【作者】 王静；

【导师】 文爱东； 吴寅；

【作者基本信息】 第四军医大学 ， 药剂学， 2009， 硕士

【摘要】 【背景】注射用重组人CTLA4-抗体融合蛋白（rhCTLA4-Ig）是目前世界上第一个选择性共刺激调节剂,它通过与CD80和CD86结合,阻断CD28与之结合,从而抑制T淋巴细胞激活;CD28与CD80和CD86的相互作用,为T淋巴细胞的完全激活提供必要的共刺激信号。T淋巴细胞的激活与类风湿关节炎（RA）的病理进程相关,在RA患者的关节滑液中可以找到激活的T淋巴细胞。本药适用于治疗一种或一种以上改善病情的抗风湿药（DMARDs）治疗无效的中、重度活动性RA的成年患者,可以减轻RA患者的临床症状、延缓关节结构损伤,并改善其生理功能。RhCTLA4-Ig是采用重组DNA技术生产的,是由一种人细胞毒性T淋巴细胞相关抗原4（CTLA4）胞外区和人免疫球蛋白G1（IgG1）修饰的Fc片段（铰链区、CH2和CH3）相连接的融合蛋白。根据我国食品药品监督管理局（SFDA）通过《药品注册管理办法》中界定的生物制品分类,rhCTLA4-Ig属于治疗用生物制品第7类新药,其同类产品在国外已经上市销售[1],但在国内尚无此类药物。本实验室已经完成了rhCTLA4-Ig I期临床健康人体耐受性[2]和健康人体药代动力学试验[3],为进一步考察本药物在RA患者体内药物代谢动力学规律、了解该药物多次给药后药代动力学改变过程,是否在RA患者体内发生改变、是否存在药物蓄积作用等。因此我们进行rhCTLA4-Ig在RA患者体内多次给药药代动力学研究。【目的】建立酶联免疫吸附法（ELISA）测定RA患者体内血清中rhCTLA4-Ig浓度的方法;进行多次静脉滴注rhCTLA4-Ig在RA患者体内的药代动力学研究;比较健康志愿者与RA患者药物代谢动力学规律以及临床初步疗效;为指导临床制定安全、合理的用药方案提供理论依据,为新药审批和临床用药提供试验依据,也为II期临床研究提供参考依据。【方法】本研究共分三部分。第一部分建立特异性强、灵敏度高、准确度和精密度良好ELISA法测定RA患者血清中rhCTLA4-Ig浓度分析方法学;通过对方法特异性、精密度和准确度、稳定性、线性范围以及定量检测限等效能指标的考核并对方法学进行评价。用稀释液将待检测样品稀释至标准曲线的定量检测范围,即用稀释液将待测样品以2倍序列稀释至200μg·L^-1到3.125μg·L^-1;将配备的酶标板已预先用鼠抗人sCTLA4单抗包被好;测定前先用洗涤液清洗3次,然后加入标准品/待检样品与生物素标记二抗,孵育2h,洗涤3次;加入HRP-亲和素,孵育1h后,洗板3次,加入TMB显色溶液,避光显色10min后终止,以450 nm为检测波长,630 nm为参比波长测定光密度值。以浓度对数为横坐标,光吸收值对数值为纵坐标进行线性回归设置标准曲线,用回归的标准曲线计算血清样品里药物的浓度,经稀释倍数校正后求得血清的最终浓度。第二部分研究多次静脉滴注10mg·Kg^-1 rhCTLA4-Ig在RA患者体内药代动力学特征。本试验经国家食品药品监督管理局批准、第四军医大学西京医院伦理委员会成员一致通过,经过严格入选确定9例RA患者进行试验,在试验开始前均自愿签属知情同意书。按试验设计要求,受试者均符合美国风湿病协会1987年提出的诊断标准[4]确诊为RA患者;采用国外已上市产品Orencia临床给药方案[1],于第0,2,4,8,12,16周静脉滴注10mg·kg^-1 CTLA4-Ig,输液在60±5分钟内完成,并在每次静脉滴注前和静脉滴注结束后即刻,并在最后一次滴注前和滴注后第0、2、4、12、24h、2、3、4、7、14、28、42、56、70和84天取血2mL,置于预先已经贴好标签并无菌干燥试管内,2200×g离心分离血清,置-80℃保存待测。试验中随时观察受试者症状、体征以及各种不良反应,并定期进行实验室检查:血尿常规、肝肾功能、血液生化及血液电解质变化等,综合评价rhCTLA4-Ig在RA患者体内的药动学特征,整个试验期间配备有经验的医师和护士监护。采用ELISA法测定血清中rhCTLA4-Ig的浓度,T max、Cmax采用实测值,AUC用统计矩方法计算,采用DAS2.1软件计算主要药代动力学参数,所有计量资料均采用Excel或SPSS软件进行计算,以?X±SD表示,并进行t检验或方差分析。第三部分RA患者多次给药后药代动力学参数与同等剂量健康志愿者单次给药药代动力学参数的比较;评价RA患者的初步临床疗效。将RA患者多次给药后药代动力学参数与健康志愿者单次给药药代动力学参数进行比较;RA患者试验前后关节疼痛个数及免疫学指标进行比较,均采用t检验进行分析,研究其规律和差异。为指导临床制定安全、合理的用药方案提供理论依据,为新药审批和临床用药提供试验依据,并为II期临床研究提供参考依据。【结果】1.本试验采用ELISA法测定RA患者血清中rhCTLA4-Ig的浓度,最低定量限6.25μg·L-1,检测浓度范围3.125～200μg·L^-1,在6.25～100μg·L^-1范围内重现性良好,准确度在100.4～112.1%之间,精密度RSD%在0.73～4.6%之间,各项指标均达到药代动力学研究的要求。2.入选10例RA患者,男3例,女7例;其中因右束枝传导阻滞剔除1例;给药结束后观察期因受试者自身原因脱落1例,全部受试者均无严重不良事件脱落。试验期间,受试者连续多次静脉滴注本药物后均未有严重不良事件发生;呼吸、心率、血压、脉搏、体温等生命体征未见异常;心电图、胸片前后均正常。试验期间进行血液生化指标、尿常规、电解质指标检测,每次与试验前比较未发现有临床意义上的改变,检查结果虽然有阳性指标,经统计分析t检验,p>0.05,无临床意义。注射药物局部未发现皮肤红肿、瘀斑或皮疹等刺激性反应。试验观察期间出现轻度不适反应:颜面潮红2例、瞬间眩晕1例、视力下降1例、口干1例,均未使用其它药物自行痊愈。3. RA患者于第0,2,4,8,12,16周连续静脉滴注CTLA4-Ig10mg·kg^-1,随着给药次数增加,血药浓度不断增加,到第8周时基本达到稳态水平。达到稳态时基本药动学参数为:最后一次测得稳态时峰浓度Cssmax为247.1±50.34mg·L^-1,稳态时谷浓度Cssmin为19.7±9.5mg·L^-1,平均稳态浓度Css?av为63.4±25.mg·L^-1,AUCss为1830±708mg·d·L^-1,波动系数DF为3.48±1.61。