Cyclosporine A transfer between high- and low-density lipoproteins: independent from lipid transfer protein I-facilitated transfer of lipoprotein-coated phospholipids because of high affinity of cyclosporine a for the protein component of lipoproteins.

The objectives of this study were to determine if lipid transfer protein I (LTP I)-facilitated phospholipid (PC) transfer activity regulates the plasma lipoprotein distribution of cyclosporine (CSA) and if the association of CSA with high-density lipoproteins (HDL) is due to the high protein and/or alterations in coat lipid content of HDL. To assess if LTP I-facilitated PC transfer activity regulates the plasma lipoprotein distribution of CSA, (14)C-PC- or (3)H-CSA-enriched HDL or low-density lipoproteins (LDL) were incubated in T150 buffer [pH 7.4, containing a (14)C-PC- or (3)H-CSA-free lipoprotein counterpart +/- exogenous LTP I (1.0 microg protein/mL)] or in delipidated human plasma that contained 1.0 microg protein/mL of endogenous LTP I in the presence or absence of a monoclonal antibody TP1 (30 microg protein/mL) directed against LTP I for 90 min at 37 degrees C. To assess the influence of HDL subfraction lipid composition and structure on the plasma distribution of CSA, CSA at 1000 ng of drug/mL of plasma was incubated in human plasma pretreated for 24 h with a lecithin:cholesterol acyltransferase (LCAT) inhibitor, dithionitrobenzoate (DTNB; 3 mM). To assess the binding of CSA to apolipoproteins AI, AII, and B, increasing concentrations of CSA were added to a constant concentration of either apolipoprotein AI, AII, or B. Equilibrium dialysis was used to determine free and bound fractions and Scatchard plot analysis was used to determine binding coefficients. To assess the influence of hydrophobic core lipid volume on the plasma distribution of CSA, CSA was incubated in plasma from patients with well-characterized dyslipidemias. The hydrophobic core lipid volume (CE + TG) within each lipoprotein subfraction was correlated to the amount of CSA recovered in each plasma sample from the different human subjects. The percent transfer of PC from LDL to HDL was different than the percent transfer of CSA in T150 buffer or human plasma source. In the presence of TP1, only PC transfer from LDL to HDL decreased. For plasma incubated with CSA and separated into HDL(2) and HDL(3), 35-50% of drug originally incubated was recovered in the HDL(3) fraction, with the remaining drug being found within the other fractions. When CSA was incubated in plasma pretreated with DTNB, the percentage of CSA recovered in the HDL(3) and HDL(2) fractions was not significantly different compared with that in the HDL(3) and HDL(2) fractions from untreated control plasma. CSA distribution into HDL inversely correlated with the hydrophobic core lipid volume of HDL, whereas distribution into LDL and triglyceride-rich lipoproteins directly correlated with their respective hydrophobic core lipid volumes. We further observed that CSA has high binding affinity and multiple binding sites with apolipoproteins AI (k(d) = 188.9 nM; n = 2), AII (k(d) = 184.7 nM; n = 2), and B (k(d) = 191 nM; n = 3). These findings suggest that the transfer of CSA between different lipoprotein particles is not influenced by LTP I-facilitated PC transfer activity probably because of the high affinity of CSA for the protein components of HDL and LDL.

The effect of serum albumin on amphotericin B aggregate structure and activity.

PURPOSE: Mild heat treatment of Fungizone (FZ, an amphotericin B:deoxycholate preparation) leads to a new self-associated form (HFZ) that demonstrates improved therapeutic index in vivo. The origin of the improvement may lie in the differential stability in the presence of serum proteins. The purpose of this study is to assess the effect of human serum albumin (HSA) on the structure and stability and in vitro channel forming ability of these two preparations against model fungal and mammalian membrane vesicles. METHODS: Kinetic absorption and CD spectroscopy were used to assess the kinetic and equilibrium stability of the characteristic amphotericin B complexes in the presence of HSA. Kinetic fluorescence spectroscopy of pyranine entrapped in model fungal and mammalian membrane vesicles was used to measure the cation-selective channel forming ability of HZ and HFZ delivered from HSA. RESULTS: It is shown that FZ is rapidly converted from its aggregated form to a protein-bound monomer in the presence of HSA, whereas HFZ demonstrates greater stability by persisting as a stable inactive aggregate. Fluorescence measurements of ion currents show that HSA attenuates the membrane-activity of both preparations. However, the activity of both HFZ and FZ remains significant against ergosterol-containing membranes. This is the first direct measurement of the intrinsic channel forming abilities of these amphotericin B preparations in the presence of serum proteins. CONCLUSION: These data provide a mechanistic rationale for the similar efficacy and lower toxicity of HFZ.

