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.     

Comparative efficacies of conventional amphotericin b, liposomal amphotericin B (AmBisome), caspofungin, micafungin, and voriconazole alone and in combination against experimental murine central nervous system aspergillosis

Central nervous system (CNS) aspergillosis is a severe disease that responds poorly to current therapies. The current studies examined the efficacies of several antifungal agents alone or in combination with a murine model of CNS aspergillosis. Immunosuppressed mice were infected intracerebrally with Aspergillus fumigatus and treated with an amphotericin B preparation, an echinocandin, or voriconazole (VCZ) given alone or in combination. Monotherapy studies showed that micafungin (MICA), caspofungin (CAS), VCZ, conventional amphotericin B (AMB), Abelcet (ABLC) (a lipid-carried AMB formulation; Enzon Pharmaceuticals, Inc.), and AmBisome (AmBi) (liposomal AMB; Gilead Sciences, Inc.) were efficacious. However, doses of AmBi above 15 mg/kg of body weight showed reduced efficacy. Neither MICA nor CAS showed dose responsiveness at the doses tested (1, 5, or 10 mg/kg). Only the 40-mg/kg dose of VCZ was effective. AmBi and ABLC showed dose responsiveness, with 10-mg/kg doses causing a significant reduction in fungal burden; they had equivalent activities at the 10-mg/kg dose. Suboptimal dosages of AmBi in combination with MICA, CAS, or VCZ were effective in prolonging survival. However, significantly enhanced activity was demonstrated only with AmBi and VCZ in combination. AmBi in combination with MICA or CAS showed a trend toward enhanced activity, but the combination was not significantly superior to monotherapy. The use of AmBi with CAS or VCZ at optimal doses did not improve efficacy. Cure was not attained with any dosage combinations. These results indicate that AmBi in combination with VCZ may be superior for treatment of CNS aspergillosis; combinations of AmBi and MICA or CAS were not antagonistic and may have a slight benefit.     

Dose-dependent antifungal activity and nephrotoxicity of amphotericin B colloidal dispersion in experimental pulmonary aspergillosis.

We investigated the safety and efficacy of amphotericin B colloidal dispersion (ABCD) for the treatment of invasive pulmonary aspergillosis in persistently granulocytopenic rabbits. Treatment groups included ABCD in dosages of 1, 5, and 10 mg/kg/day intravenously or conventional desoxycholate amphotericin B (DAmB) at 1 mg/kg/day intravenously. Antifungal activity was directly related to increasing dosage of ABCD as determined by the concentration of Aspergillus fumigatus organisms in lungs and the frequency of hemorrhagic pulmonary lesions. At 5 and 10 mg/kg/day, there was a significant reduction in the tissue burden of A. fumigatus as measured by percent culture-positive lobes and CFU per gram of tissue (P < or = 0.001), whereas at 1 mg/kg/day measured by percent culture-positive lobes and CFU per gram of tissue (P < or = 0.001), whereas at 1 mg/kg/day the tissue burden of A. fumigatus was not significantly different from that in untreated controls. Microbiological clearance was significantly greater at 1 mg of DAmB per kg per day than at 1 mg of ABCD per kg per day (P < or = 0.001). There was no difference in microbiological clearance of bronchoalveolar lavage fluid among the treatment groups as measured by CFU per milliliter. As determined by survival, ABCD at 5.0 mg/kg/day was more effective than DAmB at 1.0 mg/kg/day and ABCD at 10 mg/kg/day. ABCD at 10 mg/kg/day was more nephrotoxic than the lower dosages of ABCD and resulted in higher mortality. Impairment of glomerular filtration developed as a direct function increasing the ABCD dosage (r = 0.77; P < 0.001).(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Efficacy and safety of amphotericin B colloidal dispersion compared with those of amphotericin B deoxycholate suspension for treatment of disseminated murine cryptococcosis.

