聚乙二醇修饰尿酸酶的研究

中圃昙开火事学报Journal of China Pharmaceutical University 2008,39(6):557-562Research on PEG modification of uricaseCAI Lei, GAO Xiang-dong, ZHU Shu, WANG Hua, YAO Wen-bingSchool of Life Science and Technology, China Pharmaceutical University, Nanying 210009, ChinaAbstract Aim: To modify uricase with PEG reagent in order to decrease uricase immunogenicity and increase itsstability. Methods: The branched PEG of 40 kd was chosen to modify native uricase. The properties of the mod-ified uricase including the stabilities to protease pH and temperature, in vivo half-life time, as well as the immu-nogenicity were evaluated. The pharmacokinetic profiles of the midfied uricase were studied in mice. ResultsIt is demonstrated that the conjugation of PEG to lysine residues of Candida utilis uricase resulted in higher tryp-sin resistance, reduced immune response, and prolonged in vivo half-life. PEG modified uricase retained 80% ofthe enzymatic activity of native uricase. In addition, it was found that half-life in serum of the intravenously injec-ted PEGylated uricase of up to 696 min was longer thatthat of native uricase of 45 min. Higher plasma drug con-centrations were also reached with dosing of the PEGylated uricase to mice Furthermore, the binding affinity wasshown to be reduced for the PEG-uricase using ELISA assay, and it was one-eighth that of native uricase. Finally, it was indicated that the PEG uricase induced a delayed immunoresponse in mice following repeated adminis-trations. Conclusion: These findings demonstrate that this chemically modified form of uricase may serve as aally effective drug to treat gout paKey words uricase; PEGylation; immunogenicity; half-lifeCLC NumberDocument code A Article ID 1000-5048(2008)06-0557-06聚乙二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京2000)摘要目的:为了降低尿酸酶的免疫原性并提高其定性,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、阳H穗定性及温度稳定性方面的差异,以及PEG修饰对体内半衰期,免疫原性的彩响。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,体内半衰期从45min延长至696min。PEG化尿酸酶与抗体的结合能力为原型蛋白的1β8。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疔痛风的有效药物。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 jointsregular diet. Because uric acid is excreted as the end thereby resulting in gout. As a widely used drug, allo-Received date 2008-03-27 Corresponding author Tel: 8683271218E-mail:wbvao@cnu.edu.cnFOUNDATION ITEM This study was supported by the Natio中国煤化工。306725930772679), the National High Technology ReCNMHGChina(“863gram)(No. 2007 AA02Z101), Program forEXceuen tents in university(NCET-04-0506)and Jiangsu Province Six Talent Peak Project中■暴普火事学报 Journal of China pharmaceutical UniversityVol 39purinol, 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 wereuse!-2. Uricase, catalyzing uric acid to a more solu- supplied by Pharmacia Biotech. All other reagentsble substance-allantoin has been developed as an ef- were of analytical gradefective drug such as Eliketto treat gout-suffering pa-tients. Although uricase is an effective agent to demPEG\ ocrease in vito uric acid level in patients, its use ismPEGgreatly limited because of high immunological reactiondue to its foreign protein characterFigure 1 Structure of branched PEG N-hydroxysuccinimideTo avoid or reduce foreign protein immunogenic- 1. 2 Purification of ity, various methods, including PEGylation, site-specif-Escherichia coli containing plasmid pBV220 exic mutagenesis, co-expression with fusion proteins pressing Candida utilis uricase were cultured in LBhave been exploited". In reality, PEG modification is medium at 37C until Agoo reached 1 and induced atthe most popular and effective method in practical temperature 42C. Harvest E coli were centrifuged at8000 r/min for 10 min. Next the precipitate was re-In order to reduce the immunological response suspended in Tris-Gly buffer(0. I mol/L, pH 8. 