CN116036041A - 多价靶向蛋白降解前药和形成的纳米粒、其制备方法及用途 - Google Patents
多价靶向蛋白降解前药和形成的纳米粒、其制备方法及用途 Download PDFInfo
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- CN116036041A CN116036041A CN202111260956.1A CN202111260956A CN116036041A CN 116036041 A CN116036041 A CN 116036041A CN 202111260956 A CN202111260956 A CN 202111260956A CN 116036041 A CN116036041 A CN 116036041A
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Abstract
Description
技术领域
本发明属于医药领域,具体涉及一种多价靶向蛋白降解前药和所形成的纳米递送系统、及其制备方法和在肿瘤治疗中的应用。
背景技术
靶向蛋白降解剂是一种双功能小分子,包含可高特异性连接目标蛋白(POI)的“弹头”部分,可劫持内源性E3泛素连接酶的配体以及连接二者的连接链(linker)。靶向蛋白降解剂的作用下,POI可被E3泛素连接酶识别并打上泛素化标记,从而通过泛素-蛋白酶体途径高效降解特定蛋白质——包括不可成药的靶标,例如转录因子、支架蛋白或通过蛋白-蛋白相互作用发挥功能的蛋白等,这极大的拓展了成药空间。另外,靶向蛋白降解剂由于具有循环催化降解的特性,仅需较短暴露时间和低剂量即可发挥强大的降解整个目标蛋白的作用,故可克服耐药性。
然而,靶向蛋白降解剂分子通常具有较大的分子量,其透膜性相对较差,生物利用度相对较低,且其持续保持的高效催化降解的生物特性可能会导致非靶部位POI蛋白的不可控降解,从而导致严重的毒副作用。故此,针对上述挑战提出新的策略以拓宽靶向蛋白降解剂技术在临床的应用迫在眉睫。
有鉴于此,特提出本发明。
发明内容
发明的目的之一在于提供一种多价靶向蛋白降解前药。
本发明的目的之二在于提供一种上述多价靶向蛋白降解前药的制备方法。
本发明的目的之三在于提供一种多价靶向蛋白降解纳米粒。
本发明的目的之四在于提供一种上述多价靶向蛋白降解纳米粒的制备方法。
本发明的目的之五在于提供一种上述多价靶向蛋白降解纳米粒的应用。
为了实现本发明的上述目的,特采用以下技术方案:
本发明第一方面提供了一种多价靶向蛋白降解前药,所述多价靶向蛋白降解前药包括聚合物骨架、多个连接基团和多个靶向蛋白降解剂,其中
所述聚合物骨架具有如式I所示结构:
其中,x为10-145的整数,优选为110-130,进一步优选为113;
y为30-70的整数,优选为50-65,进一步优选为60;
z为2-6的整数,优选为4-6,进一步优选为4;
R1为对基质金属蛋白酶2和/或基质金属蛋白酶9响应的氨基酸片段,氨基酸片段的C端与亚氨基-NH-相连、N端与羰基-C(=O)-相连;
R2和R3分别为同一RAFT聚合链转移剂的不同官能团;
R4为N,N-二乙基氨基、N,N-二丁基氨基、N,N-二异丙基氨基、五亚甲基氨基或六亚甲基氨基中的一种;
所述聚合物骨架中的每个甲基丙烯酰基端通过所述连接基团与所述靶向蛋白降解剂以共价键连接;
所述聚合物骨架中的每个甲基丙烯酰基分别与一个所述连接基团一端以共价键连接;每个所述连接基团另一端分别与一个所述靶向蛋白降解剂中的脯氨酸的羟基以共价键连接;
所述靶向蛋白降解剂为能够与含Von Hippel-Lindau受体的E3泛素连接酶结合的靶向蛋白降解剂。
本发明第二方面提供了一种上述多价靶向蛋白降解前药的制备方法,包括以下步骤:
将Fmoc-氨基酸片段与NH2-PEG-OCH3发生酰胺化反应,得到CH3O-PEG-NH-氨基酸片段-Fmoc;产物CH3O-PEG-NH-氨基酸片段-Fmoc脱Fmoc保护后与RAFT聚合链转移剂发生酰胺化反应,得到CH3O-PEG-NH-氨基酸片段-CO-RAFT聚合链转移剂;
将所述连接基团一端甲基丙烯化,另一端任选地活化,得到一端甲基丙烯化、另一端活化或未活化的连接基团;将所述靶向蛋白降解剂与一端甲基丙烯化、另一端活化或未活化的的连接基团反应,得到甲基丙烯化的连接基团-靶向蛋白降解剂;
将所述CH3O-PEG-NH-氨基酸片段-CO-RAFT聚合链转移剂、甲基丙烯化的连接基团-靶向蛋白降解剂和甲基丙烯酸乙酯发生RAFT聚合反应,得到所述多价靶向蛋白降解前药;
