CN104961831B - 一种修饰的重组人内皮抑素及其应用 - Google Patents
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Abstract
本申请是200710131562.X的分案申请，属于生物制品制药技术领域，公开了一种修饰的重组人内皮抑素及其应用。该修饰的重组人内皮抑制素结构为：CH3O‑(CH2CH2O)m‑CH2CH2CH2‑NαH‑Endostar，中CH3O‑(CH2CH2O)m‑CH2CH2CH2‑的重均分子量为20kD或40kD。该修饰的重组人内皮抑制素增强了体内稳定性，提高了血药浓度，延长了半衰期，抗内皮细胞增殖活性显著增加，从而能够减少给药剂量，降低给药频率，减轻病人身体痛苦及经济负担。该修饰的重组人内皮抑制素可用于制备抗肿瘤药物。
Description
本申请是分案申请，原申请的申请号为200710131562.X，申请日为2007年9月5日，发明名称为“一种修饰的重组人内皮抑素及其应用”。
技术领域
本发明属于生物制品制药技术领域，涉及一种修饰的重组人内皮抑素及其应用。
背景技术
1971年，Folkman教授提出“肿瘤的生长依赖于新生血管生成”这一观点，通过阻断新生血管生成，就有可能控制肿瘤生长，达到遏制肿瘤侵袭、复发、转移的目的，从此开创了肿瘤治疗的新领域。
1997年，哈佛大学O’Reilly等发现小鼠血管内皮瘤细胞的培养基具有内皮细胞抑制作用，通过纯化发现该活性物质在一定浓度范围内(100ng/ml～600ng/ml)特异性对内皮细胞表现出剂量依从的生长抑制作用。通过N端氨基酸测序表明该物质为胶原XⅧ的C末端片段，分子量20kD左右，这个蛋白就被命名为内皮抑素(Endostatin，ES)。
研究表明内皮抑素能够有效控制非小细胞性肺癌、结肠癌、脑胶质瘤、纤维瘤、间皮瘤及淋巴瘤等各种实体瘤的生长、浸润和转移。目前，内皮抑素的获得都是通过基因工程的方法。由于真菌表达内皮抑素成本太高，人们更倾向于利用大肠杆菌作为表达体系，但这样表达出的蛋白存在复性率低等问题。为此，人们开始尝试对天然内皮抑素进行结构改造。其中罗永章等在天然Endostatin的N末端添加了9个氨基酸，不仅使ES稳定性提高，半衰期延长，而且生物活性增加，蛋白的复性率也高于一般生产方法。该药物已正式上市，命名为恩度(Endostar)。所生产的重组人内皮抑制素由192个氨基酸构成，其氨基酸序列为：
(M)GGSHHHHHHSHRDFQPVLHLVALNSPLSGGMRGIRGADFQCFQQARAVGLAGTFRAFLSSRLQDLYSIVRRADRAAVPIVNLKDELLFPSWEALFSGSEGPLKPGARIFSFDGKDVLRHPTWPQKSVWHGSDPNGRRLTESYCETWRTEAPSATGQASSLLGGRLLGQSAASCHHAYIVLCIENSFMTASK，其中当由大肠杆菌表达时其N末端的Met有时会被部分删除。
Ⅲ期临床实验表明改造后的内皮抑素与化疗药物NP联用可能有一定的协同作用，可延长患者的肿瘤进展时间。
临床实验证实内皮抑素毒副作用很小，适合与放疗或化疗联用。但是内皮抑素作为小分子蛋白，性质不稳定，有研究表明，内皮抑素在缺氧条件下容易降解，当内皮抑素与内皮细胞在低氧环境下共孵育48小时后，含量会下降20％左右。而肿瘤内部恰是低氧环境，对于其活性的发挥很不利。另一方面，目前Endostar临床用药量很大，为7.5mg/m2，需要每天静脉滴注3～4小时完成给药。实验表明，给药方式为腹腔注射时，内皮抑素在2小时内清除。这些现象表明，内皮抑素在进入体内后，尤其是在发挥药效的肿瘤局部可能会被蛋白水解酶水解。如何对蛋白分子进行保护，防止酶解，降低清除率从而减少给药剂量是急待解决的问题。