The efficacy and safety of amphotericin B colloidal dispersion (ABCD) were compared with those of amphotericin B deoxycholate suspension (ABDS) (Fungizone) in a murine model of disseminated cryptococcosis. Mice were treated intravenously with either ABDS at 0.2, 0.8, or 3.2 mg/kg of body weight per dose or ABCD at 0.8, 3.2, 6.4, 12.8, or 19.2 mg/kg dose three times per week for 2 weeks. Excluding mice treated with ABDS at 3.2 mg/kg, which was acutely lethal in 100% of mice, and ABCD at 19.2 mg/kg, which also resulted in two early deaths, the survival of ABCD- and ABDS-treated groups was prolonged over survival of controls (P < or = 0.05). Survival of ABCD (3.2 mg/kg)-treated mice was improved over that of ABDS (0.2 mg/kg)-treated mice (P < 0.05); however, comparisons of mice given all other dosages of ABCD with mice given sublethal dosages of ABDS did not demonstrate differences in survival. Comparative fungal burdens in organs showed a decrease in liver (P < 0.05) and spleen (P < 0.05) burdens for ABCD with the 19.2-mg/kg therapy versus those with ABDS with the 0.8-mg/kg therapy and liver burdens for ABCD with the 12.8-mg/kg therapy versus ABDS with the 0.8-mg/kg therapy (P < 0.05). There was no difference in organ burdens between therapy with ABCD at 0.8 mg/kg and ABDS at 0.8 mg/kg. These data show that the efficacy of ABCD is equal to that of ABDS on a milligram-per-kilogram basis for murine disseminated cryptococcosis. Because of its decreased toxicity, greater efficacy with ABCD could be achieved through doses fourfold higher than the 100% lethal dose for ABDS. Thus, ABCD shows promise as an effective but less toxic alternative to ABDS for the treatment of disseminated cryptococcosis.     

Amphotericin B Colloidal Dispersion Combined with Flucytosine with or without Fluconazole for Treatment of Murine Cryptococcal Meningitis

Studies with animals and in vitro studies have demonstrated that flucytosine plus amphotericin B or fluconazole has significantly improved mycologic activity against meningitis caused by Cryptococcus neoformans compared to the activity of amphotericin B or fluconazole used alone. However, few doses have been tested in combination. This study evaluated the antifungal efficacy of amphotericin B colloidal dispersion (ABCD) combined with flucytosine with and without fluconazole in a murine model of cryptococcal meningitis. The following dosages were tested: ABCD at 0 to 12.5 mg/kg of body weight given intravenously 3 days/week, flucytosine at 0 to 110 mg/kg/day, and fluconazole at 0 to 50 mg/kg/day. Meningitis was established in male BALB/c mice by intracerebral injection of C. neoformans. Treatment with flucytosine with or without fluconazole dissolved in the sole source of drinking water was started on day 2; animals were sacrificed at 16 days, and the numbers of fungal colonies in the brain were quantified. A survival rate of 100% was achieved with ABCD plus flucytosine without fluconazole; however, the addition of fluconazole was required to prevent weight loss (P < 0.00001) and to achieve the maximum antifungal effect (P < 0.00001). The only region of dose combinations for which the 99% confidence intervals were less than 100 CFU/g of brain was defined by ABCD at 5.0 to 7.5 mg/kg combined with flucytosine at 20 to 60 mg/kg/day and fluconazole at 30 to 40 mg/kg/day. The triple combination of ABCD plus flucytosine and fluconazole was necessary to achieve the greatest antifungal activity.     

Comparison of a New Triazole Antifungal Agent, Schering 56592, with Itraconazole and Amphotericin B for Treatment of Histoplasmosis in Immunocompetent Mice

