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1.  Hyperthermia Sensitizes Rhizopus oryzae to Posaconazole and Itraconazole Action through Apoptosis 
The high mortality rate of mucormycosis with currently available monotherapy has created interest in studying novel strategies for antifungal agents. With the exception of amphotericin B (AMB), the triazoles (posaconazole [PCZ] and itraconazole [ICZ]) are fungistatic in vitro against Rhizopus oryzae . We hypothesized that growth at a high temperature (42°C) results in fungicidal activity of PCZ and ICZ that is mediated through apoptosis. R. oryzae had high MIC values for PCZ and ICZ (16 to 64 μg/ml) at 25°C; in contrast, the MICs for PCZ and ICZ were significantly lower at 37°C (8 to 16 μg/ml) and 42°C (0.25 to 1 μg/ml). Furthermore, PCZ and ICZ dose-dependent inhibition of germination was more pronounced at 42°C than at 37°C. In addition, intracellular reactive oxygen species (ROS) increased significantly when fungi were exposed to antifungals at 42°C. Characteristic cellular changes of apoptosis in R. oryzae were induced by the accumulation of intracellular reactive oxygen species. Cells treated with PCZ or ICZ in combination with hyperthermia (42°C) exhibited characteristic markers of early apoptosis: phosphatidylserine externalization visualized by annexin V staining, membrane depolarization visualized by bis-[1,3-dibutylbarbituric acid] trimethine oxonol (DiBAC) staining, and increased metacaspase activity. Moreover, terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) assay and DAPI (4′,6-diamidino-2-phenylindole) staining demonstrated DNA fragmentation and condensation, respectively. The addition of N-acetylcysteine increased fungal survival, prevented apoptosis, reduced ROS accumulation, and decreased metacaspase activation. We concluded that hyperthermia, either alone or in the presence of PCZ or ICZ, induces apoptosis in R. oryzae. Local thermal delivery could be a therapeutically useful adjunct strategy for these refractory infections.
doi:10.1128/AAC.00571-13
PMCID: PMC3754336  PMID: 23817366
2.  Species-Specific and Drug-Specific Differences in Susceptibility of Candida Biofilms to Echinocandins: Characterization of Less Common Bloodstream Isolates 
Candida species other than Candida albicans are increasingly recognized as causes of biofilm-associated infections. This is a comprehensive study that compared the in vitro activities of all three echinocandins against biofilms formed by different common and infrequently identified Candida isolates. We determined the activities of anidulafungin (ANID), caspofungin (CAS), and micafungin (MFG) against planktonic cells and biofilms of bloodstream isolates of C. albicans (15 strains), Candida parapsilosis (6 strains), Candida lusitaniae (16 strains), Candida guilliermondii (5 strains), and Candida krusei (12 strains) by XTT [2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide] assay. Planktonic and biofilm MICs were defined as ≥50% fungal damage. Planktonic cells of all Candida species were susceptible to the three echinocandins, with MICs of ≤1 mg/liter. By comparison, differences in the MIC profiles of biofilms in response to echinocandins existed among the Candida species. Thus, C. lusitaniae and C. guilliermondii biofilms were highly recalcitrant to all echinocandins, with MICs of ≥32 mg/liter. In contrast, the MICs of all three echinocandins for C. albicans and C. krusei biofilms were relatively low (MICs ≤ 1 mg/liter). While echinocandins exhibited generally high MICs against C. parapsilosis biofilms, MFG exhibited the lowest MICs against these isolates (4 mg/liter). A paradoxical growth effect was observed with CAS concentrations ranging from 8 to 64 mg/liter against C. albicans and C. parapsilosis biofilms but not against C. krusei, C. lusitaniae, or C. guilliermondii. While non-albicans Candida planktonic cells were susceptible to all echinocandins, there were drug- and species-specific differences in susceptibility among biofilms of the various Candida species, with C. lusitaniae and C. guilliermondii exhibiting profiles of high MICs of the three echinocandins.
doi:10.1128/AAC.02541-12
PMCID: PMC3716187  PMID: 23529739
3.  Synergy, Pharmacodynamics, and Time-Sequenced Ultrastructural Changes of the Interaction between Nikkomycin Z and the Echinocandin FK463 against Aspergillus fumigatus 
Antimicrobial Agents and Chemotherapy  2001;45(12):3310-3321.
