Plasma lipoprotein distribution of amphotericin B formulations : potential role of phospholipid transfer protein

• Main Author: Patankar, Nilesh A.
• Language: English
• Published: 2010
• Online Access: http://hdl.handle.net/2429/18119

The objectives of this study were to compare the plasma distribution profile of Amphotericin B (AmpB) following incubation of three commonly used formulations, Fungizone®, Abelcet® and Ambisome® and to determine if phospholipid transfer protein (PLTP) facilitates the transfer of AmpB into high density lipoproteins (HDL) following incubation of AmpB-phospholipid complex within human plasma. Methods: Plasma lipoprotein distribution of AmpB was studied following incubation of Fungizone®, Abelcet® and Ambisome® at an AmpB concentration of 20 pg1mL within human plasma at 37OC for a range of incubation times. These plasmas were subsequently fractionated into their lipoprotein and lipoprotein deficient fractions by gradient density ultracentrifugation. Each fraction was analyzed for AmpB by high performance liquid chromatography (HPLC). Plasma phospholipid transfer protein (PLTP) assay was established and characterized in vitro using a commercially available kit. Plasma samples from six individual donors were analyzed for PLTP activity. PLTP activity observed from plasmas was then plotted against the percentage of AmpB recovered from the HDL fractions of the respective plasmas. HDL fractions of the same six plasmas were analyzed for the phospholipid content. HDL-phospholipid content was plotted against the percentage of AmpB recovered from the HDL fractions. In order to inhibit plasma PLTP activity, three different approaches were used. 1. Incubation of plasma with polyclonal antibody to PLTP. 2. Incubation of plasma with thimerosal. 3. Heating of plasma at 56°C for 1 hour. Results: Following incubation of Fungizone®, within human plasma, the majority (- 50-60%) of the incubated AmpB was recovered from the lipoprotein deficient fraction (LPDP). However, following incubation of lipid based formulations (i.e. Abelcet® and Ambisome® ), the majority (- 60-85%) of incubated AmpB was recovered from the HDL fraction. Plasmas containing elevated total cholesterol (TC) and triglycerides (TG) levels (plasmas with TC and TG >200 mg/dL) showed a four-fold higher AmpB-LDL association than plasmas with TC and TG levels <200 mg/dL following incubation of Fungizone® did not show any difference in the distribution profile following incubation of Abelcet® and Ambisome® . statistically significant positive correlation was observed between the plasma PLTP activity and the percentage of AmpB recovered from the HDL fraction following the incubation of Abelcet® not following the incubation of Ambisome®. addition, a positive correlation was observed between the percentage of AmpB recovered from HDL and HDLphospholipids concentration. Among the various approaches used to inhibit PLTP activity, the only approach that did show significant inhibition (80%) in the PLTP activity was heat treatment of plasma. Conclusion: Incubation of phospholipid based formulations of AmpB within human plasma resulted in the majority of drug being recovered in the HDL fraction. Elevated plasma TC and TG levels altered the plasma distribution of AmpB following the incubation of Fungizone® but not following the incubation of Abelcet® and Ambisome® Positive correlations reported between plasma PLTP activity and the percentage of AmpB recovered in the HDL fraction and between HDL-phospholipid concentration and the percentage of AmpB recovered in the HDL fraction together suggested indirect evidence that PLTP may co-transfer AmpB along with the phospholipids into HDL following incubation of Abelcet® ~Among the different methods evaluated to inhibit plasma PLTP activity, heat treatment of plasma was the only approach that showed significant inhibition in the plasma PLTP activity. However, due to limitations associated with this approach (i.e. non-specific denaturing of other plasma proteins and enzymes), it was not used in this set of studies. Future studies could employ following strategies, 1. Depletion of human plasma of PLTP by immunoprecipitation. This PLTP-depleted plasma will be used to study the distribution profile of AmpB following incubation of Abelcet®. 2. Development of PLTP antibody that will have PLTP neutralizing capacity. === Pharmaceutical Sciences, Faculty of === Graduate