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Pegylation of Recombinant Uricase from Bacillus Fastidiosus and Evaluation of mPEG5k Modified Uricase

Author: ZhangChun
Tutor: YangXiaoLan
School: Chongqing Medical University
Course: Medicinal Chemistry
Keywords: Uricase Polyethylene glycol-modified Druggability
CLC: R914
Type: Master's thesis
Year: 2011
Downloads: 105
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Abstract


E. coli recombinant expression demanding Bacillus subtilis (ATCC29604) uricase homologous tetramer, the sequence of each polypeptide chain containing 17 amino purified by DEAE-Cellulose column, twice a specific activity of 6.0 IU / mg. Metabolism in rats in vivo half-life of only 10-15min, and shows strong immunogenicity. Polyethylene glycol modified to improve protein druggability the primary means of. This article optimized single-methoxy polyethylene glycol (MPEG) modified this restructuring express uricase conditions, and preliminary evaluation of MPEG-modified uricase druggability. PEGylation reagent preparation combined with the structural characteristics of the uricase protein, prepared using two methods mainly in the free amino group of the surface of the uricase proteins as modified target modification reagents. A method for using monomethoxy polyethylene glycol 5000 (mPEG5k) and 350 (mPEG350) first reaction with succinic anhydride in tetrahydrofuran to generate polyethylene glycol succinic acid mono-ester and then with N-hydroxy-succinimidyl amine dehydrated by DCC, the single methoxy polyethylene glycol succinimidyl succinate (SS-of mPEG5k, SS-mPEG350), wherein the SS-mPEG5k closing rate of 60-75%, the yield of the SS-mPEG350 approximately 56%. Another type of method mPEG5k first trichloromethyl carbonate in methylene chloride reacts monomethoxy polyethylene glycol phosgene, succinimide, and then reacted with N-hydroxysuccinimide monomethyl group of polyethylene glycol carbonate (SC-mPEG5k), yield of approximately 87%. Difference for the comparison of different sizes N-hydroxysuccinimide ester modified amino group, with acetic acid and N-hydroxysuccinimide DCC in obtaining the acetate (Ac-NHS), the yield of about 90% at the same time. Naftifine ethylenediamine reactive with the amino group of the activated ester derivative detect and estimate its content. 2 polyethylene glycol-modified uricase conditions to optimize the a TNBS determination of amino, a ScS polyethylene glycol modifier uricase polypeptide chain quantities (molar ratio), uricase degree of modification of the amino group on the relationship between the residual activity of the modified uricase. Molar ratio in 0 1 00 between, uricase amino modification degree modifier usage was a good linear relationship (y = 0.67 X-4.96, R 2 = 0.99 ); when the molar ratio of close to 200, the modification degree of 80%; molar ratio in 200 5 00 modified flat close to 85%. With the increase in the degree of modification, the uricase activity continued to decline to 17% of the original activity. Compare SS-MPEG 5 k, SS-mPEG Post 3 50 and acetic acid succinimide ester derivative the modified product activity, found SS-mPEG Post k maximum modification degree of 75% and corresponding residual activity 60 7 0%, the SS-mPEG350 modification degree of 86% and the corresponding residual activity of 23% acetic acid succinimide ester modification degree of 94% corresponds to a residual activity of 10%. The of two uricase inhibitor oxonic acid and xanthine, and its substrate uric acid ligands can significantly improve the SC-mPEG 5 k modified uricase activity retention ratio. In a molar ratio of 200 for the amount of modifier, no ligand under uricase modification degree up to 80%, but only 20-30% active reserved. 30μM oteracil acid modified uricase can retain activity reached 70%, 150μM xanthine modified uricase can be retained activity reached 59%, and (initial concentration) 500μM uric acid modified uricase can retain activity reached 53%. 3 mPEG5k modification that uricase druggability the preliminary evaluation of mPEG5k modified uricase pH, Mg 2 Effect respects with the unmodified uricase similar with its thermal stability and resistance to protease hydrolysis significantly improved. With the uricase inhibitor of potassium oxonate (Ki <3 micromolar) per day 0.20mg / g dose of the SD rats by intraperitoneal injection for 4-6 weeks successfully established hyperuricemia rat model of rat plasma uric acid levels continued to remain above 0.42 mM, and the total amount of uric acid in vivo enzyme inhibitor (oteracil acid equivalent) is also maintained at a high level, generate significant interference uricase pharmacodynamic evaluation. Therefore, we have designed a drop in vitro ability of uric acid metabolism half-life investigation and comprehensive evaluation of MPEG 5 k modified uricase pharmacodynamic and pharmacokinetic properties. mPEG 5 k modified uricase with an unmodified uricase in vitro plasma uric acid showed good ability; decay half-life of HF ≈ 12min in the healthy rats in vivo activity of the unmodified uricase, mPEG modification uricase metabolic half-life of about 20 hours. Poultry body lacks endogenous uricase. MPEG-modified uricase longer the activity decay half-life and maintain plasma uric acid in the body for a long time at a low level in the chicken body. The unmodified uricase strong immunogenicity of uricase in rabbits anti-IgG antibody titers reached more than 1:781250; rats, mPEG 5 k the modified uricase original antigen reaction activity significantly decreased to below 5%, uric acid with rabbit anti-enzyme antibody, antigen reactivity is lower than the unmodified uricase 10%, and the emergence of new major immunogenic for mPEG. In summary, there are certain structural properties of the modifier, the amount of impact modifying effect of uricase, uric acid inhibitors have a significant impact modified uricase activity retained, the mPEG 5 k modified uricase druggability improved.

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