Safety, Tolerance, and Pharmacokinetics of a Small Unilamellar Liposomal Formulation of Amphotericin B (AmBisome) in Neutropenic Patients

The safety, tolerance, and pharmacokinetics of a small unilamellar liposomal formulation of amphotericin B (AmBisome) administered for empirical antifungal therapy were evaluated for 36 persistently febrile neutropenic adults receiving cancer chemotherapy and bone marrow transplantation. The protocol was an open-label, sequential-dose-escalation, multidose pharmacokinetic study which enrolled a total of 8 to 12 patients in each of the four dosage cohorts. Each cohort received daily doses of either 1.0, 2.5, 5.0, or 7.5 mg of amphotericin B in the form of AmBisome/kg of body weight. The study population consisted of patients between the ages of 13 and 80 years with neutropenia (absolute neutrophil count, <500/mm³) who were eligible to receive empirical antifungal therapy. Patients were monitored for safety and tolerance by frequent laboratory examinations and the monitoring of infusion-related reactions. Efficacy was assessed by monitoring for the development of invasive fungal infection. The pharmacokinetic parameters of AmBisome were measured as those of amphotericin B by high-performance liquid chromatography. Noncompartmental methods were used to calculate pharmacokinetic parameters. AmBisome administered as a 1-h infusion in this population was well tolerated and was seldom associated with infusion-related toxicity. Infusion-related side effects occurred in 15 (5%) of all 331 infusions, and only two patients (5%) required premedication. Serum creatinine, potassium, and magnesium levels were not significantly changed from baseline in any of the dosage cohorts, and there was no net increase in serum transaminase levels. AmBisome followed a nonlinear dosage relationship that was consistent with reticuloendothelial uptake and redistribution. There were no breakthrough fungal infections during empirical therapy with AmBisome. AmBisome administered to febrile neutropenic patients in this study was well tolerated, was seldom associated with infusion-related toxicity, was characterized by nonlinear saturation kinetics, and was effective in preventing breakthrough fungal infections.     

Pharmacokinetics of Liposomal Amphotericin B in Pleural Fluid

We report the penetration of liposomal amphotericin B into the pleural fluid of a patient with pulmonary zygomycosis and empyema. The ratio of area under the concentration-versus-time curve in pleural fluid (AUC_{pleural fluid}) to that in serum (AUC_serum) for liposomal amphotericin B over 24 h was 9.4%, with pleural fluid concentrations of 2.12 to 4.91 μg/ml. Given the relatively low level of intrapleural penetration of liposomal amphotericin B, chest tube drainage may be warranted for successful treatment of zygomycotic empyema.Invasion of the pleural space is a serious complication of fungal pneumonias (9). There is limited information on the penetration of liposomal amphotericin B (LAmB) into the pleural fluid of humans (20). We report herein a detailed pharmacokinetic sampling and analysis of the penetration of LAmB into the pleural fluid of a patient with pulmonary zygomycosis (mucormycosis) and empyema.     

Comparative Toxicological Studies of Amphotericin B Methyl Ester and Amphotericin B in Mice, Rats, and Dogs

In acute and subacute toxicological studies, amphotericin B methyl ester was shown to be much less toxic than the parent antibiotic. As a single intravenous dose in mice, the methyl ester was approximately 20 times less toxic than amphotericin B. Also, the acute toxicity of the methyl ester in mice was not enhanced by the presence of chemically induced hepatic or renal damage or by the concurrent administration of amphotericin B or flucytosine. In a 1-month intraperitoneal study in rats, the methyl ester was about one-fourth as nephrotoxic as amphotericin B. In a 1-month intravenous study in dogs, the methyl ester was about one-eighth as nephrotoxic and one-fourth to one-half as hepatotoxic as the parent compound. In addition, the methyl ester, unlike amphotericin B, produced minimal renal effects, which did not increase in severity with increasing dosage. Based on the results of these studies, it is concluded that amphotericin B methyl ester has the potential for an improved therapeutic ratio in the treatment of systemic mycoses.     