其中,所述氨基酸片段为R1,所述RAFT聚合链转移剂含有不同官能团R2和R3,所述甲基丙烯酸乙酯含R4基团。
本发明第三方面提供了一种由上述多价靶向蛋白降解前药自组装而成的多价靶向蛋白降解纳米粒。
本发明第四方面提供了一种上述多价靶向蛋白降解纳米粒的制备方法,包括以下步骤:
将所述多价靶向蛋白降解前药溶于有机相,然后超声分散于水中,所得溶液通过超滤或透析得到多价靶向蛋白降解纳米粒。
本发明第五方面提供了上述多价靶向蛋白降解纳米粒在制备用于治疗恶性肿瘤的药物中的用途,所述恶性肿瘤优选自乳腺癌、宫颈癌、肝癌、胃癌、胰腺癌、卵巢癌、结肠癌或前列腺癌。
本发明的技术方案至少具有以下技术效果:
本发明的多价靶向蛋白降解前药经过自组装所得到的纳米粒,可改善靶向蛋白降解剂在生物体内的组织分布情况及药代动力学性质。其可选择性地靶向至肿瘤部位,实现在肿瘤组织的高效蓄积滞留及渗透,提高肿瘤细胞对靶向蛋白降解剂的摄取能力,然后可控地释放其荷载的多个蛋白降解剂分子,在增强治疗效果的同时降低毒副作用,为靶向蛋白降解剂的临床应用奠定基础。
附图说明
为了更清楚地说明本发明具体实施方式或现有技术中的技术方案,下面将对具体实施方式或现有技术描述中所需要使用的附图作简单地介绍,显而易见地,下面描述中的附图是本发明的一些实施方式,对于本领域普通技术人员来讲,在不付出创造性劳动的前提下,还可以根据这些附图获得其他的附图。
图1示出了在本发明实施例2中制备的甲基丙烯化-SS-蛋白降解剂还原响应释放结果图,其中,771-SH表示二硫键响应之后蛋白降解剂片段,Me-771表示甲基丙烯化-SS-771;
图2示出了本发明实施例5中制备的多价靶向蛋白降解纳米粒在不同pH条件下的流体力学粒径和透射电镜图,其中,左边图为pH=7.4,右边图为pH=6.0;
图3示出了在本发明的实施例5中制备的多价靶向蛋白降解纳米粒在体外模拟肿瘤细胞生理微环境中的蛋白降解剂累计释放图,其中,坐标点方形的表示模拟环境中含有10mM的GSH,坐标点圆形的表示模拟环境中不含GSH;
图4示出了在本发明的实施例5中制备的多价靶向蛋白降解纳米粒在MDA-MB-231乳腺癌细胞中的摄取能力,其中,背景颜色为黑色的表示加入了MMP-2酶,白色的表示没加入MMP-2酶;
图5示出了在本发明的实施例5中制备的多价靶向蛋白降解纳米粒在不同浓度下与MDA-MB-231细胞共孵育72h后的细胞毒性测定,命名中带G表示纳米粒中含对MMP-2酶响应的氨基酸片段GPLGLAG,命名中不带G表示纳米粒中不含对MMP-2酶响应的氨基酸片段GPLGLAG;
图6示出了在本发明的实施例4中制备的多价靶向蛋白降解纳米粒分别对MDA-MB-231乳腺癌细胞中BRD4蛋白降解情况,命名中带G表示纳米粒中含对MMP-2酶响应的氨基酸片段GPLGLAG,命名中不带G表示纳米粒中不含对MMP-2酶响应的氨基酸片段;
图7示出了PBS溶液、蛋白降解剂溶液、本发明实施例5中制备的多价靶向蛋白降解纳米粒在雌性Balb/c裸鼠中的抑瘤情况,其中,坐标点为圆形表示PBS,正方形表示游离的靶向蛋白降解剂分子771,三角形表示多价靶向蛋白降解纳米粒;
图8示出了PBS溶液、蛋白降解剂溶液、本发明实施例5中制备的多价靶向蛋白降解纳米粒对雌性Balb/c裸鼠的体重影响,其中,坐标点为圆形表示PBS,正方形表示游离的靶向蛋白降解剂分子771,三角形表示多价靶向蛋白降解纳米粒。
具体实施方式
为使本发明实施例的目的、技术方案和优点更加清楚,下面将对本发明实施例中的技术方案进行清楚、完整地描述,显然,所描述的实施例是本发明一部分实施例,而不是全部的实施例。基于本发明中的实施例,本领域普通技术人员在没有作出创造性劳动前提下所获得的所有其他实施例,都属于本发明保护的范围。另外,为了更好的说明本发明,在下文的具体实施方式中给出了众多的具体细节。本领域技术人员应当理解,没有某些具体细节,本发明同样可以实施。在一些实施例中,对于本领域技术人员熟知的原料、元件、方法、手段等未作详细描述,以便于凸显本发明的主旨。
除非另有其它明确表示,否则在整个说明书和权利要求书中,术语“包括”或其变换如“包含”或“包括有”等等将被理解为包括所陈述的元件或组成部分,而并未排除其它元件或其它组成部分。
