CN115244064A - 治疗性分子的靶向递送 - Google Patents
治疗性分子的靶向递送 Download PDFInfo
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- CN115244064A CN115244064A CN201980092916.5A CN201980092916A CN115244064A CN 115244064 A CN115244064 A CN 115244064A CN 201980092916 A CN201980092916 A CN 201980092916A CN 115244064 A CN115244064 A CN 115244064A
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Abstract
本发明涉及将治疗性分子靶向递送至人和其他哺乳动物的器官、组织和细胞。本发明涉及用于递送这样的治疗性分子的化学构建体,以及制造和使用所述构建体的方法。
Description
相关专利申请的交叉引用
本申请要求于2018年12月28日提交的美国临时专利申请第62/786,213号的权益和优先权,该申请通过引用整体并入本文。
序列表
本申请含有序列表,其已以ASCII格式以电子方式提交,并通过引用整体并入本文。所述ASCII副本创建于2020年2月14日,文件名为4690_0024i_SL.txt,大小为9,268字节。
技术领域
本发明涉及将治疗性分子靶向递送至人和其他哺乳动物的器官、组织和细胞。
背景技术
将治疗性化合物递送至人体内的特定位置(例如,至所需的器官、组织或细胞)具有许多益处,不仅增强治疗效果,而且在剂量和清除率方面改善安全性特征。在通过提高疗效和减少副作用来改善肿瘤治疗的驱动下,靶向递送治疗剂引起了极大兴趣[1]。此外,将治疗性化合物有效地递送至体内的特定位置将最小化或避免非期望的副作用,如需要高得多的剂量以确保将适量的材料递送至作用部位并预期在这些较高剂量下产生有害的副作用。
已证明可有效地将治疗性化合物递送至目标位置的方法之一是将该化合物连接至靶向配体[2]。选择配体以识别并结合其归巢受体(存在于待靶向细胞的质膜外部),并且该归巢受体在与连接了治疗性化合物的配体结合后将该化合物转移至细胞中以发挥其治疗作用。
在哺乳动物中功能性RNAi通路的发现,作为选择性沉默特定基因和减少作为疾病病因学的内在原因的蛋白质产生的方法,为反向遗传学提供了强大的工具,因此,在包括基于核苷酸的药物如microRNA(miRNA)、小干扰RNA(siRNA)和DNA疫苗的新型生物药物之中,RNAi沉默任何基因的潜力使其成为一种有吸引力的治疗方式。最近,由于其序列特异性的转录后基因沉默能力,siRNA已成为治疗许多疾病的有前途的新型治疗性候选物,所述疾病如癌、感染、黄斑变性、心血管疾病、神经***疾患和其他基因相关疾病。由于其降低(knockdown)任何基因表达的能力,siRNA被认为是治疗多种疾病的理想候选物,所述疾病包括那些具有“无成药性(undruggable)”靶点的疾病(即那些单克隆抗体无法接触的疾病,或不具有小分子可以在该处阻断蛋白质活性的清晰位点的疾病)。
由于未修饰的siRNA被血清核酸酶降解、被快速清除、被内体截留和用于递送的纳米颗粒产生的先天免疫刺激,因此体内靶向递送siRNA的方法一直具有挑战性[3]。
附图说明
图1:一般构建体示意图。构建体含有接头-1和接头-2、连接配体部分和有效载荷部分的连接桥、以及靶向配体和递送的有效载荷。所述有效载荷是治疗性分子。
图2:配体偶联的siRNA示意图。N-乙酰半乳糖胺(GalNAc)偶联的siRNA(TGFβ1或cox2)由25个核苷酸的有义链和25个核苷酸的反义链组成,其中该反义链的3'末端具有零核苷酸的突出(zero-nucleotide overhang)。GalNAc分别与有义链的3'或5'末端偶联。在此呈现三种类型的配体,分别为三价-GalNAc偶联物、二价-GalNAc偶联物和单价-GalNAc偶联物,其中在每种情况下连接三个、两个和一个配体。
图3:可选的配体偶联的siRNA有义链示意图。N-乙酰半乳糖胺(GalNAc)偶联的siRNA(TGFβ1或Cox2)由25个核苷酸的有义链和25个核苷酸的反义链组成,其中该反义链的3'末端具有零核苷酸的突出。GalNAc分别通过脂肪链与有义链的3'或5'末端偶联。在此呈现三种类型的配体,分别为三价-GalNAc偶联物(n=3)、二价-GalNAc偶联物(n=2)和单价-GalNAc偶联物(n=1),其中在每种情况下连接三个、两个和一个配体。