A murine model of intratracheally induced histoplasmosis was used to evaluate a new triazole antifungal agent, Schering (SCH) 56592, for treatment of histoplasmosis. MICs were determined for SCH 56592, amphotericin B, and itraconazole by testing yeast-phase isolates from 20 patients by a macrobroth dilution method. The MICs at which 90% of the isolates are inhibited were for 0.019 μg/ml for SCH 56592, 0.5 μg/ml for amphotericin B, and ≤0.019 μg/ml for itraconazole. Survival studies were done on groups of 10 B6C3F₁ mice with a lethal inoculum of 10⁵. All mice receiving 5, 1, or 0.25 mg of SCH 56592 per kg of body weight per day, 2.5 mg of amphotericin B per kg every other day (qod), or 75 mg of itraconazole per kg per day survived to day 29. Only 44% of mice receiving 5 mg of itraconazole/kg/day survived to day 29. Fungal burden studies done in similar groups of mice with a sublethal inoculum of 10⁴ showed a reduction in CFUs and Histoplasma antigen levels in lung and spleen tissue in animals treated with 2 mg of amphotericin B/kg qod, 1 mg of SCH 56592/kg/day, and 75 mg of itraconazole/kg/day, but not in those treated with lower doses of the study drugs (0.2 mg of amphotericin B/kg qod, 0.1 mg of SCH 56592/kg/day, or 10 mg of itraconazole/kg/day). Serum drug concentrations were measured 3 and 24 h after the last dose in mice (groups of five to seven mice), each treated for 7 days with SCH 56592 (10 and 1 mg/kg/day) and itraconazole (75 and 10 mg/kg/day). Mean levels measured by bioassay were as follows: SCH 56592, 10 mg/kg/day (2.15 μg/ml at 3 h and 0.35 μg/ml at 24 h); SCH 56592, 1 mg/kg/day (0.54 μg/ml at 3 h and none detected at 24 h); itraconazole, 75 mg/kg/day (22.53 μg/ml at 3 h and none detected at 24 h); itraconazole, 10 mg/kg/day (1.33 μg/ml at 3 h and none detected at 24 h). Confirmatory results were obtained by high-pressure liquid chromatography assay. These studies show SCH 56592 to be a promising candidate for studies of treatment of histoplasmosis in humans.     

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.     

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.     

Efficacies of amphotericin B lipid complex (ABLC) and conventional amphotericin B against murine coccidioidomycosis.

The comparative activities of two preparations of amphotericin B against Coccidioides immitis were investigated. These preparations were a deoxycholate suspension (conventional amphotericin B) and a lipid-based formulation, amphotericin B lipid complex (ABLC). In-vitro susceptibility testing demonstrated that the MICs of ABLC were < or = 0.25 mg/L and of conventional amphotericin B were 0.5 mg/L for C. immitis. However, conventional amphotericin B was at least four-fold more fungicidal, with a minimum fungicidal concentration of 4.0 vs > 16 mg/L for ABLC. The therapeutic efficacies were tested in murine models of acute systemic coccidioidomycosis. Female CD-1 mice were infected iv with C. immitis arthroconidia to establish high (> 50%) or low (< 50%) mortality models. Therapy with conventional amphotericin B or ABLC was given three times per week for two weeks starting three days post-infection. Controls received no therapy or drug-free diluent only. Survival was tallied up to 49 days post-infection and the fungal cfu counts in spleen, liver, and lungs of all survivors were determined. In the low mortality study all treated mice survived and all therapy regimens reduced infection in all organs. All mice given ABLC 6.6 or 13.2 mg/kg/dose and 80% given ABLC 16.5 mg/kg/dose, as well as 90% given conventional amphotericin B 0.66 mg/kg/dose were free of infection; all controls remained infected. In two high mortality studies, all mice given ABLC 0.66-20 mg/kg/dose or conventional amphotericin B 0.22 or 0.66 mg/kg/dose survived compared with 0-20% of controls. Thirty per cent of uninfected mice given ABLC 20 mg/kg/dose and 40% given conventional amphotericin B 2.0 mg/kg/dose died due to drug toxicity. Mice given ABLC or conventional amphotericin B had lower residual cfu counts of C. immitis in all organs than did controls. Sixty to one hundred per cent of mice given ABLC regimens > or = 6.6 mg/kg/dose were cured, whereas all controls and 50-60% of mice receiving the highest non-toxic conventional amphotericin B regimen (0.66 mg/kg/dose) remained infected. At equal non-toxic amphotericin B doses, conventional amphotericin B was more effective than ABLC in reducing cfu in infected organs.(ABSTRACT TRUNCATED AT 400 WORDS)     

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 analysis of amphotericin B lipid complex and liposomal amphotericin B kinetics of lung accumulation and fungal clearance in a murine model of acute invasive pulmonary aspergillosis

