聚乙二醇修饰尿酸酶的研究

中国科大李学报 Journal of China Pharmaceutical University 2008,39(6) :557-562557 Research on PEG modification of uricase CAI Lei, GAO Xiang-dong, ZHU Shu, WANG Hua, YAO Wen-bing' Sche iversity, Narjing 210009, China th PEG reagent in order to decrease uricase immunogenici and increase its bility. Methods: The branched PEG of 40 kD was chosen to modify native uricase. The properties of the mod- and temperature, in vivo half-life time, as well as the immu- the midofied uricase were studied in mice. Results: of Candida utilis uricase resulted in higher tryp alf-life. PEG modified uricase retained 80% of d that half-life in serum of the intravenously injec- native uricase of 45 min. Higher plasma drug con- cent e. Furthermore, the binding affinity was ISA assay, and it was one-eighth that of native uricase. Final- d a delayed immunoresponse in mice following repeated adminis- trations. Conclus chemically modified form of uricase may serve as a potentially effectiv Key words uricase; PEGylation; immunogenicity; half-life CLC Number Q55 Document code A Article ID 1000-5048(2008)06-0557-06聚乙醇修饰尿酸酶的研究才蕾,高向东,朱姝,王华,姚文兵(中国药科大学生命科学与技术学院,南京210009摘要目的:为了降低尿酸酶的免疫原性并提高其稳定性,利用PEG修饰剂对尿酸酶进行修饰,以期获得性质更优的治疗痛风的药物。方法:用相对分子质量为40kD,活化基团为羟基琥珀酰亚胺的PEG修饰剂对尿酸酶进行修饰,并比较修饰前后在酶解稳定性、pH稳定性及温度稳定性方面的差异,以及PEG修饰对体内半表期免疫原性的影响。结果:发现PEG修饰后尿酸酶的酶解稳定性显著提高,PEG化尿酸酶保留了原有尿酸酶80%的活性,体内半表期从45min延长至696min。PEg化尿酸酶与抗体的结合能力为原型蛋白的1/8。体内的免疫原性也明显降低。结论:化学修饰后的尿酸酶可望成为潜在的治疗痛风的有效药物。关键词尿酸酶;PEG化修饰;免疫原性;半衰期 Gout is becoming more and more common be-product of purine metabolism, excessive uric acid usu- cause of the growing consumption of purine-rich foods ally precipitate and its low solubility in serum is re- such as meat and alcoholic beverages in consumers' sponsible for the formation of crystal at bone joints, regular diet. Because uric acid is execreted as the end thereby resulting in gout. As a widely used drug, allo- Received date 2008-03-27 Corresponding author Tel: 86-25-83271218 E-mail: wbyao@ epu. edu. cn FOUNDATION ITEM This study was supported by the National Natural Science Foundation No. 30672559. No. 30772679), the National High Technology Research and Development Program of China(863'pro- gram)(No. 2007AA02Z101), Program for New Century Excellent Talents in University NCET-04- 0506)and Jiangsu Province Six Talent Peak Project558中国科大学学报 Journal of China Pharmaceutical University Vol.39 purinol, a purine inhibitor of enzyme xanthine oxi- peroxidase conjugated goat antimouse IgG and 0-Phe- dase, inhibits the formation of uric acid in vivo but was nylenediamine were obtained from Sigma. The DEAE- associated with serious side effects in clinical Sepharose, Sephacryl S-200 and Q-Sepharose were Uricase, catalyzing uric acid to a more solu- supplied by Pharmacia Biotech. All other reagents ble su ed as an ef- were of analytical grade. g pa- crease in vivo uric acid level in patients, its use mPEG re Structure of branched PEG N-hydroxysuccinimide 2 Purification of uricase Escherichia coli containing plasmid pBV220 ex ing Candida utilis uricase were cultured in LB medium at 37 C until Ago reached 1 and induced at the temperature 42 C. Harvest E. coli were centrifuged at use 0 r/min for 10 min. Next the precipitate was re- ed in Tris-Gly buffer (0. mol/L, pH 8.4) by sonication. Then it was centrifuged 0 000/min for 10 min. Supernatant was subjec- sulfate at concen- was then dia- fate and molecular cut in and elu- m borate buffer (pH 8.4) s were acid was ffer (pH f 59. 48 umol/L. Af- 293 nm was o 5 mg/mL purified H8.4 sodium borate ).The Branched PEG N-h (NHS-PEG, indicated as Figure 1) was purcha to Q-Sepharose from Nektar Huntsville, Al, USA). Uric acid. complete F Cl gradient. The eluate was Freund's adjuvant (IFA), 3, 5-di be ulfonic acid, 4-a peroxidase conjugated goat antirabbit IgG,horseradish ng the PEGylated No.6 CAI Le et al: Research on PEG modification of uricase559 uricase were pooled and purified by exclusion chroma- rated and diluted for later activity determination. One- compartmental pharmacokinetic modeling was per- 1.5 Electrophoresis formed. SDS polyacrylamide gel electrophoresis was per- 1. 