4)caused by this enzyme, the native uricase was chemi- and processed by sonication. Then it was centrifugedcally modified with PEG. When modified with PEG at 10 000 r/min for 10 min. Supematant was subjec-5 kD, Candida utilis uricase containing 32 lysine resi- ted to be precipitated by ammonium sulfate at concen-dues retained 23% of the original activity with 57% tration of 30%-60%. The precipitate was then dia-modification ratel). While, according to another lyzed to remove ammonium sulfate and molecular cutsource of data, 71% modification rate brought out off of the dialysis membrane was 3 kD. The crude ex-11% activity retain. It was speculated that PEGylation tract was loaded on DEAE-Sepharose column and eluwith low molecular weight would greatly affect enzyme ted with 20 mmol/L sodium borate buffer(pH 8. 4)activity due to conformational change caused by high and 0-0. 3 mol/L NaCl, and effective fractions weremodification rate. In this study, branched PEG 40 kD collected. To obtain further purified uricase, Sephacrwas adopted to be conjugated to primary amino groups yl-S 200 column(1 cm x 100 cm) was used.of uricase with an amide linkage.1.3 Determination of uricase activityHere, the aim was to examine the effect of PEGyTo detect uricase activity, at 25C, uric acid waslation on chemical properties, pharmacokinetic profile dissolved in 0. 1 mol L, sodium borate buffer ( phand immunogenic properties of uricase. In detail, tryp- 8. 4)at the final concentration of 59. 48 umol/ L. AfpH and temperature stability of PEG-uricase was ter the enzyme was added, absorbance at 293 nm wasexamined and in vivo pharmacokinetic behavior was monitored at 30 s intervals for 5 minstudied following dosing of the PEG-uricas to mice. In 1.4 Preparation and purification of PEG-uricaseaddition, in vitro antibody binding ability and in vivoNHS-PEG was added to 5 mg/mL purifiedantigenic reaction were studied in this research.uricase solution(0. 1 mol/L, pH 8. 4 sodium borate1 Materials and methodsbuffer) with molar ratio of 4: 1( PEG: uricase). Thereaction mixture was gently stirred for 1 h at 4C, and1.1 Materialswas terminated by adding 10 mmol/L glycine.Branched PEG N-hydroxysuccinimide 40 kDReaction mixture was diluted with 20 mmol/LNHS-PEG, indicated as Figure 1)was purchased sodium borate(pH 8. 4)and rendered to Q-Sepharosefrom Nektar( Huntsville, Al, USA). Uric acid, tryp- column that was eluted with 20 mmol/L sodium boratesin,complete Freunds adjuvant( CFA), incomplete (pHN灬C~:^ t. The eluateFreund's adjuvant( IFA), 3, 5-dichloro-2-hydroxy. anal中国煤化工 or protein con-benzene sulfonic acid, 4-amino-antipyrine, horseradish tent,CNMH Gemination o, andperoxidase conjugated goat antirabbit IgG, horseradish enzyme activity Fractions containing the PEGylatedNo 6CAI Le ef al: Research on PEG modification of uricaseuricase were pooled and purified by exclusion chroma- rated and diluted for later activity determination.1.5 Electrophformed.SDS polyacrylamide gel electrophoresis was1. 11 Antigenicity activityformed according to the method of Laemmli on aELISA was performed to determine whether PEGgel containing 12%(w/v) polyacrylamide running gel ylated uricase shields the protein from antibody bindnd 4%(w/v)stacking gel. The protein bands were ing 96-well microplates were coated overnight withstained with commasim brilliant blue100 FL of uricase or PEG-uricase. The wells were1.6 Kinetic analysisblocked with 10% FBS in PBS followed by incubationteady-state kinetic measurements were per- with rabbit anti-uricase serum. Binding ability was de-formed in 0. 1 mol/L sodium borate(pH 8. 4)at 25 tected with HRP-labeled goat anti-rabbit IgG andC, by varying the concentration of the substrate uric OPD was utilized for colorimetric detection. The platesacid at 0. 000 125%, 0. 