CN1298444A - 胰岛素样生长因子ii反义寡核苷酸序列以及使用该序列调节细胞生长的方法 - Google Patents
胰岛素样生长因子ii反义寡核苷酸序列以及使用该序列调节细胞生长的方法 Download PDFInfo
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Abstract
本发明涉及与IGFⅡ基因互补的寡核苷酸,该寡核苷酸可调节哺乳动物的肿瘤细胞生长。本发明还涉及使用该化合物抑制哺乳动物肿瘤细胞生长的方法。本发明还涉及含有药物可接受的赋形剂和有效量的本发明化合物的药物组合物。
Description
使用该序列调节细胞生长的方法可供参考的相关申请
本申请要求1998年4月23日提交的美国临时申请流水号60/082,791的优先权,该临时申请全文列入本文作为参考。
发明背景
发明领域
本发明涉及与哺乳动物胰岛素样生长因子Ⅱ(IGFⅡ)基因互补的寡核苷酸,该寡核苷酸可调节哺乳动物的肿瘤细胞生长。本发明还涉及使用该化合物抑制哺乳动物肿瘤细胞生长的方法。本发明还涉及含有药物可接受的赋形剂和有效量的本发明化合物的药物组合物。
参考文献
本申请中提及下列出版物,专利申请和专利:1.Toretsky,J.A和Helman,L.J,IGF-Ⅱ与人类癌症有关,内分泌学杂志,149:367-72,1996。2.Werner,H和LeRoith,D.胰岛素样生长因子系统对人类癌症的作用,Adv Cancer Res.68:183-223,1996。3.Rogler,C.E.,Yang,D.,Rossetti,L.,Donohoe,J.,Alt,E.,Chang,C.J.,Rosenfeld,R.,Neely,K.,和Hintz,R.胰岛素样生长因子Ⅱ转基因小鼠体内经改变的身体组成和频率增加的多种恶性肿瘤,生物化学杂志,269:13779-84,1994。4.Bates,P.,Fisher,R.,Ward,A.,Richardson,L.,Hill,D.J.,和Graham,C.F.表达胰岛素样生长因子Ⅱ(IGF-Ⅱ)的转基因小鼠的乳腺癌[见评论],英国癌症杂志,72:1189-93,1995.5.Cullen,K.J.,Lippman,M.E.,Chow,D.,Hill,S.,Rosen,N.,和Zwiebel,J.A,MCF-7细胞中胰岛素样生长因子Ⅱ的过量表达诱导与恶性肿瘤进程相关的表型变化,分子内分泌学,6:91-100,1992.6.Werner,H.,Adamo,M.,Roberts,C.T.,Jr.,和LeRoith,D.胰岛素样生长因子作用的分子和细胞方面的机理,Vitam Horm.48:1-58,1994。7.Curcio,L.D.,Bouffard,D.Y.,和Scanlon,K.J.用作癌症基因表达调节剂的寡核苷酸,药物治疗,74:317-32,1997。8.Narayanan,R.和Allhtar,S.反义疗法,Curr Opin Oncol。8:509-15,1996。9.Ho,P.T.和Parkinson,D.R.用作恶性肿瘤疾病治疗剂的寡核苷酸,Semin Oncol.24:187-202,1997。10.Crooke,S.T.和Bennett,C.F.反义寡核苷酸治疗剂进展,AnnuRev Pharmacol Toxicol.36:107-29,1996。11.Christofori,O.,Naik,P.,和Hanahan,D.在癌基因-诱导的肿瘤生成中由胰岛素样生长因子Ⅱ提供的第二信号,自然,369:414-8,1994。