Toxicity, stability and pharmacokinetics of amphotericin B in immunomodulator tuftsin-bearing liposomes in a murine model

OBJECTIVES: In the present study we evaluated the pharmacokinetics and toxicity of amphotericin B in immunomodulator tuftsin-loaded liposomes in a murine model. METHODS: Stability of amphotericin B liposomes was tested by incubating one volume of liposomal formulations of amphotericin B with nine volumes of serum. The pharmacokinetics of amphotericin B in Candida albicans-infected mice treated with conventional and tuftsin-loaded amphotericin B liposomes was evaluated over a period of 24 h. In vitro toxicity of amphotericin B deoxycholate, as well as amphotericin B liposomes, was tested by incubation with human erythrocytes for 1 h at 37 degrees C. To assess amphotericin B-induced in vivo toxicity, BALB/c mice were injected with three doses of amphotericin B deoxycholate, as well as amphotericin B liposomal formulations on days 1, 2 and 3 post C. albicans infection. Blood from treated mice was taken by retro-orbital puncture to test renal function parameters such as serum creatinine and urea. RESULTS: In vitro stability studies revealed that tuftsin-bearing amphotericin B liposomes released only 11% of the total liposomal amphotericin B in the serum, while it was found to be 19% from identical tuftsin-free amphotericin B liposomes. Both tuftsin-loaded as well as tuftsin-free liposomal formulations of amphotericin B induced approximately 20% haemolysis of erythrocytes at a dose of 40 mg/L, while the same amount of drug in amphotericin B deoxycholate caused 100% lysis of the erythrocytes. Pharmacokinetic studies revealed that subsequent to administration of various formulations of amphotericin B, there was 32 mg/L amphotericin B in the systemic circulation of mice treated with tuftsin-bearing amphotericin B liposomes, while it was 25 mg/L for amphotericin B liposomes, 4 h post drug administration. In vivo toxicity studies demonstrated that the amphotericin B deoxycholate formulation induced elevations in serum creatinine (approximately 300% of control) and blood urea (approximately 380% of control) values, while these values were substantially less (blood urea approximately 150% of control and serum creatinine approximately 210% of control) in the animals treated with the tuftsin-loaded amphotericin B liposomal formulation. Further, the administration of amphotericin B deoxycholate (1 mg/kg) in BALB/c mice at a dose of 1 mg/kg body weight led to the accumulation of 18.6 +/- 5.25 g/kg (of amphotericin B) in kidneys. On the other hand, administration of liposomal amphotericin B and tuftsin-bearing liposomal amphotericin B at a dose of 5 mg/kg body weight resulted in accumulation of 8.8 +/- 2.0 and 4.0 +/- 1.6 g/kg of amphotericin B, respectively, in the kidneys of treated animals. CONCLUSIONS: Co-administration of immunomodulator tuftsin along with liposomal formulations of amphotericin B successfully minimizes toxicity, as well as other side effects of the drug. Interestingly, tuftsin also increased the stability of liposomal amphotericin B. Superior efficacy, reliable safety and favourable pharmacodynamics of tuftsin-loaded amphotericin B liposomes suggest their potential therapeutic value in the management of fungal infections.     

Amphotericin B--not so terrible

OBJECTIVE: To describe a patient who developed adverse reactions to two different lipid formulations of amphotericin B: liposomal amphotericin B (AmBisome) and amphotericin B colloidal dispersion (ABCD, Amphocil), yet tolerated amphotericin B deoxycholate (Fungizone) despite renal toxicity. CASE SUMMARY: A 72-year-old woman with acute myelomonocytic leukemia was treated with amphotericin B deoxycholate for suspected pulmonary aspergillosis; the drug was well tolerated but resulted in renal failure. Antifungal therapy was then changed to liposomal amphotericin B. Within 10 minutes of liposomal amphotericin B infusion, the patient developed severe dyspnea, chest pain, and a feeling of imminent death. On the following day, liposomal amphotericin B was switched to amphotericin B colloidal dispersion. Again, within 10 minutes of this infusion, the patient developed fever, chills, hypotension, severe chest pain, dsypnea, and a feeling of imminent death. The patient refused any further treatment with these drugs and insisted on switching back to amphotericin B deoxycholate, which was then administered for 10 days and was well tolerated. DISCUSSION: Severe adverse reactions, such as anaphylaxis, cardiac toxicity, and respiratory failure, following administration of all three lipid formulations of amphotericin B have been reported. In most reported cases, switching to a different lipid formulation of amphotericin B was well tolerated. This is in contrast to our case, where a severe reaction was repeated when another lipid preparation was given, necessitating switching back to amphotericin B deoxycholate despite its nephrotoxicity. CONCLUSIONS: In some patients, paradoxically, lipid formulations of amphotericin B may be less tolerable than conventional amphotericin B.     