Pharmacokinetics and Pharmacodynamics of Amphotericin B Deoxycholate,Liposomal Amphotericin B,and Amphotericin B Lipid Complex in an In Vitro Model of Invasive Pulmonary Aspergillosis

The pharmacodynamic and pharmacokinetic (PK-PD) properties of amphotericin B (AmB) formulations against invasive pulmonary aspergillosis (IPA) are not well understood. We used an in vitro model of IPA to further elucidate the PK-PD of amphotericin B deoxycholate (DAmB), liposomal amphotericin B (LAmB) and amphotericin B lipid complex (ABLC). The pharmacokinetics of these formulations for endovascular fluid, endothelial cells, and alveolar cells were estimated. Pharmacodynamic relationships were defined by measuring concentrations of galactomannan in endovascular and alveolar compartments. Confocal microscopy was used to visualize fungal biomass. A mathematical model was used to calculate the area under the concentration-time curve (AUC) in each compartment and estimate the extent of drug penetration. The interaction of LAmB with host cells and hyphae was visualized using sulforhodamine B-labeled liposomes. The MICs for the pure compound and the three formulations were comparable (0.125 to 0.25 mg/liter). For all formulations, concentrations of AmB progressively declined in the endovascular fluid as the drug distributed into the cellular bilayer. Depending on the formulation, the AUCs for AmB were 10 to 300 times higher within the cells than within endovascular fluid. The concentrations producing a 50% maximal effect (EC₅₀) in the endovascular compartment were 0.12, 1.03, and 4.41 mg/liter for DAmB, LAmB, and ABLC, respectively, whereas, the EC₅₀ in the alveolar compartment were 0.17, 7.76, and 39.34 mg/liter, respectively. Confocal microscopy suggested that liposomes interacted directly with hyphae and host cells. The PK-PD relationships of the three most widely used formulations of AmB differ markedly within an in vitro lung model of IPA.Aspergillus fumigatus is an environmentally ubiquitous mold that is a leading cause of morbidity and mortality in immunocompromised patients (18). Despite the advent of newer diagnostic and therapeutic modalities, the mortality rate remains approximately 50% (22). An improved understanding of the pharmacology of existing agents represents an important strategy to improve the outcomes of patients with this rapidly progressive and frequently lethal infectious syndrome.Amphotericin B (AmB) is a polyene derived from Streptomyces nodosus. This compound was discovered in the mid-1950s and remains a first-line agent for the treatment of invasive aspergillosis and other life-threatening invasive fungal infections (23, 24). Amphotericin B is amphipathic; i.e., it has both hydrophilic and hydrophobic moieties that render it insoluble in water. Aqueous solubility is achieved by formulation with deoxycholate or a variety of lipid carriers. Amphotericin B deoxycholate (DAmB) is a highly potent antifungal formulation, but its clinical utility is limited by a high frequency of adverse effects, such as infusional toxicity and nephrotoxicity (3, 27). Lipid formulations are better tolerated than DAmB and are increasingly used for the treatment of invasive pulmonary aspergillosis (IPA). Three licensed lipid-based formulations have been developed for clinical use: liposomal amphotericin (LAmB), amphotericin B lipid complex (ABLC), and amphotericin B colloidal dispersion (ABCD). These formulations differ significantly in their structures and pharmacological properties (1).Here, we describe the pharmacokinetics and pharmacodynamics (PK-PD) of the frequently used clinical formulations of amphotericin B by the use of an in vitro model of IPA. This model enabled assessment of the extent of drug penetration into a number of tissue subcompartments that are relevant to the pathogenesis of IPA.     

Pharmacodynamics of Amphotericin B Deoxycholate,Amphotericin B Lipid Complex,and Liposomal Amphotericin B against Aspergillus fumigatus

Amphotericin B is a first-line agent for the treatment of invasive aspergillosis. However, relatively little is known about the pharmacodynamics of amphotericin B for invasive pulmonary aspergillosis. We studied the pharmacokinetics (PK) and pharmacodynamics (PD) of amphotericin B deoxycholate (DAMB), amphotericin B lipid complex (ABLC), and liposomal amphotericin B (LAMB) by using a neutropenic-rabbit model of invasive pulmonary aspergillosis. The study endpoints were lung weight, infarct score, and levels of circulating galactomannan and (1→3)-β-d-glucan. Mathematical models were used to describe PK-PD relationships. The experimental findings were bridged to humans by Monte Carlo simulation. Each amphotericin B formulation induced a dose-dependent decline in study endpoints. Near-maximal antifungal activity was evident with DAMB at 1 mg/kg/day and ABLC and LAMB at 5 mg/kg/day. The bridging study suggested that the “average” patient receiving LAMB at 3 mg/kg/day was predicted to have complete suppression of galactomannan and (1→3)-β-d-glucan levels, but 20 to 30% of the patients still had a galactomannan index of >1 and (1→3)-β-d-glucan levels of >60 pg/ml. All formulations of amphotericin B induce a dose-dependent reduction in markers of lung injury and circulating fungus-related biomarkers. A clinical dosage of liposomal amphotericin B of 3 mg/kg/day is predicted to cause complete suppression of galactomannan and (1→3)-β-d-glucan levels in the majority of patients.     