000 25%,0.000 5%, were measured at an absorbance of 492 nm.0.001% and 0. 002%(1/0). The kinetic parametersThe amount of the adsorbed protein on the wellK and K were calculated by non-linear regression surface was estimated as follows": To 96-well micro-plate coated with uricase or the PEG-uricase, 50 uL ofnumbers were calculated on the basis of one active uric acid solution(59. 48 umol/L) was added. Thensite per 34 kDa subunit.100 uL of detection mixture containing 0. 2 mov/L1. 7 Stability to proteolytic digestionsodium phosphate(pH 6), I mmol/L 3, 5-dichloro-2Samples of native uricase and PEG-uricase(0hydroxybenzene sulfonic acid. 0. 25 mmol/L 4-amino-mg/mL) in I mL of 0. 02 mol/L, pH 8. 4 sodium bo. antipyrine, and 25 U/mL horseradish peroxidase wasrate were incubated at 37oC with 1. 8 mg of tryp added, and microplate was placed in 37C condisin. Aliquots were taken at the intervals and assayed tion. Absorbance at 510 nm was recorded.for residual enzymatic activity1.12 Immunogenicity evaluation1.8 Stability to pHTwenty male Balb/C mice were randomly dividedNative uricase and the PEG-uricase (0.1 into 2 groups and immunized on weeks 1, 2, 3 and 4mg/mL)were dissolved in the following buffers: 0. 1 with 8 Ag of native uricase or equimolar amounts ofmol/L sodium acetate(pH 3. 6-5. 2),0. 1 mol/L so- PEG-uricase, respectively. The immunizing solutionsdium phosphate( pH 5.6-7.2),0. 1 molL sodiwere prepared by dissolution of uricase or the PEGborate(pH 7. 68. 4)and 0. 1 mol/ L glycine- NaOH uricase in 50 uL of PBs and 50 ul of Freunds adju-(pH 8.8-10. 4). After 24-h incubation, pH of the vant( complete adjuvant was used in the first immtmixture was adjusted to 8. 4 and enzyme activities zation; incomplete adjuvant was used for boosting)ons were intraperitoneally injected, and1.9 Stability to temperatureblood for antibody determination were taken just beThermal stability of native uricase and the PEG- fore treatment on days I(predose)and after dose 8uricase were evaluated on the basis of the residual en- 15, 22, and 29. Blood samples were centrifuged forzyme activity of protein samples(0. 2 mg/mL in 0. 1 5 min at 3 500 r/min, and the separated serum wasmol/L, pH 8. 4 sodium borate) heated for 30 min at properly diluted in PBS. Anti-native uricase antibodiestemperatures 4, 20, 30, 40, 50, 60, 70 and 80 C, re- in serum were estimated by ELISA.spectively, and cooled to room temperature.2 Resul1. 10 Half-life determination of pEg-uricase in vitHalf-life of uricase and the PEG-uricase were an- 2. 1 Preparation of PEG-uricase conjugatesalyzed in Balb/C mice(n=6).50 ug of the purifiedThe purified uricase from Sephacryl S-200, withsamples were injected into the tail vein. The blood specif中国煤化工 purity of moresamples were drawn at 10 min, 30 min, 1, 2, 4, 6.8 thanIPLC (Figure 3and 24 h after injection. Blood samples were centri- andCNMHication. After thefuged for 5 min at 3 500 r/min, and serum was sepa- reaction, the PEGylated uricase was purified by ion560中圃暴科火事学报 Journal of China Pharmaceutical UniversityVol 39exchange chromatography. The PEGylation solution, Table I Kinetic parameters of PEG-unicase and native unicawhich contained PEG reagent, unreacted protein andmplV/(μmouL·min)k-(uno/L)k/(L/)PEG-uricase, was separated by Q-Sepharose chromato-Uricase128.4PEG-uricase7.45113.0The PEGylated uricase eluted firstly, followed byun PEGylated uricase. Figure 2 tells that under this de- 2.3 Stability of PEG-uricase to proteaseined reaction condition, after purification, there is on-In Figure 4, the activity of the PEG-uricase un-ly mono-PEG modified conjugate in SDS-PAGE analy- der the proteolysis condition was determined. PEGysis. And according to protein and PEG content deteclation often increases protease resistance, It was foundtion, the molar ratio of PEG: protein is between 2: 1 to that PEGylation provide considerable protection effect3: 1. it is concluded that every tetrameric uricase molto protease, especially, after 325-min incubationNative uricase retained 10% of the initial activity,ecule is conjugated to 2-3 PEG molecules on average. while the activity of the PEG-uricase was 70% rela-tive to that before digestion.PEG-uncaseUricase004014450100150200250300350Figure 2 SDS-PAGE of uricase and PEG-uricaseI: Purified PEG-uricase: 2: Molecular mass marker: 3: Native uricase-o-Unicase: -.