Quantitative determination of nystatin in human plasma using LC-MS after inhalative administration in healthy subjects.

The antifungal polyene antibiotics nystatin was tested in a clinical trial to describe pharmacokinetics and safety after repeated administration of Nystatin "Lederle" sterile powder in healthy volunteers. To monitor the nystatin concentration-time profile in plasma we developed a sensitive method in the range of 1-100ng/ml based on liquid chromatography coupled with tandem mass spectrometry. The target substance was separated from the biological matrix on C(18) solid-phase extraction cartridges with methanol. The Chromatography was performed isocratically using a reversed phase Caltrex Resorcinearene column. The mobile phase consisted of 5mM ammonium formate buffer and acetonitrile (40:60, v/v). The mass spectrometer works with electrospray ionization in its positive selected ion monitoring (SIM) mode using the respective MH(+) ions, m/z 926.6 for nystatin and m/z 924.4 for amphotericin B as internal standard. The method validation was performed according to the demands and international criteria for validation of bioanalytical methods and was successfully applied to the quantification of nystatin in human plasma in the pharmacokinetic trial.

Liposomal nystatin against experimental pulmonary aspergillosis in persistently neutropenic rabbits: efficacy, safety and non-compartmental pharmacokinetics.

The activity of liposomal nystatin against invasive pulmonary aspergillosis was investigated in persistently neutropenic rabbits. Treatment groups included liposomal nystatin at dosages of 1, 2 and 4 mg/kg/day intravenously, or amphotericin B deoxycholate 1 mg/kg/day administered intravenously after normal saline loading. As compared with untreated controls, liposomal nystatin administered at 2 and 4 mg/kg/day prolonged survival and reduced fungus-mediated tissue injury and excess lung weight at post-mortem in a similar manner to amphotericin B. Although amphotericin B was superior in clearing infected lung tissue, treatment with all regimens of liposomal nystatin led to a significant reduction in pulmonary fungal tissue burden. During treatment, ultrafast CT-scan demonstrated ongoing resolution of pulmonary lesions at 2 and 4 mg/kg/day, but not at 1 mg/kg/day. With the exception of mild increases in blood urea nitrogen (BUN) and serum creatinine values during treatment at 2 and 4 mg/kg/day, which were similar to those found in amphotericin B-treated rabbits, liposomal nystatin was well tolerated. Preliminary pharmacokinetic studies in non-infected animals established linear drug disposition of liposomal nystatin in plasma over the investigated dosage range and peak plasma levels above the MIC for the test strain after multiple daily dosing for 7 days. Liposomal nystatin increased survival and provided reduced tissue injury, effective microbiological clearance and tolerable side effects in experimental pulmonary aspergillosis in persistently neutropenic rabbits, thus providing a rational basis for further investigations in clinical trials.

Species differences in the proportion of plasma lipoprotein lipid carried by high-density lipoproteins influence the distribution of free and liposomal nystatin in human, dog, and rat plasma.

The objective of this study was an interspecies comparison of free nystatin (NYS) and liposomal NYS (Nyotran) distribution in plasma. NYS and liposomal NYS at concentrations of 5, 10, and 20 microg of NYS/ml were incubated in human, dog, and rat plasma for 5, 60, and 180 min at 37 degrees C. Following these incubations, plasma samples were separated into their high-density lipoprotein (HDL), triglyceride-rich lipoprotein, low-density lipoprotein, and lipoprotein-deficient plasma (LPDP) fractions by density-gradient ultracentrifugation, and each fraction was assayed for NYS by high-pressure liquid chromatography. Total plasma and lipoprotein cholesterol, triglyceride, and protein concentrations in each human, dog, or rat plasma sample were determined by enzymatic assays. When NYS and liposomal NYS were incubated in human, dog, or rat plasma, the majority of the NYS was recovered in the LPDP fraction. For the 5- and 60-min incubation times for all plasmas measured, a significantly greater percentage of NYS was recovered in the lipoprotein fraction (primarily HDL) following the incubation of liposomal NYS than following the incubation of NYS. There was a significant correlation between the lipoprotein lipid and protein profiles in human, dog, and rat plasmas and the distribution of NYS and liposomal NYS in plasma. In particular, differences in the proportion of plasma lipoprotein cholesterol, triglyceride, and apolar lipids (cholesteryl ester and triglycerides) carried by HDL influenced the distribution of NYS and liposomal NYS within plasmas of different species. These findings suggest that the distribution of NYS among plasma lipoproteins of different species is defined by the proportion of lipid carried by HDL, and this is possibly an important consideration when evaluating the pharmacokinetics, toxicities, and activities of these compounds following administration to different animal species.