达到稳态后基本药动学参数为:达峰时间Tmax为0.063±0.039d,峰浓度Cmax为247.1±50.3mg·L^-1, t1/2为12.6±4.7d,AUC（0-t）为2347±901mg·d·L^-1,AUC（0-∞）为2362±906mg·d·L^-1,清除率CL为0.0050±0.0023L·d·kg^-1,表观分布容积Vss为0.074±0.031L·kg^-1。4.比较RA患者和健康志愿者的药动学参数,RA患者的半衰期t1/2为12.6±4.7d,健康志愿者为14.2±2.3d,但是差异无统计意义（p>0.05）。RA患者清除率CL为0.0050±0.0023L·d·kg^-1 ,健康志愿者为0.0067±0.0010L·d·kg^-1（p>0.05）。RA患者表观分布容积Vss明显比健康人小,为0.074±0.031L·kg^-1,而健康者为0.138±0.033 L·kg^-1。根据美国风湿病学会的评价标准进行评判,第3次给药后,给药前和给药后的临床症状有所改善,关节改善达到20%（ADR20）为78%,ACR50有效率分别为11%。第6次给药后临床症状进一步改善,ADR20有效率分别为100%,ACR50有效率分别为78%。试验期间进行免疫学指标血沉、C反应蛋白、类风湿因子指标检测,均与试验前比较,结果显示各项指标均有所下降。血沉平均降低16.67±19.83 mm·L^-1;C反应蛋白平均降低204.41±300.91 IU·mL^-1;类风湿因子平均降低1.55±2.30 mg·L^-1,但经t检验,p>0.05,无临床意义。【结论】1. ELISA分析方法具有特异性强、灵敏度高、准确度和精密度良好的特点。高、中、低三种浓度的质量控制样品在盒内、盒间精密度的变异均小于10 %,符合生物样品分析要求;三种浓度的质控样品分别置于4℃26小时,室温20小时,-80℃9个月稳定性良好,表明该方法适合定量分析测定RA患者体内血清中rhCTLA4-Ig浓度,全部指标均符合2005年版的《中国药典》以及SFDA颁布的《化学药物临床药代动力学研究技术指导原则》关于进行药动学研究的试验要求。2. RA患者多次静脉滴注rhCTLA4-Ig后,RA患者连续6次静脉滴注rhCTLA4-Ig 10mg·kg^-1后,随着给药次数增加血药浓度不断增加,到第4次基本达到稳态水平;每次给药后血药浓度迅速达到峰值,但消除缓慢,在体内不存在蓄积现象。本研究得到的主要药代动力学参数与国外文献报道基本相同。对肝、肾功能、血常规、尿常规和血液生化检查均无明显影响,安全性良好。因此我们推荐治疗国人RA患者,rhCTLA4-Ig的给药剂量为10 mg·Kg^-1,给药方式为静脉滴注,于wK 0、2、4给药一次,以后每4 wK给药1次,该方案在临床推广使用。建议临床按此方案用药时,使用时间不宜过长,以减少药物不良反应。3.静脉滴注rhCTLA4-Ig 10mg?Kg^-1后,药物在RA患者和健康人体内血药浓度迅速达到最大值后缓慢清除。RA患者与健康人有所区别的是,前者有半衰期缩短而清除率增加的趋势,但无统计学差异。本试验药物有明显改善血沉和C反应蛋白的作用,对RA的症状有所缓解。更多还原

【Abstract】 【Background】Recombinant human Cytotoxic T Lymphocyte associate Antigen-4 Fusion Protein （rhCTLA4-Ig）, a selective costimulation modulator, inhibits T cell （T lymphocyte ） activation by binding to CD80 and CD86, thereby blocking interaction with CD28. This interaction provides a costimulatory signal necessary for full activation of T lymphocytes found in the synovium of parients with RA. It is indicated for reducing signs and symptoms, slowing the progression of strural damage, and improving physical function in adult patients with moderately to senerely active rheumatoid arthritis who have had an inadequate response to one or more DMARDs. RhCTLA4-Ig is produced by recombinant DNA technology, and a fusion protein combining human cytotoxic T lymphocyte-associated antigen 4 （CTLA4） and the extracellular region of human immunoglobulin G1（IgG1）modified Fc fragment （hinge region, and CH2 zone CH3）. According to the regulations of drug registration issued from State Food and Drug Administration（SFDA）,rhCTLA4-Ig belongs to the national therapy new drug,class seven, namely, has been on market on aboard but not yet in China. Our Phase I clinical trial of this new drug is to assess the clinical tolerance and pharmacokinetics in healthy volunteers, and the in Chinese rheumatoid arthritis patients.