Study On The Self-Assembled Systems Of Phosphoryl N-Fatty Acyl Gemcitabin Lipid Prodrugs

The prodrugs that could be actively targeted to liver were designed and synthesized in this study. It based on the theory of self-assembled drug delivery systems (SADDS) and HepDirect technology. The HepDirect prodrugs were synthesized by the nucleoside drug gemcitabine (GEM) as start material at first, then linked with the long fatty chains by covalent bond to get amphiphilic prodrugs. The molecules formed the nanoscale self-assemblies in the aqueous media. The in vitro and in vivo properties of the self-assemblies were explored to evaluate their anti-cancer activity.1. Synthesis and characteristics of phosphoryl N-fatty acyl gemcitabine derivative self-assembliesThe lipid prodrugs were prepared from GEM as the hydrophilic segments, and the fatty acids of different chains and phosphoryl as the hydrophobic segments. The chemical structure of the prodrugs were characterized by the method of’H-NMR,13C-NMR and31P-NMR, The prodrugs could form the stable molecular monolayers at the air/water interface2. Preparation and properties of the self-assembliesThe self-assemblies were prepared with the methanol injection method. The morphology and size of phosphoryl N-fatty acyl gemcitabine (CPFG) self-assemblies were evaluated by transmission electron microscope (TEM) and dynamic light scattering instrument. With the increase of fatty chains, the morphology of self-assemblies varied from vesicle to rod-shape, and the size increased from100nm to400nm. Zeta potential of CPFG was all about-30mV.3. Physical stability of the self-assembliesThe influence of storage at room temperature, high pressure, centrifugation and heating on the physical stability of CPFG self-assemblies was investigated. High pressure could destroy the vesicle structure.The mean size of self-assemblies was stable and redispersed easily after speed centrifugation. The self-assemblies of low concentration were stable at room temperature.4. Chemical stability of the self-assembliesThe influence of the buffer solutions of different pH and mice plasma on the chemical stability of CPFG self-assemblies were determined with HPLC. The results indicated that the degradation of self-assemblies were faster in the buffer solution of pH2.0(t1/2=27.72h), but stable in the buffer solution of pH5.0and pH7.4(t1/2=138.6h and t1/2=173.25h). The t1/2of self-assemblies in the plasma of mice was1.3h.5. In vitro pharmacodynamics of phosphoryl N-fatty acyl gemcitabine self-assembliesThe cytotoxic activities of phosphoryl N-fatty acyl gemcitabine self-assemblies were evaluated on HepG2cells by MTT method. The results indicated that the cytotoxic effect of phosphoryl N-octanoyl gemcitabine (CPOG) self-assemblies, phosphoryl N-dodecanoyl gemcitabine (CPDG) self-assemblies and phosphoryl N-hexadecanoyl gemcitabine (CPHG) self-assemblies were higher than the positive control drug Gem. The high concentration of phosphoryl N-tetradecanoyl gemcitabine (CPTG) self-assemblies and the phosphoryl N-octadecanoyl gemcitabine derivative (CPODG) self-assemblies were similar cytotoxic effect to Gem.6. Pharmacodynamics of phosphoryl N-dodecanoyl gemcitabine self-assemblies in vivoTumor-bearing mice model was established by subcutaneous injection with H22cells. The self-assemblies were investigated the activity of anti-tumor by oral and injection administration to tumor-bearing. The oral groups of self-assemblies had no apparently activity of anti-tumor. However, the injection groups of self-assemblies have equal activity to Gemcitabine in half molarity. When adding to long circulation metrial, the activity of anti-tumor will increase apparently.7. Pharmacokinetics and tissue distribution of CPDG self-assemblies After iv administration to Kunming mice, CPDG were eliminated quickly to liver, speen. The eliminated t1/2was84±5.53min.The content of CPDG in liver and tumor were much higher than other tissues. The results indicated that CPDG self-assemblies could target to the liver system.In this paper, the self-assemblies of CPFG were designed and prepared. They were stable in physical and chemical conditions to some extent. The self-assemblies exhibited obvious anti-cancer activity in vivo and it may be a novel promising formulation for targeted liver therapy.