Amphotericin B Pharmacokinetics in Humans

The pharmacokinetics of amphotericin B were studied in two patients at the conclusion of long-term therapy for disseminated histoplasmosis. The distribution kinetics of this drug were adequately described by a three-compartment mamillary model with a total distribution volume averaging 4 liters/kg. The elimination phase half-life of amphotericin B was approximately 15 days, reflecting slow release of amphotericin B from a peripheral compartment. In accordance with previous reports, renal excretion accounted for only 3% of total amphotericin B elimination. The pharmacokinetic model for one of the patients also was used to compare the simulated amphotericin B serum levels that would be expected if initial therapy followed two recommended regimens.     

Comparative Pharmacodynamics of Amphotericin B Lipid Complex and Liposomal Amphotericin B in a Murine Model of Pulmonary Mucormycosis

We compared the kinetics of amphotericin B (AMB) lung accumulation and fungal clearance by liposomal amphotericin B (L-AMB) and amphotericin B lipid complex (ABLC) in a neutropenic murine model of invasive pulmonary mucormycosis (IPM). Immunosuppressed BALB/c mice were inoculated with 1 × 10⁶ Rhizopus oryzae spores and administered L-AMB or ABLC at daily intravenous doses of 1, 5, or 10 mg/kg of body weight for 5 days starting 12 h after infection. At a dose of 10 mg/kg/day, both L-AMB and ABLC were effective at reducing the R. oryzae lung fungal burden and achieved lung tissue concentrations exceeding the isolate mean fungicidal concentration (MFC) of 8 μg/ml by 72 h. When ABLC was dosed at 5 mg/kg/day, the ABLC-treated animals had significantly higher AMB lung concentrations than the L-AMB treated animals at 24 h (6.64 and 1.44 μg/g, respectively; P = 0.013) and 72 h (7.49 and 1.03 μg/g, respectively; P = 0.005), and these higher concentrations were associated with improved fungal clearance, as determined by quantitative real-time PCR (mean conidial equivalent of R. oryzae DNA per lung, 4.44 ± 0.44 and 6.57 ± 0.74 log₁₀, respectively; P < 0.001). Analysis of the AMB tissue concentration-response relationships revealed that the suppression of R. oryzae growth in the lung required tissue concentrations that approached the MFC for the infecting isolate (50% effective concentration, 8.19 μg/g [95% confidence interval, 2.81 to 18.1 μg/g]). The rates of survival were similar in the animals treated with L-AMB and ABLC at 10 mg/kg/day. These data suggest that higher initial doses may be required during L-AMB treatment than during ABLC treatment of experimental IPM.Invasive pulmonary mucormycosis (IPM) is an uncommon but frequently fatal angioinvasive mold infection that has increased in incidence over the last decade, especially in patients with hematological malignancies and recipients of hematopoietic stem cell transplantation (HSCT) (23). In a recent multicenter, prospective observational study of invasive fungal infections in HSCT recipients, mucormycosis was the third most common invasive fungal infection (7.2%), behind invasive aspergillosis (59.2%) and invasive candidiasis (24.8%) (21). Data from the Centers for Disease Control and Prevention Transplant Associated Infection Surveillance Network (TRANSNET) reported that the incidence of mucormycosis in U.S. transplant centers increased nearly sixfold from 2001 to 2004, with Rhizopus being the most frequently isolated genus (22).Although new diagnostic and treatment options have improved the survival rates in patients with invasive pulmonary aspergillosis (IPA) over the last decade, the prognosis for patients with IPM remains poor, as only one-third of the patients survive beyond 12 weeks after the diagnosis (13, 21, 23). The outcome of IPM is heavily dependent on a timely diagnosis, as the initial clinical manifestations and radiographic appearance of IPM are often indistinguishable from those of IPA, and the first-line antifungals used to treat aspergillosis, such as voriconazole, lack activity against members of the order Mucorales (24). In one case series, 84% of leukemia and HSCT patients were receiving ineffective antifungal therapy at the time of diagnosis of IPM (15). Similarly, we found that delays in the administration of lipid amphotericin B (AMB) formulations as few as 6 days from the time of the initial appearance of symptoms was associated with a doubling of the 12-week mortality rate for IPM (48.6% and 82.9%, respectively; P = 0.029) (6). These data suggest that the rapid delivery to infected organs of antifungals active against Mucorales is critical to suppress fungal proliferation and reduce the potential for angioinvasion and subsequent dissemination (6).Although no prospective randomized trials have compared antifungals for the primary treatment of IPM, lipid formulations of AMB are considered the first-line treatment during the acute phases of infection due to their spectra of activity and predictable pharmacokinetics (12). Currently, two lipid formulations are frequently prescribed for the treatment of IPM: AMB lipid complex (ABLC) and liposomal AMB (L-AMB). These formulations differ in their compositions, particle sizes, and pharmacokinetic behaviors. L-AMB consists of small unilamellar particles (60 to 70 nm) that avoid uptake by the mononuclear phagocytic system (MPS) (28). Hence, the intravenous administration of L-AMB results in sustained, high concentrations of encapsulated AMB in the bloodstream and a somewhat delayed distribution of free drug into tissue. Conversely, the intravenous administration of the larger-particle ABLC formulation (1,600 to 11,000 nm) results in relatively lower AMB bloodstream concentrations due to the rapid uptake and distribution to tissues rich in mononuclear phagocytic cells, including lung tissue (9, 18, 19). The clinical relevance of these pharmacokinetic differences between L-AMB and ABLC, however, remains unknown.In a previous study, we examined how pharmacokinetic differences between L-AMB and ABLC affected the rate and extent of fungal clearance in a neutropenic murine model of IPA (16). At daily doses of <10 mg/kg of body weight per day, ABLC treatment achieved significantly higher concentrations of AMB in lung tissue at earlier time points of therapy, and the higher concentrations were associated with the more rapid clearance of Aspergillus fumigatus. However, both formulations were effective after 5 days of therapy for the treatment of this AMB-susceptible isolate. Nevertheless, formulation-dependent differences in antifungal pharmacokinetics may be more critical in the treatment of pulmonary infections caused by more angioinvasive, amphotericin B-tolerant pathogens, such as Rhizopus orzyae. Therefore, we examined whether the differences in the pharmacokinetics between L-AMB and ABLC may require the use of different dosing approaches for the treatment of mucormycosis. To explore this question, we compared (i) the patterns of AMB accumulation in lung tissue following intravenous treatment with L-AMB or ABLC and (ii) the dose-dependent patterns of Rhizopus oryzae clearance from the lung in an experimental model of IPM.(This work was previously presented at the 48th Interscience Conference on Antimicrobial Agents and Chemotherapy [15a].)     

