Antifungal Activity of LY303366, a Novel Echinocandin B, in Experimental Disseminated Candidiasis in Rabbits

The safety and antifungal activity of LY303366 (LY), a new broad-spectrum semisynthetic echinocandin, were studied against disseminated candidiasis in persistently neutropenic rabbits. In vitro time-kill assays demonstrated that LY has concentration-dependent fungicidal activity. The pharmacokinetics of LY in the plasma of nonneutropenic rabbits suggested a linear relationship between dose and area under the curve (AUC). The times spent above the MIC during the experimental dosing interval of 24 h were 4 h for LY at 0.1 mg/kg of body weight/day (LY0.1), 8 h for LY at 0.25 mg/kg/day (LY0.25), 12 h for LY at 0.5 mg/kg/day (LY0.5), and 20 h for LY at 1 mg/kg/day (LY1). Antifungal therapy was administered to infected rabbits for 10 days starting 24 h after the intravenous (i.v.) inoculation of 10(3) Candida albicans blastoconidia. Study groups consisted of untreated controls (UCs) and animals treated with amphotericin B (AmB; 1 mg/kg/day i.v.), fluconazole (FLU; 10 mg/kg/day i.v.), and LY0.1, LY0.25, LY0.5, or LY1 i.v. Rabbits treated with LY0.5, LY1, AmB, and FLU had similarly significant clearance of C. albicans from the liver, spleen, kidney, lung, vena cava, and brain in comparison to that for UCs. There was a dose-dependent clearance of C. albicans from tissues in response to LY. Among rabbits treated with LY0.1 there was a significant reduction of C. albicans only in the spleen. In animals treated with LY0.25 there was a significant reduction in all tissues but the brain. By comparison, LY0.5 and LY1 cleared all tissues, including the brain, of C. albicans. These in vivo findings were consistent with the results of in vitro time-kill assays. A dose-dependent effect of altered cell wall morphology was observed among UCs and animals treated with LY0.1, and LY0.25, with a progressive transition from hyphal structure to disrupted yeast forms. Serum creatinine levels were higher and serum potassium levels were lower in AmB-treated rabbits than in UCs and LY- and FLU-treated rabbits. LY0.5 and LY1 were well tolerated, displayed predictable pharmacokinetics in plasma, and had activities comparable to those of AmB and FLU in the treatment of disseminated candidiasis in persistently neutropenic rabbits.     

Antifungal Efficacy of Caspofungin (MK-0991) in Experimental Pulmonary Aspergillosis in Persistently Neutropenic Rabbits: Pharmacokinetics, Drug Disposition, and Relationship to Galactomannan Antigenemia

The antifungal efficacy, pharmacokinetics, and safety of caspofungin (CAS) were investigated in the treatment and prophylaxis of invasive pulmonary aspergillosis due to Aspergillus fumigatus in persistently neutropenic rabbits. Antifungal therapy consisted of 1, 3, or 6 mg of CAS/kg of body weight/day (CAS1, CAS3, and CAS6, respectively) or 1 mg of deoxycholate amphotericin B (AMB)/kg/day intravenously for 12 days starting 24 h after endotracheal inoculation. Prophylaxis (CAS1) was initiated 4 days before endotracheal inoculation. Rabbits treated with CAS had significant improvement in survival and reduction in organism-mediated pulmonary injury (OMPI) measured by pulmonary infarct score and total lung weight (P < 0.01). However, animals treated with CAS demonstrated a paradoxical trend toward increased residual fungal burden (log CFU per gram) and increased serum galactomannan antigen index (GMI) despite improved survival. Rabbits receiving prophylactic CAS1 also showed significant improvement in survival and reduction in OMPI (P < 0.01), but there was no effect on residual fungal burden. In vitro tetrazolium salt hyphal damage assays and histologic studies demonstrated that CAS had concentration- and dose-dependent effects on hyphal structural integrity. In parallel with a decline in GMI, AMB significantly reduced the pulmonary tissue burden of A. fumigatus (P < or = 0.01). The CAS1, CAS3, and CAS6 dose regimens demonstrated dose-proportional exposure and maintained drug levels in plasma above the MIC for the entire 24-h dosing interval at doses that were > or =3 mg/kg/day. As serial galactomannan antigen levels may be used for therapeutic monitoring, one should be aware that profoundly neutropenic patients receiving echinocandins for aspergillosis might have persistent galactomannan antigenemia despite clinical improvement. CAS improved survival, reduced pulmonary injury, and caused dose-dependent hyphal damage but with no reduction in residual fungal burden or galactomannan antigenemia in persistently neutropenic rabbits with invasive pulmonary aspergillosis.     

