Targeted liposomes in fungi: Modifying the therapeutic index of amphotericin b by its incorporation into negatively charged liposomes

Abstract

The mechanism of action of liposome—incorporated amphotericin B (ABLC) is not well understood. Most studies to date have dealt with the role of liposome size, lipid composition, and fluidity on ABLC toxicity and activity. However, little is known about the behavior of ABLC once injected into the circulation. This review describes the behavior of ABLC, both as a particle and in a lipophilic system, following intravenous administration in an attempt to better understand the mechanisms involved in ABLC's enhanced therapeutic index. Our data suggest that once ABLC is injected into the circulation, two major processes probably occur: (1) exchange with lipoproteins: both the liposomal lipid and the liposome—associated AmpB are actively exchanged with lipoproteins; and (2) phagocytosis: liposomes are taken up by phagocytes that potentially carry the internalized ABLC to infection sites. As a result of these interactions, ABLC is more effectively delivered to fungal cells where ABLC is released from the phagocyte and AmpB is liberated from the lipid by fungal lipases. Furthermore, ABLC predominantly distributes into high—density lipoproteins (HDL) following incubation in human serum for 1 hour at 37°C. This HDL—associated ABLC is less toxic to renal cells than either AmpB or LDL—associated AmpB because of the low level of expression of HDL receptors on renal cells. These findings demonstrate that the interaction of ABLC with plasma lipoproteins and blood phagocytes may be responsible for ABLC's enhanced therapeutic index.     

Development and Characterization of Liposomal Tumor Necrosis Factor (Tnf)

Abstract

Free and liposomal formulations of caprylated TNF-SAM2, a TNF mutant, were evaluated in both a canine model for TNF-induced hypotension and in the murine Meth A sarcoma model for efficacy. The liposomal formulations consisted of caprylated TNF-SAM2 either bound to the outer leaflet of small unilamellar vesicles (SUVs) or encapsulated in multilamellar vesicles (MLVs). Whereas systemic administration of parental TNF-a to anesthesized mongrel dogs resulted in acute shock with a precipitous fall in systemic arterial pressure (SAP), the SUV-TNF-SAM2 resulted in no net hypotension. Both the free and liposomal-TNF-SAM2 elicited a hyperdynamic response with tachycardia and elevated cardiac output and SAP, which was not observed with parental TNF-a. Two tail-vein injections of these formulations were administered 4–5 days apart to BALB/c mice in which Meth A sarcoma cells were previously implanted subcutaneously or intradermally. Free TNF-SAM2 at 4 × 10 U/mouse caused highly significani tumor control compared to PBS on days 5 and 12; however, substantial toxicity (-24% lethality; was observed at this level. The liposomal formulations of caprylated TNF-SAM2 demonstratec significant tumor control at all dose levels (up to 8 × 10 U/mouse) on day 12 (p<0.025) wit! either no or reduced lethality (0–15%); the tumor responses were comparable to those with fret TNF-SAM2. We conclude that the liposomal caprylated-TNF-SAM2 formulations exhibit; therapeutic index superior to that of free parental TNF-a or free TNF-SAM2.     

Aerosolized Delivery of Antifungal Agents

Pulmonary infections caused by Aspergillus species are associated with significant morbidity and mortality in immunocompromised patients. Although the treatment of pulmonary fungal infections requires the use of systemic agents, aerosolized delivery is an attractive option in prevention because the drug can concentrate locally at the site of infection with minimal systemic exposure. Current clinical evidence for the use of aerosolized delivery in preventing fungal infections is limited to amphotericin B products, although itraconazole, voriconazole, and caspofungin are under investigation. Based on conflicting results from clinical trials that evaluated various amphotericin B formulations, the routine use of aerosolized delivery cannot be recommended. Further research with well-designed clinical trials is necessary to elucidate the therapeutic role and risks associated with aerosolized delivery of antifungal agents. This article provides an overview of aerosolized delivery systems, the intrapulmonary pharmacokinetic properties of aerosolized antifungal agents, and key findings from clinical studies.     

