a Immunocompromised Host Section, Pediatric Oncology Branch, National Cancer Institute b Department of Radiology, Warren Grant Magnuson Clinical Center c Surgery Service, Veterinary Resources Program, National Center for Research Resources d Pharmacokinetics Research Laboratory, Pharmacy Department, Warren Grant Magnuson Clinical Center, National Institutes of Health, Bethesda, MD 20892, USA
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Abstract |
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Introduction |
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Nystatin, a tetraene diene macrolide, was discovered as the first polyene antibiotic in the early 1950s. 7 The primary mechanism of action is identical to that of amphotericin B and, like amphotericin B, nystatin possesses potent, broad-spectrum fungicidal activity in vitro. 8 Although the compound has been widely used topically for both treatment and prevention of superficial fungal infections, problems with solubilization and toxicity after parenteral administration have limited its use for systemic treatment. 8 More recent studies, however, have shown that incorporation of nystatin into a stable multilamellar liposome preparation of 0.13 µm particle diameter consisting of dimyristoyl phosphatidylcholine (DMPC) and dimyristoyl phosphatidylglycerol (DMPG) in a 7:3 molar ratio significantly reduces its toxicity to mammalian cells while preserving its antifungal activity in vitro. 9 This liposomal formulation demonstrated promising in-vivo activity in murine models of both disseminated candidiasis 10 and disseminated aspergillosis, 11 and it was well tolerated at dosages of up to 8 mg/kg/day in a phase I clinical trial in patients with haematological malignancies and refractory febrile neutropenia. 12 Little is known, however, about the antifungal efficacy of liposomal nystatin in this patient population.
In order to define better the potential clinical usefulness of this novel formulation in neutropenic patients, we investigated the safety, pharmacokinetics and efficacy of liposomal nystatin in a persistently neutropenic rabbit model of invasive pulmonary aspergillosis.
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Materials and methods |
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Female New Zealand White rabbits (Hazleton, Denver, PA, USA) weighing 23 kg at the time of inoculation were used in all experiments. They were individually housed and maintained with water and standard rabbit feed ad libitum according to National Institutes of Health guidelines for laboratory animal care 13 and in fulfilment of American Association for Accreditation of Laboratory Animal Care criteria. For repeated non-traumatic administration of medications and sampling of blood, vascular access was established in each rabbit before experimentation by the surgical placement of a central venous silastic catheter. The catheter was inserted in the right external jugular vein, positioned into the right atrium and externalized via a subcutaneous tunnel at the interscapular region with a heparin lock device under general anaesthesia as previously described. 14
Immunosuppressive regimen and supportive care
Cytosine arabinoside (kindly provided by Upjohn, Kalamazoo, MI, USA) was administered intravenously at 525 mg/m2 on days 15 and at 484 mg/m2 on days 8 and 9 to produce profound and persistent neutropenia, such as is encountered in patients at highest risk for invasive fungal infections. Body weight W (in kg) was converted to body surface area A (in m 2) using the following formula: A = 0.05W + 0.05. 15Granulocyte counts were closely monitored and maintained below 0.5 x 10 9/L; they were <0.1x 10 9/L from day 5 onwards. Concomitant platelet counts ranged from 1x 10 10 to 2.5x 10 10 /L. Starting on day 4, ceftazidime (Glaxo, Research Triangle Park, NC, USA) 75 mg/kg iv twice daily, gentamicin (Baxter Health Care Corp., Deerfield, IL, USA) 5 mg/kg iv every other day, and vancomycin (Eli Lilly, Indianapolis, IN, USA) 15 mg/kg iv daily were administered to prevent the occurrence of invasive bacterial infections during neutropenia.
Organism and preparation of inoculum
On day 2 of the experiment, after the second dose of cytosine arabinoside, rabbits were inoculated intratracheally with a well characterized strain of Aspergillus fumigatus (NIH4215) obtained from a fatal case of pulmonary aspergillosis.
The inoculum was prepared from a frozen isolate that was subcultured onto potato dextrose slants, which were incubated for 24 h at 37°C and then kept at room temperature for 5 days. Conidia were harvested under a laminar air flow hood with a solution of 0.025% Tween 20 (Fisher Scientific, Fair Lawn, NJ, USA) in normal saline, transferred to a 50 mL conical tube and counted in a haemocytometer. The concentration was adjusted to give each rabbit a predetermined inoculum of 1 x 10 8 conidia of A. fumigatus in a volume of 200350 µL. The concentrations of the inoculum were confirmed retrospectively by serial dilution and culture on Sabouraud dextrose agar check plates.