The reformulation of amphotericin B (AMB) into a lipid complex (AMB lipid complex [ABLC]) or liposomal carrier (liposomal AMB [L-AMB]) changes the rate and extent of drug distribution to the lung. The importance of pharmacokinetic differences among the various lipid AMB formulations in the treatment of invasive pulmonary aspergillosis (IPA) remains unknown. We compared the kinetics of AMB lung accumulation and fungal clearance of ABLC- and L-AMB-treated mice with acute IPA. BALB/c mice were immunosuppressed with cyclophosphamide and cortisone before intranasal inoculation with 1.5x10(6) Aspergillus fumigatus 293 conidia. ABLC or L-AMB was administered in daily intravenous doses (1, 5, or 10 mg/kg of body weight), starting 12 h after infection and continuing until day 5. At predetermined times (0, 24, 72, and 120 h), mice were euthanized, and lungs were harvested for determinations of lung fungal burdens (quantitative PCR) and total AMB lung tissue concentrations. Both ABLC and L-AMB were effective at reducing lung fungal burdens at doses of >or=5 mg/kg/day. Clearance of A. fumigatus during the first 24 h was associated with AMB tissue concentrations of >4 microg/g. At 5 mg/kg/day, ABLC produced a more rapid fungal clearance than did L-AMB, but at the end of therapy, fungal burden reductions were similar for both formulations and were not improved with higher dosages. These data suggest that ABLC delivers active AMB to the lung more rapidly than does L-AMB, resulting in faster Aspergillus clearance in an experimental model of IPA. However, pharmacodynamic differences between the two formulations were less apparent when mice were dosed at 10 mg/kg/day.     

Comparative efficacies of four amphotericin B formulations--Fungizone, amphotec (Amphocil), AmBisome, and Abelcet--against systemic murine aspergillosis

We compared various amphotericin B formulations (no treatment or 0.8 mg of Fungizone [conventional deoxycholate amphotericin B] per kg of body weight, or 0.8, 4, or 8 mg of Amphocil, AmBisome, or Abelcet per kg of body weight) for treatment of systemic murine aspergillosis. In two studies, all formulations prolonged survival, with the results for AmBisome nearly equivalent to those for Fungizone; Amphocil and Abelcet were less effective or equivalent depending on the severity of infection. No survivors were cured in both kidneys and brain, but each formulation showed efficacy, especially in the kidneys. Although higher doses could be given, no lipid-based formulation showed consistent superiority over Fungizone or over each other.     

Efficacy of LY303366 against Amphotericin B-Susceptible and -Resistant Aspergillus fumigatus in a Murine Model of Invasive Aspergillosis

LY303366 is a novel antifungal echinocandin with excellent in vitro activity against Aspergillus spp. We compared four doses (1, 2.5, 10, and 25 mg/kg of body weight) of LY303366 with amphotericin B (0.5 to 5 mg/kg) in a temporarily neutropenic murine model of invasive aspergillosis against an amphotericin B-susceptible (AF210) and an amphotericin B-resistant (AF65) Aspergillus fumigatus isolate based on in vivo response. Mice were immunosuppressed with cyclophosphamide (200 mg/kg) and infected 3 days later. Treatment started 18 h after infection and lasted for 10 days. LY303366 was given once daily intravenously for 10 days, and amphotericin B (at 0.5, 2, and 5 mg/kg) was given once daily intraperitoneally for 10 days, or only on days 1, 2, 4, and 7 (at 5 mg/kg). Kidneys and lungs from survivors were cultured on day 11. Control mice in both experiments had 90 to 100% mortality. Amphotericin B at 0.5 mg/kg and LY303366 at 1 mg/kg yielded 10 to 20% survival rates for mice infected with either AF210 or AF65. Amphotericin B at 2 and 5 (both regimens) mg/kg yielded a 70 to 100% survival rate for mice infected with AF210 but a 10 to 30% survival rate for mice infected with AF65 (P = 0.01 to 0.04 compared with AF210). Against AF210 and AF65, LY303366 at 2.5, 10, and 25 mg/kg produced a survival rate of 70 to 80%, which was as effective as amphotericin B for AF210, but superior to amphotericin B for AF65 (P < 0.03 to 0.0006). For AF65, LY303366 at 10 and 25 mg/kg/day was superior to amphotericin B at 2 and 5 mg/kg/day in reducing tissue colony counts (P = 0.01 to 0.003), and for AF210, amphotericin B at 5 mg/kg/day and at 5 mg/kg in four doses was more effective than all four regimens of LY303366 in reducing renal culture counts (P = 0.01 to 0.0001). The present study shows, for the first time, that in vivo resistance of A. fumigatus to amphotericin B exists, although this could not be detected by in vitro susceptibility assays. Furthermore, LY303366 appears to be effective against amphotericin B-susceptible and -resistant A. fumigatus infection in this model and should be further evaluated clinically.     