Comparative Efficacies of Lipid-Complexed Amphotericin B and Liposomal Amphotericin B against Coccidioidal Meningitis in Rabbits

In separate previous studies, we have shown that lipid-complexed amphotericin B (Abelcet [ABLC]) and liposomal amphotericin B (AmBisome [AmBi]) are efficacious against coccidioidal meningitis in rabbits. Here, we compared ABLC and AmBi directly in a coccidioidal meningitis model. Male New Zealand White rabbits were infected with 5 × 10⁴ Coccidioides posadasii arthroconidia by direct cisternal puncture. Therapy with intravenous ABLC or AmBi at 7.5 or 15 mg/kg of body weight or sterile 5% dextrose water (D5W) began 5 days later. Clinical assessments were done daily; cerebrospinal fluid and blood samples were obtained on day 15 and upon euthanasia. Survivors to day 25 were euthanatized, the numbers of CFU in their tissues were determined, and histology analyses of the brains and spinal cords were done. Controls showed progressive disease, whereas animals treated with either dose of either drug showed few clinical signs of infection. All ABLC- or AmBi-treated rabbits survived, whereas eight of nine D5W-treated rabbits were euthanatized before day 25 (P < 0.0001). Numbers of CFU in the brains and spinal cords of ABLC- or AmBi-treated animals were 100- to 10,000-fold lower than those in the corresponding tissues of D5W-treated animals (P < 0.0006 to 0.0001). However, only two or fewer given a regimen of ABLC or AmBi were cured of infection in both tissues. Fewer ABLC-treated rabbits (four of eight treated with 7.5 mg/kg and five of eight treated with 15 mg/kg) than controls (nine of nine) had meningitis at any level of severity (P, 0.015 or 0.043 for animals treated with ABLC at 7.5 or 15 mg/kg, respectively). Although groups of rabbits treated with AmBi regimens did not have significantly fewer animals with meningitis than the control group (P > 0.05), ABLC and AmBi were not significantly different. In this model, intravenous ABLC and AmBi were similarly highly effective, with few clinical signs of infection, 100% survival, and significantly reduced fungal burdens among treated animals. There appeared to be little benefit in using the 15-mg/kg dosage of either formulation. There was no significant advantage of one drug over the other for this indication. Further studies are required to determine the lowest effective doses of these formulations.Coccidioidal meningitis remains a severe and lethal infection (4, 7, 9-11). If left untreated, it is considered uniformly fatal within 2 years. Despite advances in oral azole therapy over the last decade, therapy is prolonged, often considered to be lifelong, and relapses can occur (3, 8). In addition, cure of the disease may not be attainable using oral azoles, regardless of the treatment duration (3). The use of conventional amphotericin B is limited, since the intravenous administration of conventional amphotericin B is ineffective for treatment. The intrathecal administration of amphotericin B is effective in many instances but is suboptimal because of associated side effects and toxicities and because of technical issues of administration. Thus, improvements in therapy are needed for the treatment of this devastating manifestation of coccidioidomycosis.In previous individual studies, we have shown that intravenous lipid-complexed amphotericin B (Abelcet [ABLC]) and liposomal amphotericin B (AmBisome [AmBi]) are efficacious in a rabbit model of coccidioidal meningitis (1, 2). Dosages of amphotericin B as ABLC or AmBi of 7.5 and 15 mg per kg of body weight given three times per week for 3 weeks were most effective and resulted in cures in some animals, whereas 1 mg/kg of conventional amphotericin B was much less effective and noncurative in the rabbit. These results suggest that intravenous therapy with ABLC or AmBi may be effective, whereas no dose of conventional amphotericin B is effective by this route in humans. Although both lipid formulations are effective in the model, the question of whether one formulation was superior to the other when the two were given at equivalent doses was unanswered. Comparing drug efficacies across studies done at different times does not account for possible variations in the inoculum level or the virulence of the organism, induced disease severity, or variation in the experimental animals and may lead to an incorrect conclusion. Slight differences in disease severity may result in a drug''s being efficacious in a moderately severe infection model but ineffective in a highly severe infection model. Therefore, it is important to do comparisons of the efficacies of drugs side by side, with the variables of the experimental system constant. Thus, the aim of the present study was to compare ABLC and AmBi directly in the same experiment to determine their efficacies against experimental coccidioidomycosis.     