11 Antigenicity activity formed according to the method of Laemmli on a slab ELISA was performed to determine whether PEG- gel containing 12% w/) polyacrylamide running gel ylated uricase shields the protein from antibody bind- and 4% (w/) stacking gel. The protein bands were ing. 96-well microplates were coated overnight with stained with commasim brilliant blue. 100 uL of uricase or PEG-uricase. The wells were 1.6 Kinetic analysis blocked with 10% FBS in PBS followed by incubation Steady-state kinetic measurements were per- with rabbit anti-uricase serum. Binding ability was de- formed in 0. mol/L sodium borate (pH 8.4)at 25 tected with HRP-labeled goat anti-rabbit IgG and f the substrate uric OPD was utilized for colorimetric detection. The plates 0.000 125%, 0.000 25%, 0.000 5%, were measured at an absorbance of 492 nm. 0.001% and 0. 002% w/). The kinetic paramet The amount of the adsorbed protein on the well surface was tate data. Turnover plate coated with uricase or the PE50lof uric acid solution(59.48 umol/L)was added. Then 100 uL of detection e was d in 37 C condi- Twenty male Balb/C mice were randomly divided1,2,3and4 amounts of uricase or the PEG ting) aken just be- fore treatment on days (predose) and after dose 8 15, 22, and 29. Blood samples were centrifuged for n at 3 500 r/min, and the separated serum was y diluted in PBS. Anti-native uricase antibodies ELISA. 2 Results 2.1 Preparation of PEG-uricase conjugates alyzed in Ba The purified uricase from Sephacryl S-200, with blood specific activity of 15 U/mg, exhibited purity of more samples were drawn at 10 min, 30 min, 1, 2, 4, 6. than 95% when detected by SEC-HPLC(Figure 3) and then it was used to PEG modification. After the fuged for 5 min at 3 500/min, and serum was sepa-reaction, the PEGylated uricase was purified by ion-560中国科大学学报 Journal of China Pharmaceutical University Vol.39 exchange chromatography. The PEGylation solution, ters of PEG-uricase and native uricase which contained PEG reagent, unreacted protein a(umo/L min)/( umol/L)/(/s) PEG-uricase, was separated by Q-Sep9.3128.42.47.45113.01.9 grapher. The PEGylated 2.3 Stability of PEG-uricase to protease In Figure 4, the activity of the PEG-uricase un- der the proteolysis condition was determined. PEGy- lation often increases protease resistance, It was found tion, the molar r n 2: to that PEGylation provide considerable protection effect 3: 1. it is concluded that every tetrameric uricase m to protease, especially, after 325-min incubation. ecule is conjugated to 2-3 PEG molecules on average. Native uricase retained 10% of the initial activity, while the activity of the PEG-uricase was 70% rela- tive to that before digestion. PEG-uncase kD97466210043380 Uricase Uncase604020120144°50100150200250300350 t/min Figure 2 SDS-PAGE of uricase and PEG-uricase I: Purified PEG-uricase: 2: Molecular mass marker; 3: Native uricase -O-Uricase;--PEG-uricase Flgure 4 Trypain resistance of uricase and PEG-uricase Figure 3 shows the SEC-HPLC result of tetramer- ic uricase and PEG-uricase. It tells that the tetramer. 2.4 Stability of PEG-uricase to various pH conditions ic uricase was eluted at 27. 112 min, while the elution The stability of the PEG-uricase to pH was indi time of PEG-uricase was 21. 964 min. PEG-uricase cated in Figure 5. PEG-uricase was more stable than showed a purity of >95% in this detection. that of native uricase at pH starting from 5.5 to 8. 5.120100 PEG-urica Uricase806040200102304034567891011 t/min Figure 3 SEC-HPLC of PEG-uricase (=27. 11 min) and o-Uricase: -PEG- uricase(=21.96 min) Figure 5 pH-stability of PEG-uricase 2.2 Kinetic parameters 2.5 Stability of PEG-uricase to temperatures Table 1 shows kinetic parameters profiles of the Themostability studies demonstrated that after in- PEG-uricase and uricase by measuring enzyme activi- cubation in specific temperature, the PEG-uricase re- ty. After conjugation, maximum velocity of the PEG- mained greater enzymatic activity relative to native uricase remained 80% of the initial value compared to uricase at temperature 20-60 C. However, when temper- native uricase and value was diminished a littleature increased to 65 C, the activity of PEG-uricase de- after the modification. creased more quickly than that of uricase(Figure 6). No.6 CAI Le et al: Research on PEG modification of uricase561120 standard antibody binding response at 0. 