聚乙二醇(polyethylene glycol，PEG)是一种安全的、无活性、无毒的聚合物。通过共价连接至蛋白质上进行化学修饰，聚乙二醇化(pegylation)在保留天然蛋白质的内在生物学活性的同时，能有效地克服蛋白质类药物在临床应用中的一些缺点，如可以提高血浆半衰期，增强生物利用度，减低蛋白质免疫原性、提高药物疗效及安全性等。目前为止，成功上市的PEG修饰蛋白多肽药物有酰苷脱胺酶
天冬酰胺酶 
干扰素α-2b
干扰素α-2a
等，而更有尿酸酶、水蛭素、白介素-2、白介素-6等20多种药物正进行临床研究。
在蛋白质PEG化过程中，活化的PEG分子偶联到蛋白分子中的自由氨基上，主要是Lys上。由于蛋白质链中自由氨基不只一个，反应产物中不仅存在单分子、双分子甚至三分子等多类修饰产物，而且各类修饰产物中还包括多种异构体。与单分子修饰产物相比，多分子修饰产物活性损失大，不仅浪费了反应底物，而且降低了反应产物的总体活性。通过控制反应条件和采用纯化手段可以克服多分子修饰产物的问题，而获得单分子修饰产物。但是单分子修饰还存在异构体的问题，异构体在生物活性上可能存在较大差异且异构体的混合物通常很难进行纯化。所以最好的是能通过控制反应条件，实现定点修饰，以帮助产物的纯化和性质研究。而且定点修饰对蛋白活性的保持更为有利。Kinstler利用N末端的α氨基比赖氨酸的氨基有更低的pKa值发明了一种方法。用PEG乙醛修饰粒细胞集落刺激因子(G-CSF)得到了
发明内容
本发明的目的是提供一种修饰的重组人内皮抑素CH3O-(CH2CH2O)m-CH2CH2CH2-NαH-Endostar，使得Endostar在体内的稳定性增强，从而可以提高有效血药浓度，延长半衰期，降低给药剂量。CH3O-(CH2CH2O)m-CH2CH2CH2-的重均分子量在20kD～40kD，优选CH3O-(CH2CH2O)m-CH2CH2CH2-的重均分子量为 20kD(以下采用m＝20kD的方式表示)，另一优选CH3O-(CH2CH2O)m-CH2CH2CH2-的重均分子量为40kD(以下采用m＝40kD的方式表示)，分散度≤1.2，优选分散度为1.1。
本发明的另一个目的是提供上述修饰的重组人内皮抑素CH3O-(CH2CH2O)m-CH2CH2CH2-NαH-Endostar在制备抗肿瘤药中的应用。
本发明的目的是通过下列措施实现的：
1、Endostar的N-末端化学修饰。反应式为：
或
(1)Endostar的来源：本发明中所用的重组人血管内皮抑素是来自江苏先声麦得津生物制药有限公司。是大肠杆菌表达的N末端带有(Met)GlyGlyXaa HisHisHisHisHis附加氨基酸序列的Endostatin，其中Xaa代表中性氨基酸或不存在，当由大肠杆菌表达时其N末端的Met有时会被部分删除，分子量为21kD。
(2)本发明中所用的重组人血管内皮抑素和单甲基聚乙二醇衍生物在还原剂氰基硼氢化钠存在的条件下(终浓度10mM～30mM)，于0℃～25℃反应10h～40h，生成CH3O-(CH2CH2O)m-CH2CH2CH2-NαH-Endostar。
本发明的有益效果：
本发明提供的修饰的重组人内皮抑素(CH3O-(CH2CH2O)m-CH2CH2CH2-NαH-Endostar)，具有下列优点：
(1)与Endostar相比，CH3O-(CH2CH2O)m-CH2CH2CH2-NαH-Endostar的体内有效血药浓度更高；
(2)与Endostar相比，CH3O-(CH2CH2O)m-CH2CH2CH2-NαH-Endostar的抗内皮细胞增殖活性显著增加；
(3)与Endostar相比，CH3O-(CH2CH2O)m-CH2CH2CH2-NαH-Endostar的体内半衰期更长；
(4)与Endostar相比，CH3O-(CH2CH2O)m-CH2CH2CH2-NαH-Endostar的AUC更大。