Liposomal amphotericin B: clinical experience and perspectives

While amphotericin B deoxycholate (Fungizone, Apothecon Pharmaceuticals) has been considered by many to be the gold standard for the treatment for numerous invasive fungal infections for over 45 years, toxicities associated with its use often necessitate treatment modification or discontinuation. Lipid-based formulations, including liposomal amphotericin B (AmBisome, Fujisawa Healthcare, Inc.), were developed to decrease many of these toxicities while retaining broad antifungal spectrum and potency of amphotericin B. In clinical trials, liposomal amphotericin B has demonstrated efficacy comparable to that of amphotericin B deoxycholate while reducing the incidence of treatment-related nephrotoxicity, electrolyte-wasting, and infusion-related reactions. In addition, recent clinical trials have also compared liposomal amphotericin B with other antifungal classes. Acquisition costs of liposomal amphotericin B are substantially higher than those of amphotericin B deoxycholate and other antifungals. While pharmacoeconomic analyses consider outcomes and other treatment-related costs, they have yet to clearly demonstrate the cost-effectiveness of liposomal amphotericin B when compared with amphotericin B deoxycholate or other antifungal agents. This review will focus primarily on recent liposomal amphotericin B experience and attempt to put its use into perspective considering other available antifungal agents.     

Liposomal amphotericin B: clinical experience and perspectives

While amphotericin B deoxycholate (Fungizone^®, Apothecon Pharmaceuticals) has been considered by many to be the gold standard for the treatment for numerous invasive fungal infections for over 45 years, toxicities associated with its use often necessitate treatment modification or discontinuation. Lipid-based formulations, including liposomal amphotericin B (AmBisome^®, Fujisawa Healthcare, Inc.), were developed to decrease many of these toxicities while retaining broad antifungal spectrum and potency of amphotericin B. In clinical trials, liposomal amphotericin B has demonstrated efficacy comparable to that of amphotericin B deoxycholate while reducing the incidence of treatment-related nephrotoxicity, electrolyte-wasting, and infusion-related reactions. In addition, recent clinical trials have also compared liposomal amphotericin B with other antifungal classes. Acquisition costs of liposomal amphotericin B are substantially higher than those of amphotericin B deoxycholate and other antifungals. While pharmacoeconomic analyses consider outcomes and other treatment-related costs, they have yet to clearly demonstrate the cost-effectiveness of liposomal amphotericin B when compared with amphotericin B deoxycholate or other antifungal agents. This review will focus primarily on recent liposomal amphotericin B experience and attempt to put its use into perspective considering other available antifungal agents.     

Distribution of lipid formulations of amphotericin B into bone marrow and fat tissue in rabbits

The distribution of the three currently available lipid formulations of amphotericin B (AmB) into bone marrow and fat tissue was evaluated in noninfected rabbits. Groups of four animals each received either 1 mg of AmB deoxycholate (D-AmB) per kg of body weight per day or 5 mg of AmB colloidal dispersion, AmB lipid complex, or liposomal AmB per kg per day for seven doses. Plasma, bone marrow, fat, and liver were collected at autopsy, and AmB concentrations were determined by high-performance liquid chromatography. At the investigated dosages of 5 mg/kg/day, all AmB lipid formulations achieved at least fourfold-higher concentrations in bone marrow than did standard D-AmB at a dosage of 1 mg/kg/day. Concentrations in bone marrow were 62 to 76% of concurrent AmB concentrations in the liver. In contrast, all AmB formulations accumulated comparatively poorly in fat tissue. The results of this study show that high concentrations of AmB can be achieved in the bone marrow after administration of lipid formulations, suggesting their particular usefulness against disseminated fungal infections involving the bone marrow and against visceral leishmaniasis.     

Pharmacokinetics of liposomal amphotericin B (Ambisome) in critically ill patients.

