| Uricase (UOX) plays an important role in the metabolism of purine in vivo, and it can catalyze uric acid into allantoin. Unlike nearly all other mammals, humans lack the enzyme uricase, which catalyzes the oxidation of uric acid to allantoin, a more soluble product that is readily excreted in the urine. The lack of uricase in humans results in plasma uric acid concentrations that are much higher than in most mammals. When less uric acid is excreted than is produced, the plasma urate concentration rises and may cause hyperuricemia. Treatment with uricase can dramatically lower the levels of urate in plasma and has potential for therapy of gout and uric acid nephropathy resulting from hyperuricemia.Liposomes are commonly recognized as a superior drug delivery vehicles which show the advantages of delayed-release, target effects and lower drug toxicity.In this study, lipid vesicles containing uricase were designed and prepared in an attempt that a general strategy to improve its pharmacokinetics characters such as half-life, increase its enzyme activity, stabilize and deliver multimeric enzymes through entrapping them in the lipid vesicles might be affirmed. Meanwhile it can reduce immunogenicity and antigenicity.Method:1. Uricase-containing lipid vesicles were separated from free uricase by gel exclusion chromatography performed with Sephadex G-200 column. And then the protein content was determined by the Coomassie blue method initially reported by Bradford.2. UOXLVs were prepared by reverse-phase evaporation method. Single factor tests were conducted to optimize the reaction conditions.3. Determinating the optimum temperature, optimum pH and the Michaelis constant of UOXLVs and UOX by measuring the enzyme activity. The FITC fluorescence in lipid vesicle suspensions was measured in the presence and absence of uricase using a fluorescence Spectrophotometer to examine a lipid vesicle–uricase interaction.4. Measuring the storage stability, thermal stability, pH stability, stability to proteolytic digestion and effect of various chemical agents on uricase activity of UOXLVs and UOX by measuring the enzyme activity.5. The rat antiserum was prepared by inoculating the rat with UOXLVs and UOX. The antibody against UOX was measured through indirect ELISA using UOXLVs or UOX as coating antigen to analyze the immunogenicity of UOXLVs and UOX in rats.6. Hypoxanthine (at the dose of 500 mg/kg) combined with oxonic acid potassium salt (at the dose of 100 mg/kg) were used to prepare the hyperuricemia rat model.Result:1. Uricase-containing lipid vesicles were separated from free uricase by gel exclusion chromatography performed with Sephadex G-200 column, and the average recovery rate of gel column of UOX is 98.29%. The method is simple, sensitive, and reproducible, which could accurately determinate the contents of UOX and entrapment efficiency in UOXLVs.2. The best formula for UOXLVS by optimize process including: the volume ratio of diethyl ether and buffer was worked out to be 3:1; the molar ratio of SPC and CH is 1:1; and the dosage is 2 mg. In the condition of the optimal formulation, UOX was encapsulated is 64.24%, average particle size is 206.73 nm, the polydispersity index is 0.247, and zeta potential is -37.33 mV.3. The results show that, the optimum temperature of UOXLVs and free uricase were the same as 40℃. The temperature range at which UOXLVs remained a higher activity than that of free uricase. UOXLVs did shift the optimal pH of enzyme activity from 8.5 (for free uricase) to 8.0, and the activity of UOXLVs was significantly higher than free UOX. The Km of UOXLVs is lower than free UOX, which shows the affinity increasing between UOXLVs and the substrate.The FITC fluorescence in lipid vesicle suspensions was measured in the presence and absence of uricase using a fluorescence Spectrophotometer to examine a lipid vesicle–uricase interaction. It was found that the FITC and uricase molecules competitively interacted with the interfacial region of zwitterionic lipid vesicle membranes through electrostatic and hydrophobic interactions.4. Stability experiments showed, in the aspect of its storage stability, thermal stability, pH stability, resistance to trypsin hydrolysis and some organic compounds the effects on the enzyme activity, all the stability of UOXLVs was higher than that of UOX.5. The results of experimental on Immunogenicity showed that the antiserum titer which was based on the antigen of UOXLVs and UOX was separately 1: 500 and 1: 8000, indicating that UOXLVs can reduce immunogenicity effectively.6. Preliminary pharmacodynamic study in vivo show that it took less than 3 h to decrease the uric acid from the high concentration (489.61μmol/L) to the normal uric acid level (240μmol/L in human) after intravenous injection of UOXLVs, while the time was about more than 6 h in the case of free uricase group. UOXLVs suppressed uric acid levels up to 88.72%. The lowest uric acid level (55.24μmol/L) was achieved at 12 h after intravenous injection of UOXLVs (compared with the 189.94μmol/L in the case of the free uricase group and 271.67μmol/L in the model group). In vivo pharmacodynamic studies explicitly suggested that UOXLVs distinctively decreased the uric acid level on the hyperuricemia rat model.Conclusion:The uricase-containing lipid vesicles with high entrapment efficiency were prepared and characterized in terms of their stability and activity. All the results obtained showed that the UOXLVs were advantageous to maintain higher stability and activity than that of free uricase. The immunogenicity of free uricase can be notablely decreased when delivered in the form of UOXLVs. Moreover, in vivo pharmacodynamic studies demonstrated that UOXLVs could decrease the uric acid level obviously and reduce immunogenicity protection at the same time.
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