Aerosolized Antifungals for the Prevention and Treatment of Invasive Fungal Infections

Invasive fungal infections result in significant morbidity and mortality, most notably in immunosuppressed patients. Aerosolized antifungal agents have been utilized primarily as prophylaxis (either alone or in combination with systemic antifungals) in patients at highest risk of invasive infections in attempts to optimize drug delivery while minimizing the potential for systemic toxicity and/or drug interactions. Published clinical experience with aerosolized antifungals most frequently involves various formulations of the polyene amphotericin B in patients undergoing lung transplantation and/or select patients with hematologic malignancy. Adverse events are infrequent and generally limited to dyspnea, dysgeusia, and cough. Existing data suggests lipid-based amphotericin B formulations may be better tolerated than amphotericin B deoxycholate. Published clinical experience with aerosolized antifungals as adjunctive treatment of invasive fungal infections is limited to case reports. Currently, there is insufficient evidence to support use of aerosolized echinocandins and azoles in clinical practice. Outstanding questions regarding comparative efficacy, optimal dose, duration and drug delivery present a continuing challenge when utilizing these agents in clinical practice.     

Refractory disseminated fusariosis by Fusarium verticillioides in a patient with acute myeloid leukaemia relapsed after allogeneic hematopoietic stem cell transplantation: A case report and literature review

BackgroundFusarium species are among the leading fungal pathogens to cause invasive mould infections in patients with hematopoietic malignancy. The Fusarium species most frequently involved in human infections are Fusarium solani, Fusarium oxysporum and Fusarium verticillioides. However, identification is a cumbersome and time-consuming task. Fusarium is resistant in vitro to many of the antifungal agents and the management of fusariosis is not well defined.ObjectivesTo emphasise the difficulty of identifying Fusarium spp. by conventional methods and the need of new rapid molecular tests to achieve earlier diagnosis and appropriate therapy.MethodsA disseminated Fusarium infection due to F. verticillioides was documented in a neutropenic refractory patient with acute myeloid leukaemia, relapsed after allogeneic hematopoietic stem cell transplantation.ResultsThe patient died despite liposomal amphotericin B and voriconazole combination and “in vitro” susceptibility of agents employed. Morphological and molecular identification of F. verticillioides was obtained only after the death of the patient.ConclusionsThis case highlights the poor outcome of an invasive fungal disease caused by Fusarium in aplastic patients. Identification of members of Fusarium genus remains restricted to selected laboratories and should be introduced into routine mycological diagnostics. In immunocompromised patients, diagnosis of fusariosis is directly related to prompt diagnosis and to patient's status. Current diagnosis methods and therapeutic options are discussed.     

The role of echinocandins, extended-spectrum azoles, and polyenes to treat opportunistic moulds and Candida

Three classes of antifungals—polyenes, extended-spectrum azoles, and echinocandins—are now available for treating systemic fungal infections. Guidance for the appropriate use of this expanded variety of antifungals may come from recent clinical trials. Extended-spectrum azoles have excellent in vitro activity against Aspergillus and have been shown to improve clinical outcomes. For Zygomycetes, along with the lipid formulations of amphotericin, of the new agents, only posaconazole has activity. For Candida, the echinocandins offer a broad spectrum of activity. These new agents offer less toxicity and potentially improved efficacy in these difficult infections.     

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.     

Possible Trichosporon asahii urinary tract infection in a critically ill COVID-19 patient

BackgroundTrichosporon asahii, an emerging fungal pathogen, has been frequently associated with invasive infections in critically ill patients.Case reportA 74-year-old male patient diagnosed with COVID-19 was admitted to an Intensive Care Unit (ICU). During hospitalization, the patient displayed episodes of bacteremia by Staphylococcus haemolyticus and a possible urinary tract infection by T. asahii. While the bacterial infection was successfully treated using broad-spectrum antibiotics, the fungal infection in the urinary tract was unsuccessfully treated with anidulafungin and persisted until the patient died.ConclusionsWith the evolving COVID-19 pandemic, invasive fungal infections have been increasingly reported, mainly after taking immunosuppressant drugs associated with long-term broad-spectrum antibiotic therapy. Although Candida and Aspergillus are still the most prevalent invasive fungi, T. asahii and other agents have emerged in critically ill patients. Therefore, a proper surveillance and diagnosing any fungal infection are paramount, particularly in COVID-19 immunocompromised populations.     

Systemic antifungals. Pharmacodynamics and pharmacokinetics

Invasive fungal infections are important causes of morbidity and mortality in critically ill non neutropenic patients. For many years, amphotericin B and flucytosine have been the only available antifungal agents for invasive fungal infections. Fortunately, the antifungal armamentarium has increased during the past two decades with the addition of several new agents. In addition to itraconazole and fluconazole, lipid formulations of amphotericin B, voriconazole, and caspofungin have been recently licensed. These various antifungal agents differ in their pharmacokinetic and pharmacodynamic profile.     

