CN107987132A - 一种抗her2多肽-阿霉素复合物及其制备方法和应用 - Google Patents
一种抗her2多肽-阿霉素复合物及其制备方法和应用 Download PDFInfo
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Abstract
一种抗HER2多肽‑阿霉素复合物及其制备方法和应用，结构式如下所示：该复合物具有治疗HER2阳性乳腺癌的作用。
Description
技术领域
本发明属于生物与医药技术领域，具体涉及一种具有治疗HER2阳性乳腺癌作用的抗HER2多肽-阿霉素复合物及其制备方法和应用。
背景技术
有研究发现在20％到30％的乳腺癌患者中存在HER2过表达的现象，人表皮生长因子受体-2(human epidermal growth factor receptor-2,HER2)作为人表皮生长因子受体家族成员，它的过表达与肿瘤更快的恶性生长、不良预后和总体生存率的降低都息息相关。与人表皮生长因子受体家族其他成员一样，HER2具有三个域：胞外配体结合域、跨膜域、胞内酪氨酸激酶域。这三个域对受体二聚化和下游信号通路的激活都至关重要，下游信号通路的激活可能导致肿瘤细胞的增殖与存活(如PI3K/AKT信号通路)，最终会增加乳腺癌的风险。
目前，曲妥珠单抗(赫赛汀)是治疗HER2阳性乳腺癌最为重要的药物之一。这种人源化单克隆抗体可以结合HER2的胞外部分从而阻止HER2的二聚化进而抑制HER2介导的恶性转化。曲妥珠单抗可能是通过下调HER2表达和加快HER2的翻转从而降低HER2信号来达到治疗作用。与此同时，有研究证据表明曲妥珠单抗与化疗药物例如阿霉素(DOX)联用可以获得更好的治疗效果。众所周知，阿霉素通过简单扩散进入细胞后插入到DNA结构中从而抑制拓扑异构酶II进而阻止DNA的复制。曲妥珠单抗与阿霉素的联合应用尽管取得了一定的临床治疗效果，但因其缺乏选择性和由曲妥珠单抗和阿霉素蓄积导致的严重心脏毒性使得这样简单的药物联用通常并不推荐使用。在这一背景下，曲妥珠单抗-阿霉素复合物被成功研制出来并证明复合物通过曲妥珠单抗对HER2的特异性识别可以提高药物的选择性、降低药物的心脏毒性。与大多数抗体药物复合物一样，由于“结合位点屏障”作用曲妥珠单抗-阿霉素复合物也缺乏很好的肿瘤穿透性。
相比于抗体，多肽具有分子量低、突出的组织/细胞穿透性、易于合成、便于化学修饰和免疫原性低等优点。有研究以曲妥珠单抗的补体决定域重链3(complementarity-determining region heavy chain 3,CDR-H3)的环状结构为基础设计了具有12个氨基酸的抗HER2环肽(exocyclic anti-HER2/neu peptide mimic,AHNP)，这一环肽能很好地与HER2胞外域相结合。与此同时，有研究发现AHNP与曲妥珠单抗具有相似的药物作用效果。
发明内容
解决的技术问题：本发明提供一种抗HER2多肽-阿霉素复合物及其制备方法和应用，该复合物具有治疗HER2阳性乳腺癌的作用。
技术方案：一种抗HER2多肽-阿霉素复合物，结构式如式I所示：
抗HER2多肽-阿霉素复合物的制备方法，包括以下步骤：步骤1，将阿霉素溶于N,N’-二甲基甲酰胺(DMF)中，在氮气保护下避光室温搅拌10分钟；加入芴甲氧羰酰氯(Fmoc-Cl)后滴加N,N’-二异丙基乙胺(DIPEA)，阿霉素与DIPEA的摩尔比为1:8，避光室温搅拌4小时，阿霉素与Fmoc-Cl摩尔比为1:2；步骤2，使用真空冷冻浓缩仪除去溶剂直至剩余油状液体后加入0.1％三氟乙酸(TFA)后离心收集沉淀，以除去未反应的阿霉素，再将沉淀用乙醚洗两次离心收集沉淀以除去未反应的Fmoc-Cl，制得N-Fmoc-DOX；步骤3，将N