Antifungal Activity of the Pradimicin Derivative BMS 181184 in the Treatment of Experimental Pulmonary Aspergillosis in Persistently Neutropenic Rabbits

The activity of the pradimicin derivative BMS 181184 was evaluated in a model of invasive pulmonary aspergillosis in persistently neutropenic rabbits and compared with that of amphotericin B deoxycholate. BMS 181184 at total daily doses of 50 and 150 mg/kg of body weight was at least as effective as amphotericin B at 1 mg/kg once a day in conferring survival and had comparable activity in reducing organism-mediated tissue injury and excess lung weight. Although treatment at all dosing regimens of BMS 181184 resulted in significant reductions in fungal tissue burden compared to untreated controls, equivalence to amphotericin B occurred only at the higher dosage level. Similar observations were made in bronchoalveolar lavage fluid cultures obtained postmortem. Monitoring of the animals through ultrafast computerized tomography scan revealed a marked resolution of pulmonary lesions during treatment with BMS 181184. The compound was well tolerated at all dosing regimens, and no toxicity was noted. Pharmacokinetic studies revealed nonlinear drug disposition with increased clearance at higher dosages and some evidence for extravascular drug accumulation. BMS 181184 had excellent activity in the treatment of experimental invasive pulmonary aspergillosis in persistently neutropenic rabbits, thus underscoring the potential of pradimicin derivatives in therapy of invasive aspergillosis in the neutropenic host.     

Safety and Efficacy of Multilamellar Liposomal Nystatin against Disseminated Candidiasis in Persistently Neutropenic Rabbits

The activity of liposomal nystatin (L-Nys) against subacute disseminated candidiasis was investigated in persistently neutropenic rabbits. Antifungal therapy was administered for 10 days starting 24 h after intravenous inoculation of 10(3) blastoconidia of Candida albicans. Responses to treatment were assessed by the quantitative clearance of the organism from blood and tissues. Treatments consisted of L-Nys at dosages of 2 and 4 mg/kg of body weight/day (L-Nys2 and L-Nys4, respectively) amphotericin B deoxycholate at 1 mg/kg/day (D-AmB), and fluconazole at 10 mg/kg/day (Flu). All treatments were given intravenously once daily. Compared to the results for untreated but infected control animals, treatment with L-Nys2, L-Nys4, D-AmB, and Flu resulted in a significant clearance of the residual burden of C. albicans from the kidney, liver, spleen, lung, and brain (P < 0.0001 by analysis of variance). When the proportion of animals infected at at least one of the five tissue sites studied was evaluated, a dose-dependent response to treatment with L-Nys was found (P < 0.05). Compared to D-AmB-treated rabbits, mean serum creatinine and blood urea nitrogen levels at the end of therapy were significantly lower in animals treated with L-Nys2 (P < 0.001) and L-Nys4 (P < 0.001 and P < 0.01, respectively). L-Nys was less nephrotoxic than conventional amphotericin B and had dose-dependent activity comparable to that of amphotericin B for the early treatment of subacute disseminated candidiasis in persistently neutropenic rabbits.     

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.     

Pharmacokinetics and Concentration-Dependent Efficacy of Isavuconazole for Treatment of Experimental Invasive Pulmonary Aspergillosis

We studied the pharmacokinetics and efficacy of the broad-spectrum triazole isavuconazole for the treatment of experimental invasive pulmonary aspergillosis (IPA) in persistently neutropenic rabbits. Treatment started 24 h after endotracheal administration of Aspergillus fumigatus inoculum; study subjects included rabbits receiving orally administered prodrug isavuconazonium sulfate (BAL8557) equivalent to active moiety isavuconazole (ISA; BAL4815) at 20 (ISA20), 40 (ISA40), and 60 (ISA60) mg/kg (of body weight)/day, with an initial loading dose of 90 mg/kg (ISA90), and untreated rabbits (UC). There were significant concentration-dependent reductions of residual fungal burden (log CFU/gram) and of organism-mediated pulmonary injury, lung weights, and pulmonary infarct scores in ISA40- and ISA60-treated rabbits in comparison to those of UC (P < 0.001). ISA20-treated (P < 0.05), ISA40-treated, and ISA60-treated (P < 0.001) rabbits demonstrated significantly prolonged survival in comparison to that of UC. ISA40- and ISA60-treated animals demonstrated a significant decline of serum (1→3)-β-d-glucan levels (P < 0.05) and galactomannan indices (GMIs) during therapy following day 4 in comparison to progressive GMIs of UC (P < 0.01). There also were significantly lower concentration-dependent GMIs in bronchoalveolar lavage (BAL) fluid from ISA40- and ISA60-treated rabbits (P < 0.001). There was a direct correlation between isavuconazole plasma area under the concentration-time curve from 0 to 24 h (AUC_0–24) and residual fungal burdens in lung tissues, pulmonary infarct scores, and total lung weights. In summary, rabbits treated with isavuconazole at 40 and 60 mg/kg/day demonstrated significant dose-dependent reduction of residual fungal burden, decreased pulmonary injury, prolonged survival, lower GMIs in serum and BAL fluid, and lower serum (1→3)-β-d-glucan levels.     