Miconazole nitrate bearing ultraflexible liposomes for the treatment of fungal infection

Miconazole nitrate is a widely used antifungal agent, but its use in topical formulations is not efficacious because deep seated fungal infections are difficult to treat with conventional topical formulation. Miconazole nitrate loaded ultraflexible liposomes have been prepared and their topical performance has been compared with conventional liposomes containing miconazole nitrate. Various ultraflexible liposomal formulations were prepared and extensively characterized for vesicular shape, size, entrapment efficiency, degree of deformability and in-vitro skin permeation through rat skin. Higher rate of drug transfer across the skin with ultraflexible liposomal formulations of miconazole nitrate suggests that the drug in its lipo-solubilized state might have gained facilitated entry into the tough barrier consisting of subcutaneous. In-vivo study showed better antifungal activity as compared to traditional liposomes and plain drug solution. This was confirmed through fluoroscence microscopy. It is concluded that prepared ultraflexible liposomes can facilitate improved and localized drug action in the skin, thus providing a better option to deal with deep seated skin problems.     

AmBisome,tres retos: infección por Candida auris,infección del sistema nervioso central e infección asociada a biopelículas

The treatment of invasive fungal infections remains a challenge, both for the diagnosis and for the need of providing the appropriate antifungal therapy. Candida auris is a pathogenic yeast that is responsible for hospital outbreaks, especially in intensive care units; it is characterized by a high resistance to the antifungal agents and can become multidrug-resistant. At present, the recommended antifungal agents for the invasive infections with this pathogen are echinocandins, always after carrying out an antifungal susceptibility testing. In case of no clinical response or persistent candidemia, the addition of liposomal amphotericin B or isavuconazole may be considered. Both fungal infection of the central nervous system and that associated with biomedical devices remain rare entities affecting mainly immunocompromised patients. However, an increase in their incidence in recent years, along with high morbidity and mortality, has been shown. The treatment of these infections is conditioned by the limited knowledge of the pharmacokinetic properties of antifungals. A better understanding of the pharmacokinetic and pharmacodynamic parameters of the different antifungals is essential to determine the efficacy of the antifungal agents in the treatment of these infections.     

Coronavirus Disease (Covid-19) Associated Mucormycosis (CAM): Case Report and Systematic Review of Literature

Severe coronavirus disease (COVID-19) is currently managed with systemic glucocorticoids. Opportunistic fungal infections are of concern in such patients. While COVID-19 associated pulmonary aspergillosis is increasingly recognized, mucormycosis is rare. We describe a case of probable pulmonary mucormycosis in a 55-year-old man with diabetes, end-stage kidney disease, and COVID-19. The index case was diagnosed with pulmonary mucormycosis 21 days following admission for severe COVID-19. He received 5 g of liposomal amphotericin B and was discharged after 54 days from the hospital. We also performed a systematic review of the literature and identified seven additional cases of COVID-19 associated mucormycosis (CAM). Of the eight cases included in our review, diabetes mellitus was the most common risk factor. Three subjects had no risk factor other than glucocorticoids for COVID-19. Mucormycosis usually developed 10–14 days after hospitalization. All except the index case died. In two subjects, CAM was diagnosed postmortem. Mucormycosis is an uncommon but serious infection that complicates the course of severe COVID-19. Subjects with diabetes mellitus and multiple risk factors may be at a higher risk for developing mucormycosis. Concurrent glucocorticoid therapy probably heightens the risk of mucormycosis. A high index of suspicion and aggressive management is required to improve outcomes.

     

A 12-year study of fungal infections in Rio Grande do Sul,Southern Brazil

BackgroundThe number of fungal infections has increased in recent years in Rio Grande do Sul (RS), Brazil. Epidemiological studies are important for proper control of infections.AimsTo evaluate the etiology of fungal infections in patients in RS, from 2003 to 2015.MethodsThis is a retrospective and longitudinal study carried out at Mycology Department of Central Laboratory of RS; 13,707 samples were evaluated. The variables sex, age, site of infection, and etiologic agent were analyzed. Susceptibility of Candida to fluconazole was tested in isolates from samples collected in 2015 from 51 outpatients.ResultsOf the 13,707 samples, 840 cases (6.12%) of fungal infections were found and included in the analyses; female gender accounted for the 55.9% of the cases. The main fungus was Candida albicans (450 cases, 53.38%; p < 0.001). Onychomycosis was the most frequent infection in superficial mycoses. Systemic mycoses accounted for 54.05% of the cases, from which 68.8% occurred in males, mainly HIV-positive (33.11%), and the main etiologic agent in these cases was Cryptococcus neoformans (73.13%). Among 51 samples tested for susceptibility to fluconazole, 78.43% of Candida isolates were susceptible; 5.88% were susceptible in a dose-dependent manner, and 15.69% were resistant.ConclusionsC. albicans is a common cause of fungal infections in RS, accounting for half of the cases; resistance to antifungals was found in non-hospitalized patients. In addition, women seem to be more susceptible to fungal infections than men, however men show more systemic mycoses than women. The nails are the most common site of infection.     