We investigated the potential synergy between two cell wall-active agents, the echinocandin FK463 (FK) and the chitin synthase inhibitor nikkomycin Z (NZ), against 16 isolates of filamentous fungi. Susceptibility testing was performed with a broth macrodilution procedure by NCCLS methods. The median minimal effective concentration (MEC) of FK against all Aspergillus species was 0.25 μg/ml (range, 0.05 to 0.5 μg/ml). For Fusarium solani and Rhizopus oryzae, MECs of FK were >512 μg/ml. The median MEC of NZ against Aspergillus fumigatus was 32 μg/ml (range, 8 to 64 μg/ml), and that against R. oryzae was 0.5 μg/ml (range, 0.06 to 2 μg/ml); however, for the other Aspergillus species, as well as F. solani, MECs were >512 μg/ml. A checkerboard inhibitory assay demonstrated synergy against A. fumigatus (median fractional inhibitory concentration index = 0.312 [range, 0.15 to 0.475]). The effect was additive to indifferent against R. oryzae and indifferent against other Aspergillus spp. and F. solani. We further investigated the pharmacodynamics of hyphal damage by MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay and examined the time-sequenced changes in hyphal ultrastructure. Significant synergistic hyphal damage was demonstrated with the combination of NZ (2 to 32 μg/ml) and FK (0.03 to 0.5 μg/ml) over a wide range of concentrations (P < 0.001). The synergistic effect was most pronounced after 12 h of incubation and was sustained through 24 h. Time-sequenced light and electron microscopic studies demonstrated that structural alterations of hyphae were profound, with marked transformation of hyphae to blastospore-like structures, in the presence of FK plus NZ, while fungi treated with a single drug showed partial recovery at 24 h. The methods used in this study may be applicable to elucidating the activity and interaction of other cell wall-active agents. In summary, these two cell wall-targeted antifungal agents, FK and NZ, showed marked time-dependent in vitro synergistic activity against A. fumigatus.
doi:10.1128/AAC.45.12.3310-3321.2001
PMCID: PMC90831  PMID: 11709302
4.  Compartmental Pharmacokinetics and Tissue Distribution of the Antifungal Echinocandin Lipopeptide Micafungin (FK463) in Rabbits 
Antimicrobial Agents and Chemotherapy  2001;45(12):3322-3327.
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 μg/ml at 0.5 mg/kg to 16.8 μg/ml at 2 mg/kg, the area under the concentration-time curve from 0 to 24 h ranged from 5.66 to 21.79 μg · 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 μg/g), liver (2.05 to 8.82 μg/g), spleen (1.87 to 9.05 μg/g), and kidney (1.40 to 6.12 μg/g). While micafungin was not detectable in cerebrospinal fluid, the concentration in brain tissue ranged from 0.08 to 0.18 μg/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.
doi:10.1128/AAC.45.12.3322-3327.2001
PMCID: PMC90832  PMID: 11709303
5.  Safety, Tolerance, and Pharmacokinetics of High-Dose Liposomal Amphotericin B (AmBisome) in Patients Infected with Aspergillus Species and Other Filamentous Fungi: Maximum Tolerated Dose Study 
Antimicrobial Agents and Chemotherapy  2001;45(12):3487-3496.
We conducted a phase I-II study of the safety, tolerance, and plasma pharmacokinetics of liposomal amphotericin B (L-AMB; AmBisome) in order to determine its maximally tolerated dosage (MTD) in patients with infections due to Aspergillus spp. and other filamentous fungi. Dosage cohorts consisted of 7.5, 10.0, 12.5, and 15.0 mg/kg of body weight/day; a total of 44 patients were enrolled, of which 21 had a proven or probable infection (13 aspergillosis, 5 zygomycosis, 3 fusariosis). The MTD of L-AMB was at least 15 mg/kg/day. Infusion-related reactions of fever occurred in 8 (19%) and chills and/or rigors occurred in 5 (12%) of 43 patients. Three patients developed a syndrome of substernal chest tightness, dyspnea, and flank pain, which was relieved by diphenhydramine. Serum creatinine increased two times above baseline in 32% of the patients, but this was not dose related. Hepatotoxicity developed in one patient. Steady-state plasma pharmacokinetics were achieved by day 7. The maximum concentration of drug in plasma (Cmax) of L-AMB in the dosage cohorts of 7.5, 10.0, 12.5, and 15.0 mg/kg/day changed to 76, 120, 116, and 105 μg/ml, respectively, and the mean area under the concentration-time curve at 24 h (AUC24) changed to 692, 1,062, 860, and 554 μg · h/ml, respectively, while mean CL changed to 23, 18, 16, and 25 ml/h/kg, respectively. These data indicate that L-AMB follows dose-related changes in disposition processing (e.g., clearance) at dosages of ≥7.5 mg/kg/day. Because several extremely ill patients had early death, success was determined for both the modified intent-to-treat and evaluable (7 days of therapy) populations. Response rates (defined as complete response and partial response) were similar for proven and probable infections. Response and stabilization, respectively, were achieved in 36 and 16% of the patients in the modified intent-to-treat population (n = 43) and in 52 and 13% of the patients in the 7-day evaluable population (n = 31). These findings indicate that L-AMB at dosages as high as 15 mg/kg/day follows nonlinear saturation-like kinetics, is well tolerated, and can provide effective therapy for aspergillosis and other filamentous fungal infections.
