| Lipid-based multivesicular carrier loaded with uricase(UOMVLs) was prepared, and used as a new preparation for enzyme. In order to evaluate the clinical application value of UOMVLs, it is necessary to study its pharmacology. The metabolic enzyme kinetics, pharmacokinetics, pharmacodynamics, immunogenicity and hemolysis and vascular stimulation of UOMVLs were studied in this research. Results showed that UOMVLs could overcome low activity, low half-life and immunogenicity of uricase. High blood uric acid level of rats could effectively be reduced by UOMVLs and no hemolysis or vascular stimulation was observed. Theoretical basis of clinical application value of UOMVLs was provided in this study.Uricase (UOX) plays a very important role in the metabolism of purine in vivo, and it can catalyze uric acid into allantoin. Most mammals have the enzyme uricase, which catalyzes the oxidation of uric acid to allantoin. Allantoin is a much soluble product that can be readily excreted in the urine. Unlike nearly all other mammals, humans lack the enzyme uricase, which results in plasma uric acid concentrations that are much higher than in most mammals. The solubility of uric acid is very low in the water, when less uric acid is excreted than is producted, the plasma urate concentration rises and may cause high uric acid hematic disease, which in turn leads to gout syndrome, kidney stones, etc. Multivescular liposomes (MVLs), which are designed to serve as a hydrophilic drug carrier, which show the advantages of delayed-release, target effects and lower drug toxicity.In this study, lipid-based multivesicular carrier loaded with UOX (UOMVLs) was designed and the content of this study includes the following several parts:In Chapter I, the UOMVLs was prepared by a double emulsion method, and evaluated by encapsulation efficiency rate of drug, size and zeta. The optimum temperature, optimum pH and the Michelis constant of UOMVLs and free uricase were determined by measuring the enzyme activity. UOMVLs emerge as spheroids with granular structure under the light microscope. The encapsulation efficiency of UOMVLs is 63.75± 3.65%, the average diameter of them was (26.64±1.70)μm, and the zeta potential was (-41.81±6.59) mV. The optimum activity of UOMVLs was measured when the temperature was 40℃, and the pH was 8.0.In Chapter Ⅱ, effects of high temperature, low temperature, pH and trypsin on UOMVLs and free uricase, and the activity of them in the plasma were studied. The results showed that the activity of UOMVLs was significantly superior to the free uricase. And in the same circumstances, the activity of uricase in UOMVLs was significantly higher than that of the free uricase.In Chapter III, the related mechanisms of UOMVLs were studied. Fluorescence spectrophotometer was used to investigate the thermal stability of UOMVLs and free uricase. The results showed that the structure of uricase could be easily changed at a high temperature. The lipid membrane might prevent hydrophobic interactions among molecules, so it could protect uricase in UOMVLs. And the results also showed that the activity of uricase increased when packed in MVLs. Through long-time observing of the morphological changes of UOMVLs during the release process, we speculated that the main ways of UOMVLs release were exocytosis and splitting directly.In chapter IV, enzyme kinetics and pharmacokinetics of UOMVLs were studied in SD rats through intravenous injection and subcutaneous injection. The Km of UOMVLs and free uricase were (12.29±3.5)μmol/L and (14.27±3.1)μmol/L, which showed that the affinity of UOMVLs was better than that of free uricase. DAS2.1.1 pharmacokinetic software was used for processing and statistical results. The main pharmacokinetic parameters of UOMVLs though intravenous injection were (446.27±70.60) U/L·h, MRT(0.72h) (4.17±0.31) h, Cmax (73.04±6.35) U/L, Tmax (1.00±0.00) h. The main pharmacokinetic parameters of free uricase though intravenous injection were AUC(0-72h) (27.80±10.36) U/L·h, MRT (0.72h, (0.91±0.20) h, Cmax (13.91±.03) U/L, Tmax (0.67±0.29) h. These results showed that UOMVLs and free uricase were not bioequivalent, and the bioavailability of UOMVLs was 1605.3%. The main pharmacokinetic parameters of UOMVLs though subcutaneous injection were AUC(0.72h) (179.13±17.76) U/L-h, MRT(0-72h) (4.37±0.52) h, Cmax (60.33±6.42) U/L, Tmax (4.00±0.88) h. The main pharmacokinetic parameters of free uricase though subcutaneous injection were AUC(0-72h) (87.61±10.54) U/L·h, MRT(0-72h) (2.26±0.03) h, Cmax (38.26±6.03) U/L, Tmax (1.00±0.00) h. These results showed that UOMVLs and free uricase were not bioequivalent, and the bioavailability of UOMVLs was 204.5%.Chapter V mainly focused on the pharmacodynamics of UOMVLs in SD rats through intravenous injection and subcutaneous injection. The time needed for UOMVLs to reach the normal of rats is 8.96 h, which was 0.2 of that needed for free uricase after intravenous injection. In comparison, UOMVLs was remarkably better than free uricase when used to catalyze uric. Once again, UOMVLs was proved to be somehow better to catalyze uric through subcutaneous injection.In Chapter VI, the immunogenicity of UOMVLs was envaluated. ELISA method was used to determine of the results. The antibodies in mice of UOMVLs group producing significantly lower than that in rats of free uricase group. The antibody titer results also showed that UOMVLs could effectively reduce the immunogenicity of uricase.In Chapter W, the evaluation of hemolysis test and vascular stimulation of UOMVLs were studied. The results showed that no hemolysis or vascular stimulation was observed in UOMVLs group. So UOMVLs can be used through intravenous injection. |
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