Influence of Liposomal Amphotericin B on CD8 T-Cell Function

Liposomal amphotericin B was immunosuppressive on target cell lysis in vitro and on protection mediated by cytotoxic CD8 T cells in murine listeriosis. When dosages usually used for therapy in humans were compared, the immunosuppressive effect of 5 mg of liposomal amphotericin B/kg of body weight/day was similar to that of standard amphotericin B at 1 mg/kg/day, but a dosage of liposomal amphotericin B of 1 mg/kg/day was not suppressive in vivo.     

Efficacy of Liposomal Amphotericin B and Posaconazole in Intratracheal Models of Murine Mucormycosis

Mucormycosis is a life-threatening fungal infection almost uniformly affecting diabetics in ketoacidosis or other forms of acidosis and/or immunocompromised patients. Inhalation of Mucorales spores provides the most common natural route of entry into the host. In this study, we developed an intratracheal instillation model of pulmonary mucormycosis that hematogenously disseminates into other organs using diabetic ketoacidotic (DKA) or cyclophosphamide-cortisone acetate-treated mice. Various degrees of lethality were achieved for the DKA or cyclophosphamide-cortisone acetate-treated mice when infected with different clinical isolates of Mucorales. In both DKA and cyclophosphamide-cortisone acetate models, liposomal amphotericin B (LAmB) or posaconazole (POS) treatments were effective in improving survival, reducing lungs and brain fungal burdens, and histologically resolving the infection compared with placebo. These models can be used to study mechanisms of infection, develop immunotherapeutic strategies, and evaluate drug efficacies against life-threatening Mucorales infections.     

Case-Control Study and Case Series of Pseudohyperphosphatemia during Exposure to Liposomal Amphotericin B

Pseudohyperphosphatemia due to an interaction between liposomal amphotericin B and the Beckman Coulter PHOSm assay occurs sporadically and remains underrecognized in clinical practice. This retrospective case-control study compares the incidences of hyperphosphatemia in adult inpatients exposed to liposomal amphotericin B or a triazole. A case series of patients with confirmed pseudohyperphosphatemia is described. A total of 80 exposures to liposomal amphotericin B and 726 exposures to triazoles were identified. Among subjects without chronic kidney disease and no concomitant acute kidney injury, hyperphosphatemia occurred more often during liposomal amphotericin B therapy than during triazole therapy (40% [14/35 cases] versus 10% [47/475 cases] of cases; P < 0.01; adjusted odds ratio, 5.2 [95% confidence interval {CI}, 2.3 to 11.9]). Among individuals with chronic kidney disease and no concomitant acute kidney injury, hyperphosphatemia also occurred more often during liposomal amphotericin B exposure (59% [10/17 cases] versus 20% [34/172 cases] of cases; P < 0.01; adjusted odds ratio, 6.0 [95% CI, 2.0 to 18.0]). When acute kidney injury occurred during antifungal exposure, the frequencies of hyperphosphatemia were not different between treatments. Seven episodes of unexpected hyperphosphatemia during liposomal amphotericin B exposure prompted a confirmatory test using an endpoint-based assay that found lower serum phosphorus levels (median difference of 2.5 mg/dl [range, 0.6 to 3.6 mg/dl]). Liposomal amphotericin B exposure confers a higher likelihood of developing hyperphosphatemia than that with exposure to a triazole antifungal, which is likely attributable to pseudohyperphosphatemia. Elevated phosphorus levels in patients receiving liposomal amphotericin B at institutions using the Beckman Coulter PHOSm assay should be interpreted cautiously.     