Superior Efficacy of Liposomal Amphotericin B with Prolonged Circulation in Blood in the Treatment of Severe Candidiasis in Leukopenic Mice

In leukopenic mice with severe systemic candidiasis, single-dose treatment (5 mg of amphotericin B [AMB]/kg of body weight) with long-circulating polyethylene glycol-coated AMB liposomes (PEG-AMB-LIP) resulted in zero mortality and a significant reduction in the number of viable Candida albicans in the kidney, whereas 70% mortality was seen in mice treated with five daily doses of AmBisome (5 mg of AMB/kg · day). When the first of five daily doses of AmBisome was combined with a single low dose of Fungizone (0.1 mg of AMB/kg), the efficacy was equal to that of PEG-AMB-LIP.     

Comparison of the efficacies of various formulations of amphotericin B against murine visceral leishmaniasis.

The antileishmanial efficacies of four proprietary amphotericin B (AmB) formulations (Fungizone, AmBisome, Abelcet, and Amphocil) and an experimental nonionic surfactant vesicle (NIV) formulation were compared in a murine model of acute visceral leishmaniasis. By a multiple-dosing regimen, groups of Leishmania donovani-infected BALB/c mice were treated (2.5 mg of AmB per kg of body weight) on days 7 to 11 postinfection with one of the AmB formulations, and parasite burdens were determined on day 18 postinfection. All of the formulations caused significant suppression parasite burdens in spleens (P < 0.01 to 0.0005) and livers (P < 0.0005) compared with those in the spleens and livers of the controls. In addition, a significant suppression of parasite burdens in bone marrow (P < 0.0005) compared to the burdens in the bone marrow of the controls was obtained for all the formulations except Abelcet, which was inactive at this site. On the basis of their overall efficacies (activity against liver, spleen, and bone marrow parasites), the formulations could be ranked as follows: Amphocil = AmBisome > AmB-NIV > Abelcet >> Fungizone. On the basis of spectrophotometric measurements, AmB was shown to exist in a predominantly aggregated state in all of the formulations. Although incubation in 50% serum altered the degree of aggregation, the AmB remained predominantly aggregated, indicating that the AMB-lipid complex in all of the formulations was physically stable. The results of the study showed that antiparasitic efficacy is associated positively with the degree of AmB aggregation in the presence of serum.     

Visceral Leishmaniasis in the BALB/c Mouse: A Comparison of the Efficacy of a Nonionic Surfactant Formulation of Sodium Stibogluconate with Those of Three Proprietary Formulations of Amphotericin B

In this study, treatment efficacies of a nonionic surfactant vesicle formulation of sodium stibogluconate (SSG-NIV) and of several formulations of amphotericin B were compared in a murine model of visceral leishmaniasis. Treatment with multiple doses of AmBisome, Abelcet, and Amphocil (total dose, 12.5 mg of amphotericin B/kg of body weight) resulted in a significant suppression of parasite burdens in liver (P < 0.0005) and spleen (P < 0.0005) compared with those of controls, with Abelcet having the lowest activity. Only AmBisome and Amphocil gave significant suppression of parasites in bone marrow (compared to control values, P < 0.005). In the acute-infection model, single-dose treatments of SSG-NIV (296 mg of Sb^V/kg), SSG solution (296 mg of Sb^V/kg), or AmBisome (8 mg of amphotericin B/kg) were equally effective against liver parasites (compared to control values, P < 0.0005). SSG-NIV and AmBisome treatment also significantly suppressed parasites in bone marrow and spleen (P < 0.005), with SSG-NIV treatment being more suppressive (>98% suppression in all three sites). Free-SSG treatment failed to suppress spleen or bone marrow parasites. Infection status influenced treatment outcome. In the chronic-infection model, the AmBisome single-dose treatment was less effective in all three infection sites and the SSG-NIV single-dose treatment was less effective in the spleen. The results of this study suggest that the antileishmanial efficacy of SSG-NIV compares favorably with those of the novel amphotericin B formulations.     