【Objectives】The aim of present study is to establish an Enzyme-linked immunoassay assay （ ELISA ） method to monitor the concentration of rhCTLA4-Ig in rheumatoid arthritis patients blood serum samples, to assess clinical pharmacokinetics after successive intravenous injection of rhCTLA4-Ig in rheumatoid arthritis patients, and provide safety and reasonable administration for Phase II clinical trial.【Methods】This study comprises the following three parts:The first part was to establish an ELISA method with high specificity, high precision and sensitivity for rhCTLA4-Ig detection in rheumatoid arthritis patients’blood serum. An ELISA blood serum with high precision,high specifity and sensibility was validated for rhCTLA4-Ig determination in blood samples. RSD of inter-plate precision and intra-plate precision,selective and ruggedness to proved whether it could study the pharmacokinetics of rhCTLA4-Ig. Procedure: CTLA4 monoclonal mouse anti-human antibody was diluted with a proportional buffer and added to presidium on the enzyme scale plate. The enzyme scale plate was then incubated at 4℃and closed by buffer at room temperature. The plate was washed with detergent solution. The standards of the different concentration and the diluted samples were loaded to the plate. The diluted biotin-labeled anti-secondary antibody was then immediately added. Enzyme scale plate was placed, shocked and incubated for 2 hours at room temperature. After incubation with the HRP-labeled enzyme linked avidin, enzyme scale plate placed and shocked, incubated at room temperature, kept from light. Upoun termination of reaction absorbance （A） values was read at 450 nm by enzyme scale equipment, results were recorded. Duplicate experiments were carried out, and a set standard curve for every kit was used to measure concentration of unknown samples.The second part was to study the pharmacokinetics of recombinant human Cytotoxic T Lymphocyte associate Antigen-4 Fusion Protein injection after multiple-dose administration to Chinese rheumatoid arthritis patients. 9 subjects voluntarily signed an informed consent agreement that was in compliance with Chinese Food and Drug Administration regulation and approved by the Ethic Committee of Xijing Hospital of the fourth military medical university.According to the experimental design,9 subjects were food forbidden for 12 hours and blood sampled just before drug administration and then at 0、2、4、12、24h、2、3、4、7、14、28、42、56、70、84 days after drug administration. Plasma samples were immediately centrifuged at 3,600 rpm for 10 min at 4℃, and then stored at -80℃until analysis. RhCTLA4-Ig was extracted from blood samples and quantified by ELISA. The T max and C max were actual data,and the AUC calculated by statistical moment methods and other