Study On Self-Assembled Systems Of Anti-HBV Lipid Prodrug

Hepatitis B virus is the first cause of chronic hepatitis,hepatic cirrhosis and liver cancer worldwide.Nucleoside drugs are able to inhibit the deoxyribonucleic acid polymerase, terminate the extension and synthesis of DNA chain,so that it can inhibit the replication of HBV.Poor liver targeting was one of the most important reasons that caused the deficiency of effective drugs. HepDirect prodrugs are a novel class of phosphate and phosphonate prodrugs.The HepDirect prodrugs are oxidized specifically by the P450 isoenzyme family CYP3A4,and release the active phosphates and phosphonates.In both animal and clinical studies, liver-targeting have been observed and have no side effect-related toxicities.Self-assembled drug delivery systems(SADDS) were prepared from amphiphilic conjugates of hydrophilic drugs and lipids through self-assembling into highly ordered dispering aggregates in the aqueous media.The SADDS is more stable than other microparticles in vivo.The amphiphilic nature of SADDS lead to strong affinity and permeability with biomembranes.Meanwhile,the property of high disrersion lead to targeting efficiently.In this paper, a novel drug delivery system (DDS) was designed and prepared with the combination of the phosphate ester prodrugs , self-assembled drug delivery systems, liver-targeting and long-circulation.The new system may have more antiviral effect and lower side effect-related toxicities.The antiviral drug - adefovir was selected as the model drug.With esterification and cyclization,we get the adefovir phosphate ester prodrugs(APE),then lipophilic derivated the self-assemblies of lauroyl adefovir phosphate ester prodrugs (LAPE) were prepared. The main contents were described in details as follows:1. Synthesis and characteristics of anti-HBV prodrug.An anti-HBV prodrug was designed and synthesized, that was lauroyl adefovir phosphate ester prodrugs (LAPE).After purification,its structure was identified.The hydrogen bonding and hydrophobic interaction were the key factors that affected the solubility and self-assembly ability.The lipophilicity of the prodrug increased greatly. Meanwhile, the formation and characterization of its langmuir monolayers were investigated.As a typical amphipathic prodrug,LAPE formed the stable and flexible monolayer.It showed well amphipathic.2. Synthesis and identify of G₂₀DE.For liver-targeting and long-circulation,G₂₀DE was designed and synthesized.And its purification was identified.The PEG moieties give the long-circulation to the LAPE self-assembly system and the galactose moieties make it target to the liver.Added with the G₂₀DE, LAPE can form highly dispersed homogeneous suspension self-assembly system..3. Preparation and characteristics of anti-HBV prodrug self-assemblies.Many methods were used to prepare LAPE self-assemblies. A kind of highly dispersed homogeneous suspension system with high concentration was prepared by MeOH injection method.The morphology and size of LAPE self-assembly system was evaluated with transmission electron microscope (TEM) and the dynamic light scattering mehtod.LAPE molecules self-assembled to bilayer membrane by the hydrophobic interaction of lauric acid moieties and under the action of the hydrogen bonding of adefovir, and then formed to the vesicles. The lipid chain of G₂₀DE could insert into the Vesicle structure to form a new system.The mean particle size of LAPE self-assemblies was about 68 nm. The surface Zeta potential was -20.4 mV.4. Physical stability of anti-HBV prodrug self-assemblies.Physical stability was investigated through heating, high pressure, centrifugation (≤10000 rpm) and storing at room temperature. The results indicated the content of LAPE almost have no change when LAPE self-assemblies were heated and under high pressure. Moreover, LAPE self-assemblies were stable to a great extent through high speed centrifugation and storing at room temperature. Self-assemblies of high concentration could redisperse easily in water.5. Chemical stability of anti-HBV prodrug self-assemblies.Chemical stability of LAPE self-assemblies in the buffer solutions of different pH, plasma of different animals and tissue homogenates of mice were all investigated with HPLC.Self-assemblies of LAPE hydrolyzed quickly in the buffer solution of pH 2.0, but kept stable for a long time in the neutral and weak acid buffer solutions with t1/2 more than 633 h and 277 h, respectively. Furthermore, the degradation of LAPE in the plasma of animals was different. The degradation rate of LAPE in the plasma of mice, rat and human were faster than that of dogs.The degradation rate of LAPE in liver homogenates of mice was very fast, and the t_1/2 was less than 1h. However,the degradation of LAPE was slowly in kidney.The main degradation products in liver were adefovir phosphate ester prodrugs.The results indicated that the prodrug could degrade to the active compound efficiently, which could exert antiviral efficiency in vivo.6. Pharmacodynamics of anti-HBV prodrug self-assemblies.With intragastric administration LAPE self-assemblies to DNA transfection mice,we find the HBV DNA copy action was inhibited obviously. The results indicated that the prodrug could degrade to the active compound-adefovir phosphate ester prodrugs, which could play a part in pharmacodynamic action.7. Pharmacokinetics and tissue distribution of anti-HBV prodrug self-assemblies.After single bolus of iv. administration to mice, LAPE self-assemblies were eliminated gradually from the blood circulation, The distribution and elimination t1/2 were 1.87 and 136.8min, respectively. It was suggested that LAPE self-assemblies had some long-circulation characteristics in the blood. LAPE quickly distribute to spleen, liver, lung, heart and kidney. The LAPE detected in liver was much higher than other tissues due to the liver targeting.We can also find LAPE self-assemble system had sustained releasing effects. The AUC of LAPE self-assemblies after iv. administration to mice were 5377.6 mg/L*min.Under the effect of amidase, the LAPE molecule degraded to adefovir phosphate ester prodrugs. The results indicated that LAPE self-assemblies could target to the liver system, then degrade to the active compound efficiently in the targeting tissues and played an important role in antiviral action.In general,a highly dispersed,homogeneous and stable self-assemblies of lauroyl adefovir phosphate ester prodrugs (LAPE) was prepared in this paper. A nano-size, ordered structure formed under the action of hydrophobic interaction and hydrogen bonding was proved. The self-assemblies were uptaken by tissues quickly in mice and degraded sustainedly into adefovir phosphate ester. The LAPE self-assemblies could target to the liver system as the detected LAPE in liver was much higher than other tissues obviously. Moreover,it had sustained releasing effects.LAPE self-assemblies could make the liver-targeting and long-circulation come true in vivo and played an important part in the antiviral action.