The liposomal formulation of amphotericin B (AmBisome) greatly reduces the acute and chronic side effects of the parent drug. The present study describes the pharmacokinetic characteristics of AmBisome applied to 10 patients at a dose of 2.8 to 3.0 mg/kg of body weight and compares them to the pharmacokinetics observed in 6 patients treated with amphotericin B deoxycholate at the standard dose of 1.0 mg/kg. Interpatient variabilities of amphotericin B peak concentrations (Cmax) and areas under concentration-time curves (AUC) were 8- to 10-fold greater for patients treated with AmBisome than for patients treated with amphotericin B deoxycholate. At the threefold greater dose of AmBisome, median Cmaxs were 8.4-fold higher (14.4 versus 1.7 microg/ml) and median AUCs exceeded those observed with amphotericin B deoxycholate by 9-fold. This was in part explained by a 5.7-fold lower volume of distribution (0.42 liters/kg) in AmBisome-treated patients. The elimination of amphotericin B from serum was biphasic for both formulations. However, the apparent half-life of elimination was twofold shorter for AmBisome (P = 0.03). Neither hemodialysis nor hemofiltration had a significant impact on AmBisome pharmacokinetics as analyzed in one patient. In conclusion, the liposomal formulation of amphotericin B significantly (P = 0.001) reduces the volume of drug distribution, thereby allowing for greater drug concentrations in serum. The low toxicity of AmBisome therefore cannot readily be explained by its serum pharmacokinetics.     

A comparative review of conventional and lipid formulations of amphotericin B

Over the past 15 years, factors suh as corticosteroid treatment, cytotoxic chemotherapy, excessive use of broad spectrum antibiotics and HIV have led to an increased risk of serious fungal infections in both adults and pediatric patients. This increase in invasive fungal infections poses increasing difficulty in their treatment. Three new lipid formulations of amphotericin B are now available in the U.S.: amphotericin B lipid complex (Abelcet), amphotericin B colloidal dispersion (Amphotec), and liposomal amphotericin B (AmBisome). 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 amphotericin B. The properties of these new agents are summarized in this review. Discussion of current national guidelines as well as those used at our institution are presented to provide guidance for the development of institution specific guidelines for the most cost-effective drug for most patients, some may benefit more from one of the newer lipid formulations.     

Efficacy of high doses of liposomal amphotericin B in the treatment of experimental aspergillosis

OBJECTIVE: Differences in efficacy between deoxycholate amphotericin B (d-AmB) and escalating doses of liposomal amphotericin B (L-AmB) were evaluated in a model of invasive pulmonary aspergillosis in persistently steroid-immunosuppressed rats. METHODS: Animals were infected intratracheally with a conidial suspension of a clinical isolate of Aspergillus fumigatus and randomized to receive intravenously 5% dextrose, 1 mg/kg/day of d-AmB or 3, 5 or 10 mg/kg/day of L-AmB. RESULTS: All the antifungal treatments improved survival, although no differences were found among the groups, perhaps because of treatment-related toxicity. In animals surviving long enough to receive at least five doses of antifungal treatment, there were significant reductions in paired lung weight in the 5 and 10 mg/kg/day L-AmB groups as compared with the controls (P=0.004 and 0.001, respectively) and with the 3 mg/kg/day L-AmB group (P=0.007 and 0.002, respectively). Significant decreases in fungal biomass, measured indirectly by chitin quantification, were found only in the 10 mg/kg/day L-AmB group as compared with controls (P=0.003), d-AmB (P=0.007) and 3 mg/kg/day L-AmB (P=0.001). CONCLUSION: Infusion of L-AmB doses as high as 10 mg/kg/day may be a good therapeutic option for the management of invasive pulmonary aspergillosis developing in the context of steroid immunosuppression, although further studies are needed to assess this approach.     

Liposomal Amphotericin B and Echinocandins as Monotherapy or Sequential or Concomitant Therapy in Murine Disseminated and Pulmonary Aspergillus fumigatus Infections

