Oxidation Matrine Liposome Research

Oxymatrine is the major active alkaloid constituent extracted from the root of Sophora flavescens Ait (Kushen) and the terrestrial part of Sophora alopecuroides (Kudouzi). In recent years, oxymatrine has been found posses remarkable anti-hepatic activity and has been used for treating hepatitis B in clinical therapy in China. However, oxymatrine administered via parenteral route will be distributes to all over the body and eliminated rapidly. Furthermore, duration of drug concentration in liver is very short too. As drug carriers, liposomes could preferably distribute in liver, maintain effective drugs concentration level in liver over longer time periods. In order to facilitate the delivery of oxymatrine to liver and enhance the therapeutic efficiency for hepatitis, oxymatrine liposomal preparation was investigated in this study.Preformulation investigation was performed to obtain the drug physicochemical properties for rational liposomes preparation design. The water solubility for oxymatrine is more than 1g/ml. The pk_a value of oxymatrine determined by potential titration and spectrophotometric indicator method were 6.98 and 6.71 respectively. The n-octanol/water partition coefficient (Po/w) of oxymatrine was 0.2. Those result indicatd that oxyamtrine is a very hydrophilic weak base. A procedure of separating free drug from the liposomes by ultrafiltration for liposomes entrapment efficiency determination was developed.Passive loading methods including ethanol injection, film dispersion, reverse evaporation, dehydration-rehydration, proliposome and MVL (Depofoam) procedure were introduced to oxymatrine liposomes preparation. At the same time, the factors affecting the entrapment efficiencies were investigated and compared. In ethanol injection method, the lipid composition influenced the entrapment efficiency. In thin-film hydration method, it has been shown that oxymatrine had some interaction with negatively charged lipid PS. For multivesicular liposomes, the pH of aqueous medium is critical for liposomes preparration with good appearance. It was difficult to obtain oxymatrine liposomes with encapsulation efficiency above 30% by passive loading methods mentioned above.pH gradient active loading method was introduced and investigated for oxymatrine liposomes preparation. Drug to lipid mass ratio, lipid composition and the buffer capacity of internal media greatly influenced the encapsulation efficiency. For SPC liposomes, encapsulation efficiency above 50% could been obtained with 150raM citric acid when drug to lipid ratio below 0.6; For HSPC liposomes, encapsulation efficiency above 50% could been obtained with 200mM tartaric acid below drug to lipid ratio 0.4; The incubation temperature had almost no effect on SPC liposomes in active loading procedure, but have a remarkable effect for HSPC liposomes. The in vitro release data show that oxymatrine was leaked more from SPC liposomes than from HSPC liposomes. The HSPC liposomes were stable when incubated with serum or dialysis in PBS solution.Pharmacokinetics and tissue distribution of Oxymatrine liposomes prepared by active loading method and oxymatrine solution in mice were studied. The half-life time(t_1/2)of liposomes A(HSPC liposomes), liposomes B (SPC liposomes)and oxymatrine solution was 17.1, 3.89 and 2.58h respectively, the MRT₍(0-t)_for three formulation above was 10.9, 2.7 and 1.17h, and the AUC_(0-t)for three formulation was 402.61, 206.37 and 63.11mg·h/ml respectively. Compared with oxymatrine solution, two kinds of oxymatrine liposomes greatly enhanced blood concentration of oxymatrine and prolonged the drug duration in blood. Tissue distributions were investigated at 0.833, 1 and 8 hour. HSPC liposomes have a remarkable effect on enhancing drug concentration and release duration in liver.To obtain a more stable oxymatrine liposomes preparation, freeze-drying processes for liposomes and Tert butyl alcohol (TBA)/water cosolvent system were investigated. It was shown that sucrose could maintain the liposomes particle size in liposomes freeze-drying process. In TBA/water cosolvent system, low temperature microscope and low temperature DSC were used to investigate the frozen status of TBA/water monophase solution. Results of DSC determination shown Vitrification temperature (T_g') of excipents decreased with the addition of TBA. It is critical to control the temperature and vacummn of freeze-drying process for obtaining product without collapse. Two freeze drying processes combined with pH gradient method would be used in oxyamtrine liposomal preparation.