Randomized Comparison of Safety and Pharmacokinetics of Caspofungin,Liposomal Amphotericin B,and the Combination of Both in Allogeneic Hematopoietic Stem Cell Recipients

The combination of liposomal amphotericin B (LAMB) and caspofungin (CAS) holds promise to improve the outcome of opportunistic invasive mycoses with poor prognosis. Little is known, however, about the safety and pharmacokinetics of the combination in patients at high risk for these infections. The safety and pharmacokinetics of the combination of LAMB and CAS were investigated in a risk-stratified, randomized, multicenter phase II clinical trial in 55 adult allogeneic hematopoietic stem cell recipients (aHSCT) with granulocytopenia and refractory fever. The patients received either CAS (50 mg/day; day 1, 70 mg), LAMB (3 mg/kg of body weight/day), or the combination of both (CASLAMB) until defervescence and granulocyte recovery. Safety, development of invasive fungal infections, and survival were assessed through day 14 after the end of therapy. Pharmacokinetic sampling and analysis were performed on days 1 and 4. All three regimens were well tolerated. Premature study drug discontinuations due to grade III/IV adverse events occurred in 1/18, 2/20, and 0/17 patients randomized to CAS, LAMB, and CASLAMB, respectively. Adverse events not leading to study drug discontinuation were frequent but similar across cohorts, except for a higher frequency of hypokalemia with CASLAMB (P < 0.05). Drug exposures were similar for patients receiving combination therapy and those randomized to monotherapy. There was no apparent difference in the occurrence of proven/probable invasive fungal infections and survival through day 14 after the end of therapy. CASLAMB combination therapy in immunocompromised aHSCT patients was as safe as monotherapy with CAS or LAMB and had similar plasma pharmacokinetics, lending support to further investigations of the combination in the management of patients with invasive opportunistic mycoses.Invasive opportunistic fungal infections are an important cause of morbidity and mortality following allogeneic hematopoietic stem cell transplantation. Depending on the presence of well-characterized risk factors, rates of infection by opportunistic fungal pathogens are between 10 and 25%. The case fatality rates vary between 35 and 50% for invasive candidiasis and 65 and 90% for invasive aspergillosis and infections by other filamentous fungi (8, 15).The existence of antifungal agents with different molecular targets is providing new opportunities to improve the efficacy of antifungal chemotherapy through combination therapy. Potential target populations include profoundly immunocompromised patients with prolonged granulocytopenia or following allogeneic hematopoietic stem cell transplantation, patients with fulminant or refractory infections, or those with infections in compartments that are difficult to treat and infections by fungal pathogens with decreased microbiological susceptibility (23, 31, 39, 48, 49, 50).Based on its favorable microbiologic and pharmacokinetic properties (16), well-documented efficacy against Candida and Aspergillus infections, and excellent safety (24, 28, 32, 45), the echinocandin lipopeptide caspofungin (CAS) is a suitable candidate for antifungal combination therapies with liposomal amphotericin B (LAMB), a standard agent with broad-spectrum antifungal activity (18) and first-line indications against major opportunistic fungal infections (12, 22, 39). While preclinical studies of the combination of caspofungin and amphotericin B (CASLAMB)) have documented the absence of antagonistic effects in vitro and in animal infection models (3, 4, 5, 10, 19, 20, 30, 38), the clinical experience with combination therapies of caspofungin and lipid formulations of amphotericin B is more difficult to assess (1, 9, 21, 24, 34). Particularly in the setting of allogeneic hematopoietic stem cell transplantation and immunosuppression with cyclosporine, the safety of caspofungin alone and in combination with liposomal amphotericin B and the pharmacokinetic interactions of the combination have not been systematically studied.     

Biofilm Production and Antibiofilm Activity of Echinocandins and Liposomal Amphotericin B in Echinocandin-Resistant Yeast Species