Efficacy of the Investigational Echinocandin ASP9726 in a Guinea Pig Model of Invasive Pulmonary Aspergillosis

ASP9726 is an investigational echinocandin with in vitro activity against Aspergillus species. We evaluated the pharmacokinetics and efficacy of this agent in an established guinea pig model of invasive pulmonary aspergillosis. ASP9726 plasma concentrations were measured in guinea pigs administered either a single dose or multiple doses of this agent at 2.5, 5, and 10 mg/kg of body weight/day by subcutaneous injection. Immunosuppressed guinea pigs were inoculated with A. fumigatus AF293, and ASP9726 (2.5, 5, and 10 mg/kg/day), voriconazole (10 mg/kg by oral gavage twice daily), or caspofungin (3 mg/kg/day by intraperitoneal injection) was administered for 8 days. Changes in fungal burden were measured by enumerating CFU and by quantitative PCR of specimens from within the lungs, as well as by analysis of serum (1→3)-β-d-glucan and galactomannan. Lung histopathology was also evaluated. ASP9726 plasma concentrations increased in a dose-proportional manner, and the drug was well tolerated at each dose. Each dose of ASP9726, voriconazole, and caspofungin significantly reduced pulmonary fungal burden as measured by quantitative PCR and by determining (1→3)-β-d-glucan and galactomannan levels, but only voriconazole significantly reduced numbers of CFU. ASP9726 at 5 mg/kg also significantly improved survival. Histopathology demonstrated morphological changes in hyphae in animals exposed to ASP9726 and caspofungin, consistent with the activities of the echinocandins. These results suggest that ASP9726 may be efficacious for the treatment of invasive pulmonary aspergillosis.     

Compartmental Pharmacokinetics and Tissue Distribution of the Antifungal Echinocandin Lipopeptide Micafungin (FK463) in Rabbits

The plasma pharmacokinetics and tissue distribution of the novel antifungal echinocandin-like lipopeptide micafungin (FK463) were investigated in healthy rabbits. Cohorts of three animals each received micafungin at 0.5, 1, and 2 mg/kg of body weight intravenously once daily for a total of 8 days. Serial plasma samples were collected on days 1 and 7, and tissue samples were obtained 30 min after the eighth dose. Drug concentrations were determined by validated high-performance liquid chromatographic methods. Plasma drug concentration data were fit to a two-compartment pharmacokinetic model, and pharmacokinetic parameters were estimated using weighted nonlinear least-square regression analysis. Micafungin demonstrated linear plasma pharmacokinetics without changes in total clearance and dose-normalized area under the concentration-time curve from 0 h to infinity. After administration of single doses to the rabbits, mean peak plasma drug concentrations ranged from 7.62 microg/ml at 0.5 mg/kg to 16.8 microg/ml at 2 mg/kg, the area under the concentration-time curve from 0 to 24 h ranged from 5.66 to 21.79 microg x h/ml, the apparent volume of distribution at steady state ranged from 0.296 to 0.343 liter/kg, and the elimination half-life ranged from 2.97 to 3.20 h, respectively. No significant changes in pharmacokinetic parameters and no accumulation was noted after multiple dosing. Mean tissue micafungin concentrations 30 min after the last of eight daily doses were highest in the lung (2.26 to 11.76 microg/g), liver (2.05 to 8.82 microg/g), spleen (1.87 to 9.05 microg/g), and kidney (1.40 to 6.12 microg/g). While micafungin was not detectable in cerebrospinal fluid, the concentration in brain tissue ranged from 0.08 to 0.18 microg/g. These findings indicate linear disposition of micafungin at dosages of 0.5 to 2 mg/kg and achievement of potentially therapeutic drug concentrations in plasma and tissues that are common sites of invasive fungal infections.     