doi:10.1128/AAC.45.12.3487-3496.2001
PMCID: PMC90858  PMID: 11709329
6.  Pharmacokinetic and Pharmacodynamic Modeling of Anidulafungin (LY303366): Reappraisal of Its Efficacy in Neutropenic Animal Models of Opportunistic Mycoses Using Optimal Plasma Sampling 
Antimicrobial Agents and Chemotherapy  2001;45(10):2845-2855.
The compartmental pharmacokinetics of anidulafungin (VER-002; formerly LY303366) in plasma were characterized with normal rabbits, and the relationships between drug concentrations and antifungal efficacy were assessed in clinically applicable infection models in persistently neutropenic animals. At intravenous dosages ranging from 0.1 to 20 mg/kg of body weight, anidulafungin demonstrated linear plasma pharmacokinetics that fitted best to a three-compartment open pharmacokinetic model. Following administration over 7 days, the mean (± standard error of the mean) peak plasma concentration (Cmax) increased from 0.46 ± 0.02 μg/ml at 0.1 mg/kg to 63.02 ± 2.93 μg/ml at 20 mg/kg, and the mean area under the concentration-time curve from 0 h to infinity (AUC0–∞) rose from 0.71 ± 0.04 to 208.80 ± 24.21 μg · h/ml. The mean apparent volume of distribution at steady state (Vss) ranged from 0.953 ± 0.05 to 1.636 ± 0.22 liter/kg (nonsignificant [NS]), and clearance ranged from 0.107 ± 0.01 to 0.149 ± 0.00 liter/kg/h (NS). Except for a significant prolongation of the terminal half-life and a trend toward an increased Vss at the higher end of the dosage range after multiple doses, no significant differences in pharmacokinetic parameters were noted in comparison to single-dose administration. Concentrations in tissue at trough after multiple dosing (0.1 to 10 mg/kg/day) were highest in lung and liver (0.85 ± 0.16 to 32.64 ± 2.03 and 0.32 ± 0.05 to 43.76 ± 1.62 μg/g, respectively), followed by spleen and kidney (0.24 ± 0.65 to 21.74 ± 1.86 and <0.20 to 16.92 ± 0.56, respectively). Measurable concentrations in brain tissue were found at dosages of ≥0.5 mg/kg (0.24 ± 0.02 to 3.90 ± 0.25). Implementation of optimal plasma sampling in persistently neutropenic rabbit infection models of disseminated candidiasis and pulmonary aspergillosis based on the Bayesian approach and model parameters from normal animals as priors revealed a significantly slower clearance (P < 0.05 for all dosage groups) with a trend toward higher AUC0–24 values, higher plasma concentrations at the end of the dosing interval, and a smaller volume of distribution (P < 0.05 to 0.193 for the various comparisons among dosage groups). Pharmacodynamic modeling using the residual fungal tissue burden in the main target sites as the primary endpoint and Cmax, AUC0–24, time during the dosing interval of 24 h with plasma drug concentration equaling or exceeding the MIC or the minimum fungicidal concentration for the isolate, and tissue concentrations as pharmacodynamic parameters showed predictable pharmacokinetic-pharmacodynamic relationships in experimental disseminated candidiasis that fitted well with an inhibitory sigmoid maximum effect pharmacodynamic model (r2, 0.492 to 0.819). However, no concentration-effect relationships were observed in experimental pulmonary aspergillosis using the residual fungal burden in lung tissue and survival as parameters of antifungal efficacy. Implementation of optimal plasma sampling in discriminative animal models of invasive fungal infections and pharmacodynamic modeling is a novel approach that holds promise of improving and accelerating our understanding of the action of antifungal compounds in vivo.
