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Characterization and formulation of liposomal disodium ascorbyl phytostanyl phospates (FM-VP4) Ng, Agatha Wai Kwan.

Abstract

High plasma cholesterol levels, smoking, and high blood pressure are some of the major risk factors for atherosclerosis. However, the risk factor that has received the most attention over the past two decades is cholesterol. Recently, disodium ascorbyl phytostanyl phosphates (FM-VP4), which are derived from plant stanols, have been developed. FMVP4 is a mixture of two compounds, disodium ascorbyl sitostanyl phosphate and disodium ascorbyl campestanyl phosphate. Even though the mechanisms of FM-VP4 remains unknown, it has been shown to be effective in lowering LDL-cholesterol levels and total cholesterol levels in animals. One of the major obstacles in examining the activity of FMVP4 in animal models is the poor systemic absorption of FM-VP4 when given orally. Oral administration of FM-VP4 has been reported to have an absorbability of only 6.5%. Also, poor cellular uptake has been demonstrated in cell culture studies. In vitro, only about 25% of FM-VP4 was shown to associate with IEC-6 cells after 1 hour of incubation. To overcome these obstacles, it has been proposed that FM-VP4 can be administered intraveneously using liposomes as its delivering vehicles. Liposomes have been shown to enhance the therapeutic activity of various pharmaceutical agents and it has been proposed that liposomal FM-VP4 can enhance the cellular uptake of FM-VP4 in vitro. The specific objectives of this project were (1) to develop liposomal FM-VP4 formulations, (2) to develop an LC/MS/MS assay for quantification of FM-VP4 in liposomal formulations and plasma samples, (3) to characterize liposomal FM-VP4 formulations by finding the optimal drug-to-lipid ratios and determining the degradation of FM-VP4 in liposomes, and (4) to compare the hemolytic activity of FM-VP4 and liposomal FM-VP4. Section 4 describes the identification and quantification of FM-VP4 in liposomal formulations using an LC/MS/MS assay, which provides estimates of the concentrations of FM-VP4 and its encapsulation efficiency in liposomes. For FM-VP4 samples in plasma, extra steps are employed to remove plasma proteins prior to LC/MS/MS assay. The limit of detection (LOD) and limit of quantification (LOQ) of this LC/MS/MS assay were found to be 0.17μg/mL and 0.625 μg/mL, respectively, for liposomal samples; and 0.51 μg/mL and 1.25 μg/mL for plasma samples. Section 5 describes experiments designed to find the optimal drug-to-lipid ratio for liposomal FM-VP4 formulations through the comparison of the encapsulation efficiencies of various DMPC/cholesterol compositions. FM-VP4 was loaded in the liposomes at various drug-to-lipid molar ratios of 0.1, 0.3, 0.6, and 1.0. An encapsulation efficiency of approximately 80% was achieved up to a drug-to-lipid ratio of 0.6, representing a postformulation drug-to-lipid ratio of 0.5. After the manufacturing processes, the dimyristoylphosphatidylcholine (DMPC)/cholesterol ratios (55/45, 70/30, 85/15, and 100/0) remained unchanged when compared to the original preparations. Additionally, section 5 details our degradation studies to determine if liposomes have any protective effects on FM-VP4 in rabbit plasma incubated at 37°C. The results showed that there was minimal FM-VP4 degradation in the 55/45 formulation, as more than 95% of FM-VP4 remained in the first four days; in contrast, the concentration of the free FM-VP4 decreased to 79% over the first two days. Liposomal encapsulation prevents FM-VP4 from degradation in rabbit plasma. Furthermore, the hemolytic effects of FM-VP4 and liposomal FM-VP4 were also investigated. Free FM-VP4 at 50uM induces about 92% hemolysis within 1 hour of incubation with rabbit red blood cells, but its hemolytic activity was decreased when encapsulated; only 40% hemolysis was observed with liposomal FM-VP4 at equivalent FM-VP4 concentration. Encapsulating FM-VP4 in liposomes prevents FM-VP4 from lyzing the rabbit red blood cells. The mechanism of how FM-VP4 lowers LDL cholesterol levels and total cholesterol levels in various animal models is presently unknown. However, before the mechanism of action could be studied, FM-VP4 first had to be delivered efficiently into plasma or cultured cells. The low systemic bioavailability and cellular uptake of FM-VP4 further suggested the importance of finding an efficient delivery vehicle for this drug. This project proposes a framework for such delivery and paves the way for further investigation into how FM-VP4 works in vivo and in vitro.

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