图4:可选的配体偶联的siRNA有义链示意图。N-乙酰半乳糖胺(GalNAc)偶联的siRNA(TGFβ1)由25个核苷酸的有义链和25个核苷酸的反义链组成,其中该反义链的3'末端具有零核苷酸的突出。该RNA被甲基化为OMe(或部分修饰的)官能团以提高稳定性。GalNAc分别通过磷酸酯与有义链的5'(或3')末端偶联。在此呈现三种类型的配体,分别为三价-GalNAc偶联物(n=3)、二价-GalNAc偶联物(n=2)和单价-GalNAc偶联物(n=1),其中在每种情况下连接三个、两个和一个配体。另一个3'(或5')末端与胆固醇官能团偶联以增强膜穿透能力。图4公开了SEQ ID NO:7。
图5:可选的配体偶联的siRNA示意图。N-乙酰半乳糖胺(GalNAc)偶联的siRNA(COX-2)由25个核苷酸的有义链和25个核苷酸的反义链组成,其中该反义链的3'末端具有零核苷酸的突出。该RNA被甲基化为OMe官能团以提高稳定性。GalNAc分别通过磷酸酯与有义链的5'(或3')末端偶联。在此呈现三种类型的配体,分别为三价-GalNAc偶联物(n=3)、二价-GalNAc偶联物(n=2)和单价-GalNAc偶联物(n=1),其中在每种情况下连接三个、两个和一个配体。另一个3'(或5')末端与胆固醇官能团偶联以增强膜穿透能力。图5公开了SEQ ID NO:21。
图6:siRNA有义链和接头-配体之间的连接类型和合成路线图示。通过几个合成步骤进行化学转化来共价修饰有义链的3'末端,以连接官能化聚乙二醇(Fun PEG)基团。2'位置可以是H或具有保护基团如TOM或TBDMS的OR基团。
图7:siRNA(TGFβ1)和配体(例如GalNAc)之间的接头-1的设计。在此描述了两种类型的接头,其用于将siRNA连接至配体。一种是使用可作为接头的具有末端硫醇的水溶性PEG,另一种是使用也具有末端硫醇的聚(L-丙交酯)以提供用于连接至其他部分的位点。具有不同长度的这两种产品都便捷可得,并随时可以使用标准马来酰亚胺连接化学与硫醇基团偶联[4]。
图8:以马来酰亚胺官能团封端的配体GalNAc分子的结构图示。显示了单价GalNAc分子、二价GalNAc分子和三价GalNAc分子。通过使用叠氮化物和炔烃之间的“点击(click)”反应,将三个GalNAc配体通过***环连接至三足(tripodal)接头[5]。该分子的另一末端被马来酰亚胺功能基序封帽(capped)以允许进一步的化学修饰。
图9:GalNAc-siRNA在HepG2细胞系中的体外测试。这项对人肝细胞癌HepG2细胞生存力的研究使用图4中m=0的GalNAc-TGFβ1。以GalNAc-TGFβ1(25nM、50nM、100nM)以及对照空白、非沉默siRNA(NC,100nM)、HKP(100nM)、脂质体(分别为25nM、50nM和100nM)和脂质体-GalNAc-TGFβ1(分别为25nM、50nM、100nM)表示细胞死亡siRNA处理的作用。将GalNAc-TGFβ1(分别为25nM、50nM、100nM)、空白对照、非沉默siRNA(NC、100nM)、HKP(100nM)、脂质体(分别为25nM、50nM和100nM)和脂质体-GalNAc-TGFβ1(分别为25nM、50nM、100nM)的混合物与细胞在100μL OPTI-MEM培养基中孵育。6h后用10%FBS/DMEM或EMEM替换转染培养基。在转染后72h,通过实时定量逆转录QRT-PCR测定评估活细胞的数量,以量化TGF-β1mRNA的相对表达。将源自未处理细胞(空白)的值设为100%。NC-非沉默siRNA。
图10:GalNAc-siRNA在小鼠模型中的体内测试。本研究使用了图4中GalNAc-TGFβ-1(m=0)的结构。剂量为每只小鼠的siRNA,对于GalNAc/siRNA-H(高浓度)至GalNAc/siRNA-L(低浓度),一次注射施用200μg、100μg或50μg,PC(HKP/siRNA=4:1)为40μg。小鼠按设计剂量通过尾静脉注射施用。药物施用24小时后,收集经处理动物的肝脏右叶并匀浆以提取RNA。然后进行QRT-PCR以测量相关mRNA的沉默程度。显示的数据是4只小鼠的平均值。*-P<0.05对比空白,**-P<0.01对比空白。PC是指阳性对照。总之,在阳性对照(PC)中,HKP/siRNA被递送至整个肝脏,然而,GalNAc-H、-M、-L仅对肝脏的肝细胞具有特异性。因此GalNAc案例肝脏中表达的总mRNA略高于PC案例。我们还观察到GalNAc-H、-M和-L的剂量依赖性效应。总体而言,其强烈表明GalNAc已成功递送siRNA并显示出沉默作用。
图11:单价GalNAc配体的合成路线。显示了单价GalNAc配体的合成。该方法采用几个步骤,主要利用两个分子之间的“点击”反应,随后在NHS基团和胺之间形成酰胺。最后,其以马来酰亚胺基团封端。
图12:二价GalNAc配体的合成路线。在此显示了二价GalNAc配体的合成,通过五个步骤进行,主要是通过引入炔基,叠氮化物-GalNAc之间的“点击”反应,随后在NHS基团和胺之间形成酰胺。最后,其以马来酰亚胺基团封端。