为了缓解靶向蛋白降解剂分子透膜性较差,以及非特异持续的高效催化降解导致的毒副作用,本发明实施例提出了一种多价靶向蛋白降解纳米粒及其前药,通过形成载药纳米粒,借助肿瘤细胞对纳米粒的内吞作用增加细胞对药物的摄取,降低药物分子自身透膜性能对细胞摄取药物的影响;同时在纳米粒的保护作用下,药物的半衰期可有效延长,药物组织分布及药代动力学性质均可改善。本发明实施例的多价靶向蛋白降解纳米粒是由多价靶向蛋白降解前药自组装而成。本发明实施例的多价靶向蛋白降解前药通过连接基团将靶向蛋白降解剂与聚合物骨架共价连接,聚合物骨架引入肿瘤微环境响应性的功能基团(肿瘤微环境响应性的功能基团是指对金属基质蛋白酶2/9相应的氨基酸片段、酸响应基团R4以及连接基团的一些谷胱甘肽、活性氧响应键),可实现纳米粒在肿瘤部位的特异性蓄积滞留及高效渗透,以及药物在肿瘤部位的可控释放。本发明是先将氨基酸片段的羧基与CH3O-PEG-NH2的氨基反应,再将RAFT聚合链转移剂的羧基与氨基酸片段的氨基对接,所得产物再进行RAFT聚合反应连接含R4基团的甲基丙烯酸乙酯及甲基丙烯化的连接基团-靶向蛋白降解剂。因为其多重响应的功能,可以增加纳米粒在肿瘤部位的蓄积并在特定条件下释放蛋白降解剂。另外该多价靶向的纳米粒通过聚合物合成方法的调整,可灵活控制靶向蛋白降解剂的荷载数量,进一步提高药物在靶部位的暴露浓度,从而在增加药效的同时可降低耐药性产生的风险。
本发明实施例提供的多价靶向蛋白降解前药的结构如式1所示:
多价靶向蛋白降解前药包括聚合物骨架、连接基团(Linker)、靶向蛋白降解剂(Degrader,D),彼此之间通过共价键连接,即聚合物骨架一端通过Linker与D以共价键连接,具体的,聚合物骨架一端通过聚合物骨架中的聚甲基丙烯酰基与Linker一端以共价键连接,D通过D中的脯氨酸的羟基与Linker另一端以共价键连接。
因为聚合物骨架中聚甲基丙烯酰基具有多个甲基丙烯酰基(z的数量),也就是聚合物骨架一端是具有多个甲基丙烯酰基端的,每个甲基丙烯酰基端均连接一个Linker,每个Linker再连接一个D,因此,聚合物骨架一端可以连接多个Linker-D。
所述聚合物骨架一端RAFT聚合链转移剂通过可逆加成-断裂链转移聚合反应(Reversible Addition-Fragmentation Chain Transfer Polymerization Reaction,简称RAFT聚合反应)连接多个Linker-D;
聚合物骨架
聚合物骨架的结构如式I所示:
波浪线表示聚合物骨架与连接基团反应后的化学键连接位置;或,
星号表示Linker-D;
聚合物骨架包括如下片段:甲氧基、聚乙二醇、基质金属蛋白酶2或基质金属蛋白酶9响应的氨基酸片段、RAFT聚合链转移剂、含有不同基团的聚甲基丙烯酸乙酯、聚甲基丙烯酰基;
其中,在式I中,
x为10-145的整数,优选为110-130,进一步优选为113;
y为30-70的整数,优选为50-65,进一步优选为60;
z为2-6的整数,优选为4-6,进一步优选为4;
R1为对基质金属蛋白酶2和/或基质金属蛋白酶9响应的氨基酸片段,氨基酸片段的C端与聚乙二醇片段中亚氨基-NH-相连、N端与RAFT聚合链转移剂片段中羰基-C(=O)-相连;
优选地,R1选自氨基酸序列GPLGLAG、GGKGPLGLPG或PVGLIG中的一种;
R2和R3为同一RAFT聚合链转移剂的不同官能团;
聚合反应前R2和R3为某一RAFT聚合链转移剂上的不同官能团,聚合反应时RAFT聚合链转移剂会打开以和其他基团进行连接,聚合反应后形成具有带有R2和R3的两部分RAFT聚合链转移剂片段。
聚合物骨架的RAFT聚合链转移剂与氨基酸片段N端连接,再通过RAFT聚合连接甲基丙烯化的Linker-D和含有不同功能基团的甲基丙烯酸乙酯。
RAFT聚合链转移剂的结构如下:
具体可选自如下结构:
优选地,R2和R3选自以下两种情况中的一种:
(a)R2为氰基时,R3为十二烷硫基、苄基或(2-苯乙基)硫基;
(b)R2为甲基时,R3只能为十二烷硫基。
R4为聚甲基丙烯酸乙酯的衍生基团,选自N,N-二乙基氨基、N,N-二丁基氨基、N,N-二异丙基氨基、五亚甲基氨基或六亚甲基氨基中的一种,优选选自N,N-二异丙基氨基。
连接基团(Linker)
Linker为连接聚合物骨架与D的连接基团。
在某些实施方式中,连接基团选自如式i1、式i2、式i3和式i4所示的基团;
其中,M1、M2、R7各自独立地选自碳原子数为1~4的亚烷基;R5、R6各自独立地选自羰基-C(=O)-、亚氨基-NH-、-O-;
优选地,R7为-C(CH3)2-,
进一步优选地,M1与M2相同。
在某些实施方式中,式i1所示的基团可通过硫代二酸为原料反应得到,例如硫代二乙酸。式i2所示的基团可通过二硫代二酸为原料反应得到,例如4,4-二硫代二丁酸。式i3所示的基团可通过缩硫酮二酸为原料反应得到,例如2,2'-[丙烷-2,2-二基双(硫代)]二乙酸。式i4所示基团可通过二硒代二酸为原料反应得到,例如3,3’-二硒二丙酸。