-Fmoc-DOX和戊二酸酐溶于DMF后滴加DIPEA，N-Fmoc-DOX与戊二酸酐摩尔比为1:8，N-Fmoc-DOX与DIPEA的摩尔比为1:8，在氮气保护下避光室温搅拌16小时；通过制备型高效液相色谱对粗产物进行分离纯化得到N-Fmoc-DOX-14-O-hemiglutarate；步骤4，在氮气保护下将N-Fmoc-DOX-14-O-hemiglutarate、2-(7-氧化苯并三氮唑)-N,N,N’,N’-四甲基脲六氟磷酸酯(HATU)和DIPEA溶于DMF反应15分钟来活化N-Fmoc-DOX-14-O-hemiglutarate上的羧基，N-Fmoc-DOX-14-O-hemiglutarate与DIPEA的摩尔比为1:8，N-Fmoc-DOX-14-O-hemiglutarate与2-(7-氧化苯并三氮唑)-N,N,N’,N’-四甲基脲六氟磷酸酯(HATU)的摩尔比为2:3；将多肽GPLGLAGDDYCDGFYACYMDV-NH2(MAHNP,disulfide-bridged)溶于DMF，将多肽溶液加入到N-Fmoc-DOX-14-O-hemiglutarate溶液中，避光室温搅拌过夜，N-Fmoc-DOX-14-O-hemiglutarate与多肽摩尔比为1:1；步骤5，使用真空冷冻浓缩仪除去溶剂直至剩余油状液体，加入乙酸乙酯离心收集沉淀以除去未反应的N-Fmoc-DOX-14-O-hemiglutarate；步骤6，将沉淀溶于溶剂搅拌30分钟以脱去保护基团Fmoc，所述溶剂为哌啶和DMF的混合物，哌啶和DMF的体积比为1:9；滴加TFA与DMF的混合物直至溶液颜色变为淡红色以终止反应，TFA和DMF的体积比为1:9；使用真空冷冻浓缩仪除去溶剂，通过制备型高效液相色谱进行纯化得到精制品MAHNP-DOX。
上述制备型高效液相色谱使用Waters XBridge C18色谱柱在室温下进行色谱分离，色谱柱具体参数为19mm×150mm,5μm；流动相A为0.01％甲酸-水溶液，流动相B为100％乙腈；梯度洗脱，洗脱程序为：0～15min，30％～98％流动相B；15～18min，98％～30％流动相B；18～20min，30％～30％流动相B。
上述抗HER2多肽-阿霉素复合物在制备抗肿瘤药物中的应用。
治疗HER2阳性乳腺癌的药物，有效成分为式I所示的抗HER2多肽-阿霉素复合物。
上述抗HER2多肽-阿霉素复合物在制备治疗HER2阳性乳腺癌制剂中的应用。
有益效果：1)本发明所述的MAHNP为多肽，可化学合成，稳定性好；2)本发明所述的MAHNP-DOX在肿瘤微环境中，多肽MAHNP被MMP-2剪切，释放出药理活性的阿霉素和抗HER2多肽AHNP；3)本发明的抗HER2多肽-阿霉素复合物通过各部分结合，达到一个综合性的效果，相比单一药物具有更高肿瘤生长抑制特征，以HER2高表达的人乳腺癌细胞BT474和SKBR3为细胞模型，以荷瘤小鼠为动物模型，MAHNP-DOX可在肿瘤微环境中通过MMP-2的剪切作用在细胞外释放出药理活性的阿霉素和抗HER2多肽AHNP，多肽AHNP降低AKT表达活性，阿霉素与多肽AHNP协同作用抑制肿瘤细胞增殖，诱导其凋亡，将肿瘤细胞阻滞于G1期，抑制裸鼠移植肿瘤的生长；4)本发明的多靶点多肽-阿霉素复合物整体效果能综合各组成部分(如DOX，MAHNP等)的作用，并且各组成部分之间不会相互影响，能够使得各组成部分发挥最佳效果。
附图说明
图1为本发明的MAHNP-DOX复合物的合成流程示意图；
图2为本发明的MAHNP-DOX的LC/MS/MS图谱；其中，A：MAHNP-DOX的母离子图；B：MAHNP-DOX的子离子图；
图3为本发明的MAHNP-DOX及游离DOX对BT474和SKBR3细胞生长抑制曲线；