Preclinical Evaluation of the Stability,Safety, and Efficacy of CD101, a Novel Echinocandin

Fungal infections pose a significant public health burden with high morbidity and mortality. CD101 is a novel echinocandin under development for the treatment and prevention of systemic Candida infections. Preclinical studies were conducted to evaluate the metabolic stability, plasma protein binding, pharmacokinetics, toxicity, and efficacy of CD101 at various dose levels. CD101 was stable to biotransformation in rat, monkey, and human liver microsomes and rat, monkey, dog, and human hepatocytes. In vitro studies suggest minimal interaction with recombinant cytochrome P450 enzymes (50% inhibitory concentrations [IC₅₀s] of >10 μM). Similar to anidulafungin, CD101 bound avidly (>98%) to human, mouse, rat, and primate plasma proteins. In a 2-week repeat-dose comparison study, CD101 was well tolerated in rats (no effects on body weight, hematology, coagulation, or urinalysis). In contrast, administration of anidulafungin (at comparable exposure levels) resulted in reduced body weight, decreases in red blood cell, hemoglobin, hematocrit, mean cell volume, mean corpuscular hemoglobin, platelet, and reticulocyte counts, increases in neutrophil and eosinophil counts, polychromasia, and decreased activated partial thromboplastin time. Elevated plasma transaminases, total bilirubin, cholesterol, and globulin, dark and enlarged spleens, and single-cell hepatocyte necrosis were also observed for anidulafungin but not CD101. Hepatotoxicity may be due to the inherent chemical lability of anidulafungin generating potentially reactive intermediates. A glutathione trapping experiment confirmed the formation of a reactive species from anidulafungin, whereas CD101 did not exhibit instability or reactive intermediates. CD101 showed antifungal activity against Candida and Aspergillus infections in neutropenic mice. These preclinical studies demonstrated that CD101 is chemically and metabolically stable, well tolerated with no hepatotoxicity, and efficacious as an antifungal agent.     

Susceptibilities of Candida Species Isolated from the Lower Gastrointestinal Tracts of High-Risk Patients to the New Semisynthetic Echinocandin LY303366 and Other Antifungal Agents

Fifty-two percent of stool specimens collected from 1,200 high-risk patients were colonized with yeasts, primarily Candida albicans (53.6%) and Candida glabrata (35.7%). Susceptibilities to all antifungal agents tested, including LY303366, were similar to those reported previously for Candida species isolated from blood.     

Efficacy of Combination Antifungal Therapy with Intraperitoneally Administered Micafungin and Aerosolized Liposomal Amphotericin B against Murine Invasive Pulmonary Aspergillosis