-PEG-uricaseFIgure 4 Trypsin resistance of uricase and PEG-uricaseFigure 3 shows the SEC-HPLC result of tetramer-uricase and PEG-uricase. It tells that the tetramer- 2.4 Stability of PEG-uricase to warious pH conditionsic uricase was eluted at 27. 112 min, while the elutionThe 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 thanshowed a purity of >95% in this detectionthat of native uricase at pH starting from 5. 5 to 8.5.Uricase000030Figure 3 SEC-HPLC of PEG-uricase(4R=27 11 min) and native -o-Unicase:-.-PEG-uricase2. 5 Stability of PEG-uricase to temperaturesTable I shows kinetic parameters profiles of theThermostability studies demonstrated that after in-PEG-uricase and uricase by measuring enzyme activi- cubation in specific temperature, the PEG-uricase re-ty.njugation, maximum velocity of the PEG- me中国煤化工icase remained 80% of the initial value compared to ucasever, when temper-native uricase and K value was diminished a little atureCN MH GPEG-unicase de-after the modification.creased more quickly than that of uricase( Figure 6)No 6CAI Le et al Research on PEG modification of uricasestandard antibody binding response at 0. 2-5 ug/mL,and the PEG-uricase showed a standard antibodyamount of uricase or the PEG-uricase attached to microplates, the best shielding resulted in 8-fold reduction in antibody affinity to the PeGylated uricase at0. 1 ug/mL It was indicated that antibody binding tothe 40 kD conjugate was sufficiently reduced in com-2030405060708parson to native uricase. Its combination with the re-tained enzyme activity and the improvement in phar-igure 6 Thermostability of native uricase and PEG-uricasemacokinetics profile, making PEG-uricase an attractivecandidate for a new derivative of uricase.2.6 Prolonged serum drug half-life of PEG-ylated uricasePharmacokinetic profiles of the PEG-uricase inmice were determined and compared to that of nativeuricase. The concentration-time curves of nativeuricase and the PEG-uricase were generated as serumenzyme activity(%) versus time after a single administration at dose of 50 ug per mouse. Figure 7 shows05101.5202.5that native uricase was eliminated rapidly from themice had a half-life of 45 min and PEGylated uricase --Unicase--PEG-UrncaseFigure 8 Deterof antigenicity of uricase and PEGylatedhas a half-life of 696 min. Pharmacokinetic parameterwere listed in Table 22.8 Reduced immunogenic activityRepeated administration of this therapeutic pro-tein would elicit immune response no matter PEG-uricase and the PEG-uricase were repeatedly adminis-tered to mice at weekly interval, and in vivo antiuricase antibody formation was monitored. Nativeuricase was found to elicit rapid antibody levels12001600whereas PEG-uricase stimulated a significantly lowerimmunoresponse Figure 9 indicates that the PEG-. -PEG-Uricase:-o-UnicaseFigure 7 Plasma concentration-timeof unicase and PEG-uricaseuricase consistently elicited low antibody productionrelative to native uricase strongly suggesting that PEGTable 2 Pharmacokinetic parameters of PEG-uricase and uricase inBalb/C mice(n=6)conjugates effectively lower in vivo immune reaction.ParameterPEG-uricasee/(U/mL)0.858000AUC/(U. min/mL)cL(μLmin)13.661.16三1002. 