Monotherapy and combination therapy were compared using optimal doses of liposomal amphotericin B, micafungin, or caspofungin in Aspergillus fumigatus pulmonary and disseminated infections. Mice were challenged intravenously (2.8 × 10⁴ to 5.7 × 10⁴ conidia) or intranasally (5.8 × 10⁷ conidia) with A. fumigatus. Drugs (5, 10, or 15 mg/kg of body weight) were given for 3 or 6 days as single, concomitant, or sequential therapy (i.e., days 1 to 3 and then days 4 to 6). Mice were monitored for survival, and tissues were assayed for fungal burden and drug concentrations. Treatments starting 24 h postchallenge significantly prolonged survival in disseminated aspergillosis (P < 0.002), but only liposomal amphotericin B treatments or treatments beginning with liposomal amphotericin B increased survival to 100% in the pulmonary aspergillosis model. Fungi in kidneys and spleens (disseminated) and lungs (pulmonary) were significantly decreased (P ≤ 0.04) by liposomal amphotericin B, liposomal amphotericin B plus echinocandin, or liposomal amphotericin B prior to echinocandin. In the disseminated infection, liposomal amphotericin B and micafungin (10 or 15 mg/kg) had similar kidney drug levels, while in the spleen, 5 and 15 mg/kg liposomal amphotericin B gave higher drug levels than micafungin (P < 0.02). In the pulmonary infection, drug levels in lungs and spleen with 5-mg/kg dosing were significantly higher with liposomal amphotericin B than with caspofungin (P ≤ 0.002). In summary, treatment of A. fumigatus infections with liposomal amphotericin B plus echinocandin or liposomal amphotericin B prior to echinocandin was as effective as liposomal amphotericin B alone, and a greater decrease in the fungal burden with liposomal amphotericin B supports using liposomal amphotericin B prior to echinocandin.Invasive aspergillosis (IA) is a life-threatening fungal infection that occurs in 10 to 28% of immunosuppressed patients (42, 43, 44), and the mortality rate for untreated IA is virtually 100% (16, 34, 45). Aspergillus fumigatus is responsible for 66 to 70% of IA infections (19, 36, 43). In a prospective, randomized clinical trial comparing 3 mg/kg of body weight and 10 mg/kg AmBisome, a liposomal form of amphotericin B, the response rates for IA were reported to be 50% and 46%, respectively (14). These are similar to the response rate (53%) for voriconazole reported in another prospective randomized clinical trial of IA comparing conventional amphotericin B and voriconazole (22). In a more recent clinical trial for primary treatment of IA, the investigators reported a 33% response rate for caspofungin (Cancidas) at 50 mg/day with a 70-mg loading dose on day 1 (53).To further improve the treatment outcome for IA, combination antifungal therapy of polyenes with the echinocandins has begun to be investigated in nonclinical studies. Amphotericin B targets ergosterol in the fungal cell membrane, binding with ergosterol to create a pore in the membrane, which disrupts membrane integrity and leads to fungal-cell lysis (5, 15). In comparison, the echinocandins target the enzyme 1,3-β-glucan synthetase, required for the synthesis of 1,3-β-glucan, an important fungal cell wall component (18, 20). The fungal specificity of the echinocandins for the cell wall results in these drugs having minimal toxic side effects. Thus, by combining a drug which targets the cell wall with one directed at the cell membrane, it is possible that there could be additive or synergistic antifungal effects.Nonclinical studies have shown no antagonistic effects with the combination of amphotericin B and the echinocandins for the treatment of Aspergillus infections (10, 24, 47). In fact, improved efficacy has been reported in mice with chronic granulomatous disease and pulmonary aspergillosis following administration of micafungin (Mycamine) and amphotericin B (17), as well as for murine systemic aspergillosis with a combination of caspofungin plus amphotericin B or amphotericin B-intralipid (2, 47). In contrast, although there was no antagonism, Clemons and Stevens (10) reported no synergistic activity of micafungin and amphotericin B for pulmonary aspergillosis in immunosuppressed DBA/2 mice.Treatment of aspergillosis with the combination of lipid amphotericin B formulations and the echinocandins has also begun to be studied, but the reports are limited. This approach has the added advantage of the reduced toxicity of the amphotericin B lipid formulations (2, 51). In one study of a systemic murine aspergillosis model (21), the investigators reported limited additive effects of suboptimal doses of liposomal amphotericin B and micafungin with significantly reduced fungal burden in the spleens, but only when liposomal amphotericin B was given before micafungin.The initial site of Aspergillus infection in humans is primarily the lungs, with hematogenous dissemination of the fungus to the spleen, kidneys, liver, and brain, which occurs as the disease progresses, making the infection even more difficult to treat. Leenders et al. (33) reported that in a pulmonary aspergillosis infection in rats, liposomal amphotericin B monotherapy at 5 and 10 mg/kg was effective in preventing dissemination from the lungs to the kidneys, liver, and spleen. To extend these observations, in the present study, we wanted to determine if concomitant or sequential therapy with optimal doses of liposomal amphotericin B and either caspofungin or micafungin would be more or less effective than monotherapy in decreasing the fungal burden and increasing survival in an A. fumigatus pulmonary and/or disseminated infection. There are at least one randomized pilot clinical trial and several case reports in which clinicians have begun using combination regimens of liposomal amphotericin B and echinocandins for the treatment of aspergillosis, although there are no published large-scale, randomized, double-blind clinical-trial data in which these combinations have been used. In the pilot randomized clinical trial (8), investigators compared concomitant liposomal amphotericin B (3 mg/kg) plus standard caspofungin therapy (70-mg/kg loading dose; 50 mg/kg daily) with high-dose (10-mg/kg) liposomal amphotericin B monotherapy. The overall response of the combination was significantly better than the monotherapy (P = 0.028). In case reports, some investigators have used the drugs together as concomitant therapy (9), and in other case reports, the investigators have used sequential treatment, with one drug replacing the other drug (25) or initiating treatment with one drug and then adding another drug to the regimen (3, 26, 41). Thus, it is important to evaluate whether there will be any antagonism or synergism when liposomal amphotericin B and an echinocandin are used together at optimal doses or if it is better to begin therapy with one class of drug and then switch to another class of drug. To address these questions, we used two infection models. We challenged some groups of mice intranasally to produce a lung infection, and for the disseminated model, we infected other mice intravenously (i.v.) to target the fungus to the nonlung tissues (i.e., kidneys and spleen). By using survival and fungal burden (i.e., tissue culturing and tissue galactomannan levels), we were able to evaluate the efficacy of the drugs given as monotherapy or concomitant or sequential therapy in murine pulmonary and disseminated aspergillosis.     