2-5 ug/mL,100 and the PEG-uricase showed standard antibody binding response at 0. 1-2 ug/mL. Considerable80 hed t amount of uricase or the PEG-uricase attached to mi-60 croplates, the best shielding resulted in 8-fold reduc-40 tion in antibody affinity to the PEGylated uricase at200.1g/mlit as indicated that a tibody binding to the 40 kD conjugate was sufficiently reduced in com-420304050607080T℃ parison to native uricase. Its combination with the re- tained enzyme activity and the improvement in phar- --PEG-Uricase: --Uricase Figure 6 Thermostability of native uricase and PEG-uricase macokinetic profile, making PEG-uricase an attractive candidate for a new derivative of uricase. 2.6 Prolonged serum drug half-life of PEG1.6 Pharmacokinetic profiles of the PEG-uricase in1.2 determined and compared to that of native0.8 The concentration-time curves of native the PEG-uricase were generated as serum0.4 (%)versus time after a single admin- istration at dose of 50 ug per mouse. Figure7 shows000.51.01.52.02.5 c(Antigen)/(ug/mL) that native uricase was eliminated rapidly from the -0-Uricas: --PEC-Uricase mice had a half-life of 45 min and PEGylated uricase Figure 8 Determination of antigenicity of uricase and PEGylated has a half-life of 696 min. Pharmacokinetic parameters Uricase. were listed in Table 2. 2. 8 Reduced immunogenic activity120 Repeated administration of this therapeutic pro-100 tein would elicit immune response no matter PEG-80 uricase or native uricase was used. In a month,60 uricase and the PEG-uricase were repeatedly adminis-E40 tered to mice at weekly interval, and in vito anti-20 uricase antibody formation was monitored. Native40080012001600 uricase was found to elicit rapid antibody levels whereas PEG-uricase stimulated a significantly lower immunoresponse. Figure indicates that the PEG- --PEG-Uiase:--Unicase Figure7 Plasma concentration-time profile of uricase and PEG-uricase uricase consistently elicited low antibody production relative to native uricase, strongly suggesting that PEG Table 2 Pharmacokinetic parameters of PEG-uricase and uricase in conjugates effectively lower in vivo immune reaction. Balb/C mice (=6)100000 ameter Uriease PEG-uriease0.8581.1471000 AUC/(. min/mL)74861.813.661.161000(min)45696100 2.7 Anti-uricase antibody activity for PEG-uricase1 The ability of PEG to shield uricase from anti- bodies was tested in vitro with immunoassay(Figure8152229 8). To examine the antigenic properties of the sam- PEG-uricase uricase ples, different amounts of antigen were incubated with Flgure9 Immunological time coure of native uricase and PEG-uricase rabbit anti-uricase antiserum. Native uricase showed a562中国科大学学报 Jourmal of China Pharmaceutical University Vol.39 In conclusion, the data reported here are in fa- 3 Discussion vour of PEG conjugation for an improved use of PEG modification has long been regarded as an uricase in treatment of gout. Especially, the prolonged effective method to reduce immunogenic reaction and half-life and the reduced immune response make it an other properties of protein drugs. Modification attractive candidate in clinical utility. h 40 kD banched PEG exerted little Refe 80% activity of nativ It can be explained [1] Bardin T. Currer ive or ergic to allopurinol )] Joint Bone Spine, 2004.71 (6):481 aS-485.2】 Nuki.t []. Rheum Dis Clin North Am, 2006,32(2):333-357. [3] Brogard JM, Coumaros D, Frankhauser J, et al. Enzymatic uricoly. sis: study of the effect of a fungal urate-oxydase J]. Rev Eur Etud Clin Biol ,197217(9):890-895 [4] Schellekens H. eity of therapeutic proteins: clinical [J]. Clin Ther, 2002,24(11): [5] Roberts MJ, Bentley MD, Harris JM. Chemistry for peptide and protein PEGylation )J]. Adr Drug Deliver Rer, 2002 54(4): 4596] dRB, Choe YH, McGuire,et al. Effective drug deliver- y by PEGylated d []. Ade Drug Deliver Rev,2003,5(2):217-250. h to dnug delivery]. Drug Discou Today,2005,10(21):1451-1458. 8] Robert, Chen L, Abuchowski A, et al. Properties of two urate attachment of poly (ethylene1981,660(2):293-298 effect of].ane,1981,2 pegylated proteins ic 96-well plate assay for the d of urate oxidase activi- body production was greatly reduced after ty and its kinetic parameters [)] Anal Biochem, 2002, 309(2): tion of PEG-conjugated uricas instead of native173-179 urica