12.El-Badry,O.M.,Minniti,C.,Kohn,E.C.,Houghton,P.J.,Daughaday,W.H.,和Helman,L.J.胰岛素样生长因子Ⅱ用作人横纹肌肉瘤中的自泌生长和能动因子,细胞生长与分化,1:325-31,1990。13.Kim,K.W.,Bae,S.K.,Lee,O.H.,Bae,M.H.,Lee,M.J.,和Park,B.C.由低氧诱导的胰岛素样生长因子Ⅱ导致人肝细胞癌血管生成,癌症研究,58:348-51,1998。14.Volpert,O.,Jackson,D.,Bouck,N.,和Linzer,D.I.增殖蛋白-诱导的血管生成需要胰岛素样生长因子Ⅱ/甘露糖6-磷酸受体,内分泌学,137:3871-6,1996。15.Lin,S.B.,Hsieh,S.H.,Hsu,H.L.,Lai,M.Y.,Kan,L.S.,和Au,L.C.,ⅡGF-Ⅱ的反义寡脱氧核苷酸选择性抑制过量产生IGF-Ⅱ的人肝癌细胞生长,生物化学杂志(东京).122:717-22,1997。16.Steller,M.A.,Delgado,C.H.,Bartels,C.J.,Woodworth,C.D.,和Zou,Z.人子宫颈癌细胞中胰岛素样生长因子-1受体的过量表达和自泌刺激,癌症研究,56:1761-5,1996。17.Steller,M.A.,Delgado,C.H.,和Zou,Z.胰岛素样生长因子Ⅱ介导子宫颈癌细胞中由表皮生长因子诱导的细胞分裂发生,ProcNatl Acad Sci USA.92:11970-4,1995。18.Choy等,″涉及核糖核苷酸还原酶的药物抗性分子机制:存在逐步增加的药物浓度时在一系列克隆相关的小鼠细胞系中选择的羟基脲抗性″,癌症研究.48:2029-2035(1988)。19.Fan等,″核糖核苷酸还原酶R2成分是新的恶性肿瘤决定子,它与癌基因合作决定转化和恶变能力″Proc.Nat1.Acad.Sci USA93:14036-40(1996)。20.Huang和Wright,″成纤维细胞生长因子介导的药物抗性的变化和基因扩增的证据″癌基因,9:491499(1994)。21.Uhlmann等。Chem Rev.90:534-583(1990)。22.Agrawal等。Trends Biotechnol 10:152-158(1992)。23.Remington’s Pharmaceutical Sciences Mace PublishingCompany,hiladelphia PA17th编。(1985)。24.Sambrook等,分子克隆-实验室手册,冷泉港实验室,纽约(1989,1992)。25.Ausubel等,最新分子生物学方法,John Wiley和Sons,Baltimore Maryland(1989)。26.Perbal,分子克隆实验指南,John Wiley&Sons,纽约(1988)。27.Hurta和Wright,″H-ras所致的恶变导致转化生长因子-β异常调节核糖核苷酸还原酶的基因表达″,细胞生物化学杂志,57:543-556(1995)。28.Dreeley等,科学,258:1650-1654(1992)。29.Nielsen等;科学(1991)354:1497。30.Good和Nielsen;″通过靶向核糖体RNA的肽核酸抑制翻译和细菌生长″,PNAS USA(1998)95:2073-2076。31.Buchardt,deceased,等,美国专利5,766,855。32.Buchardt,deceased,等,美国专利5,719,262。33.