Targeted intrapulmonary delivery of drugs may reduce systemic toxicity and improve treatment efficacy. In the current study, we evaluated the effects of a combination treatment consisting of inhalation of aerosolized liposomal amphotericin B (L-AMB) with intraperitoneal administration of micafungin (MCFG) against murine invasive pulmonary aspergillosis. The combination of aerosolized L-AMB with intraperitoneal MCFG significantly improved the survival rate, and the fungal burdens and histopathology findings after this treatment were superior to those of the control and both monotherapy groups.Invasive pulmonary aspergillosis (IPA) results in significant morbidity and mortality in severely immunocompromised patients (6). Targeted intrapulmonary delivery of antifungals has the potential to reduce systemic toxicity and improve treatment efficacy as well as prophylaxis (1, 8) and may be used as an optional route in combination with other systemic antifungals. In the current study, we evaluated the efficacy of aerosolized liposomal amphotericin B (L-AMB) both singly and in combination with intraperitoneally administered micafungin (MCFG) in a murine model of IPA.Aspergillus fumigatus MF13 was clinically obtained from a patient admitted to the Nagasaki University Hospital. The minimum effective concentration of MCFG (Astellas Pharmaceuticals Inc., Tokyo, Japan) and the MIC of AMB (Sigma, St. Louis, MO) were determined using the microdilution method in accordance with Clinical Laboratory Standards Institute document M38-A2 (2). Drug interactions were assessed using the checkerboard titration broth microdilution-based method (3), and the fractional inhibitory concentration index was determined as previously described (5).Six-week-old female ICR mice (Charles River Breeding Laboratories, Shiga, Japan) were immunosuppressed and then challenged on day 0 with 5 × 10⁶ conidia of A. fumigatus MF13 intratracheally for monitoring of survival, as previously described (7, 11). Eight-week-old female ICR mice were used to determine fungal burdens and for histopathological examination. Mice were immunosuppressed by subcutaneous injection of cortisone acetate (Sigma, Tokyo, Japan) at 250 mg/kg of body weight and intraperitoneally administered cyclophosphamide (Sigma) at 200 mg/kg on days −2 and 0 for the survival study. Only cortisone acetate (200 mg/kg) was used on days −1, 0, and 1 for fungal-burden analysis and histopathological examination. Mice were assigned into the following groups: (i) control mice, (ii) mice receiving MCFG intraperitoneally, (iii) mice receiving aerosolized L-AMB, and (iv) mice receiving a combination treatment of intraperitoneally administered MCFG and aerosolized L-AMB. Each group consisted of 11 and 10 mice for survival and fungal-burden analyses, respectively. MCFG was administered intraperitoneally once daily at 1 mg/kg/day. L-AMB was administered once daily in an 8-ml suspension (at 1.2 mg/ml) per inhalation. Antifungals were initiated 16 h after inoculation and continued for 5 and 3 days for survival and fungal-burden analyses, respectively. The L-AMB solution was aerosolized using a nebulizer (Muromachi Kikai Co., Ltd., Tokyo, Japan), and mice were exposed to aerosol treatment for 60 min as previously described (9). Control mice were treated with sterile saline. Survival was observed until 11 days following the challenge. For fungal-burden and histopathological examinations, mice were sacrificed 4 h after the treatment on day 3. Numbers of CFU per lung tissue were calculated, and removed lungs were fixed and stained with Grocott''s methenamine silver nitrate and hematoxylin-eosin as previously described (11). Survival and fungal burden data are presented from a combination of two sets of experiments. The concentration in blood and the pharmacokinetics of aerosolized L-AMB were evaluated. Uninfected mice were also exposed to several concentrations of aerosolized L-AMB for 5 days, and blood samples and lungs were collected. AMB concentration was quantified as previously described (10). Survival curves were generated using the Kaplan and Meier method, and statistical differences were evaluated by the log rank test. To assess fungal burden in lung tissue, geometric means of numbers of CFU per organ were compared by Student''s t test. Statistical significance was defined as a P of <0.05.The MIC of AMB against A. fumigatus MF-13 was 1.0 μg/ml, and the minimum effective concentration of MCFG was 0.0315 μg/ml. The fractional inhibitory concentration index of AMB and MCFG was 1.5, and drug interaction was classified as indifferent (5).Survival periods of monotherapy groups, in which mice either were treated with intraperitoneally administered MCFG or inhaled aerosolized L-AMB were significantly longer than that of the control group (MCFG alone versus the control, P = 0.006; L-AMB versus the control, P < 0.001) (Fig. ​(Fig.1).1). The combination treatment group showed significantly longer survival than the intraperitoneal-MCFG (P < 0.001), aerosolized-L-AMB (P = 0.037), and control (P < 0.001) groups. Numbers of CFU in the lungs of mice in the combination treatment group were significantly reduced compared to those in each of the intraperitoneal-MCFG (P < 0.001), aerosolized-L-AMB (P = 0.027), and control (P < 0.001) groups (Fig. ​(Fig.2).2). The lungs of aerosolized-L-AMB-administered and combination treatment mice showed obviously smaller numbers of hyphae and fewer foci of inflammation than the intraperitoneal-MCFG and control groups (Fig. ​(Fig.3).3). The mean AMB concentrations in the lung tissue following L-AMB inhalation at 1.2, 2.6, and 4.0 mg/ml were 35.5, 73.2, and 94.2 μg/g, respectively. Recorded levels in sera were 0.02, 0.06, and 0.06 μg/ml when inhaled-L-AMB suspensions were administered at 1.2, 2.6, and 4.0 mg/ml, respectively.Open in a separate windowFIG. 1.Survival curves for mice with IPA (Kaplan-Meier plot). Groups of 11 mice were treated with a combination of intraperitoneal administration of MCFG (1 mg/kg/day) and inhalation of aerosolized L-AMB (8 ml at 1.2 mg/ml [open squares]), inhalation of aerosolized L-AMB (8 ml at 1.2 mg/ml [filled triangles]), intraperitoneal administration of MCFG (1 mg/kg/day [open triangles]), and no therapy (control [filled circles]). *, P < 0.05 versus the control; **, P < 0.05 versus the control group, intraperitoneal-MCFG group, or aerosolized-L-AMB group (log rank test). The survival times for all treatment groups were longer than that for controls (P < 0.05). The survival time for the combination treatment group was significantly longer than those of the intraperitoneal-MCFG group and the aerosolized-L-AMB group (P < 0.05).Open in a separate windowFIG. 2.Numbers of CFU from homogenized lung tissues of mice with IPA. Groups of 10 mice were treated once per day with a combination of intraperitoneally administered MCFG (1 mg/kg/day) and inhalation of aerosolized L-AMB (8 ml at 1.2 mg/ml), aerosolized L-AMB (8 ml at 1.2 mg/ml), intraperitoneal MCFG (1 mg/kg/day), and saline (control). CFU counts, as a parameter of A. fumigatus burden in the lungs of IPA mice at 4 h after day 3 of treatment, are shown. *, P < 0.05 (Student''s t test).Open in a separate windowFIG. 3.Histopathology of lung tissues. Both lungs were obtained from IPA mice 4 h after 3 days of treatment with a combination of intraperitoneally administered MCFG and inhalation of aerosolized L-AMB, aerosolized L-AMB, intraperitoneal MCFG, and saline alone as a control. The lungs obtained from aerosolized-L-AMB-treated and combination treatment mice showed obviously smaller numbers of hyphae and fewer foci of inflammation than intraperitoneal-MCFG and control mice. HE, hematoxylin-eosin; GMS, Grocott''s methenamine silver nitrate stain.The current study demonstrated the efficacy of monotherapy of aerosolized L-AMB in a murine IPA model. The AMB concentrations in lung tissue in our study were relatively higher but extremely lower in serum than those from another report of a murine model of intravenously administered L-AMB, although experimental conditions were not the same (10). These results suggested that systemic toxicity generally caused by AMB treatment may be reduced by L-AMB inhalation therapy.The effect of combined intraperitoneal-MCFG and aerosolized-L-AMB treatment was an enhanced survival rate, even though this drug interaction was classified as indifferent in vitro. Since 78% of all control mice died in first 3 days in a survival analysis, we changed the experimental conditions for analysis of fungal burden and histopathological examination. In this model, no mice died before euthanasia, a prerequisite for the organ CFU assay. Both fungal-burden data and histopathological findings supported the survival data in our study.Unlike in our study, Graybill et al. previously reported that combination therapy demonstrated a lack of synergistic effects following intravenous-L-AMB and intraperitoneal-MCFG treatment in a model of murine IPA (4). These discrepancies are likely due to differences between our model and Graybill et al.''s model, including (i) the route of infection, (ii) the status of immunosuppression, and (iii) the administration route of antifungal drugs. These differences also suggest that targeted intrapulmonary delivery of drugs by inhalation raises the drug concentration at the active site of infection in the lungs, thus contributing to the efficacy of combination therapy. Further comparative efficacy studies in a clinical setting are warranted.     