图13:三价GalNAc配体的合成路线。该合成路线与图11中的非常相似,仅将底物改为三(羟甲基)-氨基甲烷。
图14:三价GalNAc配体修饰的siRNA(TGFβ1)有义链的合成路线。将GalNAc偶联至由25个核苷酸有义链组成的siRNA有义链的5'末端,其中该有义链的3'末端可以进一步以其他官能团修饰。图14按出现顺序分别公开了SEQ ID NO 7和7。
图15:三价GalNAc配体修饰的siRNA(COX-2)有义链的合成路线。将GalNAc偶联至由25个核苷酸有义链组成的siRNA有义链的5'末端,其中该有义链的3'末端可以进一步以其他官能团修饰。图15按出现顺序分别公开了SEQ ID NO 21和21。
图16:三价GalNAc配体修饰的siRNA的制备。如图16中所示,在siRNA双链体有义链或反义链的3'(或5')末端,用含有硫醇的接头进行化学修饰。然后在pH 7.4-9.0的缓冲液中,通过硫醇/马来酰亚胺化学,以1.2比1的摩尔比将该构建体与预先制备的三价GalNAc配体偶联,形成硫醇-碳键。得到的GalNAc偶联的siRNA可以直接用作体外研究的缓冲液形式,或通过膜透析除去盐并冻干成固体形式。
具体实施方式
本发明涉及用于将治疗性分子递送至哺乳动物细胞的化学构建体,所述哺乳动物细胞优选人细胞,最优选人体内的人细胞。该构建体由式(I)表示:
A—B-[-C—D]n(I)
其中A是第一接头(接头1),B是桥,C是第二接头(接头2),D是靶向配体,n是1至4的整数。接头1和接头2可以相同或不同。在一个实施方案中,n=1。在另一个实施方案中,n=3。
选择适合在本文公开的构建体中使用的接头,其包括足够稳定并限制一个或多个目标部分之间的潜在相互作用的水溶性和柔性的聚乙二醇(PEG)。此外,已通过临床研究验证了PEG安全且适用于治疗目的。在一些实施方案中,所述接头可以是具有选定范围的分子量的聚(L-丙交酯),其用于合适地递送在酯键中具有生物可降解性质的靶向化合物。接头反应性连接部分包括但不限于硫醇-马来酰亚胺连接、炔-叠氮连接的***和胺-NHS连接的酰胺。
在一个实施方案中,接头1是如下面第一个结构所示的直链聚乙二醇,其中n1是介于1至50之间的整数,或接头1是如下面第二个结构所示的聚(L-丙交酯),其中n2是1至70的整数,并且其中Z(显示在下面两个结构中)是官能团,如硫醇或羧酸,其将与马来酰亚胺或胺反应以与桥共价偶联。
在另一个实施方案中,接头1具有如下所示的包含硫醇-马来酰亚胺键的亚化学基团Z:
在该实施方案的一方面,Z是化学连接接头1和桥的对接位点(docking site)。
在一个实施方案中,接头2是三-、四-或五-乙二醇。在该实施方案的一方面,将包含接头2和1至3个靶向配体的化学结构连接至桥,其中所述化学结构包含以下结构之一:
其仅允许在对位偶联一个包含接头2和靶向配体的化学构建体,或桥是具有下式
其允许在两个间位偶联两个包含接头2和靶向配体的化学构建体,或桥是具有下式
其允许在两个间位和一个对位偶联三个包含接头2和靶向配体的化学构建体。
在化学构建体的一个实施方案中,接头1是由内部酰胺键偶联的直链脂肪链,并且接头2和桥已被磷酸酯键替换(replaced),如下结构所示:
在构建体的一个实施方案中,所述靶向配体是N-乙酰-半乳糖胺(GalNAc)、半乳糖、半乳糖胺、N-缩甲醛-半乳糖胺、N-丙酰-半乳糖胺或N-丁酰半乳糖胺。在该实施方案的一方面,靶向配体是N-乙酰-半乳糖胺(GalNAc)。
构建体(I)可以通过接头1直接与治疗性分子偶联,形成具有式(II)的新构建体:
TM—A—B-[-C—D]n (II)
其中TM是治疗性分子,A是第一接头(接头1),B是桥,C是第二接头(接头2),D是靶向配体,n是1至4的整数。如本文所用,治疗性分子是在人体内具有治疗作用的分子。这样的治疗性分子包括表达抑制性寡核苷酸、治疗肽、具有治疗效果的抗体和具有治疗效果的小分子。
在该第二构建体(II)的一个实施方案中,所述表达抑制性寡核苷酸是RNAi、反义RNA或cDNA。在该实施方案的一方面,所述RNAi是siRNA或miRNA。在该实施方案的另一方面,所述RNAi是siRNA。
在另一个实施方案中,第二构建体由式(III)表示:
O—A—B-[-C—D]n (III)
其中O是寡核苷酸,A是第一接头(接头1),B是桥,C是第二接头(接头2),D是靶向配体,n是1至4的整数。这样的寡核苷酸包括RNAi、反义RNA或cDNA。A、B、C和D如上所述。在该实施方案的一方面,所述寡核苷酸是双链的。在另一方面,所述寡核苷酸是单链的。在该实施方案的一方面,所述寡核苷酸被部分化学修饰。
在该实施方案的一方面,所述RNAi是siRNA或miRNA。在该实施方案的另一方面,所述RNAi是siRNA。在另一方面,所述RNAi是双链的,并通过该RNAi有义链的3'末端处的磷酸酯、硫代磷酸酯或膦酸酯基团与接头1共价结合。