The echinocandins and liposomal amphotericin B are active against biofilm produced by echinocandin-susceptible Candida strains. However, few data have been reported on the production of biofilm by echinocandin-resistant isolates and their antifungal susceptibility. We studied the production of biofilm by fks mutant Candida strains and intrinsically echinocandin-resistant non-Candida isolates and the susceptibility of both entities to liposomal amphotericin B and echinocandins. We analyzed the production of biofilm by isolates from patients with fungemia (fks mutant Candida, n = 5; intrinsically echinocandin-resistant non-Candida, n = 12; and Candida wild type, n = 10). Biofilm formation was measured to classify strains according to biomass (crystal violet assay) and metabolic activity (XTT reduction assay). Preformed biofilms were tested against liposomal amphotericin B, caspofungin, micafungin, and anidulafungin. The sessile MIC was defined as the antifungal concentration yielding a 50% or 80% reduction in the metabolic activity of the biofilm compared to that of the growth control (SMIC₅₀ and SMIC₈₀, respectively). fks mutant Candida isolates formed biofilms in a fashion similar to that of Candida wild-type strains. The echinocandins had the highest activity against biofilms formed by wild-type Candida isolates, followed by fks mutant Candida isolates and non-Candida isolates. Liposomal amphotericin B had the highest activity against fks mutant Candida biofilms. The formation of biofilm by echinocandin-resistant strains was similar to that of wild-type strains, although resistance to echinocandins remained high.     

Amphotericin B in Children with Malignant Disease: a Comparison of the Toxicities and Pharmacokinetics of Amphotericin B Administered in Dextrose versus Lipid Emulsion

In a prospective, randomized clinical trial, the toxicity of 1 mg of amphotericin B (AmB) per kg of body weight per day infused in 5% dextrose was compared with that of AmB infused in lipid emulsion in children with malignant disease. In an analysis of 82 children who received a full course of 6 days or more of AmB (117 courses), it was shown that there were significant increases in plasma urea and creatinine concentrations and in potassium requirement after 6 days of therapy with both AmB infused in dextrose and AmB infused in lipid emulsion, with there being no difference between the two methods of AmB administration. An intent-to-treat comparison of the numbers of courses affected by acute toxicity (fever, rigors) and chronic toxicity (nephrotoxicity) also indicated that there was no significant difference between AmB infused in dextrose (78 courses) and AmB infused in lipid emulsion (84 courses). The pharmacokinetics of AmB were investigated in 20 children who received AmB in dextrose and 15 children who received AmB in lipid emulsion. Blood samples were collected up to 24 h after administration of the first dose, and the concentration of AmB in plasma was analyzed by a high-performance liquid chromatography assay. The clearance (CL) of AmB in dextrose (0.039 +/- 0.016 liter. h-1. kg-1) was significantly lower (P < 0.005) than the CL of AmB in lipid emulsion (0.062 +/- 0. 024 liter. h-1. kg-1). The steady-state volume of distribution for AmB in dextrose (0.83 +/- 0.33 liter. kg-1) was also significantly lower (P < 0.005) than that for AmB in lipid emulsion (1.47 +/- 0.77 liter. kg-1). Although AmB in lipid emulsion is apparently cleared faster and distributes more widely than AmB in dextrose, this study did not reveal any significant advantage with respect to safety and tolerance in the administration of AmB in lipid emulsion compared to its administration in dextrose in children with malignant disease.     

Dose Range Evaluation of Liposomal Nystatin and Comparisons with Amphotericin B and Amphotericin B Lipid Complex in Temporarily Neutropenic Mice Infected with an Isolate of Aspergillus fumigatus with Reduced Susceptibility to Amphotericin B

Using an isolate of Aspergillus fumigatus that is less susceptible in vivo to amphotericin B than most other isolates, we compared different doses of liposomal nystatin (L-nystatin), liposomal amphotericin B (L-amphotericin), and amphotericin B lipid complex (ABLC) with amphotericin B deoxycholate. Four experiments with intravenously infected neutropenic mice were conducted. A dose of L-nystatin at 10 mg/kg of body weight was toxic (the mice had fits or respiratory arrest). The optimal dosage of L-nystatin was 5 mg/kg daily on days 1, 2, 4, and 7 (90% survival). This was superior to L-amphotericin (5 mg/kg [P = 0.24] and 1 mg/kg [P < 0.0001]), ABLC (5 mg/kg [P = 0.014] and 1 mg/kg [P < 0.0001]), and amphotericin B deoxycholate (5 mg/kg [P = 0.008]). In terms of liver and kidney cultures, L-nystatin (5 mg/kg) was superior to all other regimens (P = 0.0032 and <0.0001, respectively). Higher doses of L-amphotericin (25 and 50 mg/kg) in one earlier experiment were more effective (100% survival) than 1 mg of L-amphotericin per kg and amphotericin deoxycholate (5 mg/kg) in terms of mortality and both liver and kidney culture results and to L-amphotericin (5 mg/kg) in terms of liver and kidney culture results only. ABLC (25 mg/kg) given daily for 7 days was superior to ABLC (50 mg/kg [P = 0.03]) but not to ABLC at 5 mg/kg or amphotericin B deoxycholate in terms of mortality, although it was in terms of liver and kidney culture results. No dose-response for amphotericin B (5 and 1 mg/kg) was demonstrable. In conclusion, in this stringent model, high doses of L-amphotericin and ABLC could overcome reduced susceptibility to amphotericin B deoxycholate, but all were inferior to 5- to 10-fold lower doses of L-nystatin.     