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.     

Comparative Pharmacodynamics of Posaconazole in Neutropenic Murine Models of Invasive Pulmonary Aspergillosis and Mucormycosis

We used two established neutropenic murine models of pulmonary aspergillosis and mucormycosis to explore the association between the posaconazole area under the concentration-time curve (AUC)-to-MIC ratio (AUC/MIC) and treatment outcome. Posaconazole serum pharmacokinetics were verified in infected mice to ensure that the studied doses reflected human exposures with the oral suspension, delayed-release tablet, and intravenous formulations of posaconazole. Sinopulmonary infections were then induced in groups of neutropenic mice with Aspergillus fumigatus strain 293 (posaconazole MIC, 0.5 mg/liter) or Rhizopus oryzae strain 969 (posaconazole MIC, 2 mg/liter) and treated with escalating daily dosages of oral posaconazole, which was designed to achieve AUCs ranging from 1.10 to 392 mg · h/liter. After 5 days of treatment, lung fungal burden was analyzed by quantitative real-time PCR. The relationships of the total drug AUC/MIC and the treatment response were similar in both models, with 90% effective concentrations (EC₉₀s) corresponding to an AUC/MIC threshold of 76 (95% confidence interval [CI], 46 to 102) for strain 293 versus 87 (95% CI, 66 to 101) for strain 969. Using a provisional AUC/MIC target of >100, these exposures correlated with minimum serum posaconazole concentrations (C_mins) of 1.25 mg/liter for strain 293 and 4.0 mg/liter for strain 969. The addition of deferasirox, but not liposomal amphotericin or caspofungin, improved the activity of a suboptimal posaconazole regimen (AUC/MIC, 33) in animals with pulmonary mucormycosis. However, no combination was as effective as the high-dose posaconazole monotherapy regimen (AUC/MIC, 184). Our analysis suggests that posaconazole pharmacodynamics are similar for A. fumigatus and R. oryzae when indexed to pathogen MICs.     