doi:10.1128/AAC.45.10.2845-2855.2001
PMCID: PMC90741  PMID: 11557479
7.  Antifungal Activity and Pharmacokinetics of Posaconazole (SCH 56592) in Treatment and Prevention of Experimental Invasive Pulmonary Aspergillosis: Correlation with Galactomannan Antigenemia 
The antifungal efficacy, safety, and pharmacokinetics of posaconazole (SCH 56592) (POC) were investigated in treatment and prophylaxis of primary pulmonary aspergillosis due to Aspergillus fumigatus in persistently neutropenic rabbits. Antifungal therapy consisted of POC at 2, 6, and 20 mg/kg of body weight per os; itraconazole (ITC) at 2, 6, and 20 mg/kg per os; or amphotericin B (AMB) at 1 mg/kg intravenously. Rabbits treated with POC showed a significant improvement in survival and significant reductions in pulmonary infarct scores, total lung weights, numbers of pulmonary CFU per gram, numbers of computerized-tomography-monitored pulmonary lesions, and levels of galactomannan antigenemia. AMB and POC had comparable therapeutic efficacies by all parameters. By comparison, animals treated with ITC had no significant changes in outcome variables in comparison to those of untreated controls (UC). Rabbits receiving prophylactic POC at all dosages showed a significant reduction in infarct scores, total lung weights, and organism clearance from lung tissue in comparison to results for UC (P < 0.01). There was dosage-dependent microbiological clearance of A. fumigatus from lung tissue in response to POC. Serum creatinine levels were greater (P < 0.01) in AMB-treated animals than in UC and POC- or ITC-treated rabbits. There was no elevation of serum hepatic transaminase levels in POC- or ITC-treated rabbits. The pharmacokinetics of POC and ITC in plasma demonstrated dose dependency after multiple dosing. The 2-, 6-, and 20-mg/kg dosages of POC maintained plasma drug levels above the MICs for the entire 24-h dosing interval. In summary, POC at ≥6 mg/kg/day per os generated sustained concentrations in plasma of ≥1 μg/ml that were as effective in the treatment and prevention of invasive pulmonary aspergillosis as AMB at 1 mg/kg/day and more effective than cyclodextrin ITC at ≥6 mg/kg/day per os in persistently neutropenic rabbits.
doi:10.1128/AAC.45.3.857-869.2001
PMCID: PMC90385  PMID: 11181372
8.  Compartmental Pharmacokinetics of the Antifungal Echinocandin Caspofungin (MK-0991) in Rabbits 
The pharmacokinetics of the antifungal echinocandin-lipopeptide caspofungin (MK-0991) in plasma were studied in groups of three healthy rabbits after single and multiple daily intravenous administration of doses of 1, 3, and 6 mg/kg of body weight. Concentrations were measured by a validated high-performance liquid chromatography method and fitted into a three-compartment open pharmacokinetic model. Across the investigated dosage range, caspofungin displayed dose-independent pharmacokinetics. Following administration over 7 days, the mean peak concentration in plasma (Cmax) ± standard error of the mean increased from 16.01 ± 0.61 μg/ml at the 1-mg/kg dose to 105.52 ± 8.92 μg/ml at the 6-mg/kg dose; the mean area under the curve from 0 h to infinity rose from 13.15 ± 2.37 to 158.43 ± 15.58 μg · h/ml, respectively. The mean apparent volume of distribution at steady state (Vdss) was 0.299 ± 0.011 liter/kg at the 1-mg/kg dose and 0.351 ± 0.016 liter/kg at the 6-mg/kg dose (not significant [NS]). Clearance (CL) ranged from 0.086 ± 0.017 liter/kg/h at the 1-mg/kg dose to 0.043 ± 0.004 liter/kg/h at the 6-mg/kg dose (NS), and the mean terminal half-life was between 30 and 34 h (NS). Except for a trend towards an increased Vdss, there were no significant differences in pharmacokinetic parameters in comparison to those after single-dose administration. Caspofungin was well tolerated, displayed linear pharmacokinetics that fit into a three-compartment pharmacokinetic model, and achieved sustained concentrations in plasma that were multiple times in excess of reported MICs for susceptible opportunistic fungi.