pharmacokinetic parameters by software Origin. Resuls are expressed as mean±standard deviation,Analysis of Variance （ANOVA） and two-one side Student’s t test were adopted to analyze the data.The third part was the pharmacokinetics of rhCTLA4-Ig in healthy adult subjecta after a single 10 mg·kg ^-1 intravenous injection and in RA patients after multiple 10 mg·kg ^-1 intravenous infusions. Pharmacokinetics parameters was compared to those of RA patients or healthy adult. Analysis of Variance （ANOVA） and two-one side Student’s t test were adopted to statistically evaluate. 【Results】1. In this experiment,rhCTLA4-Ig was extracted from blood samples and quantified by ELISA. The limit of quantification for rhCTLA4-Ig was 6.25μg·L^-1;the linear range is 100～6.25μg·L^-1.The double-pipe was repeatable in this linear range,CV% were 100.4% to 112.1%,accuracy rating were during 0.73% to 4.6%. All indicatives to achieve pharmacokinetics test requirement.2. Ten rheumatoid arthritis patients,3 males and 7 females,other 1 rheumatoid arthritis patient was to reject because right bundle branch block. One male was enrollede experiment. During the study, no one experienced visible adverse effects at all doses of rhCTLA4-Ig. No unexpected severe adverse effect was observed, all subjects showed normal respiration,heart rate,blood pressure,pulse,body temperature and other vital things,and normal ECG. The injection part did not showed any flares,ecchymosed,rashes or any other irrigative responses. In general,there has been no abnormal physical signs in all subjects. As to the blood biochemical indicators or metaphase and urine routines indicators dielectric before or metaphase or after the test,we found no statistics significant deviations in all index between two groups by t-test in all 9 subjects and with no abnormal changes.3. The main pharmacokinetic at steady state were as follows: Cssmax were 247.1±50.3mg·L^-1, C ssmin were 19.7±9.5 mg·L^-1,C ssav were 65.4±25.3 mg·L^-1, DF were 3.48±1.61. At once every four weeks 10 mg·kg^-1 intravenous drip infusion of rhCTLA4-Ig,patients do not accumulate phenomenon?. At eighth weeks, the average drug concentrations were 27.1±8.1 mg·L^-1,and at sixteenth weeks 19.1±9.0 mg·L^-1. No difference was found between these two time points （ P<0.05 ） . Back the main pharmacokinetic at stead state were as follows:After the 0,2,4,8,12,16 weeks mult-dose 10 mg·kg ^-1 intravenous drip infusion of rhCTLA4-Ig,drug blood level peaked fast but eliminated torpidly. Tmax was 0.063±0.039d,C max was 247.1±50.3 mg·L^-1, and T1/2 was 4.7±12.6d .After serial 10 mg·kg ^-1 intravenous drip infusion of rhCTLA4-Ig,AUC（0-Z） was 2347±901mg·d^-1·L^-1 , AUC（0-∞） was 2362±906 mg·d^-1·L^-1,CL was 0.0050±0.0023 