在某些实施方式中,连接基团的结构式可选自如下化学式所示的基团,并不限于此:
在某些实施方式中,连接基团的前体选自式i1-p、式i2-p和式i3-p所示的化合物;
其中,M1、M2、R7所表示的含义与式i1~式i4同;
L1、L2各自独立地选自–COOH、–NCO、–NH2、–OH;
进一步的,i1-p所示化合物优选为硫代二乙酸,i2-p所示化合物优选为4,4-二硫代二丁酸,i3-p所示化合物优选为2,2'-[丙烷-2,2-二基双(硫代)]二乙酸,i4-p所示化合物优选为3,3’-二硒二丙酸。
靶向蛋白降解剂(Degrader,D)
靶向蛋白降解剂为能够与含Von Hippel-Lindau受体的E3泛素连接酶结合的靶向蛋白降解剂。
能与E3泛素连接酶Von Hippel-Lindau受体结合的D选自如下结构的任一种:
聚合物骨架通过聚合物骨架的聚甲基丙烯酰基连接多个Linker-D,即通过调整聚合物的合成方法,调节z的数量,可灵活控制所连接的靶向蛋白降解剂的数量。
本发明实施例还提供上述多价靶向蛋白降解前药的制备方法,包括以下步骤:
将Fmoc-氨基酸片段与NH2-PEG-OCH3发生酰胺化反应,得到CH3O-PEG-NH-氨基酸片段-Fmoc;产物CH3O-PEG-NH-氨基酸片段-Fmoc脱Fmoc保护后与RAFT聚合链转移剂发生酰胺化反应,得到CH3O-PEG-NH-氨基酸片段-CO-RAFT聚合链转移剂;
将所述连接基团一端甲基丙烯化,另一端任选地活化,得到一端甲基丙烯化、另一端活化或未活化的连接基团;将所述靶向蛋白降解剂与一端甲基丙烯化、另一端活化或未活化的的连接基团反应,得到甲基丙烯化的连接基团-靶向蛋白降解剂;
将所述CH3O-PEG-NH-氨基酸片段-CO-RAFT聚合链转移剂、甲基丙烯化的连接基团-靶向蛋白降解剂和甲基丙烯酸乙酯发生RAFT聚合反应,得到所述多价靶向蛋白降解前药;
其中,所述氨基酸片段为R1,所述RAFT聚合链转移剂含有不同官能团R2和R3,所述甲基丙烯酸乙酯含R4基团。
本发明实施例还提出一种多价靶向蛋白降解纳米粒,由上述多价靶向蛋白降解前药自组装而成。
在某些实施方式中,多价靶向蛋白降解纳米粒的流体力学半径为40-100nm,更优选为60-70nm。
本发明实施例还提供多价靶向蛋白降解纳米粒的制备方法,包括以下步骤:
将所述多价靶向蛋白降解前药溶于有机相,然后超声分散于水中,所得溶液通过超滤或透析得到多价靶向蛋白降解纳米粒。
在某些实施方式中,水为去离子水,有机溶剂为N,N-二甲基甲酰胺、N,N-二甲基乙酰胺或二甲基亚砜。
超滤过程中可采用超滤管离心超滤或超滤机超滤方式进行,该过程中所使用的超滤管或超滤膜包的截留分子量可为1500D、3kD、5kD、10KD,并合理选择其中一种。
透析过程中透析袋截留分子量可为800、1000、3500、14000,并合理选择其中一种。透析采用的溶剂为去离子水。
本发明还提供多价靶向蛋白降解纳米粒在制备用于治疗恶性肿瘤的药物中的应用,其中,恶性肿瘤包括但不限于乳腺癌、宫颈癌、肝癌、胃癌、胰腺癌、卵巢癌、结肠癌或前列腺癌。
本发明的多价靶向蛋白降解纳米粒可同时荷载多个靶向蛋白降解剂,可选择性地靶向至肿瘤部位,具有良好的肿瘤组织蓄积和渗透能力。该纳米粒被肿瘤细胞高效摄取后,其可激活型的连接子断裂以释放并恢复靶向蛋白降解剂的降解功能,从而改善靶向蛋白降解剂的肿瘤组织选择性以及肿瘤细胞对靶向蛋白降解剂的摄取能力,在实现高效肿瘤组织杀伤效果的同时降低对非靶组织蛋白降解造成的毒副作用。
下面结合实施例对本发明作进一步的说明,需要说明的是,提供以下实施例仅出于说明目的并不构成对本发明要求保护范围的限制。
以下实施例中所用试剂及仪器如下:
实施例中所用的CH3O-PEG113-NH2(PEG,Mn=5000Da)、甲基丙烯酸二异丙氨基乙酯、(S)-(+)-2-(4-(4-氯苯基)-2,3,9-三甲基-6H-噻吩并[3,2-F][1,2,4]三唑并[4,3-A][1,4]二氮杂卓-6-基)乙酸((+)-JQ1)、4-氰基-4-[[(十二烷硫基)硫酮甲基]硫基]戊酸购自西格玛奥德里奇(中国)公司,甲基丙烯酰氯购自梯希爱(上海)化成工业发展有限公司,(2S,4R)-1-[(2S)-2-氨基-3,3-二甲基丁酰]-4-羟基-N-[(1S)-1-[4-(4-甲基-1,3-噻唑-5-基)苯基]乙基]吡咯烷-2-甲酰胺盐酸购自南京优氟医药科技有限公司,Fmoc-GPLGLAG-NH2购自吉尔生化(上海)有限公司,2-(3-(2-氨基乙氧基)丙氧基)乙酸叔丁酯购自上海特伯化学科技有限公司,2-羟乙基二硫化物、二(对硝基苯)碳酸酯(NPC)、1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(EDCI)、4-二甲氨基吡啶(DMAP)、二异丙基乙胺(DIEA)、二氯甲烷(DCM)、四氢呋喃、N,N-二甲基甲酰胺(DMF)、二甲亚砜、2-(7-氮杂苯并三氮唑)-N,N,N',N'-四甲基脲六氟磷酸酯(HATU)、1-羟基苯并三唑(HOBT)、偶氮二异丁腈(AIBN)、N-甲基哌啶购自上海百灵威科技有限公司。如无特殊说明,其余所用试剂和溶剂均购自国药集团(上海)化学试剂有限公司。