Inoculation was performed under general intravenous anaesthesia with 0.51.0 mL of a 2:1 (v/v) mixture of ketamine 100 mg/mL (Fort Dodge Labs, Fort Dodge, IA, USA) and xylazine 20 mg/mL (Mobay Corp., Shawney, KA, USA) for analgesia, amnesia and muscle relaxation. Anaesthetic dosage was adjusted according to body weight to obtain similar depths of general anaesthesia. Once satisfactory anaesthesia had been reached, a Flagg 0 straight-blade laryngoscope (Welch-Allyn, Skaneateles Falls, NY, USA) was inserted and the inoculum was given intratracheally with a tuberculin syringe attached to a 13.3 cmTeflon catheter (Becton Dickinson, Sandy, UT, USA).
In-vitro antifungal susceptibility
The susceptibility of the experimental isolate to liposomal nystatin, free nystatin (Lederle Laboratories, Pearl River, NY, USA) and amphotericin B deoxycholate was tested by broth macrodilution and microdilution with an inoculum of 0.52.5 x 10 3 cfu/mL in RPMI 1640 medium as previously described. 16 With that method, the MICs of liposomal nystatin, free nystatin and amphotericin B deoxycholate were 2, 8 and 1 mg/L, respectively, and the minimum fungicidal concentrations (MFCs) were the same as the MICs.
Antifungal therapy
Liposomal nystatin, administered daily in a single dose, was studied at three different dosage levels: six rabbits received 1 mg/kg, ten received 2 mg/kg and 16 received 4 mg/kg. Ten animals treated with amphotericin B deoxycholate at 1 mg/kg daily in a single dose and ten untreated, but infected animals served as controls.
Liposomal nystatin (Nyotran; 50 mg vials; 50 mg nystatin USP incorporated into a mixture of 350 mg DMPC and 150 mg DMPG; Aronex Pharmaceuticals, The Woodlands, TX, USA) was provided as a lyophilized powder and maintained at 4°C. The drug was freshly reconstituted before use with 50 mL of sterile normal saline to a 1 mg/mL solution. Following addition of normal saline, vials were vigorously shaken for 1 min, placed in a 40°C water bath for 15 min, removed from the water bath and vigorously shaken for a further 60 s. The reconstituted drug was administered at ambient temperature as slow intravenous infusion at a rate of 2 mg/min. Amphotericin B deoxycholate (50 mg vials of Fungizone; Bristol Myers-Squibb, Princeton, NJ, USA) was reconstituted with 10 mL distilled water, maintained at 4°C and diluted 1:4 (v/v) with sterile 5% dextrose in water immediately before use. Amphotericin B preparations were sheltered from light and administered intravenously at a rate of 0.2 mg/min after normal saline loading (10 mL/kg body weight) to decrease nephrotoxicity.
Antifungal treatment was begun 24 h after endotracheal inoculation and was administered daily throughout the experiment. Surviving rabbits were killed on day 12 after inoculation.
Outcome variables
All experiments were evaluated according to the following outcome variables:
Survival analysis. Duration of survival, in days post- inoculation, was recorded for each rabbit. Surviving rabbits were killed by pentobarbital anaesthesia on day 12 after inoculation.
Morphological and microbiological post-mortem studies. The entire heartlung block was carefully dissected and removed at autopsy. The heart was then dissected away from the lungs, leaving an intact tracheobronchial tree and lung preparation. The lungs were weighed (Mettler Instrument, Hightstown, NJ, USA) and inspected by two observers blinded to the treatment group who recorded the number and type of lesions, if any, in each separate lobe.
Bronchoalveolar lavage (BAL) was performed on each lung preparation by the instillation and subsequent withdrawal of 10 mL of sterile normal saline three times into the clamped trachea. An aliquot of 0.1 mL of this fluid was cultured on Sabouraudglucose agar.