图4为BT474和SKBR3细胞对MAHNP-DOX、游离DOX及MAHNP-DOX+MMP-2 inhibitor的摄取比较示意图；
图5为本发明的MAHNP-DOX和游离DOX引起的细胞凋亡情况检测示意图；其中，分别代表DAPI染核图像，TUNEL检测凋亡的细胞图像；
图6为本发明的MAHNP-DOX和游离DOX对pHER2、pAKT和Cleaved Caspase 3表达的影响示意图；
图7为本发明的MAHNP-DOX、游离DOX及MAHNP对细胞周期阻滞的作用；
图8为本发明的MAHNP-DOX和游离DOX对移植瘤裸鼠模型瘤体积的抑制情况示意图。
具体实施方式
以下实施例进一步说明本发明的内容，但不应理解为对本发明的限制。在不背离本发明精神和实质的情况下，对本发明方法、步骤或条件所作的修改和替换，均属于本发明的范围。若未特别指明，实施例中所用的技术手段为本领域技术人员所熟知的常规手段。
实施例1多肽-阿霉素复合物的合成及纯化
(1)MAHNP-DOX的制备及纯化
1)合成N-Fmoc-DOX
称取29mg阿霉素溶于4mL的N,N’-二甲基甲酰胺(DMF)中，在氮气保护下避光室温搅拌10分钟。再加入26mg芴甲氧羰酰氯(Fmoc-Cl)后滴加60μL的N,N’-二异丙基乙胺(DIPEA),避光室温搅拌4小时，使用真空冷冻浓缩仪除去大部分溶剂直至剩余少量油状液体后加入0.1％三氟乙酸(TFA)后离心收集沉淀以除去未反应的阿霉素，再将沉淀用乙醚洗两次离心收集沉淀以除去未反应的Fmoc-Cl，制得N-Fmoc-DOX。
2)合成N-Fmoc-DOX-14-O-hemiglutarate
称取38mg的N-Fmoc-DOX和45mg的戊二酸酐溶于5mL的DMF后滴加60μL的DIPEA在氮气保护下避光室温搅拌16小时。通过制备型高效液相色谱对粗产物进行分离纯化得到N-Fmoc-DOX-14-O-hemiglutarate。
3)合成MAHNP-DOX
称取0.9mg的N-Fmoc-DOX-14-O-hemiglutarate、0.6mg的2-(7-氧化苯并三氮唑)-N,N,N’,N’-四甲基脲六氟磷酸酯(HATU)和1.4μL的DIPEA溶于400μL的DMF反应15分钟来活化N-Fmoc-DOX-14-O-hemiglutarate上的羧基。称取2.3mg的多肽GPLGLAGDDYCDGFYACYMCV-NH2(MAHNP,disulfide-bridged)溶于200μL的DMF，将多肽溶液加入到N-Fmoc-DOX-14-O-hemiglutarate溶液中，避光室温搅拌过夜，使用真空冷冻浓缩仪除去大部分溶剂直至剩余少量油状液体加入400μL的乙酸乙酯离心收集沉淀以除去未反应的N-Fmoc-DOX-14-O-hemiglutarate，将沉淀溶于600μL的溶剂搅拌30分钟以脱去保护基团Fmoc，所述溶剂为哌啶和DMF的混合物。滴加TFA与DMF的混合物约600μL直至溶液颜色变为淡红色则终止反应。使用真空冷冻浓缩仪除去溶剂，通过制备型高效液相色谱进行纯化得到精制品MAHNP-DOX。
我们设计了多肽MAHNP，它由一段可被MMP-2剪切的多肽GPLGLAGDD和抗HER2多肽AHNP组成，与DOX通过戊二酸酐连接，合成流程图见图1。
MAHNP-DOX的结构式为：
(2)LC/MS/MS法和NMR法鉴定产物