doi:10.1128/AAC.45.2.596-600.2001
PMCID: PMC90333  PMID: 11158761
9.  Dosage-Dependent Antifungal Efficacy of V-Echinocandin (LY303366) against Experimental Fluconazole-Resistant Oropharyngeal and Esophageal Candidiasis 
V-echinocandin (VER-002; LY303366) is a semisynthetic derivative of echinocandin B and a potent inhibitor of fungal (1, 3)-β-d-glucan synthase. We studied the antifungal efficacy, the concentrations in saliva and tissue, and the safety of VER-002 at escalating dosages against experimental oropharyngeal and esophageal candidiasis caused by fluconazole-resistant Candida albicans in immunocompromised rabbits. Study groups consisted of untreated controls, animals treated with VER-002 at 1, 2.5, and 5 mg/kg of body weight/day intravenously (i.v.), animals treated with fluconazole at 2 mg/kg/day i.v., or animals treated with amphotericin B at 0.3 mg/kg/day. VER-002-treated animals showed a significant dosage-dependent clearance of C. albicans from the tongue, oropharynx, esophagus, stomach, and duodenum in comparison to that for untreated controls. VER-002 also was superior to amphotericin B and fluconazole in clearing the organism from all sites studied. These in vivo findings are consistent with the results of in vitro time-kill assays, which demonstrated that VER-002 has concentration-dependent fungicidal activity. Esophageal tissue VER-002 concentrations were dosage proportional and exceeded the MIC at all dosages. Echinocandin concentrations in saliva were greater than or equal to the MICs at all dosages. There was no elevation of serum hepatic transaminase, alkaline phosphatase, bilirubin, potassium, or creatinine levels in VER-002-treated rabbits. In summary, the echinocandin VER-002 was well tolerated, penetrated the esophagus and salivary glands, and demonstrated dosage-dependent antifungal activity against fluconazole-resistant esophageal candidiasis in immunocompromised rabbits.
doi:10.1128/AAC.45.2.471-479.2001
PMCID: PMC90315  PMID: 11158743
11.  Dose-Dependent Pharmacokinetics of Amphotericin B Lipid Complex in Rabbits 
Amphotericin B lipid complex (ABLC) was recently approved by the Food and Drug Administration for treatment of patients with invasive fungal infections who are intolerant of or refractory to conventional amphotericin B therapy. Little is known, however, about the pharmacokinetics of this new antifungal compound. We therefore investigated the pharmacokinetics of ABLC in comparison with those of conventional desoxycholate amphotericin B (DAmB) in rabbits. The pharmacokinetics of DAmB in a rabbit model were similar to those previously reported in humans. The pharmacokinetics of ABLC differed substantially from those of DAmB. Plasma amphotericin B levels following ABLC administration were 10 times lower than those following administration of an equal dosage of DAmB. The levels of ABLC in whole blood were approximately 40 times greater than those in plasma. The ABLC model differed from the DAmB model by (i) a dose- and time-dependent uptake and return between the plasma compartment and apparent cellular components of the blood-sediment compartment and (ii) time-dependent tissue uptake and return to plasma from serially connected compartments. Following infusion of ABLC, there was a nonlinear uptake into the apparent cellular components of the blood-sediment compartment. This uptake was related to the reciprocal of the integral of the total amount of drug infused (i.e., the more drug infused the greater the fractional uptake between 0.5 and 5 mg/kg of body weight for ABLC). The transfer of drug from plasma to the cellular components of the blood-sediment compartment resulted in initial uptake followed by rapid redistribution back to the plasma. The study describes a detailed model of the pharmacokinetics of ABLC and characterizes a potential role of the cellular components of the blood-sediment compartment in the distribution of this new antifungal compound in tissue.
PMCID: PMC90015  PMID: 10898677
12.  Effect of Fluconazole on the Pharmacokinetics of Doxorubicin in Nonhuman Primates 
Antifungal prophylaxis in cancer patients who are undergoing chemotherapy is associated with prolonged neutropenia. We measured the effect of fluconazole on doxorubicin pharmacokinetics in nonhuman primates to determine if neutropenia is related to a pharmacokinetic interaction that delays the clearance of the chemotherapeutic agent. Fluconazole pretreatment had no effect on doxorubicin pharmacokinetics.