Thereafter, a representative region of each lobe was excised for cultures and histopathological examination. Each fragment reserved for culture was weighed individually, minced with sterile scissors and, depending on the weight, homogenized with 2 mL or 5 mL of sterile saline for 1 min per tissue sample in reinforced sterile polyethylene bags (Stomacher 80; Tekmar, Cincinnati, OH, USA). 17 Lung homogenates in 10 -2 and 10 -4 dilutions were prepared in sterile saline, and aliquots of 100 µL were plated on Sabouraudglucose agar and incubated at 37°C for the first 24 h and then at room temperature for another 24 h. After this, the cfu of A. fumigatus were counted and recorded for each lobe and the cfu/g were calculated. One colony of A. fumigatus was considered positive. In addition, the percentage of culture-positive lobes was calculated for each rabbit.
Specimens reserved for histopathological examination were sectioned and preserved in 10% neutral-buffered formalin. The fixed specimens were then embedded in paraffin, sectioned and stained with haematoxylineosin (HE), periodic acidSchiff (PAS) and Gomori methenamine silver stains (GMS) for microscopic examination. Haemorrhagic infarcts (dark-red, consolidated lesions) corresponded microscopically to coagulative necrosis and intra-alveolar haemorrhage.
Radiographic studies. Serial ultra-fast computerized tomography (UFCT) was performed inactively treated rabbitsin order to monitor the effects of antifungal treatment on the course of the infection during life. The radiographic course of untreated, but infected rabbits monitored by UFCT has been previously described and consists of a continuous progression of pulmonary lesions until death. 18
Computerized tomography (CT) was performed with a C-100XL ultrafast electron beam CT scanner (Imatron, Oyster Point, CA, USA). 18 Rabbits were transported between the laboratory animal facility and the UFCT scanner under general anaesthesia. An induction dose of 0.3 mL of a 2:1 mixture (v/v) of ketamine 100 mg/mL and xylazine 20 mg/mL was administered intravenously. Anaesthesia was maintained during the procedure by an additional dose of 0.2 mL of that mixture, if necessary. In virtually all cases, 30 slices were sufficient to scan the entire thorax of the rabbit, allowing a scanning time of <5 min per animal.
The radiographic course of experimental infection was followed on the day after inoculation and on days 3, 6 and 9 after inoculation, if feasible. At each session a mean pulmonary lesion score was established by evaluating each of the six lung lobes from each rabbit. Each lobe was evaluated independently using a score ranging from 0 to 3 with increments of 0.5, where 0 signified the absence of an infiltrate and 3 opacification of the entire lobe. Ultimately, the mean pulmonary lesion score for a given day and dosage level represents the mean of all lobes of all rabbits treated at that dosage level and scanned on that day. All CT scans were scored by the same observer, who was blinded to the identity of the study group of each rabbit.
Toxicity studies
Blood samples were collected from each rabbit on days 5, 7 and 9 after inoculation. Plasma samples were stored in Sarsted tubes (Sarsted Inc., Newton, NC, USA) at -80°C and were processed simultaneously. Levels of blood urea nitrogen (BUN), serum creatinine, potassium and hepatic transaminases were tested on the last sample drawn from each rabbit before death (Anilytics, Gaithersburg, MD, USA).
Pharmacokinetic studies in plasma
Three groups of four uninfected rabbits each were studied. Animals received liposomal
nystatin at
1, 2 and 4 mg/kg body weight once daily for a total of eight doses as a slow intravenous infusion
(2 mg/min). On days 1 and 8, plasma samples were drawn immediately before administration of
the compound and then at 10 min, 30 min and 1, 2, 4, 6, 8, 12 and 24 h after administration. An
accurate, sensitive, reproducible and specific reversed phase high-performance liquid
chromatographic (HPLC) method was used for quantification. The sample preparation involved
addition of 2:1 (v/v) methanol to standard, quality controls, or unknown rabbit plasma and
incubation at 4°C for 20 min. This was followed by two centrifugation steps (2000g for 10
min and 10,000g for 4 min, respectively), and a final centrifugation step before
injection at
4000g
for 4 min using 0.2 µm centrifugal filters (Ultra-Free MC, Millipore, Bedford, MA, USA).