Pretreatment with empty liposomes attenuates the immunopathology of invasive pulmonary aspergillosis in corticosteroid-immunosuppressed mice

In a nonneutropenic murine model of invasive pulmonary aspergillosis, pretreatment with empty liposomes (E-lipo) was nearly as effective as 10 mg/kg of body weight liposomal amphotericin B and superior to 1 mg/kg amphotericin B deoxycholate. The beneficial immunomodulatory properties of E-lipo appear to compensate for their lack of direct antifungal activity.     

Comparative efficacies of amphotericin B, triazoles, and combination of both as experimental therapy for murine trichosporonosis.

We assessed the activities of amphotericin B deoxycholate, liposomal amphotericin B, fluconazole, and SCH 39304 against 10 strains of Trichosporon beigelii in mice with hematogenous infections. Cyclophosphamide-immunosuppressed CF1 male mice were challenged intravenously with a lethal inoculum of T. beigelii (5 x 10(6) conidia per mouse) and were assigned to different treatment groups or were left untreated. Amphotericin B deoxycholate (1 mg/kg of body weight and liposomal amphotericin B (1, 5, and 10 mg/kg) were given parenterally once daily. Escalating doses (5, 10, and 20 mg/kg/day) of fluconazole and SCH 39304 were tested. We also compared the activity of amphotericin B deoxycholate plus fluconazole (1 and 10 mg/kg/day, respectively) with that of each agent alone. Fluconazole significantly prolonged the survival of mice infected with each of the 10 strains tested. Amphotericin B deoxycholate achieved various responses, improving the outcomes in mice infected with seven of the strains. Liposomal amphotericin B was not more effective than amphotericin B deoxycholate against the two strains tested. Both fluconazole and SCH 39304 reduced the kidney fungal counts in a dose-dependent pattern, with SCH 39304 being more active than fluconazole against one of the two strains tested. The activity of the combination of amphotericin B deoxycholate plus fluconazole appeared to be superior to that of either agent alone, especially in reducing the kidney fungal burden. Fluconazole is more active than amphotericin B deoxycholate against experimental murine trichosporonosis.     