美国专利5,034,506。34.Altschul,等″基本的局部序列对比检索工具″,分子生物学杂志(1990)215:403-10。35.Devereux J.等,″VAX的全套序列分析程序″,核酸研究(1984)12:387-395。36.Chang等;体细胞基因治疗,CRC Press,Ann Arbor MI(1995)。37.Vega等;基因靶向,CRC Press,Ann Arbor MI(1995)。38.载体:分子克隆载体及其用途概述,Butterworths,Boston MA(1988)。39.Sullivan,美国专利5,225,347。40.1991年6月11日公开的美国专利5,023,252。41.Felgner等,美国专利5,580,859。
上述所有出版物,专利申请和专利都全文列入本文作为参考,就象特别地和个别地指出每篇出版物,专利申请或专利都全文列入本文作为参考一样。
现有技术
胰岛素样生长因子Ⅱ(IGF-Ⅱ)是67个氨基酸多肽的生长因子,它在处于发育中的人胚胎组织中被广泛表达并与多种组织的生长和分化有关。出生之后,它在几乎所有人组织中的表达逐渐消失。在成人中,约100ng/ml的血清水平主要由肝脏产生。IGF-Ⅱ的生物学功能由其与IGF-Ⅱ受体(与糖类代谢,恶性细胞的能动性和/或肿瘤-诱导的血管生成相关)或IGF-Ⅰ受体(与信号转导途径和细胞分裂发生相关)的结合介导。
IGF-Ⅱ通过多种机理参与多种肿瘤的肿瘤进程和转移(参见(1,2))。与IGF-Ⅱ十分相关的肿瘤包括儿童易患的肿瘤,如横纹肌肉瘤,维尔姆斯瘤和成神经细胞瘤。这些肿瘤阐明了IGF-Ⅱ的过量表达显示出旁分泌或自分泌环的存在,并可通过阻断环抑制肿瘤生长或转移。在包括骨肉瘤,乳腺癌,肝胚细胞瘤,生殖细胞瘤,肝细胞癌,肾上腺皮质癌,肺癌,平滑肌肉瘤,脑瘤和结肠癌的多种肿瘤中,IGF-Ⅱ不同程度地导致肿瘤生长和转移。另外,过量表达IGF-Ⅱ的转基因小鼠和人细胞系也阐明了IGF-Ⅱ对肿瘤发生的直接作用(3-5)。
人IGF-Ⅱ基因位于染色体11p15,紧接胰岛素基因的下游并跨越30kb(参见(6);见图1)。它由9个外显子组成,其中外显子7和8以及外显子9的一部分编码前体蛋白质。外显子1,4,5和6的上游是不同的启动子P1,P2,P3和P4。启动子P1仅在成人的肝脏中有活性,而P2-4在大多数胎儿组织中具有活性。有几个成人组织由P2,3和4启动表达少量转录物(胎儿转录物)。已鉴定出4种主要的mRNA类型(胎儿转录物为6kb,4.8-5kb和2.2kb,成人转录物为5.3kb),它们是由不同启动子启动并通过不同方式的剪接产生的。在多种原发性癌症和细胞系中观察到的IGF-Ⅱ过量表达似乎是由胎儿mRNA的重新激活(在肝脏中)或过量表达(在其它器官中)引起的,所述胎儿mRNA的表达主要由P3和P4启动产生。这些胎儿转录物含有独特的5’非翻译区(含有外显子4或5或6的5’UTR),而由P1启动产生的成人转录物却不含该区域(含有外显子1,2和3的5’UTR)。
通过与靶mRNA进行序列特异性杂交,反义寡核苷酸(AS-ODN)已被广泛用于以靶-特异性的方式抑制基因表达。在大量的研究中,由反义寡核苷酸-介导的对癌基因的阻抑揭示出:这些化合物不仅对阐明控制肿瘤发生的生化机理十分有用(7),还很有希望用作新的治疗化合物以治疗人类癌症(8,9)。另外,基于寡核苷酸的治疗剂的毒性相对较小(10)。