LC/MS/MS法鉴定产物，采用Agilent 1200系列高效液相色谱系统和6410三重四级杆液质联用仪，使用Waters XBridge C18(250mm×4.6mm,5μm)色谱柱在室温下进行色谱分离。流动相A为0.01％甲酸-水溶液，流动相B为100％乙腈；梯度洗脱，洗脱程序为：0～15min，10％～90％流动相B；15～20min，90％～90％流动相B；20～25min，90％～10％流动相B；质谱系统的离子源为电喷雾电离源(ESI)；正离子扫描模式；喷雾电压4000V；雾化压力45psi；雾化温度350℃；雾化气流10L/min；检测方式为全扫描(Full Scan)和子离子扫描(Product Scan)，采集时间设在200ms；传输电压设为120V，碰撞能分别设为30eV，所有数据均通过Agilent MassHunter工作站软件(B.01.04版)采集和处理。
MAHNP-DOX母离子图见图2(A)，ESI-MS/MS m/z：1442[M+2H]2+，1453[M+Na+H]2+。当碰撞能设置为30eV时，可见质谱碎片特征。如图2(B)所示，MAHNP-DOX含有肽段序列特异性的y离子(m/z 116.1(y1)，m/z 231.1(y2)，m/z 362.1(y3))，以及DOX特征碎片(m/z 397)，可以验证该产物的合成。
实施例2MTT法测定细胞存活率试验
将状态良好的BT474和SKBR3细胞消化，用培养液稀释至2×104cells/mL细胞密度，吹匀后于96孔板中每孔加入细胞悬液200μL，置37℃、5％二氧化碳环境中孵育24h，使其贴壁。换用200μL DOX和MAHNP-DOX含药培养液，每个浓度设5个平行孔，同时设置空白对照组，均置于培养箱中孵育48h，再向每孔加入MTT液(5mg/mL)20μL，继续培养4h，吸去培养液，加入DMSO 150μL，室温下微孔板震荡器上震荡30min，在酶联免疫检测仪490nm下测定各孔吸光值。以对照细胞为100％，以给药组的细胞存活相对于对照细胞存活的百分率来测定其存活率。
通过MTT方法考察两种药物对BT474和SKBR3肿瘤细胞的细胞毒性作用，绘制细胞存活率曲线，计算IC50值，评价药物的体外细胞毒性。如图3所示，DOX对BT474和SKBR3的IC50值分别为2074.0±368.0nM和172.9±19.2nM，而MAHNP-DOX对BT474和SKBR3的IC50分别为746.8±81.5nM和110.1±12.7nM，说明相比DOX，BT474和SKBR3细胞对MAHNP-DOX更加敏感。同时，给予MMP-2抑制剂后MAHNP-DOX对BT474和SKBR3的IC50分别为1348.0±153.7nM和146.7±16.3nM要高于MAHNP-DOX组，说明MAHNP-DOX的细胞毒作用也是依赖于MMP-2的剪切作用的。
实施例3细胞内阿霉素蓄积实验
BT474和SKBR3细胞以1×105/孔的密度接种于6孔培养板中，置37℃、5％二氧化碳环境中孵育24h后，加入DOX、MAHNP-DOX和MAHNP-DOX+MMP-2inhibitor，阿霉素终浓度都为1.5μM，药物作用1h，4h，8h和12h。吸弃含药培养液，用冷PBS洗细胞3次，激光共聚焦显微镜下观察各组处理细胞，结果见图4。BT474和SKBR3细胞经过不同的给药处理后，1h，4h短时间内DOX组细胞内的荧光强度略高于MAHNP-DOX组，8h，12h时MAHNP-DOX组细胞内的荧光强度与DOX组没有明显区别，这是由于MAHNP-DOX可以被MMP-2剪切释放出游离阿霉素，而MAHNP-DOX+MMP-2inhibitor组细胞内的荧光强度一直弱于DOX组和MAHNP-DOX组。
实施例4缺口末端标记(Tunel)法检测细胞凋亡
将2×107/mL密度的细胞制成细胞涂片后，用4％多聚甲醛/PBS液，4℃固定20min。加入20μg/mL的蛋白酶K 100μL作透化处理，室温孵育5min，PBS洗涤3次。滴加100μL平衡液，室温孵育30min，再加入TdT孵育缓冲液50μL，37℃温育1h，PBS洗涤后，再用DAPI复染，荧光显微镜下分析样品，结果如图5所示，DAPI染核呈蓝色，凋亡细胞呈绿色。