MDA-MB-231乳腺癌细胞购自中科院上海细胞库,细胞培养用DMEM培养基购自大连美仑生物科技有限公司,胎牛血清购自Gibco公司。
多价靶向蛋白降解纳米粒的流体力学粒径由马尔文激光粒径仪测定仪(ZEN3690、Malven、USA)测得;纳米粒的形貌考察由120kV透射电子显微镜(Talos L120C,FEI,USA)完成。电子天平(Quintix 224-1,Sartorius Germany);旋转蒸发仪(B-40,Buchi,Switzerland);恒温磁力搅拌器(DF-101S,上海予化仪器有限公司);核磁共振谱仪(Mercury Plus 400,Varian,USA);蛋白降解剂及其衍生物的释放由Waters 2695高效液相色谱仪(waters e2695色谱泵,Xbridge C18 5μm 19*250mm色谱柱,Waters 2998紫外检测器)完成。流式实验数据由BD FACS Fortessa流式细胞仪测得。如无特殊说明,所用设备及测试方法均为本领域常规的设备和方法。
实施例1:中间体甲基丙烯化-SS-NO2合成
将2-羟乙基二硫化物(300mg,1.95mmol)、甲基丙烯酰氯(184.3mg,1.77mmol)溶于5.0ml无水二氯甲烷(DCM)中,再加入三乙胺(TEA,268.1mg,2.65mmol),室温下反应24h。反应完毕后,以二氯甲烷稀释反应液,经水洗涤、无水硫酸钠干燥,柱分离得甲基丙烯化-SS-OH(319.8mg),收率81.4%。
将甲基丙烯化-SS-OH(171mg,0.77mmol)、二(对硝基苯)碳酸酯(NPC,349.6mg,1.15mmol)溶于7.0ml无水二氯甲烷中,再加入三乙胺(233.7mg,2.31mmol),室温下反应12h。反应完毕后,以二氯甲烷稀释反应液,经水、饱和氯化铵溶液洗涤,无水硫酸钠干燥,旋干,得中间体甲基丙烯化-SS-NO2。
实施例2:甲基丙烯化-SS-771合成
将JQ1(1.03g)溶于12.0mL 50%三氟乙酸/二氯甲烷溶液中,室温下反应4h。反应完毕,减压蒸馏出去溶剂后,经柱色谱分离所得淡黄色固体粉末即为化合物1(862.4mg),收率95.45%。
将化合物1(500mg,1.25mmol)溶于5.0mL无水二氯甲烷中,并向其中加入2-(7-氮杂苯并三氮唑)-N,N,N',N'-四甲基脲六氟磷酸酯(HATU,570mg,1.5mmol),N,N-二异丙基乙胺(DIEA,483.7mg,3.75mmol)和化合物2(349.5mg,1.5mmol),室温下反应24h。反应完毕后,以二氯甲烷稀释反应液,经水、饱和氯化铵溶液洗后,无水硫酸钠干燥,柱分离得化合物3(561.2mg),收率73%。
将化合物3(199.9mg,0.325mmol)溶于5.0mL 50%三氟醋酸/二氯甲烷溶液中,室温下反应4h。反应结束后,旋去溶剂,柱分离获得化合物4(170.9mg),收率94.1%。
将化合物4(100.6mg,0.18mmol)和化合物5(87.9mg,0.198mmol)溶于5.0mL无水二氯甲烷中,并向其中加入2-(7-氮杂苯并三氮唑)-N,N,N',N'-四甲基脲六氟磷酸酯(102.6mg,0.27mmol),N,N-二异丙基乙胺(69.6mg,0.54mmol),室温下反应48h。反应完毕后,旋去溶剂,柱分离得771(134.7mg),收率76%。1H-NMR(CDCl3,500MHz)δ:1.08(s,9H),1.42(d,J=7.0Hz,3H),1.67(s,3H),1.83-1.92(m,2H),2.16-2.23(m,5H),2.40(s,3H),2.50(s,3H),2.63(d,J=7.4Hz,2H),3.38-3.67(m,12H),3.93(d,J=15.5Hz,1H),4.03(d,J=15.5Hz,1H),4.63-4.67(m,2H),4.82-4.85(m,1H),5.04-5.07(m,1H),7.28-7.41(m,8H),7.67(d,J=7.75Hz,2H),8.88(s,1H).