在又一方面,所述寡核苷酸是siRNA。优选地,所述siRNA的长度介于10至27个核苷酸之间。最优选地,其长度介于19至25个核苷酸之间。优选地,所述靶向配体是GalNAc。
在另一方面,第一构建体(I)通过接头1在3'位置或5'位置共价连接至siRNA分子,如下所示,x=O或S,y=O或S:
如本文所用,siRNA分子是双链体寡核苷酸,其是短双链多核苷酸,其在该分子引入细胞中后干扰该细胞中基因的表达。例如,其靶向并结合单链靶向RNA分子中的互补核苷酸序列。通过本领域技术人员已知的技术化学合成或以其他方式构建siRNA分子。这样的技术描述于美国专利No.5,898,031、6,107,094、6,506,559、7,056,704和欧洲专利No.1214945和1230375中,其通过引用整体并入本文。按照本领域的惯例,当通过单一、特别的核苷酸序列识别siRNA分子时,该序列是指双链体分子的有义链。可以通过本领域已知的技术化学修饰该分子包含的一种或多种核糖核苷酸。除了在其一个或多个个体核苷酸的水平上修饰之外,也可以修饰寡核苷酸的主链。其他修饰包括使用小分子(例如糖分子)、氨基酸、肽、胆固醇和其他大分子与siRNA分子偶联。
在一个特别的方面,所述siRNA是抗TGFβ1siRNA。如本文所用,抗TGFβ1siRNA是siRNA分子,其降低或阻止人或其他哺乳动物细胞中编码TGFβ1蛋白合成的基因的表达。
在另一个特别的方面,所述siRNA是抗Cox2 siRNA。如本文所用,抗-Cox2siRNA是siRNA分子,其降低或阻止人或其他哺乳动物细胞中编码Cox2蛋白合成的基因的表达。
在另一个特别的方面,所述siRNA的寡核苷酸在2'位置被完全或部分化学修饰以提高稳定性。
表1.靶向TGF-β1和Cox2的强效siRNA:
某些抗TGFβ1和抗Cox-2siRNA分子描述于美国专利9,642,873B2(2017年5月9日)和美国再颁发专利RE46,873E(2018年5月29日)中,其公开内容通过引用整体并入本文。
在第二构建体(II)的一个实施方案中,所述治疗性分子是治疗肽。这样的治疗肽包括环状(c)RGD、APRPG(SEQ ID NO:25)、NGR、F3肽、CGKRK(SEQ ID NO:26)、LyP-1、iRGD(CRGDRCPDC)(SEQ ID NO:27)、iNGR、T7肽(HAIYPRH)(SEQ ID NO:28)、可被MMP2剪切的八肽(GPLGIAGQ)(SEQ ID NO:29)、CP15(VHLGYAT)(SEQ ID NO:30)、FSH(FSH-β,33至53个氨基酸,YTRDLVKDPARPKIQKTCTF)(SEQ ID NO:31)、LHRH(QHTSYkcLRP)、胃泌素释放肽(GRP)(CGGNHWAVGHLM)(SEQ ID NO:32)、RVG(YTWMPENPRPGTPCDIFTNSRGKRASNG)(SEQ ID NO:33)、FMDV20肽序列(NAVPNLRGDLQVLAQKVART)(SEQ ID NO:34)或GLP。
在第二构建体(II)的另一个实施方案中,所述治疗性分子是用于治疗用途的抗体。这样的治疗性抗体包括IgM、IgD、IgG、IgA、IgE或抗体片段F(ab')2、Fab、Fab'或Fv。
在第二构建体(II)的又一个实施方案中,所述治疗性分子是用于治疗用途的小分子。这样的治疗性小分子包括吉西他滨、叶酸、顺铂、奥沙利铂、卡铂、多柔比星或紫杉醇。
考虑到本文公开的结构和教导,本发明的构建体可以由本领域技术人员合成。例如,当第二构建体中的治疗性分子是siRNA分子时,该构建体可以通过以下步骤合成:
1)在siRNA分子的5'或3'位点处,通过形成磷酸酯键将siRNA分子有义链与官能化接头1偶联;
2)将一至三个靶向配体-接头2分子连接至三足、二足(dipodal)或直链桥位点;在桥的另一末端是预先引入的以马来酰亚胺基团封端的短PEG基团,其用于将接头1与桥偶联;
3)通过硫醇/马来酰亚胺反应,将siRNA-接头-1构建体与接头-2-靶向配体构建体偶联,以提供具有所述一至三个靶向配体分子的siRNA分子有义链的构建体;和
4)将该有义链-靶向配体构建体与siRNA分子反义链混合,以形成具有一至三个靶向配体的双链体siRNA。
当第二构建体中的治疗性分子是抗体或肽时,该构建体可以通过以下步骤合成:
1)在抗体(或肽)分子的炔、硫醇、NHS官能化位点处,通过形成***环、硫醇-碳键或酰胺键,将抗体(或肽)分子与官能化接头-1(如叠氮基、马来酰亚胺、胺)偶联;
2)将一个(或两个或三个)靶向配体-接头2构建体连接至中心直链接头(或双足或三足桥)位点;在桥的另一末端与末端具有马来酰亚胺官能团的短PEG基团偶联,所述PEG基团用于将接头1与桥偶联;和
