMB (CAMB) is a new oral formulation of AMB and thus an attractive option for oropharyngeal candidiasis (OPC), esophageal candidiasis (EC) and vulvovaginal candidiasis (VVC). We assessed the efficacy of CAMB in mouse models of OPC and VVC and in 4 patients with azole resistant CMC manifesting as OPC, EC or VVC.Methods Act1 -/- mice were infected with C. albicans in models of OPC and VVC and were treated once daily via oral gavage with CAMB or vehicle or intraperitoneal AMB-deoxycholate (AMBd) from day 1 through 4 post-infection (pi). At day 5 pi, the tongue or vaginal tissue was harvested to quantify fungal burden. Patients with azole resistant CMC enrolled in a phase 2A CAMB dose escalation study. The primary endpoint was clinical improvement at 2 weeks based on an efficacy scale, followed by optional extension for long-term suppression of CMC to assess safety and efficacy.ResultsCAMB-treated mice had significantly reduced tongue and vaginal tissue fungal burden compared to vehicle-treated mice, while they exhibited comparable fungal control relative to AMBd-treated mice. Among 4 CAMB-treated patients, 3 reached clinical efficacy by 2 weeks at a dose of 400 mg twice daily and one reached clinical efficacy at 200 mg twice daily. Three of 4 patients continued on the extension phase past 48 months with sustained clinical improvement of OPC and EC; patient #3 had relapse of esophageal symptoms at week 24 and was withdrawn from further study. Clinical response was not seen for onychomycosis or VVC. CAMB was safe and well-tolerated without renal toxicity.ConclusionOral administration of CAMB in IL-17-signaling deficient mice resulted in reduced tongue and vaginal tissue fungal burden during mucosal C. albicans infections. A proof-of-concept clinical trial in humans with inherited CMC showed efficacy in OPC and EC with good tolerability and safety.DisclosuresBenjamin Colton, PharmD, Merck (Shareholder)Pfizer (Shareholder) Ruying Lu, n/a, Matinas BioPharma Inc. (Employee)Matinas BioPharma Inc. (Employee, Shareholder) Theresa Matkovits, PhD, Matinas BioPharma (Employee, Shareholder) Raphael J. Mannino, n/a, Matinas BioPharma Inc. (Employee, Shareholder) Michail Lionakis, MD, ScD, Matinas BioPharma (Research Grant or Support)