Amphotericin B use in children: conventional and lipid-based formulations

Invasive fungal infections (IFIs) are important causes of morbidity and mortality in immunocompromised children. The increased incidence and high mortality rates associated with IFIs has led to development of novel antifungal agents to expand the breadth and effectiveness of treatment options available to clinicians. Since its initial approval in 1958, conventional amphotericin B deoxycholate had been considered the standard in treatment for IFIs. However, because of the dose-limiting toxicity of conventional amphotericin B deoxycholate, lipid formulations of amphotericin have been developed to potentially improve outcomes and mitigate the adverse effects associated with antifungal therapy. While less frequently employed today as prophylaxis (given the expanded availability of safer alternatives), amphotericin B is still considered a treatment option in select cases of severe or life-threatening IFIs. This article reviews the clinical use of amphotericin B for the prevention and treatment of IFIs.     

Comparative studies on the efficacy of AmBisome and Fungizone in a mouse model of disseminated aspergillosis

OBJECTIVES: The efficacy of intravenous injections of a liposomal formulation of amphotericin B (AmBisome) and amphotericin B deoxycholate (Fungizone) was evaluated in immunocompetent and temporarily leucopenic mouse models of disseminated aspergillosis using seven isolates of Aspergillus. METHODS: Mice were infected with the organisms via tail veins. At 4 h after infection, antifungals were administered intravenously. For 30 days the number of mice surviving was recorded. RESULTS: AmBisome at 1 mg/kg or higher significantly prolonged the survival time of mice infected with five out of seven isolates of Aspergillus compared with the control group. There was no difference in in vivo activity between AmBisome and Fungizone at 1 mg/kg in six isolates of Aspergillus. At the maximum tolerated dose of antifungals, however, AmBisome (10 mg/kg) showed greater efficacy than Fungizone (1 mg/kg). CONCLUSIONS: These results suggest that the overall protective activity of AmBisome against disseminated aspergillosis is superior to that of Fungizone.     

Effect of lipid composition and liposome size on toxicity and in vitro fungicidal activity of liposome-intercalated amphotericin B.

Intercalation of amphotericin B into liposomes at a 10 mol% drug/lipid ratio decreased its cytotoxicity by 3- to 90-fold in cultured murine cells and reduced its lethality by 2- to 8-fold in a median lethal dose (LD50) test in mice when compared with the commercial deoxycholate-solubilized drug (LD50 = 2.3 mg/kg). The cytotoxicity and lethality of the liposomal preparations were a function of their lipid composition and diameter. There was no correlation between the reduction of toxicity in the tissue culture assay and the reduction of lethality in the LD50 test. The rank order of reduction of lethality was sterol-containing liposomes greater than solid liposomes greater than fluid liposomes. In general, small sterol-containing vesicles were less lethal than large vesicles of the same composition. Intercalation of amphotericin B in sterol or solid liposomes increased not only the LD50 but also the time to death. The organ distribution of amphotericin B 24 h after intravenous administration was similar whether the drug was given as the commercial deoxycholate preparation or in liposomes. Finally, there were no differences among any of the formulations in their fungicidal activity against Candida tropicalis and Saccharomyces cerevisiae in vitro. The lesser and slower lethality of the liposomal and detergent-solubilized drug suggests that the mechanism by which liposomes reduce the lethality of amphotericin B is by slowing its rate of transfer to a sensitive cellular target.     

Amphotericin B lipid complex therapy of experimental fungal infections in mice.

The amphotericin B lipid complex (ABLC), which is composed of amphotericin B and the phospholipids dimyristoyl phosphatidylcholine and dimyristoyl phosphatidylglycerol, was evaluated for its acute toxicity in mice and for its efficacy in mice infected with a variety of fungal pathogens. ABLC was markedly less toxic to mice when it was administered intravenously; it had a 50% lethal dose of greater than 40 mg/kg compared with a 50% lethal dose of 3 mg/kg for Fungizone, the desoxycholate form of amphotericin B. ABLC was efficacious against systemic infections in mice caused by Candida albicans, Candida species other than C. albicans, Cryptococcus neoformans, and Histoplasma capsulatum. ABLC was also efficacious in immunocompromised animals infected with C. albicans, Aspergillus fumigatus, and H. capsulatum. Against some infections, the efficacy of ABLC was comparable to that of Fungizone, while against other infections Fungizone was two- to fourfold more effective than ABLC. Against several infections. Fungizone could not be given at therapeutic levels because of intravenous toxicity. ABLC, with its reduced toxicity, could be administered at drug levels capable of giving a therapeutic response. ABLC should be of value in the treatment of severe fungal infections in humans.     

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.     