3)通过硫醇/马来酰亚胺反应,将抗体(或肽)-接头-1构建体与接头-2-靶向配体构建体偶联,以提供具有靶向配体的抗体(或肽)构建体。
当第二构建体中的治疗性分子是siRNA分子,且靶向配体是GalNAc时,该构建体可以通过以下步骤合成:
1)通过将一至三个GalNAc-接头2分子连接至三足、二足或直链桥位点,构建GalNAc-接头-2-桥;在桥的另一末端是预先引入的以马来酰亚胺基团封端的短PEG基团,其用于将接头1与桥偶联;
2)将接头1,如含有硫醇基团部分的PEG或聚(L-丙交酯),与桥-接头2-GalNAc部分的末端马来酰亚胺反应,以形成S-C共价键;
3)通过亚磷酰胺基团和羟基基团之间的磷酸酯键,将配体-接头2-接头1构建体偶联至siRNA分子有义链的5'或3'末端;和
4)将该有义链-GalNAc构建体与siRNA分子反义链混合,以形成具有一至三个GalNAc配体的双链体siRNA。
可以通过递送剂如细胞渗透肽和/或内体释放剂,将本发明的构建体(I)间接偶联至治疗性分子。构建体(I)首先与短功能肽(3至20个氨基酸,如细胞内体释放肽HHHK(SEQID NO:35)、HHHHK(SEQ ID NO:36)、(HHHK)n(n=1至5)等)(SEQ ID NO:37)偶联。然后将治疗性分子(如反义寡核苷酸、siRNA、DNA、适配体(aptamer)、肽、小分子药物等)与功能肽偶联。
本发明还包括药物组合物。在一个实施方案中,该组合物包含在药学上可接受的载体中的上述第一构建体(I)。在另一个实施方案中,该组合物包含在药学上可接受的载体中的上述第二构建体(II)或第三构建体(III)。在两个实施方案的一方面,所述药学上可接受的载体包含水和一种或多种以下盐或缓冲剂:无水磷酸二氢钾NF、氯化钠USP、七水磷酸氢二钠USP和磷酸盐缓冲盐水(PBS)。
本发明的构建体和药物组合物可用于在体外或体内向人细胞递送治疗性分子。如上所述,这样的治疗性分子包括表达抑制性寡核苷酸、治疗肽、治疗有效的抗体和治疗有效的小分子。
当在体内使用时,该构建体和药物组合物用于治疗人的疾病。在一个实施方案中,将治疗有效量的本发明的药物组合物递送至患有需要治疗的疾病的人。
一类这样的疾病是人的癌。这样的癌包括肝癌、胆管癌(CCA)、结肠癌、胰腺癌、肺癌、膀胱癌、卵巢癌、头颈癌、食道癌、脑癌和皮肤癌,所述皮肤癌包括黑色素瘤和非黑色素瘤皮肤癌。在该实施方案的一方面,所述癌是肝癌、结肠癌或胰腺癌。
在一个特别的方面,所述癌是肝癌。所述肝癌可以是原发性肝癌或从人体内的另外的组织转移至肝脏的癌。原发性肝癌包括肝细胞癌或肝母细胞癌。转移癌包括结肠癌和胰腺癌。
其他的人的疾病可用本发明的构建体和药物组合物治疗。这样的疾病包括肝炎、纤维化和原发性硬化性胆管炎(PSC)。将治疗有效量的本发明的药物组合物施用于需要治疗的患者。
本发明的构建体和药物组合物也可用于基因疗法。将治疗有效量的本发明的药物组合物施用于需要这样的疗法的人或其他哺乳动物。其他哺乳动物包括实验室动物,如啮齿动物、豚鼠和雪貂、宠物和非人灵长类动物。
以下实施例说明了本发明的某些方面,并且不应被解释为限制其范围。
实施例:
实施例1.以马来酰亚胺封端的三价GalNAc-PEG6-Mal的1H NMR谱(D2O,400MHz)。通过“点击”反应,通过***环通过三甘醇,将GalNAc连接至三足中心。在另一末端使用六-PEG与马来酰亚胺连接。
实施例2.以马来酰亚胺封端的三价GalNAc-PEG6-Mal的质谱(ESI-MS,正离子)。发现分子离子为[M+H]+=1928.1,计算为1928。
实施例3.以马来酰亚胺封端的三价GalNAc-PEG6-Mal的HPLC谱(C18柱,0.1%TFA水/0.1%TFA乙腈梯度)。
实施例4.TGFβ1和COX-2的序列和结构。有义链和反义链的序列如下所示。在完全甲基化的有义链内的所有核苷酸处进行修饰。有义链的5'末端通过接头与GalNAc配体偶联,有义链的3'末端以胆固醇进行化学修饰以提高膜穿透能力。
实施例5.GalNAc-siRNA在HepG2细胞系中的体外测试。
这项人肝细胞癌HepG2细胞生存力的研究使用图3中m=0的GalNAc-TGFβ1。用GalNAc-TGFβ1(25nM、50nM、100nM)以及对照空白、非沉默siRNA(NC,100nM)、HKP(100nM)、脂质体(分别为25nM、50nM和100nM)和脂质体-GalNAc-TGFβ1(分别为25nM、50nM、100nM)表示细胞死亡siRNA处理的作用。将GalNAc-TGFβ1(分别为25nM、50nM、100nM)、空白对照、非沉默siRNA(NC、100nM)、HKP(100nM)、脂质体(分别为25nM、50nM和100nM)、脂质体-GalNAc(分别为25nM、50nM、100nM)的混合物与细胞在100μL OPTI-MEM培养基中孵育。6h后用10%FBS/DMEM或EMEM替换转染培养基。在转染后72h,通过实时定量逆转录QRT-PCR测定评估活细胞的数量,以量化TGF-β1mRNA的相对表达。将源自未处理细胞(空白)的值设为100%。NC-非沉默siRNA。见图8。