In Vitro Activity of the Echinocandin Antifungal Agent LY303,366 in Comparison with Itraconazole and Amphotericin B against Aspergillus spp.

LY303,366 (LY) is a novel derivative of the echinocandin class of antifungal agents. The in vitro activities of LY, itraconazole (ITZ), and amphotericin B (AMB) were assessed against 60 Aspergillus isolates, including 35 isolates of A. fumigatus, eight isolates of A. terreus, eight isolates of A. flavus, eight isolates of A. niger and one isolate of A. nidulans. Four A. fumigatus isolates were resistant to ITZ. Susceptibility testing for all drugs was performed with a broth microdilution procedure. LY was tested in two media: antibiotic medium 3 (AM3) and Casitone with 2% glucose (CAS) with an inoculum of 2 × 10³ spores/ml. ITZ and AMB were tested in RPMI 1640 with 2% glucose with an inoculum of 1 × 10⁶ spores/ml. All tests were incubated at 37°C for 48 h. A novel end point was used to determine a minimal effective concentration (MEC) for LY, i.e., almost complete inhibition of growth save a few tiny spherical colonies attached to the microplate. MICs were measured for ITZ and AMB with a no-growth end point. Ranges and geometric mean (GM) MECs were from 0.0018 to >0.5 and 0.0039 mg/liter and from 0.0018 to >0.5 and 0.008 mg/liter for LY in AM3 and LY in CAS, respectively. Differences between species were apparent, with A. flavus being significantly less susceptible to LY than any other species tested with both media (P ≤ 0.05). Ranges and GM MICs were from 0.125 to >16 and 0.7 mg/liter for ITZ and from 0.25 to 16 and 1.78 mg/liter for AMB. Minimal fungicidal concentrations (MFCs) were also determined for all drugs. GM MFCs were 0.018, 0.09, 19.76, and 12.64 mg/liter for LY in AM3, LY in CAS, ITZ, and AMB, respectively. LY in AM3 and LY in CAS were fungicidal for 86.7 and 68% of isolates, respectively (98% killing). In comparison, ITZ and AMB were fungicidal for 35 and 70% of isolates, respectively (99.99% killing). A reproducibility study was performed on 20% of the isolates. For 12 isolates retested, the MEC or MIC was the same or was within 1 dilution of the original value for 11, 11, 10, and 9 isolates for LY in AM3, LY in CAS, ITZ, and AMB, respectively. In conclusion, LY seems to be a promising antifungal agent with excellent in vitro activity against Aspergillus spp.

Invasive aspergillosis is now one of the most common fungal infections found in immunocompromised patients (1) and is also one of the most fatal (2). Treatment of Aspergillus infections is still not ideal, and the two currently used antifungal drugs have a variety of associated problems. Amphotericin B (AMB) can cause serious side effects due to its toxicity and itraconazole (ITZ) is not always absorbed in high enough quantities to be therapeutic, especially in certain patient groups, e.g., AIDS patients.The rise in serious fungal infection over the past decade has prompted the development of new antifungal agents with novel modes of action. LY303,366 (LY) is a semisynthetic derivative of a natural product class of antifungal agents belonging to the new class of drugs known as echinocandins. Echinocandins are noncompetitive inhibitors of (1,3)-β-d-glucan synthase which produces glucan polymers, a major component of the fungal cell wall (3). LY has been reported to have excellent activity against a wide range of fungal pathogens, including Aspergillus species (12) and Candida species (8, 10, 12).In this study we evaluated the in vitro activity of LY against a variety of Aspergillus species and compared it with the activity of currently used antifungal agents, ITZ and AMB.     