High-performance liquid chromatographic determination of liposomal nystatin in plasma and tissues for pharmacokinetic and tissue distribution studies.

A reliable reversed-phase high-performance liquid chromatographic method was developed for the determination of liposomal nystatin in plasma. Nystatin is extracted by 1:2 (v/v) liquid-liquid extraction with methanol. Separation is achieved by HPLC after direct injection on a muBondapak C18 analytical column with a mobile phase composed of 10 mM sodium phosphate, 1 mM EDTA, 30% methanol and 30% acetonitrile adjusted to pH 6. Detection is by ultraviolet absorbance at 305 nm. Quantitation is based on the sum of the peak area concentration of the two major isomers of nystatin, which elute at 7.5-8.5 and 9.5-10.5 min. The assay was linear over the concentration range of 0.05 to 50 microg/ml. The lower limit of quantitation was 0.05 microg/ml, sufficient for investigating the plasma pharmacokinetics of liposomal nystatin in preclinical studies. Accuracies and intra- and inter-day precision showed good reproducibility. With minor modifications, this method also was used for assaying nystatin in various non-plasma body fluids and tissues.

Physical characteristics and lipoprotein distribution of liposomal nystatin in human plasma.

The physical characteristics and lipoprotein distribution of free nystatin (NYS) and liposomal NYS (L-NYS) in human plasma were investigated. To determine the percentage of NYS that was lipid associated following incubation in human plasma, C18 reverse-phase extraction columns were used. To assess plasma drug distribution, NYS and L-NYS (20 microg/ml) were incubated in human plasma for 5, 60, and 120 min at 37 degrees C. After each interval, plasma was removed and separated into its lipoprotein and lipoprotein-deficient plasma (LPDP) fractions by ultracentrifugation and assayed for NYS by high-pressure liquid chromatography. Further studies evaluated the liposome structure of L-NYS by filtering through a 0.14-microm-pore-size microfilter before and after the addition of human plasma. When reconstituted L-NYS (mean particle diameter +/- standard deviation, 321 +/- 192 nm) was applied to a C18 column, 67% +/- 4% of the initial NYS concentration was associated with the lipid. When plasma samples containing L-NYS that had been incubated for 5 to 120 min at 37 degrees C were applied to C18 columns, 66 to 76% of the NYS was lipid associated. Incubation of NYS in human plasma for 5 min at 37 degrees C resulted in 3% +/- 1% of the initial NYS concentration incubated in the low-density lipoprotein (LDL) fraction, 23% +/- 4% of that in the high-density lipoprotein (HDL) fraction, and 66% +/- 10% of that in the LPDP fraction. In contrast, the distribution of NYS following incubation of L-NYS in human plasma for 5 min was 13% +/- 2% in the LDL fraction, 44% +/- 5% in the HDL fraction, and 42% +/- 5% in the LPDP fraction. Similar results were observed following 60 and 120 min of incubation. In addition, the liposome structure of L-NYS was quickly lost when mixed with plasma. These findings suggest that rapid disruption of the L-NYS structure upon incubation in human plasma is consistent with its rapid distribution in plasma. The preferential distribution of NYS into the HDL fraction upon incubation of L-NYS may be a function of its phospholipid composition.