实施例6.GalNAc-TGFβ1在小鼠模型中的体内测试。
将一组20只4周龄的雌性小鼠分为四组。剂量为每只小鼠的siRNA,GalNAc/siRNA-H至GalNAc/siRNA-L的一次注射剂量为200μg、100μg和50μg,PC(HKP/siRNA=4:1)为40μg。每组在尾静脉注射相应药物,注射一次。施用后24小时,处死动物并收集肝组织。将肝组织的右叶匀浆,用于RNA提取。然后进行qRT-PCR。显示的数据是4只小鼠的平均值。*-P<0.05对比空白,**-P<0.01对比空白。见图9。在阳性对照(PC)中,HKP/siRNA被递送至整个肝脏,然而,GalNAc-H、-M、-L仅对肝脏的肝细胞具有特异性。因此GalNAc案例中的总体mRNA表达水平略高于PC案例,但与空白(未处理)相比很好。我们观察到GalNAc-H、-M和-L的剂量依赖性效应。总体而言,这强烈表明GalNAc已成功递送siRNA并显示出沉默作用。
实施例7.图12中的三足化合物2的制备。[7]
在剧烈搅拌下,向三(羟甲基)氨基甲烷(1)(10.0g,83.0mmol)的t-BuOH(100mL)悬浮液中缓慢加入二碳酸二叔丁酯(23.4g,107.2mmol)在MeOH:t-BuOH(160mL,V/V=1:1)中的混合物,并将反应混合物在室温搅拌15h。15h后,使用旋转蒸发仪蒸发溶剂,得到白色粗固体,在室温将其从乙酸乙酯(300mL)中重结晶。使用真空过滤收集白色针状结晶,将其用***(100mL)洗涤。将固体在真空下干燥六小时,得到白色固体状的纯产物2(17.0g,93%)。1HNMR数据与文献值非常吻合。TLC(硅胶,己烷:乙酸乙酯=5:1),1H NMR(400MHz,DMSO-d6)δ:5.77(br s,1H,NH),4.50(t,3H,J=5.2Hz,3×OH),3.50(d,6H,J=4.8Hz,CH2OH),1.37[s,9H,3×C(CH3)3]ppm。
实施例8.图12中的三足化合物3的制备。[8]
向4(13.0g,58.7mmol)的无水DMF溶液中加入炔丙基溴(80重量%的甲苯溶液)(32.0mL,364.3mmol),并在0℃搅拌反应混合物10min。随后以小份加入细粉状KOH(20.0g,364.3mmol)。然后将反应混合物整体在室温搅拌40h,此时TLC(正己烷:EtOAc=5:1)显示产生了移动较快的斑点。向所得棕色混合物中加入乙酸乙酯并再搅拌10min。进一步将反应混合物整体依次用H2O(2×30mL)和盐水(25mL)洗涤。收集有机乙酸乙酯层,用无水Na2SO4干燥并过滤。然后真空蒸发溶剂。使用正己烷:EtOAc作为洗脱剂,通过快速柱色谱法纯化由此获得的粗物质,得到黄色油状的纯化合物5(13.2g,67%)。1H NMR(500MHz,CDCl3)δ:4.9(br s,1H,NH),4.14(d,6H,3×CH2CCH),3.78(s,6H,CH2OH),2.42(t,3H,2.0Hz,CCH),1.42(s,1H,3×C(CH3)3)。
实施例9.三价GalNAc-PEG6-Mal配体的制备。
通过5个步骤合成以马来酰亚胺封端的三价GalNAc-PEG6-Mal;通过“点击”反应,将化合物9与化合物3偶联,得到化合物10。Boc脱保护得到化合物11后,将化合物11与N-羟基琥珀酰亚胺基团反应,得到目标化合物三价GalNAc-PEG6-Mal配体。详细步骤见图12,表征见实施例1至3。
实施例10.寡核苷酸-GalNAc偶联物的制备。
由RNA ABI合成仪制备具有设计的序列和功能部分的寡核苷酸。见图15中的示例。通过合成后修饰,以硫醇接头修饰有义链。然后在pH=7.5-9的磷酸盐缓冲液中,将硫醇修饰的有义链与三价β-(GalNAc)3-PEG6-MAl偶联,经gel-pak柱或反向C18柱纯化后,用乙腈和醋酸钠缓冲液洗脱,得到纯的配体偶联的寡核苷酸。siRNA双链体包含两个单寡核苷酸(连接配体的有义链和反义链),在这种情况下,通过接头和GalNAc对3'-有义链进行硫醇修饰,然后通过加热两条单链的混合物使两条链(有义:反义比=1:1.05=nmol:nmol)在90℃退火5min,然后使其以1℃/min缓慢冷却至室温。然后将所得混合物在-20℃过夜储存待用。可选地,首先使用具有配体修饰的有义链和反义链,通过类似的方法使双链体退火。然后在pH=7.5-9的磷酸盐缓冲液中,使用退火的双链体与三价β-(GalNAc)3-PEG6-MAl偶联。在去除盐后获得纯的配体偶联的siRNA,或按其原样使用。见图12至15。