将中间体甲基丙烯化-SS-NO2(58mg,0.15mmol),N,N-二甲氨基吡啶(DMAP,27.5mg,0.225mmol),1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(EDCI,43.1mg,0.225mmol)和N,N-二异丙基乙胺(58mg,0.45mmol),溶于3.0mL无水二氯甲烷,滴加771(98.6mg,0.1mmol)溶液,室温下反应48h。反应完毕后,反应液经无水二氯甲烷稀释后,依次用饱和氯化铵溶液、去离子水洗涤,收集有机层用无水Na2SO4干燥,柱分离得化合物甲基丙烯化-SS-771(102.5mg),收率83.2%。1H-NMR(CDCl3,500MHz)δ:1.08(s,9H),1.37(d,J=6.95Hz,1H),1.69(s,3H),1.82(s,3H),1.84-1.94(m,5H),2.40(s,3H),2.49(s,3H),2.58(m,4H),2.96(m,4H),3.86-4.01(m,11H),4.30(m,1H),4.39-4.42(m,4H),4.63-4.67(m,2H),4.86(t,J=7.7Hz,1H),5.04(m,1H),5.26(m,1H),5.58(s,1H),6.13(s,1H),7.23-7.32(m,8H),7.41(m,2H),7.89(d,J=7.75Hz,1H),8.65(s,1H).
实施例3:PEG-GALGLPH-CTA合成
将Fmoc-GPLGLAG(43.1mg,0.05mmol)溶于5.0mL无水二氯甲烷(DCM)中,并向其中加入1-羟基苯并三唑(HOBT,6.8mg,0.05mmol),1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(EDCI,9.6mg,0.05mmol),室温下羧基活化2h后,滴加H2N-PEG113-OCH3(253.1mg,0.05mmol)溶液,室温下反应24h。反应完毕后,选用透析法除去杂质,冷冻干燥得聚合物Fmoc-GPLGLAG-PEG113-OCH3(171.7mg)(缩写为Fmoc-GPLGLAG-PEG),收率58%。
将Fmoc-GPLGLAG-PEG(118.1mg,0.02mmol)溶于4.0mL N,N-二甲基甲酰胺(DMF)中,并向其中加入1-羟基苯并三唑(HOBT,2.7mg,0.02mmol),1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(3.8mg,0.02mmol),N-甲基哌啶(7.9mg,0.08mmol),室温下反应24h。反应完毕后,选用透析法除去杂质,冷冻干燥得聚合物NH2-GPLGLAG-PEG113-OCH3(104.4mg)(缩写为GPLGLAG-PEG),收率92%。
将4-氰基-4-[[(十二烷硫基)硫酮甲基]硫基]戊酸(6.0mg,0.015mmol)溶于2.0ml无水二氯甲烷中,并向其中加入N,N-二甲氨基吡啶(DMAP,1.8mg,0.015mmol),1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(EDCI,2.9mg,0.015mmol)和N,N-二异丙基乙胺(1.9mg,0.015mmol),室温下羧基活化2h后,滴加GPLGLAG-PEG(85.0mg,0.015mmol)溶液,室温下反应24h。反应完毕后,选用透析法除去杂质,冷冻干燥得聚合物CH3O-PEG113-GALGLPG-CTA(64.7mg)(缩写为PEG-GALGLPG-CTA),收率为68.6%。
实施例4:多价靶向蛋白降解前药合成
将PEG-GALGLPG-CTA(60.0mg,0.01mmol)、甲基丙烯酸二异丙氨基乙酯(127.8mg,0.6mmol)、甲基丙烯化-SS-771(49.3mg,0.04mmol)和偶氮二异丁腈(AIBN,1.6mg,0.01mmol)溶于2.0ml DMF中,70℃下反应24h。反应完毕后,选用透析法除去杂质,冷冻干燥得多价靶向蛋白降解前药(159.2mg),收率为67.1%。
实施例5:多价靶向蛋白降解纳米粒的制备
将2mg的多价靶向蛋白降解前药聚合物溶于100μl二甲亚砜溶剂中,然后将上述溶液在超声(功率70W,2min)作用下缓慢滴入1.0mL去离子水中。随后,将所获得的溶液于去离子水中透析(透析袋截留分子量为3500),所得即为多价靶向蛋白降解纳米粒(PGD7)。同时以相同方法制备不含对MMP-2响应的氨基酸片段GPLGLAG的纳米粒(PD7)作为对照。
实施例6:蛋白降解剂衍生物的还原测试
