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Urate oxidase (Uox), an enzyme catalyzing oxidation of uric acid to allantoin, is widely used as diagnostic reagents and for treatments of uarthritis and hyperuricemia diseases. In our study, a higher Uox producer, bacterial strain OUC-1, was isolated from soil samples. The 16S rRNA gene sequence of strain OUC-1 showed 99% identity to the homologous fragments of Bacillus fastidiosus. After purification, Uox showed the optimal pH and temperature was 10.0 and 40 °C. The Km value of Uox was (0.15±0.04) mmol/L (n=5) with uric acid as the substrate. Uox activity was enhanced by Mg²⁺, and seriously inhibited by Zn²⁺ and SDS. Then the uox gene of B. fastidiosus OUC-1 was amplified and sequenced. The 3D structures of Uox, predicted with SWISS-MODEL, showed a homotetramer structure with a subunit molecular weight of 35.38 kDa. Finally, the gene coding for the B. fastidiosus Uox was successfully cloned and heterologously expressed in E. coli, which provides theoretical basis and technical support for improvement of Uox in the future.
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Objective To screen a thermostable urate oxidase-producing strain, optimize the fermentation conditions and study the enzymatic properties.Methods A urate oxidase-producing strain was screened from high temperature starter based on transparent circle method.Its 16S rDNA sequence was then amplified and analyzed.Meanwhile, the phylogenetic trees were built.Optimization of the fermentation conditions from this strain was carried out.The enzymatic properties of urate oxidase were studied.Results A urate oxidase-producing strain, named Bacillus subtilis ZX-6 by molecular identification, was obtained.The production of urate oxidase under the optimized conditons (135.9 U/L) was 133.7%higher than before.The optimum reaction temperature and pH were 45℃ and 7.6 respectively.The residual activity of urate oxidase at 37℃ for 48 h was still 17.2%.Conclusion The successful screening of a thermostable urate oxidase-producing strain and optimization of the fermentation conditions will lay a foundation for the further research.
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Objective To assess the interference by calcium dobesilatein 7 peroxidase-baseduric acid assays and to determine its clinical significance.Methods In the in vitro experiments, uric acid in pooled serum with final concentrations of calcium dobesilate additions (0, 2, 4, 8, 16, 32, and 64μg/ml) were measured by 7 peroxidase-based assays.Percent Bias (%) was calculated relative to the drug-free specimen.In the in vivo experiments, changes in serum uric acid and calcium dobesilate concentrations were observed before and after calcium dobesilate administration ( baseline, 0 h, 1 h, 2 h, 3 h, 4 h, 6 h ) involunteers.The interference in different assays was assessed compared with LC-IDMS/MS method. Calcium dobesilate levels in 40 specimens from those taking calcium dobesilate were measured by HPLC method.Of the 40 specimens, 10 were selected to analyse the levels of uric acid by both peroxidase and UV measurement method to assess the impact in clinical status.Results In the in vitro study, concentrations of uric acid measured by 7 peroxidase-based assays were reduced by -6.3%to -21.2%compared with drug-free serum, when theconcentration of calcium dobesilate was16μg/ml.In the in vivo study, comparedto UA levels at 0 h, the biasesof serum uric acid determined by peroxidase method after calcium dobesilate administration(1 h, 2 h, 3 h, 4 h, 6 h) were of -3.33%, -6.79%, -7.49%, -6.07%, -4.09%, respectively.The observed uric acid concentrations for 8 participants measured by enzymatic assays were inhibited by -3.75% to -6.89% at 0 hour and by -16.9% to-22.22% at 2 hours relative to the concentrations measured by the LC-IDMS/MS method. Conclusions Calcium dobesilate produced a clinically significant negative interference with uric acid in all peroxidase-based uric acid assays,which may result in false evaluation of uric acid level in clinical status.Significant differences in the degree of interference were observed among the assays.
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PEGylated UOX is a kind of modified pharmaceuticals designed by coupling PEG with UOX to increase in vivo circulation half-life and reduce immunogenicity. UOX is modified by PEG at random multiple sites, which results in more complicated physical and chemical characteristics and in vivo and in vitro activities than UOX. At present, there is no standardized quality analysis method for such drugs. Through collecting and summarizing references about the quality control method of PEG-modified drugs in recent years, this article summarizes the analysis methods for three difficulties of the quality control of UOX namely the average extent of modification, consistency of modification and PEGylation sites so as to provide technical guidance for enterprises to develop the drug.