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在此标识的所有出版物,包括已发布的专利和公开的专利申请,以及由url地址或登录号标识的所有数据库条目,均通过引用整体并入本文。
尽管本发明已经结合其某些实施例进行了描述,并且为了说明的目的已经阐述了许多细节,但是对于本领域技术人员来说显而易见的是,本发明易于采用另外的实施方案,并且本文所描述的某些细节在不背离本发明的基本原理的情况下可以有变化。
Claims (60)
2.根据权利要求1所述的构建体,其中接头2包含三-、四-或五-乙二醇。
8.根据权利要求7所述的构建体,其中Z包含化学连接接头1和所述桥的对接位点。
9.根据权利要求1至8中任一项所述的构建体,其中所述靶向配体选自N-乙酰-半乳糖胺(GalNAc)、半乳糖、半乳糖胺、N-缩甲醛-半乳糖胺、N-丙酰-半乳糖胺和N-丁酰半乳糖胺。
10.根据权利要求9所述的构建体,其中所述靶向配体是N-乙酰-半乳糖胺(GalNAc)。
11.根据权利要求1至9中任一项所述的构建体,其中n是2。
12.根据权利要求1至9中任一项所述的构建体,其中n是3。
13.根据权利要求1至9中任一项所述的构建体,其中n是4。
14.根据权利要求1至13中任一项所述的构建体,其中接头1连接至治疗性分子,所述治疗性分子选自表达抑制性寡核苷酸、治疗肽、具有治疗效果的抗体和具有治疗效果的小分子。
15.根据权利要求14所述的构建体,其中所述表达抑制性寡核苷酸包括RNAi、
反义RNA或cDNA。
16.根据权利要求15所述的构建体,其中所述RNAi包括siRNA或miRNA。
17.根据权利要求15所述的构建体,其中所述RNAi包括siRNA。
19.根据权利要求14所述的构建体,其中所述治疗肽包括环状(c)RGD、APRPG(SEQ IDNO:25)、NGR、F3肽、CGKRK(SEQ ID NO:26)、LyP-1、iRGD(CRGDRCPDC)(SEQ ID NO:27)、iNGR、T7肽(HAIYPRH)(SEQ ID NO:28)、可被MMP2剪切的八肽(GPLGIAGQ)(SEQ ID NO:29)、CP15(VHLGYAT)(SEQ ID NO:30)、FSH(FSH-β,33至53个氨基酸,YTRDLVKDPARPKIQKTCTF)(SEQ IDNO:31)、LHRH(QHTSYkcLRP)、胃泌素释放肽(GRP)(CGGNHWAVGHLM)(SEQ ID NO:32)、RVG(YTWMPENPRPGTPCDIFTNSRGKRASNG)(SEQ ID NO:33)、FMDV20肽序列(NAVPNLRGDLQVLAQKVART)(SEQ ID NO:34)或GLP。
20.根据权利要求14所述的构建体,其中用于治疗用途的所述抗体包括IgM、IgD、IgG、IgA、IgE或抗体片段F(ab')2、Fab、Fab'或Fv。
21.根据权利要求14所述的构建体,其中所述具有治疗效果的小分子包括吉西他滨、叶酸、顺铂、奥沙利铂、卡铂、多柔比星或紫杉醇。
22.一种药物组合物,其包含根据权利要求14至21中任一项所述的构建体和药学上可接受的载体。
23.根据权利要求22所述的药物组合物,其中所述药学上可接受的载体包含水和一种或多种以下盐或缓冲剂:无水磷酸二氢钾NF、氯化钠USP、七水磷酸氢二钠USP和磷酸盐缓冲盐水(PBS)。
24.一种将治疗性分子递送至人细胞的方法,其包括将根据权利要求14至21中任一项所述的构建体递送至所述细胞。
25.一种将治疗性分子递送至人细胞的方法,其包括将根据权利要求22或权利要求23所述的组合物递送至所述细胞。
26.根据权利要求24或权利要求25所述的方法,其中所述治疗性分子在体内递送至所述细胞。
27.根据权利要求24至26中任一项所述的方法,其中所述治疗性分子选自表达抑制性寡核苷酸、治疗肽、具有治疗效果的抗体和具有治疗效果的小分子。
28.根据权利要求24至27中任一项所述的方法,其中所述人细胞是人的癌中的恶性细胞。
29.根据权利要求28所述的方法,其中所述癌选自肝癌、胆管癌(CCA)、结肠癌、胰腺癌、肺癌、膀胱癌、卵巢癌、头颈癌、食道癌、脑癌和皮肤癌,所述皮肤癌包括黑色素瘤和非黑色素瘤皮肤癌。
30.根据权利要求28所述的方法,其中所述癌选自肝癌、结肠癌和胰腺癌。
31.根据权利要求28所述的方法,其中所述癌是肝癌。
32.根据权利要求31所述的方法,其中所述肝癌包括原发性肝癌。
33.根据权利要求32所述的方法,其中所述原发性肝癌包括肝细胞癌或肝母细胞癌。
34.根据权利要求31所述的方法,其中所述癌从人体内另外的组织转移至肝脏。
35.根据权利要求34所述的方法,其中所述转移癌包括结肠癌。
36.根据权利要求34所述的方法,其中所述转移癌包括胰腺癌。
37.根据权利要求24或权利要求25所述的方法,其中所述治疗性分子包括siRNA分子,并且所述细胞包括肝细胞。