The mobile phase consisted of 10 mM sodium phosphate, 1 mM EDTA, 30% methanol and 30%
acetonitrile (Fisher Scientific, Fair Lawn, NJ, USA) delivered at 2 mL/min. During analysis,
samples were maintained at 4°C; the injection volume was 200 µL. Nystatin was
detected by UV absorbance at 305 nm using a C18 analytical column
maintained at 30°C (µBondapak C18, Waters Corp.,
Milford, MA, USA) in conjunction with an in-line precolumn filter (NewGuard RP-18, Perkin
Elmer, Norwalk, CT, USA). Quantification was based on the peak area of the first main isomer,
which eluted at 7.58.5 min, and the peak areaconcentration response of the
external calibration standard. Ten-point standard curves were linear from 0 to 25 mg/L with R2 values > 0.99. The lower limit of quantification was 0.1
mg/L. Using current guidelines for validation of analytical methods,
19
accuracies were
within ±12% and intra- and interday variability (precision) was 7.5. The stability of
extracted plasma samples maintained in the autosampler over 10 h at 4°C ranged from 90%
to 94%, and accuracies of spiked plasma samples after storage for 3 months at
-80°C were within ±9%.
Standard model-independent techniques were used to calculate the area under the plasma concentrationtime curve from 0 to 24 h (AUC024), half-life (t1/2), plasma clearance (Cl) and volume of distribution (Vd).20 Levels in plasma obtained at 10 min were used as maximum concentrations (Cmax).
Statistical analysis
Survival curves were estimated by the KaplanMeier product limit method and
differences
between groups were analysed by the MantelHaenszel
2 test. Comparisons between proportions were done by the
2 or Fisher's exact test, and group-to-group
comparisons of continuous variables were analysed by the KruskalWallis ANOVA test
with Dunn's correction for multiple comparisons or the MannWhitney U-test, as appropriate. Values are stated as means ± S.E.M. Except for
survival analysis, all P values were two-sided. A P value of
0.05 was
considered statistically significant.
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Results |
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Survival among the five study groups was analysed over 12 days using KaplanMeier analysis (Figure 1). Only one of ten untreated control rabbits survived beyond day 7 after inoculation. In contrast, four of ten rabbits (40%) treated with amphotericin B at 1 mg/kg/day survived for 12 days (P = 0.05 vs controls). Similarly, survival in rabbits treated with either 2 or 4 mg/kg/day of liposomal nystatin was greater than that of untreated controls (P < 0.05 for both dosage levels) with seven (70%) of ten and six (37.5%) of 16 rabbits surviving for 12 days after inoculation. In contrast, survival in animals treated with liposomal nystatin at 1 mg/kg/day (one of six at day 12) did not differ from that of untreated controls. There were no statistically significant differences in survival between rabbits treated with liposomal nystatin at 2 and 4 mg/kg/day or amphotericin B, or among the three dosage regimens of liposomal nystatin.
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As depicted in Figure 2a, rabbits treated with either amphotericin B (P < 0.001) or liposomal nystatin at 2 mg/kg (P < 0.05) and 4 mg/kg (P < 0.01) had significantly fewer haemorrhagic infarcts of lung tissue than untreated controls. No statistically significant differences in the mean number of haemorrhagic infarcts were observed between animals treated with liposomal nystatin at 2 or 4 mg/kg and amphotericin B-treated animals. Rabbits receiving liposomal nystatin at 1 mg/kg, however, did not have fewer haemorrhagic infarcts than untreated controls and had significantly more infarcts than amphotericin B-treated animals.
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Compared with untreated controls, rabbits treated with either daily amphotericin B (P < 0.001) or liposomal nystatin at 1 (P < 0.05), 2 (P < 0.01) and 4 mg/kg/day (P < 0.001) had a significant reduction in mean log(cfu/g/ lobe) (Figure 2c). Similarly, the percentage of culture- positive lobes was 76.6% in untreated controls, 6.6% in rabbits treated with amphotericin B (P < 0.001) and 47.2% (P < 0.01), 45.0% (P < 0.001) and 31.2% (P < 0.001) in rabbits treated at 1, 2 and 4 mg/kg/day of liposomal nystatin, respectively. Nevertheless, treatment with amphotericin B at the standard dose of 1 mg/kg/day was significantly more effective in reducing fungal burden than either of the investigated regimens of liposomal nystatin (P < 0.01, P < 0.001 and P < 0.05 vs the 1, 2 and 4 mg/kg/day of liposomal nystatin, respectively), and was associated with a significantly lower percentage of infected lobes (P < 0.001 for each of the investigated dosage levels of liposomal nystatin).