上述优点表明，Endostar经修饰后增强了体内稳定性，提高了血药浓度，延长了半衰期，抗内皮细胞增殖活性显著增加，从而能够减少给药剂量，降低给药频率，减轻病 人的身体上痛苦，同时还能减轻病人的经济负担。该CH3O-(CH2CH2O)m-CH2CH2CH2-NαH-Endostar可用于制备抗肿瘤药物。
附图说明
图1显示的是CH3O-(CH2CH2O)20kD-CH2CH2CH(OCH2CH3)2与Endostar的偶联。图中1～4号电泳道分别对应：1、分子量分别为14400，20100，31000，43000，66200，97400的标准蛋白；2、Endostar；3、反应混合物；4、CH3O-(CH2CH2O)20kD-CH2CH2CH(OCH2CH3)2。
图2显示的是CH3O-(CH2CH2O)20kD-CH2CH2CH2-NαH-Endostar的纯化层析图。
图3显示的是纯化后CH3O-(CH2CH2O)20kD-CH2CH2CH2-NαH-Endostar的SDS-PAGE鉴定。图中1～4号电泳道分别对应：1、纯化后的CH3O-(CH2CH2O)20kD-CH2CH2CH2-NαH-Endostar；2、反应混合物；3、CH3O-(CH2CH2O)20kD-CH2CH2CH(OCH2CH3)2；4、分子量分别为14400，20100，31000，43000，66200，97400的标准蛋白。
图4显示的CH3O-(CH2CH2O)40kD-CH2CH2CHO与Endostar的偶联。图中1～5号电泳道分别对应：1～4、反应混合物；5、CH3O-(CH2CH2O)40kD-CH2CH2CHO。
图5显示的是CH3O-(CH2CH2O)40kD-CH2CH2CH2-NαH-Endostar的纯化层析图。
图6显示的是纯化后CH3O-(CH2CH2O)40kD-CH2CH2CH2-NαH-Endostar的SDS-PAGE鉴定。图中1～3号电泳道分别对应：1～2、纯化后的CH3O-(CH2CH2O)40kD-CH2CH2CH2-NαH-Endostar；3、分子量分别为14400，20100，31000，43000，66200，97400的标准蛋白。
图7显示的是Endostar修饰前后的血药浓度和时间的关系曲线。
图8显示的是Endostar修饰前后抗内皮细胞增殖活性。
具体实施方式
以下通过实施例对本发明作进一步的说明，但不是对本发明保护范围的限制。
实施例1、重均分子量为20kd的CH3O-(CH2CH2O)20kD-CH2CH2CH(OCH2CH3)2和Endostar的偶联物(简写为PEG20-ENDO)的制备：
(1)CH3O-(CH2CH2O)20kD-CH2CH2CH(OCH2CH3)2与Endostar的偶联
20ml Endostar原液(PH5.0～5.5，HAC/NaAC Buffer，蛋白质含量为5mg/ml)，加入还原剂氰基硼氢化钠NaCNBH3至终浓度为20mM，加入单甲氧基聚乙二醇-3,3-二乙氧基丙烷100mg，使其与Endostar的摩尔比为1：1，在4℃搅拌过夜，即形成PEG20-ENDO修饰物，产物用SDS-PAGE鉴定，鉴定结果见图1。
(2)PEG20-ENDO的纯化
上述反应产物用CM－Sepharose柱，AKTA层析仪分离、纯化。样品以20mM，pH7.0的PB缓冲液稀释后上样，以20mM，pH7.0的PB缓冲液平衡至基线，再分别以含0.1M、0.2M、0.5MNaCl的20mM，pH7.0的PB缓冲液阶段洗脱(图2)，分部收集器收集，修饰产物用SDS-PAGE鉴定。(图3)
实施例2、重均分子量为40kd的CH3O-(CH2CH2O)40kD-CH2CH2CHO和Endostar的偶联物(简写为PEG40-ENDO)的制备：