38.根据权利要求14至27中任一项所述的方法,其中将所述治疗性分子递送至人以治疗疾病,所述疾病选自肝炎、纤维化和原发性硬化性胆管炎(PSC)。
39.根据权利要求38所述的方法,其中所述治疗性分子包括siRNA。
40.一种在人中进行基因疗法的方法,其包括向所述人施用治疗有效量的根据权利要求14至21中任一项所述的构建体。
41.一种在人中进行基因疗法的方法,其包括向所述人施用治疗有效量的根据权利要求22或权利要求23所述的组合物。
42.一种合成根据权利要求14所述的构建体的方法,其中所述治疗性分子是siRNA分子,所述方法包括以下步骤:
在siRNA分子的5'或3'位点处,通过形成磷酸酯键将siRNA分子有义链与官能化接头-1偶联;
将数目为一个(或二个或三个)的靶向配体-接头2构建体连接至中心直链接头(或二足或三足桥)位点,其中所述桥的另一末端与末端具有马来酰亚胺官能团的短PEG基团偶联,所述PEG基团用于将接头1与所述桥偶联;
通过硫醇/马来酰亚胺反应,将siRNA-接头-1构建体与接头-2-靶向配体构建体偶联,以提供具有一至三个靶向配体分子的siRNA分子有义链的构建体;和
将有义链-靶向配体构建体与siRNA分子反义链混合,以形成具有一至三个靶向配体的双链体siRNA。
43.一种合成根据权利要求14所述的构建体的方法,其中所述治疗性分子是抗体或肽,所述方法包括以下步骤:
在抗体(或肽)分子的炔、硫醇、NHS官能化位点处,通过形成***环、硫醇-碳键或酰胺键,将抗体(或肽)分子与官能化接头-1(叠氮基、马来酰亚胺、胺)偶联;
将数目为一个(或两个或三个)的靶向配体-接头2构建体连接至中心直链接头(或二足或三足桥)位点,其中所述桥的另一末端与末端具有马来酰亚胺官能团的短PEG基团偶联,所述PEG基团用于将接头1与所述桥偶联;
通过硫醇/马来酰亚胺反应,将抗体(或肽)-接头-1构建体与接头-2-靶向配体构建体偶联,以提供具有靶向配体的抗体(或肽)构建体。
44.一种合成根据权利要求18所述的构建体的方法,其包括以下步骤:
将数目为一个(或两个或三个)的靶向配体-接头2构建体连接至中心直链接头(或二足或三足桥)位点,其中所述桥的另一末端与另一末端具有马来酰亚胺官能团的短PEG基团偶联,其用于将接头1与所述桥偶联;
将接头1,如含有硫醇基团部分的PEG或聚(L-丙交酯),与桥-接头2-GalNAc部分的末端马来酰亚胺反应,以形成S-C共价键;
通过亚磷酰胺基团和羟基基团之间的磷酸酯键,将配体-接头2-接头1构建体偶联至siRNA分子有义链的5'或3'末端;和
将有义链-GalNAc构建体与siRNA分子反义链混合,以形成具有一至三个GalNAc配体的双链体siRNA。
45.一种用于将寡核苷酸递送至人肝细胞的构建体,其包含以下结构:
O—A—B-[-C—D]n
其中O包含寡核苷酸,A包含第一接头(接头1),B包含桥,C包含第二接头(接头2),D包含靶向配体,n是1至4的整数。
46.根据权利要求45所述的构建体,其中所述寡核苷酸是双链的。
47.根据权利要求45所述的构建体,其中所述寡核苷酸是单链的。
48.根据权利要求45至47中任一项所述的构建体,其中所述寡核苷酸包括siRNA、反义RNA、miRNA或cDNA。
49.根据权利要求48所述的构建体,其中所述寡核苷酸包括siRNA。
50.根据权利要求45至49中任一项所述的构建体,其中所述寡核苷酸的长度介于10至27个核苷酸之间。
51.根据权利要求45至49中任一项所述的构建体,其中所述寡核苷酸的长度介于19至25个核苷酸之间。
52.根据权利要求45至51中任一项所述的构建体,其中所述寡核苷酸或siRNA在2'位置或硫代磷酸酯键连接处被完全或部分化学修饰以增强稳定性。
53.根据权利要求45至52中任一项所述的构建体,其中所述靶向配体包括GalNAc。
54.一种药物组合物,其包含根据权利要求45至53中任一项所述的构建体和药学上可接受的载体。
55.根据权利要求54所述的药物组合物,其中所述药学上可接受的载体包含水和一种或多种以下盐或缓冲剂:无水磷酸二氢钾NF、氯化钠USP、七水磷酸氢二钠USP和磷酸盐缓冲盐水(PBS)。
56.一种将寡核苷酸递送至人肝细胞的方法,其包括将根据权利要求45至53中任一项所述的构建体递送至所述肝细胞。
57.一种将寡核苷酸递送至人肝细胞的方法,其包括将根据权利要求54或权利要求55所述的组合物递送至所述肝细胞。
58.根据权利要求56或权利要求57所述的方法,其中所述寡核苷酸分子在体内递送至所述肝细胞。
59.一种在人中进行基因疗法的方法,其包括向所述人施用治疗有效量的根据权利要求45至53中任一项所述的构建体。
60.一种在人中进行基因疗法的方法,其包括向所述人施用治疗有效量的根据权利要求54或权利要求55所述的组合物。
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