Cultures from BAL fluid obtained at post-mortem yielded A. fumigatus in 70% of untreated control animals vs 10% in those treated with amphotericin B (P < 0.05). In comparison with untreated controls, a significant reduction in positive BAL cultures was noted only in rabbits treated with 2 mg/kg/day (to 20%) and 4 mg/kg/day (to 12.5%) of liposomal nystatin (P < 0.05 and P < 0.01, respectively; Fisher's exact test). No differences were observed between nystatin-treated and amphotericin B-treated rabbits, or among the different dosage regimens of liposomal nystatin.
Radiographic studies
Serial monitoring of pulmonary findings by UFCT in rabbits treated with amphotericin B or liposomal nystatin at 2 and 4 mg/kg, respectively, revealed marked improvement of pulmonary infiltrates in the course of the experiment (Figure 3). In contrast, there was no evidence for resolution of pulmonary lesions in rabbits treated with liposomal nystatin at 1 mg/kg; indeed, the mean pulmonary lesion score on day 9 in that group was significantly greater than that in amphot any deaths related to procedure or anaesthesia. ericin B-treated rabbits (P < 0.05). UFCT scanning was well tolerated without
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Rabbits treated with either amphotericin B (with prior saline-loading) or liposomal nystatin at 2 and 4 mg/kg (without saline-loading) had significantly greater mean serum creatinine and BUN levels when values determined from the last sample drawn in each individual rabbit were compared with untreated controls (Table). However, there were no statistically significant differences between amphotericin B-treated animals and animals treated with liposomal nystatin at 2 and 4 mg/kg. No differences were observed between liposomal nystatin-treated rabbits, untreated controls and rabbits treated with amphotericin B with regard to serum potassium and hepatic transaminase levels.
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The plasma concentrationtime curves of liposomal nystatin after od administration to non-infected, normal rabbits over 8 days and the pharmacokinetic parameters derived from these curves are shown in Figure 4. Over the investigated dose-range, liposomal nystatin demonstrated linear disposition, with rapid initial distribution and a relatively short elimination half-life. At all dose levels, near-peak levels were at least two-fold greater than the MIC for the isolate used in the efficacy studies.
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Discussion |
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Preliminary, noncompartmental pharmacokinetic studies in uninfected animals established linear disposition of liposomal nystatin in plasma over the observed dose range after multiple dosing over 8 days. As previously described for multilamellar liposomal preparations, 21 the compound reached relatively high peak levels and was then rapidly distributed and eliminated from plasma with a relatively short half-life. These findings are consistent with pharmacokinetic data obtained from HIV-infected patients, who received the drug at dosages ranging from 0.25 to 7 mg/kg. 22 ,23 Taken together, both experimental and clinical data indicate a pharmacokinetic profile of liposomal nystatin which is markedly different from that of amphotericin B deoxycholate and the three currently available lipid formulations of amphotericin B. 6 Notably, to the best of our knowledge, no data have ever been published on the plasma pharmacokinetics of free nystatin after intravenous administration.
Peak plasma levels were above both the MIC and MFC for the aspergillusstrain used in the infection model, and above the MICs recently reported for various clinical fungal isolates. 24 ,25 Due to the linearity of pharmacokinetics and antifungal effects, no general conclusions can be drawn regarding whether maximum plasma concentrations or the amount of drug circulating in plasma over time are more important to the pharmacodynamics of liposomal nystatin. For a primarily localized infection, such as invasive pulmonary aspergillosis, sufficient tissue levels are certainly major determinants of efficacy. 26 ,27 Indeed, unpublished studies in normal rabbits performed in our laboratories revealed lung tissue levels 10- to 15-fold above the MIC for our test strain shortly after the last of multiple doses of 2 and 4 mg/kg/day, respectively. However, the ultimate distribution of free drug into infected tissue and into the fungal cell membrane is unclear.