Heat treatment of amphotericin b modifies its serum pharmacokinetics, tissue distribution, and renal toxicity following administration of a single intravenous dose to rabbits.

The purpose of this investigation was to determine the serum pharmacokinetics, tissue distribution, and renal toxicity of amphotericin B (AmpB) following administration of a single intravenous dose (1 mg/kg of body weight) of Fungizone (FZ) and a heat-treated form of FZ (HFZ) to New Zealand White female rabbits. FZ solutions were heated at 70 degrees C for 20 min to produce HFZ. Blood samples were obtained before drug administration and serially thereafter. After collection of the 48-h blood sample, each rabbit was humanely sacrificed and the right kidney, spleen, lungs, liver, and heart were harvested for AmpB analysis. Serum creatinine levels were measured before and 10 h after drug administration. AmpB concentrations in the serum and tissues were analyzed using high-performance liquid chromatography. FZ administration to rabbits resulted in a greater-than-50% increase in serum creatinine concentrations compared to baseline. However, HFZ administration resulted in no difference in serum creatinine concentrations compared to baseline. The AmpB area under the concentration-time curve (AUC) after HFZ administration was significantly lower than the AmpB AUC in rabbits administered FZ. However, AmpB systemic total body clearance was significantly greater in rabbits administered HFZ than in rabbits administered FZ without any differences in volume of distribution at steady state. Kidney tissue AmpB concentrations, although not significantly different, were greater in rabbits administered FZ than in rabbits administered HFZ. Likewise, lung and spleen AmpB concentrations, although not significantly different, were greater in rabbits administered FZ than in rabbits administered HFZ. However, liver AmpB concentrations were significantly lower in rabbits administered FZ than in rabbits administered HFZ. No significant differences in heart AmpB concentration between rabbits administered FZ and those given HFZ were found. These findings suggest that the pharmacokinetics, tissue distribution, and renal toxicity of AmpB are modified following administration of HFZ. HFZ could be an improved low-cost AmpB drug delivery system that has a potentially higher therapeutic index than FZ.

Heat-induced superaggregation of amphotericin B modifies its interaction with serum proteins and lipoproteins and stimulation of TNF-alpha.

The purpose of the present study was to examine the influence of heat-induced superaggregation of Amphotericin B (AmB) in the Fungizone (FZ) formulation on its interaction with human serum components and relate this to reduced toxicity. Whole serum distribution studies showed that a significantly lower percentage of AmB from HFZ was recovered in the high-density lipoprotein (HDL), low-density lipoprotein (LDL), and triglyceride-rich lipoprotein (TRL) fractions and a greater percentage recovered in the lipoprotein-deficient plasma (LPDP), though the majority of both preparations were recovered in LPDP. Circular dichroism (CD) and difference absorption spectroscopy were used to determine the stability of FZ and heat-treated FZ (HFZ) in the presence of HDL, LDL, serum, and albumin. The CD studies indicate that the "core" aggregate of HFZ is more stable in the presence of HDL and LDL, whereas the FZ is less stable and more dynamic with the core aggregate dissociating to a greater extent in the presence of either purified lipoprotein. Absorption studies with whole serum and purified albumin suggest that FZ aggregates are far less stable in the presence of albumin than HFZ and that interaction with serum albumin is a dominant feature for both drug preparations. HFZ also has a different effect on the cytokine response in vitro. Studies using THP-1 human monocytes show that HFZ provokes a smaller release of tumor necrosis factor (TNF)-alpha than FZ. This cytokine may be associated with the unpleasant side effects of AmB. These findings suggest that heat-induced superaggregation of AmB alters its interaction with HDL, LDL, serum proteins, and monocytes, and these findings may be important in explaining the reduced toxicity of the superaggregated form of AmB.