Infección múltiple fúngica en un paciente diabético

BackgroundMixed fungal infections although undervalued, are more common than mentioned in the scientific literature. These infections have a poor prognosis for the patient.ObjectivesWe present an unusual case of a 61-year-old diabetic male who had a rhino-orbito-sinusal zygomycosis in 2001. After surgical debridement of the infected parts, along with antifungal therapy with liposomal amphotericin B, the patient started improving. Several years later the patient was hospitalized due to a similar problem and was diagnosed of rhino-orbito-cerebral zygomycosis.MethodsIn both episodes, a histopathological examination and cultures were performed on the sinus lesions. Tissue sections were stained with haematoxylin and eosin, Giemsa, periodic acid-Schiff (PAS) and Grocott's methenamine silver, and cultures specific for fungi were performed.ResultsThe histopathology studies revealed the presence of bacteria, actinomyces and a mixed infection by at least four different fungi, all of them well differentiated by their morphology. Despite the rapid diagnosis the patient died due to spreading to the central nervous system.ConclusionsMixed infections by fungi are rare, but due to the high incidence of immunodeficiencies they could occur more often than reported. We would like to alert on the possibility of acquired mixed infection by fungi which have shown to be high aggressive and have a worse prognostic in patients with underlying diseases.     

Five cases of zygomycosis.

Combined histological and mycological study of tissue specimens established a proven diagnosis of cutaneous zygomycosis in four patients. All patients had been treated with wide spectrum antibiotics and one patient (liver transplantation) was in addition also treated with cyclosporine. All had acidosis and cutaneous breaks and four had also been treated with systemic corticosteroids. The infecting organisms were Absidia corymbifera (n=2), Rhizopus stolonifer (n=1) and Mucor circinelloides (n=1). Combined treatment with i.v. conventional and liposomal formulations of amphotericin B and surgical treatment lead to a favourable clinical and mycological cure in three patients (A. corymbifera and R. stolonifer infections). One lymphoma patient with suspected Rhizopus pusillus infection of the lungs (presence of hyphae in sputum and positive culture) had an unfavourable outcome. The patient had been treated with wide spectrum antibiotics, corticosteroids and showed severe neutropenia and acidosis. The clinical presentations are outlined, including the outcomes and predisposing factors and focus on the diagnostic procedures, treatment and preventive measures.     

Interaction of liposomes bearing a lipophilic doxorubicin prodrug with tumor cells

When used as nanosized carriers, liposomes enable targeted delivery and decrease systemic toxicity of antitumor agents significantly. However, slow unloading of liposomes inside cells diminishes the treatment efficiency. The problem could be overcome by the adoption of lipophilic prodrugs tailored for incorporation into lipid bilayer of liposomes. We prepared liposomes of egg yolk phosphatidylcholine and yeast phosphatidylinositol bearing a diglyceride conjugate of an antitumor antibiotic doxorubicin (a lipophilic prodrug, DOX-DG) in the membrane to study how these formulations interact with tumor cells. We also prepared liposomes of rigid bilayer-forming lipids, such as a mixture of dipalmitoylphosphatidylcholine and cholesterol, bearing DOX in the inner water volume, both pegylated (with polyethylene glycol (PEG) chains exposed to water phase) and non-pegylated. Efficiency of binding of free and liposomal doxorubicin with tumor cells was evaluated in vitro using spectrofluorimetry of cell extracts and flow cytometry. Intracellular traffic of the formulations was investigated by confocal microscopy; co-localization of DOX fluorescence with organelle trackers was estimated. All liposomal formulations of DOX were shown to distribute to organelles retarding its transport to nucleus. Intracellular distribution of liposomal DOX depended on liposome structure and pegylation. We conclude that the most probable mechanism of the lipophilic prodrug penetration into a cell is liposome-mediated endosomal pathway.     