The finding that the composite outcome at 1 mg/kg of liposomal nystatin was poorer than that in both higher dosage groups, despite similar residual fungal burden at post-mortem, might be explained by slower accumulation of free compound in lung tissue to critical levels. That amphotericin B-treated rabbits had relatively poor survival despite exhibiting the highest antifungal efficacy among all treatment groups may be explained by the compound's nephrotoxicity, and possibly, drug-associated anaemia. It may be somewhat surprising that rabbits treated with liposomal nystatin at 2 mg/kg had a trend towards improved survival over amphotericin B-treated animals despite being significantly less effective in clearing infected lung tissue from the organism. Nevertheless, there may be subtle differences in renal toxicity as indicated by lower mean BUN and higher mean potassium levels in the nystatin-treated animals; similarly, although this has only been demonstrated in vitro, 9 ,28 liposomal nystatin may also be less haemolytic than amphotericin B in vivo.These factors collectively may confer a survival benefit in the state of severe disease.
Liposomal nystatin was more active in vitro than its free counterpart, but less active than conventional amphotericin B against the A. fumigatus isolate used in our experiments. These observations are in accordance with the overall trend of two larger in-vitro studies on the comparative activity of liposomal nystatin against various clinical isolates. 24 ,25 The improved in-vitro activity of the multilamellar liposomal formulation of nystatin over free drug might indicate enhanced delivery to the fungal cell membrane. 29 Of note, the small unilamellar liposomal formulation of amphotericin B was overall considerably less active in vitro than conventional amphotericin B (and liposomal nystatin) in one study, 25 although it was equally active in another. 30 These still very limited findings indicate the complexity of in-vitro susceptibility testing of lipid polyene formulations, for which carrier effects 29 and pharmacokinetic properties 6 are thought to be the major determinants of antifungal efficacy in vivo. They also underscore the need for evaluating these novel formulations in well designed and clinically relevant animal models.
Thus far, the antifungal efficacy of liposomal nystatin has been evaluated in two survival models. In non-neutropenic mice with disseminated candidiasis and in transiently neutropenic mice intravenously inoculated with A. fumigatus, respectively, treatment with liposomal nystatin at dosages ranging from 2 to 16 mg/kg/day conferred a significant increase in survival when compared with untreated control animals. 10 ,11 While the latter study demonstrated in-vivo efficacy of the compound against Aspergillus spp. by the endpoint of survival, our model was designed to mimic the clinical situation of patients at highest risk for invasive aspergillosis, i.e. persistent and profound neutropenia and respiratory route of infection. Beyond survival analysis, it provides important data on direct effects of treatment such as organism-mediated tissue injury, residual fungal burden and laboratory toxicity in the situation of severe illness. Furthermore, by incorporating UFCT and BAL, it allows for the correlation of tissue data with these clinically most relevant non-invasive diagnostic studies. Indeed, the model has been highly predictive for the clinical activity of other lipid polyene formulations, such as liposomal amphotericin B 31 and amphotericin B colloidal dispersion. 32
Although treatment with liposomal nystatin was clearly associated with laboratory signs of renal toxicity in our study, this toxicity was mild and apparently not dose- proportional and there were no significant differences in comparison with rabbits receiving amphotericin B with prior normal saline loading. In unpublished toxicity studies performed by the manufacturer in dogs, hydration had a beneficial effect on kidney function following administration of liposomal nystatin (T. Wallace, personal communication). It is thus possible that administration of normal saline to liposomal nystatin-treated rabbits would have reduced renal toxicity and, if death was in part related to renal impairment, might have impacted on survival. Independent of these considerations, liposomal nystatin was tolerated without reaching the maximum tolerated dose (MTD) at dosages of up to 8 mg/kg and day in a phase I study in 32 patients with haematological malignancies and refractory febrile neutropenia who received the drug for a median of 8 days (range, 275 days); elevations of serum creatinine levels occured in ten patients (31%) altogether, but never exceeded Grade II toxicity. 12
In conclusion, this study demonstrates that liposomal nystatin at dosages of 2 and 4 mg/kg/day increases survival and provides reduced tissue injury, effective microbiological clearance and tolerable nephrotoxicity in a model of invasive pulmonary aspergillosis in persistently granulocytopenic rabbits. Our findings add to the understanding of both pharmacokinetics and pharmacodynamics of liposomal nystatin as it is being developed for treatment of patients with proven or suspected invasive fungal infections.
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Acknowledgments |
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Notes |
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References |
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Received 23 March 1998; returned 20 July 1998; revised 12 August 1998; accepted 24 August 1998