Antibacterial Efficacy against an In Vivo Salmonella typhimurium Infection Model and Pharmacokinetics of a Liposomal Ciprofloxacin Formulation

The fluoroquinolone antibiotic ciprofloxacin has been encapsulated into large unilamellar vesicles (LUV) at efficiencies approaching 100%. Drug accumulation proceeded in response to a transmembrane gradient of methylammonium sulfate and occurred concomitantly with the efflux of methylamine. A mechanism for the encapsulation process is described. LUV composed of dipalmitoylphosphatidylcholine-cholesterol (DPPC/chol), distearoylphosphatidylcholine-cholesterol (DSPC/chol), or sphingomyelin-cholesterol (SM/chol) increased the circulation lifetime of ciprofloxacin after intravenous (i.v.) administration by >15-fold. The retention of ciprofloxacin in liposomes in the circulation decreased in the sequence SM/chol > DSPC/chol > DPPC/chol. Increased circulation lifetimes were associated with enhanced delivery of the drug to the livers, spleens, kidneys, and lungs of mice. Encapsulation of ciprofloxacin also conferred significant increases in the longevity of the drug in the plasma after intraperitoneal administration and in the lungs after intratracheal administration in comparison to free ciprofloxacin. The efficacy of a single i.v. administration of an SM/chol formulation of ciprofloxacin was measured in a Salmonella typhimurium infection model. At 20 mg of ciprofloxacin per kg of body weight, the encapsulated formulation resulted in 10³- to 10⁴-fold fewer viable bacteria in the livers and spleens of infected mice than was observed for animals treated with free ciprofloxacin. These results show the utility of liposomal encapsulation of ciprofloxacin in improving the pharmacokinetics, biodistribution, and antibacterial efficacy of the antibiotic. In addition, these formulations are well suited for i.v., intraperitoneal, and intratracheal or aerosol administration.     

Pharmacokinetics, Distribution in Serum Lipoproteins and Tissues, and Renal Toxicities of Amphotericin B and Amphotericin B Lipid Complex in a Hypercholesterolemic Rabbit Model: Single-Dose Studies

The purpose of this study was to determine if a relationship exists among total serum and lipoprotein cholesterol concentration, the severity of amphotericin B (AmpB)-induced renal toxicity, and the serum pharmacokinetics of AmpB in hypercholesterolemic rabbits administered AmpB and AmpB lipid complex (ABLC). After 10 days of cholesterol-enriched diet (0.50% [wt/vol]) or regular rabbit diet (control), each rabbit was administered a single intravenous bolus of AmpB or ABLC (1.0 mg/kg of body weight). Blood samples were obtained before administration and serially thereafter for the assessment of serum pharmacokinetics, kidney toxicity, and serum lipoprotein distribution. Rabbits were humanely sacrificed after all blood samples were obtained, and tissues were harvested for drug analysis. Before drug treatment, cholesterol-fed rabbits demonstrated marked increases in total serum cholesterol and low-density lipoprotein (LDL) cholesterol levels compared with levels in rabbits on a regular diet. No significant differences in triglyceride levels were observed. A significant increase in serum creatinine levels was observed in cholesterol-fed and regular diet-fed rabbits administered AmpB. However, the magnitude of this increase was 2.5-fold greater in cholesterol-fed rabbits than in regular diet-fed rabbits. No significant differences in triglyceride levels were observed. A significant increase in serum creatinine levels was observed in cholesterol-fed and regular diet-fed rabbits administered ABLC. Whereas AmpB pharmacokinetics were significantly altered in cholesterol-fed rabbits administered free AmpB, similar AmpB pharmacokinetics were observed in both rabbit groups administered ABLC. Renal AmpB levels were significantly increased in cholesterol-fed rabbits administered AmpB compared with those in all other groups. Hepatic and lung AmpB levels were elevated in cholesterol-fed rabbits administered free AmpB compared to controls. In addition, hepatic, lung, and spleen AmpB levels were significantly decreased in cholesterol-fed rabbits administered ABLC compared to controls. An increased percentage of AmpB was recovered in LDL–very-low-density lipoprotein fraction when free AmpB was administered to cholesterol-fed rabbits compared with those in all other groups. These findings suggest that increases in cholesterol, specifically, LDL cholesterol levels, modify the disposition and renal toxicity of free AmpB. However, the pharmacokinetics and renal toxicity of ABLC were independent of elevations in total and LDL cholesterol levels.