(1)CH3O-(CH2CH2O)40kD-CH2CH2CHO与Endostar的偶联
20ml Endostar原液(PH5.0～5.5，HAC/NaAC Buffer，蛋白质含量为5mg/ml)，加入还原剂氰基硼氢化钠NaCNBH3至终浓度为20mM，加入单甲氧基聚乙二醇-3,3-二乙氧基丙烷400mg，使其与Endostar的摩尔比为1：2，在4℃搅拌过夜，即形成PEG40-ENDO修饰物，产物用SDS-PAGE鉴定。(图4)
(2)PEG40-ENDO的纯化
上述反应产物用CM－Sepharose柱，AKTA层析仪分离、纯化。样品以20mM，pH7.0的PB缓冲液稀释后上样，以20mM，pH7.0的PB缓冲液平衡至基线，再分别以含0～0.5M NaCl的20mM，pH7.0的PB缓冲液梯度洗脱(图5)，分部收集器收集，修饰产物用SDS-PAGE鉴定。(图6)
实施例3、聚乙二醇修饰的重组人内皮抑素的药代动力学研究
将15只雄性SD大鼠随机分成3组，按4.5mg/kg体重给药。A组为未修饰的Endostar，B组为PEG20-ENDO，C组为PEG40-ENDO。给药后5min、10min、15min、30min、1h、4h、6h、12h、24h、48h、96h取血，肝素抗凝，血样立即6000r/min，离心10min，得到血浆样品。采用HumanEndostatin Protein 
EIA试剂盒测定血浆中样品的含量。经测定修饰后内皮抑素有效血药浓度提高20倍以上(图7)，Endostar的半衰期为8小时，PEG20-ENDO的体内半衰期为18h，PEG40-ENDO的体内半衰期为97h。与Endostar相比，PEG20-ENDO的AUC提高至少10倍，PEG40-ENDO的AUC提高至少100倍。
(Endostar的AUC0-∞＝1065.46μg/L*h；PEG20-ENDO的AUC0-∞＝1133103.46μg/L*h；PEG40-ENDO的AUC0-∞＝19759375.66μg/L*h)
实施例4、聚乙二醇修饰的恩度的生物活性测定
1、将牛主动脉内皮细胞用含10％胎牛血清的RPMI-1640培养液培养，置37℃、5％CO2培养箱中，选对数生长期的细胞用于实验。
2、调细胞浓度(10000个/ml)后分10组接种于96孔板上,每组设5个复孔，置37℃、5％CO2培养箱培养24h待细胞贴壁后，分别加入终浓度为100μg/ml、25μg/ml、6.25μg/ml、1.56μg/ml、0.39μg/ml的Endostar、PEG20-ENDO和PEG40-ENDO。对照组加含2％FCS的1640培养基和加2％FCS+bFGF的1640培养基，最后四周孔用含2％FCS的1640培养基封闭。
3、以MTT法测定每孔的OD值。根据OD值求出细胞抑制率，计算公式为抑制率(IR)＝(对照组OD值均数-实验组OD值均数)/对照组OD值均数。
4、PEG20-ENDO和PEG40-ENDO的抗内皮细胞增殖活性有显著提高，PEG20-ENDO活性提高更多 (图8) 。
Claims (3)
1.一种修饰的重组人内皮抑制素，其特征在于其结构为：
CH3O-(CH2CH2O)m-CH2CH2CH2-NαH-Endostar，
其中CH3O-(CH2CH2O)m-CH2CH2CH2-的重均分子量为40kD；所述的修饰的重组人内皮抑制素相比于Endostar抗内皮活性显著增加；
所述Endostar的氨基酸序列如SEQ ID NO.1或SEQ ID NO.2所示。
2.根据权利要求1所述的修饰的重组人内皮抑制素，所述的CH3O-(CH2CH2O)m-CH2CH2CH2-的分散度≤1.2。
3.权利要求1-2任一项所述的修饰的重组人内皮抑制素在制备抗肿瘤药物中的应用。
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