Impact of In Vivo Triazole and Echinocandin Combination Therapy for Invasive Pulmonary Aspergillosis: Enhanced Efficacy against Cyp51 Mutant Isolates

Previous studies examining combination therapy for invasive pulmonary aspergillosis (IPA) have revealed conflicting results, including antagonism, indifference, and enhanced effects. The most commonly employed combination for this infection includes a mold-active triazole and echinocandin. Few studies have evaluated combination therapy from a pharmacodynamic (PD) perspective, and even fewer have examined combination therapy against both wild-type and azole-resistant Cyp51 mutant isolates. The current studies aim to fill this gap in knowledge. Four Aspergillus fumigatus isolates were utilized, including a wild-type strain, an Fks1 mutant (posaconazole susceptible and caspofungin resistant), and two Cyp51 mutants (posaconazole resistant). A neutropenic murine model of IPA was used for the treatment studies. The dosing design included monotherapy with posaconazole, monotherapy with caspofungin, and combination therapy with both. Efficacy was determined using quantitative PCR, and results were normalized to known quantities of conidia (conidial equivalents [CE]). The static dose, 1-log kill dose, and associated PD target area under the curve (AUC)/MIC ratio were determined for monotherapy and combination therapy. Monotherapy experiments revealed potent activity for posaconazole, with reductions of 3 to 4 log₁₀ Aspergillus CE/ml with the two “low”-MIC isolates. Posaconazole alone was less effective for the two isolates with higher MICs. Caspofungin monotherapy did not produce a significant decrease in fungal burden for any strain. Combination therapy with the two antifungals did not enhance efficacy for the two posaconazole-susceptible isolates. However, the drug combination produced synergistic activity against both posaconazole-resistant isolates. Specifically, the combination resulted in a 1- to 2-log₁₀ decline in burden that would not have been predicted based on the monotherapy results for each drug. This corresponded to a reduction in the free-drug posaconazole AUC/MIC ratio needed for stasis of up to 17-fold. The data suggest that combination therapy using a triazole and an echinocandin may be a beneficial treatment strategy for triazole-resistant isolates.     

Effects of the Hematoregulatory Peptide SK&F 107647 Alone and in Combination with Amphotericin B against Disseminated Candidiasis in Persistently Neutropenic Rabbits

The effects of the hematoregulatory peptide SK&F 107647 were examined in a persistently and profoundly neutropenic rabbit model of disseminated candidiasis in order to determine its potential to enhance resistance against infection and its role as an adjunct to conventional antifungal chemotherapy. In healthy animals, SK&F 107647 elicited a time-dependent increase in CD11b-positive monocytes and neutrophils. When administered to neutropenic rabbits infected with Candida albicans, no significant differences in the number of CFU per gram in any of the tissues tested compared with the number in untreated control rabbits were detected. However, when SK&F 107647 was administered in combination with low doses of amphotericin B, there was a significant reduction in organism burden in the lungs, liver, spleen, and kidneys compared with the burdens in the organs of untreated control animals and in the lungs and kidneys compared with the burdens in the lungs and kidneys of animals treated with amphotericin B alone. These data suggest a potential role for this peptide as adjunctive therapy in combination with conventional antifungal agents in the treatment of disseminated candidiasis in the setting of profound and persistent neutropenia.     

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.     

Phase I,Open-Label,Single-Dose Study To Evaluate the Pharmacokinetics and Safety of Telbivudine in Children and Adolescents with Chronic Hepatitis B

Telbivudine is a nucleoside analogue that has been approved for the treatment of chronic hepatitis B virus (HBV) infection in adults at 600 mg/day. We conducted a phase I, open-label, first-in-pediatrics study to investigate the safety and pharmacokinetics of a single dose of telbivudine in HBV-infected children and adolescents. Eligible patients were enrolled sequentially from older to younger groups, with evaluation of safety and available pharmacokinetic data after each stratum. Adolescent patients (>12 to 18 years) received a single dose of 600 mg telbivudine as an oral solution, while children aged 2 to 12 years received a single dose of 15 or 25 mg/kg of body weight up to a maximum of 600 mg. Telbivudine was well tolerated; all adverse events were mild, and none occurred in more than one patient. The plasma telbivudine concentration-versus-time profiles in adolescents given 600 mg were similar to the mean profile of healthy adults receiving the same oral dose. Children aged 2 to <6 and 6 to 12 years receiving a single 15-mg/kg dose showed similar plasma exposures. To predict the steady-state exposure, plasma concentration-versus-time profiles for patients aged 2 to 12 years (15 mg/kg) and >12 to 18 years (600 mg) were fitted to a two-compartment 1st-order, microconstant, lag time, 1st-order elimination pharmacokinetic (PK) model. This analysis predicted the following dosages to mimic exposures in healthy adults receiving 600 mg/day: 20 mg/kg/day for children 2 to 12 years and 600 mg/day for adolescents. Studies are ongoing to evaluate the efficacy of the recommended dose in pediatric patients. (This study has been registered at ClinicalTrials.gov under registration no. {"type":"clinical-trial","attrs":{"text":"NCT00907894","term_id":"NCT00907894"}}NCT00907894.)