几项研究(11,15-17)表明:某些特异性针对成年人或小鼠IGF-Ⅱ转录物的反义寡核苷酸能有效干扰肿瘤细胞的体外增殖。在一项研究(15)中,靶向成人转录物之翻译起始位点的反义寡核苷酸对IGF-Ⅱ生产的抑制作用导致对人肝细胞癌细胞系HuH-7和HepG2生长的抑制作用。在利用人子宫颈癌细胞系的另一些研究(16,17)中,靶向IGF-Ⅱ之蛋白质编码区的反义寡核苷酸可抑制表皮生长因子(EGF)-诱导的促分裂作用。
因此,需要鉴定针对IGF-Ⅱ,并能以较高的特异性和较低的毒性抑制IGF-Ⅱ表达和产生的反义寡核苷酸。
发明简述
本发明涉及调节哺乳动物中的IGF-Ⅱ基因表达和IGF-Ⅱ产生的反义寡核苷酸和含有该反义寡核苷酸的药物组合物。本发明还涉及使用该反义寡核苷酸抑制哺乳动物体内的肿瘤生长和转移的方法。因此,本发明一方面涉及反义寡核苷酸,该寡核苷酸长度约为3至100个核苷酸,其含有与哺乳动物胎儿IGF-ⅡmRNA互补的核苷酸。反义寡核苷酸可对核酶具有抗性,并具有一个或多个硫代磷酸酯核苷酸间连键。反义寡核苷酸可进一步含有不与IGF-ⅡmRNA互补的其它核苷酸。寡核苷酸可含有选自表1之SEQ ID NO:1至15的序列。
本发明还涉及反义寡核苷酸,该寡核苷酸长度约为20至100个核苷酸,其含有与成年哺乳动物IGF-ⅡmRNA互补的核苷酸,该核苷酸可含有选自表2之SEQ ID NO:17至31的序列。
本发明另一方面涉及含有反义寡核苷酸序列的载体,该寡核苷酸长度约为3至100个核苷酸,其含有与哺乳动物胎儿IGF-ⅡmRNA之5’非翻译区互补的序列。
本发明另一方面涉及含有反义寡核苷酸序列的载体,该寡核苷酸长度约为20至100个核苷酸,其含有选自表2之SEQ ID NO:17至31的序列。
本发明另一方面涉及含有药物可接受的赋形剂和有效量的反义寡核苷酸的药物组合物,该寡核苷酸长度约为3至100个核苷酸,其含有与哺乳动物胎儿IGF-ⅡmRNA互补的核苷酸。寡核苷酸可含有选自表1之SEQ ⅡD NO:1至15的序列。
本发明另一方面涉及含有药物可接受的赋形剂和有效量的反义寡核苷酸的药物组合物,该寡核苷酸长度约为20至100个核苷酸,其含有选自表2之SEQ ID NO:17至31的列。
本发明另一方面涉及抑制哺乳动物肿瘤生长的方法,所述方法包括:在能抑制肿瘤生长的条件下,给怀疑患有肿瘤的哺乳动物施用有效量的反义寡核苷酸,该寡核苷酸长度约为3至100个核苷酸,并与哺乳动物胎儿IGF-ⅡmRNA互补。反义寡核苷酸可与化学治疗剂一起使用。寡核苷酸可含有选自表1之SEQ ID NO:1至15的序列。
本发明另一方面涉及抑制哺乳动物肿瘤生长的方法,所述方法包括:在能抑制肿瘤生长的条件下,给怀疑患有肿瘤的哺乳动物施用有效量的选自表2之SEQ ID NO:17至31的反义寡核苷酸,该寡核苷酸长度约为20至100个核苷酸,并与成年哺乳动物的IGF-ⅡmRNA互补。
本发明另一方面涉及抑制哺乳动物肿瘤转移的方法,所述方法包括:在能抑制肿瘤转移的条件下,给怀疑患有转移性肿瘤的哺乳动物施用有效量的反义寡核苷酸,该寡核苷酸长度约为3至100个核苷酸,并与哺乳动物胎儿IGF-ⅡmRNA互补。反义寡核苷酸可与化学治疗剂一起使用。寡核苷酸可含有选自表1之SEQ ID NO:1至15的序列。
本发明另一方面涉及抑制哺乳动物肿瘤转移的方法,所述方法包括:在能抑制肿瘤转移的条件下,给怀疑患有转移性肿瘤的哺乳动物施用有效量的选自表2之SEQ ID NO:17至31的反义寡核苷酸,该寡核苷酸长度约为20至100个核苷酸,并与成年哺乳动物的IGF-ⅡmRNA互补。
附图简述
图1是人IGF-Ⅱ基因和可变转录或者剪接的mRNA的图谱。标明数字的框(1-9)表示IGF-Ⅱ基因的外显子,4个启动子(P1-P4)以箭头表示。图的下方以其相应的大小表示多种IGF-ⅡmRNA种,黑框表示IGF-Ⅱ前体蛋白质的编码区。