7 Anti- uricase antibody activity for PEG-uricaseThe ability of PEG to shield uricase from anti-bodies was tested in vitro with immunoassay( Figure中国煤化工8. To examine the antigenic properties of the sam-CNMHGSples, different amounts of antigen were incubated with Flgureume course ot native uricase and PEG-uricaserabbit anti-uricase antiserum Native uricase showed a中暴并火学报 Joumal of China Pharmaceutical UniversityIn conclusion, the data reported hein fa-3 Discussionvour of PEG conjugation for an improved use ofPEG modification has long been regarded as an uricase in treatment of gout. Especially, the prolongedffective method to reduce immunogenic reaction and half-life and the reduced immune response make it animprove other properties of protein drugs. Modification attractive candidate in clinical utilityof uricase with 40 kd benched PEG exerted littleeffect on enzyme activity, and PEG-uricase retained80% activity of native uricase. It can be explained [1] Bardin T Current management of gout in patients unresponsive orthat higher hindrance effect prevents PEG to reach theallergic to allopurinol[J3. Joint Bone Spine, 2004, 71(6): 481active site of uricase, so its activity remains a lot. Thisproperty makes this conjugation a proper candidate for [2] Nuki C Treatment of crystal arthropathy-history and advancegout therapy for PEGylation probably leads to great[J]. Rheum Dis Clin North Am, 2006, 32(2): 333-357activity decrease, while this PEG-uricase conjugate re-[3]Brogard JM, Coumaros D, Frankhauser J, et al. Enzymatic uricoly-tained most enzyme activity.sis:a study of the effect of a fungal urate-oxydase[ J]. Reo EurIn the proteolysis study, with the time progresstud Clin bio,192,17(9):890-895ng, differences in activity between uricase and PEG[4 Schellekens H Immunogenicity of therapeutic proteins: clinicalimplications and future prospect J]. Clin Ther, 2002,24(11)uricase become greater. The target site of protease di-gestion is usually the peptide bond with lysine or1720-1740.[5] Roberts M], Bentley MD, Harris JM. Chemistry for peptide andarginie on the opposite of N-terminal, therefore, proteprotein PEGylation[ J ]. Ade Drug Deliver Rew, 2002, 54(4): 459ase digestion is a proper standard to research PEGyla476.on. This suggests that PEG provides efficient protec[6]Greenwald RB, Choe YH, McGuire J, et al. Effective drug delivetion to uricase and will help prolong its in vivo hay by PEGylated drug conjugates[ J]. Adr Drug Deliver Rev, 2003lifeModification of uricase, an intrinsic high immu- [7] Veronese FM, Pasut G. PEGylation, successful approach to drugnogenicity protein, possesses obviously reduced bind-delivery[J]. Drug Discov Today, 2005, 10(21): 1 451-1 458ing activity with anti-uricase serum. The PEG moiety [8] Robert H, Chen L, Abuchowski A, e ad Properties of two urateappears to be responsible for the shielding of antigenoxidases modified by the covalent attachment of poly( ethylenesite from antibody. assay of binding capacity of antiglycol)[J]. Biochim Biophys Acta, 1981, 660(2): 293-298.serum with samples, native uricase showed 3 to 8 fold [9] Davis S, Part YK Abuchowski A, et al. Hypouricaemic effect ofhigher affinity than PEG-uricase. Because of possiblepolyethylene glycol modified urate oxidase[J]. Lancet, 1981, 2serious results arising from severe allergic response,reduced efficacy of therapeutic proteins and induce of [10] Gong XW, Wei Dz, He ML, et al. Discarded free PEG-based asautoimmunity to endogenous proteins caused by imsay for obtaining the modification extent of pegylated proteinsmune response, protein immunogenicity should be de[]. Talanta,2007,71(1):381-384[11] Fraisse L, Bonnet MC, de Farey JP, et aL A colorimetric 96-wellcreased to a proper level. Our study showed that arbody production was greatly reduced after administra-ty and its kinetic parameters[ J]. Anal Biochem, 2002, 309(2):tion of PEG-conjugated uricas instead of native中国煤化工CNMHG