PMCID: PMC89824  PMID: 10722523
13.  Compartmental Pharmacokinetics and Tissue Distribution of Multilamellar Liposomal Nystatin in Rabbits 
The plasma pharmacokinetics of multilamellar liposomal nystatin were studied in normal, catheterized rabbits after single and multiple daily intravenous administration of dosages of 2, 4, and 6 mg/kg of body weight, and drug levels in tissues were assessed after multiple dosing. Concentrations of liposomal nystatin were measured as those of nystatin by a validated high-performance liquid chromatography method, and plasma concentration data were fitted into a two-compartment open model. Across the investigated dosage range, liposomal nystatin demonstrated nonlinear kinetics with more than proportional increases in the AUC0–24 and decreasing clearance, consistent with dose-dependent tissue distribution and/or a dose-dependent elimination process. After single-dose administration, the mean Cmax increased from 13.07 μg/ml at 2 mg/kg to 41.91 μg/ml at 6 mg/kg (P < 0.001); the AUC0–24 changed from 11.65 to 67.44 μg · h/ml (P < 0.001), the Vd changed from 0.205 to 0.184 liters/kg (not significant), the CLt from 0.173 to 0.101 liters/kg · h (P < 0.05), and terminal half-life from 0.96 to 1.51 h (P < 0.05). There were no significant changes in pharmacokinetic parameters after multiple dosing over 14 days. Assessment of tissue concentrations of nystatin near peak plasma levels after multiple dosing over 15 days revealed preferential distribution to the lungs, liver, and spleen at that time point. Substantial levels were also found in the urine, raising the possibility that renal excretion may play a significant role in drug elimination. Liposomal nystatin administered to rabbits was well tolerated and displayed nonlinear pharmacokinetics, potentially therapeutic peak plasma concentrations, and substantial penetration into tissues. Pharmacokinetic parameters were very similar to those observed in patients, thus validating results derived from infection models in the rabbit and allowing inferences to be made about the treatment of invasive fungal infections in humans.
PMCID: PMC89797  PMID: 10722496
14.  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.
PMCID: PMC89692  PMID: 10639371
16.  Safety and Efficacy of Multilamellar Liposomal Nystatin against Disseminated Candidiasis in Persistently Neutropenic Rabbits 
Antimicrobial Agents and Chemotherapy  1999;43(10):2463-2467.
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 103 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.
PMCID: PMC89501  PMID: 10508025
17.  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.
PMCID: PMC89441  PMID: 10471559
18.  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 103 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.
PMCID: PMC89438  PMID: 10471556
19.  Histatin 3-Mediated Killing of Candida albicans: Effect of Extracellular Salt Concentration on Binding and Internalization 
Human saliva contains histidine-rich proteins, histatins, which have antifungal activity in vitro. The mechanism by which histatins are able to kill Candida albicans may have clinical significance but is currently unknown. Using radiolabeled histatin 3, we show that the protein binds to C. albicans spheroplasts in a manner that is dependent on time and concentration. Binding to the spheroplasts was saturable and could be competed with unlabeled histatin 3. A single histatin 3 binding site with a Kd = 5.1 μM was detected. Histatin 3 binding resulted in potassium and magnesium efflux, predominantly within the first 30 min of incubation. Studies with fluorescent histatin 3 demonstrate that the protein is internalized by C. albicans and that translocation of histatin inside the cell is closely associated with cell death. Histatin binding, internalization, and cell death are accelerated in low-ionic-strength conditions. Indeed, a low extracellular salt concentration was essential for cell death to occur, even when histatin 3 was already bound to the cell. The interaction of histatin 3 with C. albicans, and subsequent cell death, is inhibited at low temperature. These results demonstrate that the candidacidal activity of histatin 3 is not due exclusively to binding at the cell surface but also involves subsequent interactions with the cell.
PMCID: PMC89457  PMID: 10471575
20.  Pharmacokinetics of Oral Zidovudine Entrapped in Biodegradable Nanospheres in Rabbits 
The pharmacokinetic profile of oral zidovudine entrapped in a 50:50 polyactide-coglycolide matrix (nanospheres) was compared to those of standard oral and parenteral zidovudine formulations in rabbits. The bioavailability of zidovudine nanospheres at 50 mg/kg of body weight was 76%, and this dose achieved prolonged exposure to zidovudine compared to standard formulations without an increase in the drug’s peak concentration.