凋亡检测结果如图5所示。DOX组BT474和SKBR3的细胞凋亡率分别为34.2±2.1％和44.8±2.8％，而MAHNP-DOX组凋亡率明显增高，分别为55.1±3.2％和60.0±3.9％。说明相比于DOX，MAHNP-DOX可以促进肿瘤细胞的凋亡。
实施例5MAHNP-DOX对HER2信号通路的影响
将BT474和SKBR3细胞分别暴露于溶于无血清培养液的DOX和MAHNP-DOX 24h，通过western bloting检测细胞内pHER2及HER2和pAKT及AKT的表达。图6表明相比于DOX组，MAHNP-DOX组pAKT的表达显著降低，而AKT表达无变化，说明抗HER2多肽AHNP能够对HER2/AKT信号通路起到一定的抑制作用。而相比于对照组，DOX组BT474和SKBR3细胞pHER2和pAKT表达的差异可能是由于两种细胞本身HER3表达差异所引起的。
实施例6MAHNP-DOX对细胞周期阻滞的影响
BT474和SKBR3细胞以1×105/孔的密度接种于6孔培养板中，置37℃、5％二氧化碳环境中孵育24h后，暴露于MAHNP、DOX和MAHNP-DOX中48h。收集细胞用70％乙醇，4℃固定过夜，PBS洗涤3次后重悬于400μL碘化丙啶(PI)/RNase染色液，避光孵育30min后通过流式细胞仪进行检测。
如图7所示，多肽MAHNP组BT474和SKBR3的G1期的细胞数分别提高了6.58％和9.95％，虽然MAHNP-DOX对细胞G1期阻滞作用弱于单独使用MAHNP，但相比于DOX组，MAHNP-DOX组G1期细胞数有一定程度的增加。
实施例7体内抗肿瘤活性
将处于对数生长期的BT474乳腺癌细胞，用PBS调制细胞混悬液至细胞数为1×l07/mL，以每只0.1mL接种于小鼠乳腺垫中，建立小鼠乳腺癌瘤株原位接种模型。
待肿瘤体积生长至约50mm3，对荷瘤小鼠进行尾静脉注射给药。将荷瘤小鼠随机分成3组(每组6只)，分别为模型对照组(生理盐水)、DOX和MAHNP-DOX组，对照组尾静脉给予生理盐水，其余各组分别尾静脉注射给予相应药物，每7天给药3次，给药剂量为5mg DOX/kg鼠重，其中MAHNP-DOX组需以DOX为有效成分，经换算后再按5mg DOX/kg鼠重标准给药。
给药后，每天观察小鼠的存活状态，用游标卡尺测量肿瘤体积。待各给药组与荷瘤对照组间的肿瘤体积存在明显差异后，结束观察。处死小鼠，称瘤重，计算抑瘤率。
如图8所示，DOX和MAHNP-DOX组均能一定程度上抑制肿瘤体积的增长，呈现一定的抗肿瘤活性，DOX和MAHNP-DOX组抑瘤率分别为53.4±6.6％和74.7±5.1％，MAHNP-DOX的平均抑瘤率明显增加。
综上，我们设计了多肽MAHNP，它由一段可以被MMP-2剪切的多肽GPLGLAGDD和抗HER2多肽AHNP组成，与阿霉素通过戊二酸酐连接，形成抗HER2-阿霉素复合物。MAHNP-DOX可在肿瘤微环境中可通过MMP-2的剪切作用在细胞外释放出药理活性的阿霉素和抗HER2多肽AHNP，多肽AHNP降低AKT表达活性，阿霉素与多肽AHNP协同作用抑制肿瘤细胞增殖，诱导其凋亡，将肿瘤细胞阻滞于G1期，抑制裸鼠移植肿瘤的生长。
序列表
<110> 南京医科大学
<120> 一种抗HER2多肽-阿霉素复合物及其制备方法和应用
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 21
<212> PRT
<213> MAHNP(人工序列)
<400> 1
Gly Pro Leu Gly Leu Ala Gly Asp Asp Tyr Cys Asp Gly Phe Tyr Ala
1 5 10 15
Cys Tyr Met Asp Val
20
Claims (6)