Análisis de la utilización de anfotericina B liposomal

BackgroundThe increase in immunosuppressed patients and in the use of cytotoxic and immunosuppressive agents in the last few years has led to a rise in the incidence of invasive fungal infections.AimsThe objective of this study is to evaluate the use of liposomal amphotericin B according to its indication, dosage, effectiveness and nephrotoxicity.MethodsThis is a retrospective study over a 8 year-period. Indications and dosage described in the Summary of Product Characteristics were taken as reference. Effectiveness was measured in terms of resolution of infection, no recurrence or emergence, survival at 7th day, no discontinuation and no addition of another antifungal. Effectiveness was also analysed in relation to indication, dosage, treatment duration, cumulative dose and comorbidity. Nephrotoxicity was defined as a doubled serum creatinine when compared with basal values.ResultsA total of 47 episodes were analysed, with 91.5% of treatments being adequate for the indications. Effectiveness was achieved in 44.7% of cases: 33% in neutropenic patients, 50% of aspergillosis, 60% of candidiasis, and 100% of the leishmaniasis. Response rates in patients treated for 15 days or less and those for more than 15 days were 25 and 56.5% (P = .039), respectively. The main causes of failure were death (23%) and lack of effectiveness (17%), with 9% of patients suffering from nephrotoxicity.ConclusionsThe effectiveness and nephrotoxicity data agree with those obtained in other studies. Liposomal amphotericin B is a safe drug with a moderate effectiveness that is associated with the duration of the treatment.     

Systemic fungal infections: A pharmacist/researcher perspective

Systemic fungal infections are increasing in prevalence, especially in immunocompromised patients and post-surgical patients. The rise in systemic fungal infections has resulted in increased utilization of antifungal agents which, in turn, has contributed to escalating resistance rates as seen by the increasing number of fungal pathogens added to the Centers for Disease Control and Prevention Antimicrobial Resistance Threats Report in 2019. Unfortunately, there are few novel antifungal agents coming to market to combat these ever-increasing resistance rates. In this review, we cover the current climate of antifungal agents and explore agents coming through the pipeline and potentially to market. We also discuss the indicated uses for empiric and targeted antifungal therapies as well as the need to include antifungal agents as a part of antimicrobial stewardship programs that focus heavily on antibacterial agents.     

<Emphasis Type="Italic">Acr</Emphasis><Emphasis Type="Italic">emonium</Emphasis> Species: A Review of the Etiological Agents of Emerging Hyalohyphomycosis

Unusual fungal agents that exist environmentally as saprophytes can often lead to opportunistic infections. Hyalohyphomycosis is a group of fungal infections caused by fungi characterized by hyaline septate hyphae and can infect both immunocompetent as well as immunocompromised patients. Many a times it becomes difficult to distinguish a pathogenic and a contaminant fungus, because many such agents can assume clinical significance depending on circumstances. Subcutaneous and invasive fungal infection due to the emerging hyalohyphomycotic fungus, Acremonium, has drawn the attention of clinicians and microbiologists, as a potential pathogen in patients with and without underlying risk factors. Generally considered to be minimally invasive in the past, genus Acremonium has been responsible for eumycotic mycetomas and focal infections in otherwise healthy individuals. It has also been increasingly implicated in systemic fungal diseases. The management with different antifungals in various clinical situations has been very conflicting and hence needs to be carefully evaluated. This overview is an endeavor to consolidate the available clinical infections due to Acremonium and the recommendations on treatment.     

医院获得性真菌尿路感染的临床特点

目的 :了解我院真菌尿路感染现状 ,探讨防治对策。方法 :回顾分析 1998～ 2 0 0 0年我院医院感染调查中 73例真菌尿路感染的临床特点。结果 :平均年龄 5 4 4 9岁 ,≥ 6 0岁占 4 2 4 7% ,平均住院日5 4 76d ;见于多种基础疾病 ,与恶性肿瘤、长期卧床、危重疾病有关 ;发生真菌感染前抗生素使用率 10 0 % ,78 0 8%联用两种以上抗生素 ,平均使用抗生素时间为 (35± 14 )d ;感染前应用激素者 2 7,4 0 % ,化疗16 4 4 % ,导尿等泌尿操作 4 7 95 % ;临床表现不典型 ,抗真菌治疗效果欠佳 ,病死率 2 4 6 6 %。结论 :年龄大、住院时长、长期卧床及需长期应用抗生素者 ,无论是否导尿 ,均易发生真菌尿路感染 ,应随时监测 ,以便及时采取综合治疗措施 ,降低病死率。