取制备实施例2制备的甲基丙烯化-SS-771溶于含有10mM DTT的甲醇和水(1:1,v/v)的混合溶剂中,于37℃恒温摇晃下,分别在0.5、1、2、3、4、6h取样,运用高效液相色谱仪计算771、771-SH、甲基丙烯化-SS-771(Me-771)的累积释放量。
结果如图1所示,合成的甲基丙烯化-SS-771在6h时全部还原为蛋白降解剂,证明了蛋白降解剂衍生物的还原响应性。
实施例7:多价靶向蛋白降解纳米粒的酸响应能力测定
取制备实施例5制备的多价靶向蛋白纳米粒溶分别在pH=7.4、pH=6.0的条件下处理2h,利用动态光散射仪测定纳米粒流体力学半径。
将分别在pH=7.4和pH=6.0条件下处理2h的多价靶向蛋白降解纳米粒溶液(0.5mg/mL)滴加在透射电镜用碳膜支持铜网上,2min后使用滤纸吸除过量液滴,使用0.1%的醋酸铀溶液染色10s,除去过量染色液后使用白炽灯烘干。使用透射电镜对制备的铜网进行观测,考察纳米粒的形貌特征。
结果如图2所示,pH=6.0时,多价靶向蛋白降解纳米粒解离,呈现无定型状态,而pH=7.4时的纳米粒,外形圆润且均一。这说明多价靶向蛋白降解纳米粒具有良好的酸响应能力。
实施例8:多价靶向蛋白降解纳米粒中771的释放
将制备实施例5中所制备的多价靶向蛋白降解纳米粒分散于透析袋(14,000Da)中,配置缓冲溶液模拟肿瘤细胞生理微环境(0.1%吐温80+10mM GSH,pH 6.0),另配置不含GSH的缓冲溶液作为对照。在37℃恒温摇晃下,分别在0.5、1、2、4、8、12、24h收集一定体积的缓冲液,利用高效液相色谱分析荷载的蛋白降解剂的累计释放量。
结果如图3所示,构建的多价靶向蛋白降解纳米粒可在肿瘤微环境条件下有效释放靶向蛋白降解剂分子。
实施例9:肿瘤细胞摄取多价靶向蛋白降解纳米粒的能力测定
将MDA-MB-231细胞按照5×104的密度种于24孔板中,24h后,当细胞处于对数生长期时使用。将制备实施例5制备的纳米粒与MMP-2酶预孵育5h后分别与细胞共孵育2、4、8、12和24h,未与MMP-2酶预孵育组作为对照,摄取结束后,PBS洗涤3次,然后使用流式细胞仪检测其摄取情况(这里制备的纳米粒存在荧光染料PPa分子(焦脱镁叶绿素a),采用检测细胞内PPa的荧光强度)。
测试结果如图4所示,多价靶向蛋白降解纳米粒(柱状图背景为黑色)与MMP-2酶预孵育后,其被肿瘤细胞摄取的量显著提高且呈时间依赖性。这说明多价靶向蛋白降解纳米粒的MMP-酶响应“PEG脱壳”策略可有效促进细胞的摄取。
实施例10:多价靶向蛋白降解纳米粒的细胞毒性测定
将MDA-MB-231细胞按照2×103的密度种于96孔板中,24h后,将制备实施例5制备的多价靶向蛋白降解纳米粒以一定浓度(0.0001μM、0.001μM、0.01μM、0.1μM、0.5μM和1.0μM)分别与细胞共孵育72h后,去除培养基,PBS洗涤3次后,加入含有10%CCK8的培养基,共孵育适宜时间后,以酶标仪于450nm处测定吸光度,并计算该纳米粒的细胞毒性。
测试结果如图5所示,含对MMP-2响应的氨基酸片段GPLGLAG的纳米粒(PGD7)相比不含对MMP-2响应的氨基酸片段GPLGLAG的纳米粒(PD7)对MDA-MB-231乳腺癌细胞具有更强的细胞毒性,表明含对MMP-2响应的氨基酸片段GPLGLAG的纳米粒,在氨基酸片段被MMP-2酶切断后,更有利于肿瘤细胞对其的摄取,致使肿瘤细胞中的药物浓度增加,抗肿瘤细胞毒性增强。
实施例11:多价靶向蛋白降解纳米粒对BRD4蛋白降解能力测定
将MDA-MB-231细胞按照5×105的密度种于6孔板中,培养24h后,将制备实施例5制备的含对MMP-2响应的氨基酸片段GPLGLAG的多价靶向蛋白降解纳米粒和不含对MMP-2响应的氨基酸片段GPLGLAG的多价靶向蛋白降解纳米粒分别以一定浓度与细胞共孵育24h后(0.00μM、0.01μM、0.10μM、0.25μM、0.50μM、1.00μM),去除培养基,PBS洗涤后收集相应细胞。细胞经裂解液处理后获取相应蛋白样品,通过凝胶电泳方式分析相关蛋白表达量变化。
测试结果如图6所示,含对MMP-2响应的氨基酸片段GPLGLAG的多价靶向蛋白降解纳米粒(右边,PGD7)相比不含对MMP-2响应的氨基酸片段GPLGLAG的多价靶向蛋白降解纳米粒(左边,PD7)具有更强的BRD4蛋白降解能力,且蛋白降解同样具有浓度依赖性。
实施例12:多价靶向蛋白降解纳米粒对肿瘤生长抑制效果的评价
荷瘤小鼠模型建立:将购买的雌性Balb/c裸小鼠饲养一周后,皮下种植MDA-MB-231(1000万/只)。观测肿瘤体积约为100mm3后,将其随机分为3组,每组5只小鼠,分别尾静脉注射PBS缓冲液,蛋白降解剂溶液以及多价靶向蛋白降解纳米粒,每三天注射一次,共5次。每隔一天测量小鼠肿瘤体积及小鼠体重,小鼠肿瘤体积计算方法如下(L为最长直径,W为最短直径,单位为毫米):