Comparative efficacies, toxicities, and tissue concentrations of amphotericin B lipid formulations in a murine pulmonary aspergillosis model

Invasive aspergillosis, an important cause of morbidity and mortality in immunosuppressed (IS) patients, is often treated with amphotericin B lipid formulations. In the present study, liposomal amphotericin B (L-AMB) and amphotericin B lipid complex (ABLC) were compared in treatment of murine pulmonary aspergillosis. Uninfected, IS mice were treated for 4 days with 1, 4, 8, or 12 mg L-AMB or ABLC/kg of body weight, and their lungs were analyzed by high-performance liquid chromatography for drug concentrations. IS mice intranasally challenged with Aspergillus fumigatus were treated with 12, 15, or 20 mg/kg L-AMB or ABLC and monitored for survival, fungal burden (CFU), and tissue drug concentration. Blood urea nitrogen (BUN) levels and kidney histopathology were determined for uninfected and infected mice given 15 or 20 mg/kg L-AMB or ABLC. The results showed that both drugs had therapeutic levels of drug (>3.0 microg/g) in the lungs of uninfected or infected mice, and 24 h after the last dose, ABLC levels were significantly higher than L-AMB levels (P < 0.02). L-AMB and ABLC at 12 mg/kg both produced 57% survival, but only L-AMB at 15 or 20 mg/kg further increased survival to 80 to 90%, with BUN levels and kidney morphology similar to those of controls. Survival at 15 or 20 mg/kg ABLC was not significantly different than that of controls, and BUN levels were significantly elevated, with tubular alterations in uninfected animals and acute necrosis in kidney tubules of infected animals. In conclusion, although both drugs were effective in prolonging survival at 12 mg/kg, the reduced nephrotoxicity of L-AMB increased its therapeutic index, allowing for its safe and effective use at 15 or 20 mg/kg.     

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.     

Anti-Aspergillus fumigatus efficacy of pentraxin 3 alone and in combination with antifungals

The collectin pentraxin 3 (PTX3) is an essential component of host resistance to pulmonary aspergillosis. Here we examined the protective effects of administration of PTX3 alone or together with deoxycholate amphotericin B (Fungizone) or liposomal amphotericin B (AmBisome) against invasive aspergillosis in a murine model of allogeneic bone marrow transplantation. PTX3, alone or in combination with the polyenes, was given intranasally or parenterally either before, in concomitance with, or after the intranasal infection with Aspergillus fumigatus conidia. Mice were monitored for resistance to infection and parameters of innate and adaptive T-helper immunity. The results showed the following: (i) complete resistance to infection and reinfection was observed in mice treated with PTX3 alone; (ii) the protective effect of PTX3 was similar or superior to that observed with liposomal amphotericin B or deoxycholate amphotericin B, respectively; (iii) protection was associated with accelerated recovery of lung phagocytic cells and T-helper-1 lymphocytes and concomitant decrease of inflammatory pathology; and (iv) PTX3 potentiated the therapeutic efficacy of suboptimal doses of either antimycotic drug. Together, these data suggest the potential therapeutic use of PTX3 either alone or as an adjunctive therapy in A. fumigatus infections.     

Liposome-encapsulated amphotericin B in the treatment of experimental murine candidiasis

The efficacy of liposome-encapsulated amphotericin B in treating experimental murine candidiasis was compared with that of the commercially available amphotericin B (Fungizone). The LD50 of liposomal amphotericin B in ddY mice exceeded 10.0 mg/kg while that of Fungizone was 3.0 mg/kg. Experimental candidiasis was induced by injecting a clinical isolate of Candida albicans strain 0925-107-01, through the tail vein. With the injection of 1.7 x 10(6) colony forming units, the number of colonies in the kidneys remained between 2.1 x 10(5) and 1.2 x 10(6), whereas the number of colonies in blood, liver, spleen, lungs and heart decreased rapidly. Histological examination revealed severe pyelonephritis with fungal infiltration and a mild invasion of the heart, lungs, liver and spleen. The survival rate of mice with experimental candidiasis treated with Fungizone at a dose of 0.8 mg/kg was 50% (the maximum tolerated dose without acute lethality), whereas all mice treated with the liposomal amphotericin B at a dose of 5.0 mg/kg were alive even 42 days after the inoculation (p less than 0.01). Using liposome as a carrier for amphotericin B decreased this drug's systemic toxicity making it possible to administer doses higher than feasible with the commercial preparation and thus obtaining better therapeutic efficacy.