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BACKGROUND: Patients with hematological malignancies that are highly proliferative and have high tumor burden are at high risk of developing hyperuricemia and tumor lysis syndrome (TLS), spontaneously and while undergoing chemotherapy. AIM: To assess the safety and efficacy of a new generic formulation of recombinant rasburicase in prevention and treatment of malignancy‑associated hyperuricemia. MATERIALS AND METHODS: An open‑label, multicenter, phase‑III study was conducted on 100 eligible patients with high risk for TLS. Rasburicase was administered 0.2 mg/kg intravenously over 30 min, daily, for 4 days. The outcome measures were percentage of reduction in plasma uric acid at 4 h after rasburicase, plasma uric acid area under the curve (AUC)0-96 h and incidence of adverse events. RESULTS: Eighty eight patients completed the study period of 10 days. After rasburicase administration, there was a 75.3 ± 28.5% of reduction in plasma uric acid at 4 h as compared to baseline. The plasma uric acid AUC0-96 h was 259.9 ± 215.5 mg/dL h. Safety of rasburicase was assessed on the basis of changes in vitals, hematological, and biochemical parameters from baseline to termination. Except for the plasma uric acid level, there was no significant difference in any of the parameters. Mild to moderate adverse events were reported in 29 patients. Three patients had serious adverse events (SAEs) unrelated to rasburicase. CONCLUSIONS: These results demonstrated that recombinant rasburicase that is indigenously developed is effective for prevention and management of hyperuricemia in patients who are at high risk of developing TLS.
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BACKGROUND: Patients with hematological malignancies that are highly proliferative and have high tumor burden are at high risk of developing hyperuricemia and tumor lysis syndrome (TLS), spontaneously and while undergoing chemotherapy. AIM: To assess the safety and efficacy of a new generic formulation of recombinant rasburicase in prevention and treatment of malignancy‑associated hyperuricemia. MATERIALS AND METHODS: An open‑label, multicenter, phase‑III study was conducted on 100 eligible patients with high risk for TLS. Rasburicase was administered 0.2 mg/kg intravenously over 30 min, daily, for 4 days. The outcome measures were percentage of reduction in plasma uric acid at 4 h after rasburicase, plasma uric acid area under the curve (AUC)0-96 h and incidence of adverse events. RESULTS: Eighty eight patients completed the study period of 10 days. After rasburicase administration, there was a 75.3 ± 28.5% of reduction in plasma uric acid at 4 h as compared to baseline. The plasma uric acid AUC0-96 h was 259.9 ± 215.5 mg/dL h. Safety of rasburicase was assessed on the basis of changes in vitals, hematological, and biochemical parameters from baseline to termination. Except for the plasma uric acid level, there was no significant difference in any of the parameters. Mild to moderate adverse events were reported in 29 patients. Three patients had serious adverse events (SAEs) unrelated to rasburicase. CONCLUSIONS: These results demonstrated that recombinant rasburicase that is indigenously developed is effective for prevention and management of hyperuricemia in patients who are at high risk of developing TLS.
Subject(s)
Adult , Aged , Area Under Curve , Child , Female , Gout Suppressants/therapeutic use , Hematologic Neoplasms/complications , Humans , Hyperuricemia/drug therapy , Hyperuricemia/etiology , India , Male , Middle Aged , ROC Curve , Tumor Lysis Syndrome/prevention & control , Urate Oxidase/therapeutic use , Uric Acid/blood , Young AdultABSTRACT
OBJECTIVE: To construct and evaluate the stability, and in vitro bioactivity for new Y-shape PEG-labeled recombinant mammalian urate oxidase rUOX-mPEg as well as pharmacokinetics and immunogenicity compared with the line-shape rUOX-mPEG in rats. METHODS: Physicochemical characteristics and modification degree of PEG were detected by gel electrophoresis and HPLC, bio-activity by enzyme-spectrophotometric method, and immunogenicity though ELISA. Pharmacokinetic and tolerability property were assessed in vivo. RESULTS: The novel Y-shape PEG modified urate oxidase rUOX-mPEG2 presented higher molecular weight, modification degree of PEG compared to line-shape rUOX-mPEG And pharmacokinetic assay showed that rUOX-mPEG2 more excellent curative effects in vivo. Plus, the results of experimental on immunogenicity indicated that the Y-shape PEG modified urate oxidase could reduce immunogenicity effectively. CONCLUSION: The bioavailability, efficacy and stability of rUOX-mPEG2 were greater than those of line-shape rUOX-mPEG and recombinant mammalian control. Bioavailability, efficacy, and tolerability were also superior to that of line-shape and PEG-free urate oxidase. The determination of oxidase pharmacokinetic parameters revealed the linearity kinetic characteristic of rUOX-mPEg implying its potential excellent clinical supplication.