Study of the toxicity of a new lipid complex formulation of amphotericin B

OBJECTIVES: The aim of this study was to evaluate the toxicity of a new lipid complex formulation of amphotericin B (LC-AmB) produced by a simple process. METHODS: Toxicity was evaluated after daily administration for 21 consecutive days in female CD1 mice. Doses of LC-AmB up to 20 mg/kg were used, and compared with Fungizone at 0.5 mg/kg and Abelcet at 10 mg/kg. Acute toxicity after a single bolus injection was also determined, as well as the haemolytic activity and toxicity to mouse macrophages in vitro. RESULTS: LC-AmB reduced both the haemolytic activity of amphotericin B and its toxicity towards mouse peritoneal macrophages. Its acute toxicity (LD50 > 200 mg/kg in CD1 mice) was similar to that in the literature for the least toxic lipid formulations of amphotericin B. The relative liver weight increased slightly in mice treated daily with a dose of 20 mg/kg LC-AmB, as did the kidney weight in this group and the group treated with Fungizone. There was also a dose-dependent decrease in the haematocrit with all formulations. All treatments caused significant increases in transaminase levels. Total hepatic CYP 450 was slightly but not significantly increased in the groups treated with 20 mg/kg LC-AmB, Abelcet and Fungizone. However, expression of some isoforms of CYP 450 was reduced, the most marked being the hepatic CYP 3A1 after treatment with 20 mg/kg LC-AmB, Abelcet and Fungizone. The effects on hepatic function are probably related to accumulation in organs rich in phagocytic cells. CONCLUSION: LC-AmB did not induce any new toxicity compared with Abelcet and Fungizone.     

Single-dose AmBisome (Liposomal amphotericin B) as prophylaxis for murine systemic candidiasis and histoplasmosis

AmBisome is a liposomal formulation of amphotericin B that has broad-spectrum antifungal activity and greatly reduced toxicity compared to the parent drug. In this study, amphotericin B deoxycholate (Fungizone) (1 mg/kg) and AmBisome (1 to 20 mg/kg) were tested as single-dose prophylactic agents in both immunocompetent and immunosuppressed C57BL/6 mice challenged with either Candida albicans or Histoplasma capsulatum. Prophylactic efficacy was based on survival and fungal burden in the target organ (kidneys or spleen). At 9 to 10 days after histoplasma challenge, 80 to 90% of both immunocompetent and immunosuppressed mice in the control and Fungizone groups had died. All AmBisome-treated mice survived, although in the AmBisome groups given 1 mg/kg, the mice became moribund by day 10 to 12. No spleen CFU were detected in the histoplasma-challenged mice given 10 or 20 mg of AmBisome per kg. By 23 to 24 days after histoplasma challenge, fungal growth and/or death had occurred in all immunosuppressed mice except for four mice receiving 20 mg of AmBisome per kg. There were still no detectable fungi in the spleens of immunocompetent mice given 10 or 20 mg of AmBisome per kg. In the C. albicans experiment at 7 days postchallenge, all animals in both untreated and treated groups were alive with culture-positive kidneys. The kidney fungal burdens in AmBisome groups given 5 to 20 mg/kg were at least 1 log unit lower than those in the Fungizone group and significantly lower than those in the untreated control group (P < 0.05). There was a trend toward decreasing fungal growth in the kidneys as the dose of AmBisome was increased. In conclusion, these results show that a single high dose of AmBisome (5 to 20 mg/kg) had prophylactic efficacy in immunocompetent and immunosuppressed murine H. capsulatum and C. albicans models.