1.一种抗HER2多肽-阿霉素复合物，其特征在于结构式如式I所示：
2.权利要求1所述抗HER2多肽-阿霉素复合物的制备方法，其特征在于包括以下步骤：步骤1，将阿霉素溶于N,N’-二甲基甲酰胺(DMF)中，在氮气保护下避光室温搅拌10分钟；加入芴甲氧羰酰氯(Fmoc-Cl)后滴加N,N’-二异丙基乙胺(DIPEA)，阿霉素与DIPEA的摩尔比为1:8，避光室温搅拌4小时，阿霉素与Fmoc-Cl摩尔比为1:2；步骤2，使用真空冷冻浓缩仪除去溶剂直至剩余油状液体后加入0.1％三氟乙酸(TFA)后离心收集沉淀，以除去未反应的阿霉素，再将沉淀用乙醚洗两次离心收集沉淀以除去未反应的Fmoc-Cl，制得N-Fmoc-DOX；步骤3，将N-Fmoc-DOX和戊二酸酐溶于DMF后滴加DIPEA，N-Fmoc-DOX与戊二酸酐摩尔比为1:8，N-Fmoc-DOX与DIPEA的摩尔比为1:8，在氮气保护下避光室温搅拌16小时；通过制备型高效液相色谱对粗产物进行分离纯化得到N-Fmoc-DOX-14-O-hemiglutarate；步骤4，在氮气保护下将N-Fmoc-DOX-14-O-hemiglutarate、2-(7-氧化苯并三氮唑)-N,N,N’,N’-四甲基脲六氟磷酸酯(HATU)和DIPEA溶于DMF反应15分钟来活化N-Fmoc-DOX-14-O-hemiglutarate上的羧基，N-Fmoc-DOX-14-O-hemiglutarate与DIPEA的摩尔比为1:8，N-Fmoc-DOX-14-O-hemiglutarate与2-(7-氧化苯并三氮唑)-N,N,N’,N’-四甲基脲六氟磷酸酯(HATU)的摩尔比为2:3；将多肽GPLGLAGDDYCDGFYACYMDV-NH2(MAHNP,disulfide-bridged)溶于DMF，将多肽溶液加入到N-Fmoc-DOX-14-O-hemiglutarate溶液中，避光室温搅拌过夜，N-Fmoc-DOX-14-O-hemiglutarate与多肽摩尔比为1:1；步骤5，使用真空冷冻浓缩仪除去溶剂直至剩余油状液体，加入乙酸乙酯离心收集沉淀以除去未反应的N-Fmoc-DOX-14-O-hemiglutarate；步骤6，将沉淀溶于溶剂搅拌30分钟以脱去保护基团Fmoc，所述溶剂为哌啶和DMF的混合物，哌啶和DMF的体积比为1:9；滴加TFA与DMF的混合物直至溶液颜色变为淡红色以终止反应，TFA和DMF的体积比为1:9；使用真空冷冻浓缩仪除去溶剂，通过制备型高效液相色谱进行纯化得到精制品MAHNP-DOX。
3.根据权利要求2所述抗HER2多肽-阿霉素复合物的制备方法，其特征在于所述制备型高效液相色谱使用Waters XBridge C18色谱柱在室温下进行色谱分离，色谱柱具体参数为19mm×150mm，5μm；流动相A为0.01％甲酸-水溶液，流动相B为100％乙腈；梯度洗脱，洗脱程序为：0～15min，30％～98％流动相B；15～18min，98％～30％流动相B；18～20min，30％～30％流动相B。
4.权利要求1所述抗HER2多肽-阿霉素复合物在制备抗肿瘤药物中的应用。
5.治疗HER2阳性乳腺癌的药物，其特征在于有效成分为式I所示的抗HER2多肽-阿霉素复合物。
6.权利要求1所述抗HER2多肽-阿霉素复合物在制备治疗HER2阳性乳腺癌制剂中的应用。
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| CN105854025A (zh) * | 2016-04-01 | 2016-08-17 | 南京医科大学 | 多靶点多肽—阿霉素复合物及其制备方法和应用 |
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