PMCID: PMC89240  PMID: 10103214
22.  Antifungal Efficacy, Safety, and Single-Dose Pharmacokinetics of LY303366, a Novel Echinocandin B, in Experimental Pulmonary Aspergillosis in Persistently Neutropenic Rabbits 
Antimicrobial Agents and Chemotherapy  1998;42(11):2898-2905.
LY303366 is a novel semisynthetic derivative of echinocandin B and a potent inhibitor of fungal (1,3)-β-d-glucan synthase. The antifungal efficacy and safety of LY303366 were investigated in treatment and prophylaxis of primary pulmonary aspergillosis due to Aspergillus fumigatus in persistently neutropenic rabbits. Treatment study groups were either not treated (controls) or treated with amphotericin B (AmB) at 1 mg/kg of body weight per day or with LY303366 at 1, 5, 10, and 20 mg/kg/day. In rabbits treated with LY303366, there was a significant improvement in survival and a reduction in organism-mediated pulmonary injury measured by the number of infarcts, total lung weight, and ultrafast computerized tomography scan pulmonary lesion score. Rabbits receiving prophylactic LY303366 also demonstrated significant improvement in survival and reduction in organism-mediated pulmonary injury. AmB and LY303366 had comparable therapeutic efficacies by all parameters with the exception of reduction in tissue burden of A. fumigatus, where AmB was superior to LY303366. LY303366 demonstrated a dose-dependent effect on hyphal injury with progressive truncation, swelling, and vacuolization. LY303366 administered in single doses of 1, 5, 10, and 20 mg/kg demonstrated dose-proportional increases in the maximum concentration of drug in plasma and the area under the concentration-time curve from 0 to 72 h with no changes in plasma drug clearance. The 1-mg/kg dosage maintained plasma drug levels above the MIC for 18 h, and dosages of ≥5 mg/kg maintained plasma drug levels above the MIC for the entire 24-h dosing interval. There was no significant elevation of the concentrations of hepatic transaminases or creatinine in serum in LY303366-treated rabbits. In summary, LY303366 improved survival and decreased pulmonary injury with no apparent toxicity in the treatment and prevention of invasive pulmonary aspergillosis in persistently neutropenic rabbits.
PMCID: PMC105963  PMID: 9797223
23.  Compartmental Pharmacokinetics and Tissue Drug Distribution of the Pradimicin Derivative BMS 181184 in Rabbits 
Antimicrobial Agents and Chemotherapy  1998;42(10):2700-2705.
The pharmacokinetics of the antifungal pradimicin derivative BMS 181184 in plasma of normal, catheterized rabbits were characterized after single and multiple daily intravenous administrations of dosages of 10, 25, 50, or 150 mg/kg of body weight, and drug levels in tissues were assessed after multiple dosing. Concentrations of BMS 181184 were determined by a validated high-performance liquid chromatography method, and plasma data were modeled into a two-compartment open model. Across the investigated dosage range, BMS 181184 demonstrated nonlinear, dose-dependent kinetics with enhanced clearance, reciprocal shortening of elimination half-life, and an apparently expanding volume of distribution with increasing dosage. After single-dose administration, the mean peak plasma BMS 181184 concentration (Cmax) ranged from 120 μg/ml at 10 mg/kg to 648 μg/ml at 150 mg/kg; the area under the concentration-time curve from 0 to 24 h (AUC0–24) ranged from 726 to 2,130 μg · h/ml, the volume of distribution ranged from 0.397 to 0.799 liter/kg, and the terminal half-life ranged from 4.99 to 2.31 h, respectively (P < 0.005 to P < 0.001). No drug accumulation in plasma occurred after multiple daily dosing at 10, 25, or 50 mg/kg over 15 days, although mean elimination half-lives were slightly longer. Multiple daily dosing at 150 mg/kg was associated with enhanced total clearance and a significant decrease in AUC0–24 below the values obtained at 50 mg/kg (P < 0.01) and after single-dose administration of the same dosage (P < 0.05). Assessment of tissue BMS 181184 concentrations after multiple dosing over 16 days revealed substantial uptake in the lungs, liver, and spleen and, most notably, dose-dependent accumulation of the drug within the kidneys. These findings are indicative of dose- and time-dependent elimination of BMS 181184 from plasma and renal accumulation of the compound after multiple dosing.
PMCID: PMC105922  PMID: 9756780
24.  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.
PMCID: PMC105840  PMID: 9736570
25.  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.
PMCID: PMC105839  PMID: 9736569

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