A New High-performance Liquid Chromatographic Method for Fludarabine and Fludarabine Phosphate Compounded in Liposomes.

A simple and reproducible high-pressure liquid chromatography (HPLC) method was developed to measure simultaneously the concentrations of both fludarabine and liposome-compounded fludarabine in plasma. In this method, hypoxanthine 9-B-D arabinofuranoside was used as an internal standard. Fludarabine, fludarabine phosphate, and hypoxanthine 9-B-D arabinofuranoside were extracted from plasma and were separated by means of isocratic elution from a C18 reversed-phase column. The mobile phase consisted of 5% (v/v) methanol in 10mM ammonium phosphate solution. The pH of the mobile phase was adjusted to 2.2 with 85% phosphoric acid. The detection wavelength was 260 nm, and the retention times for fludarabine, fludarabine phosphate, and hypoxanthine 9-B-D arabinofuranoside were 48, 27, and 12 minutes, respectively. The recovery efficiencies varied depending on the amount spiked; however, they were 89% and 58% for 10 micrograms/mL of fludarabine and fludarabine phosphate, respectively. The limits of quantification for fludarabine and fludarabine phosphate were 0.03 micrograms/mL and 0.5 micrograms/mL, respectively. The assay was reproducible, and the within-day coefficients of variation (n=4) were less than 6.9% and less than 8.7% for fludarabine and fludarabine phosphate, respectively. The between day variabilities (n=4) were less than 6.3% and less then 6.4% for fludarabine and fludarabine phophate, respecitvley. The assays were linear within the range of 0.025 to 10 micrograms/mL (r squared = 0.999) for fludarabine and 0.5 to 10 micrograms/mL (r squared = 0.999) for fludarabine phosphate.

Liposomes, a potential immunoadjuvant and carrier for a cryptococcal vaccine.

Mice immunized with a cryptococcal culture filtrate antigen (CneF) emulsified in complete Freund's adjuvant (CFA) develop an anticryptococcal cell-mediated immune response (CMI). CMI is detected by delayed-type hypersensitivity (DTH) reactions and by enhanced clearance of Cryptococcus neoformans from infected tissues. The objective of this research was to evaluate anticryptococcal DTH reactivity and clearance of cryptococci from groups of mice immunized with CneF encapsulated into liposomes (CneF-liposome) and compare the results to results from mice immunized with CneF-CFA. CBA/J mice were injected subcutaneously with vaccines or control formulations (saline-liposome or saline-CFA). Six days later the mice were footpad tested to assess their DTH response to CneF or the animals were challenged intravenously with 10(5) viable C. neoformans to determine clearance of infection. Clearance was evaluated 7 days later by enumeration of cryptococcal colony forming units (CFU) in lungs, spleens, livers, and brains of the infected mice. The CneF-liposome formulation induced a positive anticryptococcal DTH response and elicited increased clearance of C. neoformans from tissues as compared to mice treated with saline-liposome. Even though the CneF-liposome preparation did not induce as strong of a DTH response or as much protection as did CneF-CFA, our results indicate that liposomes are promising carriers for immunization with cryptococcal antigen and that such immunization can provide some protection to subsequent infection with C. neoformans.

Disposition of aerosolized liposomal amphotericin B.

Amphotericin B (AmB) is an important drug for the treatment of fungal infection, but toxicity limits the lung tissue doses which may be achieved through intravenous administration. Although incorporation of AmB in liposomes reduces these effects and increases the therapeutic index for intravenous administration, targeted delivery to lung tissues via inhaled liposomal AmB aerosol may be a more effective approach. Aerosolization of liposomal amphotericin B targets the lungs, the organs first infested by many fungi. Development of optimal aerosolized liposomal AmB therapies requires a better understanding of the effect that liposome surface charge has on lung clearance kinetics. In this work we evaluated the clearance kinetics and organ distribution of inhaled liposomal AmB in male Balb/C mice. Mice were exposed via nose only to AmB-containing liposomal aerosols having positive, negative, or neutral surface charge characteristics. The formulations were aerosolized using a Collison nebulizer. Groups of animals were euthanized at predetermined times and the lungs and other organs were analyzed for AmB. AmB was not detected in serum and other organs such as kidneys, liver, and brain. The disposition of neutral and positive liposomal amphotericin B in lungs followed biexponential kinetics. The alpha and beta phase half-lives for positive liposomes were 1.3 and 15.1 days, respectively, and 2.3 and 22 days for neutral liposomes. AmB delivered via negative liposomes exhibited monoexponential clearance with a half-life of 4.5 days. These results suggest that toxic side effects in nontarget tissues are minimal and may indicate a potential for long term protection against fungal infections.

New high-performance liquid chromatographic method for amphotericin B analysis using an internal standard.

A simple and reproducible HPLC method for the analysis of amphotericin B (AmB) in serum, lung and liver using natamycin as the internal standard was developed. AmB and natamycin were extracted from serum, lung and liver and were separated using an isocratic elution from C18 reversed-phase column. The mobile phase consisted of acetonitrile-10 mM acetate buffer pH 4.0 (37:63, v/v). The HPLC system had two detectors in series. One was set at 303 nm and the other at 383 nm for the detection of natamycin and AmB, respectively. The retention times of AmB and natamycin were 15 and 6 min, respectively. The recovery efficiency was 96%-70%. The limit of quantification was 0.1 microgram/ml. The assay was reproducible, the within-day coefficient of variation (n = 6) was < 8% for serum, lungs and liver. The between-day variability (n = 6) was < 7.7% for serum, liver and lungs at 1 microgram/ml or 1 microgram/g tissue concentration. The assay was linear within the range 1-40 micrograms/ml (r2 = 0.99).