L·d·Kg^-1,VSS was 0.074±0.031 L·kg^-1.4. Clinical assessments was done according to the American College of Rheumatology criteria（ACR）. As compared with the control group, the etanercept treatement group had a more rapid improvement in ACR20 and ACR50 in disease activity during the first three（78%、11%）treatment?.At the end of sixth treatment, the etanercept treatement group had a more rapid improvement in ACR20 and ACR50 in disease activity during the first three（100%、78%）.As to the immunological blood sedimentation、C reactive protein and rheumatoid factor before or metaphase or after the test,we found no statistics significant difference in all index between two groups by t-test in all 9 subjects and with abnormal changes:blood sedimentation was valued at 16.67±19.83 mm·L^-1、C reactive protein 204.41±300.91 IU·mL^-1and rheumatoid factor 1.55±2.30 mg·L^-1.【Conclusions】1. This analysis method has high sensitivity,accuracy and wonderful specificity. The lineal concentrations correlate well the lineal area under curve;the linearity was in the scope of 6.25～100μg·L^-1 in plasma. The limit of quantization for rhCTLA4-Ig was all below 10%,the limit of quantization for rhCTLA4-Ig was 6.25μg·L^-1 in plasma;CV% of inter-case precision and intra-case precision were all below 10%.There was no significant difference. All the profiles meet the criterion of the SFDA. The results showed that the present method is suitable for the study of pharmacokinetics of plasma rhCTLA4-Ig.2. The ELISA was used to test concentration of rhCTLA4-Ig in plasma. Blood drug level increases dose dependently,concentrations of which of some point has statistics differences in groups of dose of homo-middle,time to steady state concentration for eighth weeks. At once four weeks 10 mg·kg ^-1 intravenous infusion of rhCTLA4-Ig,patients do not show accumulated phenomenon. At eighth weeks the average drug density were 27.1±8.1 mg·L^-1,at sixteenth weeks the average drug density were 19.1±9.0 mg·L^-1. No difference was found between these two groups（p<0.05）. Variations of concentration at each point has no statistical differences,as well as the blood drug levels at the same points are between dosage at first time and at the sixth by using matched t-test. The result of test is similar to the clinical report for foreign new drugs of Orencia and may provide safe and reasonable administration for Phase II clinical trial.3. When administrated subcutaneously in rheumatoid arthritis patients with the dose of 10 mg·kg^-1,rhCTLA4-Ig was well rated by patients and without any adverse effect’s but not drug therapy. RhCTLA4-Ig multiple administration of 10 mg·kg ^-1at the 0,2,4,8,12,16 weeks. During the study no adverse effect was observed.更多还原