V=L×W×W/2
测试结果如图7和图8所示,多价靶向蛋白降解纳米粒可有效抑制肿瘤生长,抑制率约为65%,并且小鼠体重没有差异性变化,说明该多价靶向蛋白降解纳米粒没有明显的毒副作用。
最后应说明的是:以上各实施例仅用以说明本发明的技术方案,而非对其限制;尽管参照前述各实施例对本发明进行了详细的说明,本领域的普通技术人员应当理解:其依然可以对前述各实施例所记载的技术方案进行修改,或者对其中部分或者全部技术特征进行等同替换;而这些修改或者替换,并不使相应技术方案的本质脱离本发明各实施例技术方案的范围。
SEQUENCE LISTING
<110> 中国科学院上海药物研究所
<120> 多价靶向蛋白降解前药和形成的纳米粒、其制备方法及用途
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<170> PatentIn version 3.5
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Claims (10)
1.一种多价靶向蛋白降解前药,其特征在于,所述多价靶向蛋白降解前药包括聚合物骨架、多个连接基团和多个靶向蛋白降解剂,其中
所述聚合物骨架具有如式I所示结构:
其中,x为10-145的整数,优选为110-130,进一步优选为113;
y为30-70的整数,优选为50-65,进一步优选为60;
z为2-6的整数,优选为4-6,进一步优选为4;
R1为对基质金属蛋白酶2和/或基质金属蛋白酶9响应的氨基酸片段,氨基酸片段的C端与亚氨基-NH-相连、N端与羰基-C(=O)-相连;
R2和R3为同一RAFT聚合链转移剂的不同官能团;
R4为N,N-二乙基氨基、N,N-二丁基氨基、N,N-二异丙基氨基、五亚甲基氨基或六亚甲基氨基中的一种;
所述聚合物骨架中的每个甲基丙烯酰基端通过所述连接基团与所述靶向蛋白降解剂以共价键连接;
所述聚合物骨架中的每个甲基丙烯酰基分别与一个所述连接基团一端以共价键连接;每个所述连接基团另一端分别与一个所述靶向蛋白降解剂中的脯氨酸的羟基以共价键连接;
所述靶向蛋白降解剂为能够与含Von Hippel-Lindau受体的E3泛素连接酶结合的靶向蛋白降解剂。
2.根据权利要求1所述的多价靶向蛋白降解前药,其特征在于,R1选自氨基酸序列GPLGLAG、GGKGPLGLPG或PVGLIG中的一种。
3.根据权利要求1所述的多价靶向蛋白降解前药,其特征在于,R2和R3选自以下两种情况中的一种:
(a)R2为氰基,R3为C6-C12烷硫基、苄基或(2-苯乙基)硫基;
(b)R2为甲基,R3为C6-C12烷硫基。
4.根据权利要求1所述的多价靶向蛋白降解前药,其特征在于,R4选自N,N-二异丙基氨基。
7.一种权利要求1-6任一项所述的多价靶向蛋白降解前药的制备方法,其特征在于,包括以下步骤:
将Fmoc-氨基酸片段与NH2-PEG-OCH3发生酰胺化反应,得到CH3O-PEG-NH-氨基酸片段-Fmoc;产物CH3O-PEG-NH-氨基酸片段-Fmoc脱Fmoc保护后与RAFT聚合链转移剂发生酰胺化反应,得到CH3O-PEG-NH-氨基酸片段-CO-RAFT聚合链转移剂;
将所述连接基团一端甲基丙烯化,另一端任选地活化,得到一端甲基丙烯化、另一端活化或未活化的连接基团;
将所述靶向蛋白降解剂与一端甲基丙烯化、另一端活化或未活化的连接基团反应,得到甲基丙烯化的连接基团-靶向蛋白降解剂;
将所述CH3O-PEG-NH-氨基酸片段-CO-RAFT聚合链转移剂、甲基丙烯化的连接基团-靶向蛋白降解剂和甲基丙烯酸乙酯发生RAFT聚合反应,得到所述多价靶向蛋白降解前药;
其中,所述氨基酸片段为R1,所述RAFT聚合链转移剂含有不同官能团R2和R3,所述甲基丙烯酸乙酯含R4基团。
8.一种多价靶向蛋白降解纳米粒,其特征在于,由权利要求1-6任一项所述的多价靶向蛋白降解前药自组装而成;
优选地,所述多价靶向蛋白降解纳米粒的流体力学半径为40-100nm,更优选为60-70nm。
9.一种权利要求8所述的多价靶向蛋白降解纳米粒的制备方法,其特征在于,包括以下步骤:
将所述多价靶向蛋白降解前药溶于有机相,然后超声分散于水中,所得溶液通过超滤或透析得到多价靶向蛋白降解纳米粒。
10.一种权利要求8所述的多价靶向蛋白降解纳米粒在制备用于治疗恶性肿瘤的药物中的用途,所述恶性肿瘤优选自乳腺癌、宫颈癌、肝癌、胃癌、胰腺癌、卵巢癌、结肠癌或前列腺癌。
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