图2A-D是施用所示反义寡核苷酸对不同细胞系的集落形成能力之抑制作用的百分比图。图2A显示了对人横纹肌肉瘤细胞系RD的抑制作用百分比;图2B显示了对人前列腺癌细胞系PC-3的抑制作用百分比;图2C显示了对人胰腺癌细胞系AsPC-1的抑制作用百分比;图2D显示了对人成神经细胞瘤细胞系SK-N-AS的抑制作用百分比。
图3是施用反义寡核苷酸GTI4006[SEQ ID NO:6]或GTI4011[SEQ IDNO:11]之后,人成神经细胞瘤细胞系SK-N-AS或横纹肌肉瘤细胞系(RD)之RNA的Northern印迹放射自显影图。
图4是用不同反义寡脱氧核苷酸处理之后,人成神经细胞瘤细胞之IGF-Ⅱ表达的Western印迹照片。
图5是用不同反义寡脱氧核苷酸处理之后,人横纹肌肉瘤细胞之IGF-Ⅱ表达的Western印迹照片。
图6A是用或不用(对照)多种反义寡核苷酸注射小鼠体内的人成神经细胞瘤细胞(SK-N-AS)之后的肿瘤体积图。
图6B是用或不用(对照)多种反义寡核苷酸注射小鼠体内的人成神经细胞瘤细胞(SK-N-AS)之后20天的肿瘤重量图。
图7A是用或不用(对照)多种反义寡核苷酸注射小鼠体内的人维尔姆斯瘤细胞(G401)之后的肿瘤体积图。
图7B是用或不用(对照)多种反义寡核苷酸注射小鼠体内的人维尔姆斯瘤细胞(G401)之后20天的肿瘤重量图。
图8是用反义寡核苷酸GTI4006[SEQ ID NO:6]处理之后,人成神经细胞瘤(SK-N-AS)中的IGF-ⅡmRNA水平的Northern印迹放射自显影图。
图9是用多种反义寡核苷酸处理之后,人成神经细胞瘤(SK-N-AS)之IGF-Ⅱ蛋白质水平的Western印迹照片。下面的带是经印度墨汁染色以显示上样的总蛋白质的凝胶照片。
图10是用多种反义寡核苷酸处理细胞系之后,每只小鼠中的人黑素瘤细胞系(C8161)肺转移的平均数目。
图11是人IGF-Ⅱ基因之核苷酸序列的一部分。图11A是外显子4[SEQID NO:34]的序列,图11B是外显子5[SEQ ID NO:35]的序列,图11C是外显子6[SEQ ID NO:36]的序列,和图11D是外显子7-9[SEQ ID NO:37]的序列。
发明详述
本发明涉及与哺乳动物IGFⅡ基因互补的寡核苷酸,该寡核苷酸可调节哺乳动物的肿瘤细胞生长。在多种人原发性癌症和细胞系中观察到的IGF-Ⅱ过量表达似乎是由胎儿mRNA的重新激活(在肝脏中)或过量表达(在其它器官中)引起的。因此,被设计成特异性靶向5’UTR中胎儿转录物,而使成人转录物保持原样的反义寡核苷酸可高度特异性地靶向肿瘤细胞。
不必受理论或机理的束缚,据信这些反义化合物可通过下列方式发挥其抗肿瘤活性:即不仅可抑制肿瘤细胞的自泌生长并可能会诱导细胞凋亡,还可抑制IGF-Ⅱ的自泌/旁分泌功能,如肿瘤细胞的能动性和/或诱导内皮细胞移动和血管生成。定义
本文所用的下列术语具有下列含义:
本文所用术语“反义寡核苷酸”指的是与目的mRNA互补的核苷酸序列。反义寡核苷酸与能有效用作抑制IGF-Ⅱ表达之靶的哺乳动物IGF-ⅡmRNA的任何部分互补。优选反义寡核苷酸与IGF-Ⅱ胎儿转录物的5’非翻译区互补。更优选反义寡核苷酸与图11A-C所示外显子4,5或6的核苷酸序列互补。
不必受理论或机理的束缚,一般相信反义寡核苷酸的活性取决于寡核苷酸与靶核酸的结合(例如与基因组区域,基因或其mRNA转录物的至少一部分结合),从而通过杂交抑制或通过用RNA酶H破坏靶RNA(当与RNA杂交时能激活RNA酶H)来破坏靶的功能。
术语“寡核苷酸”指的是由天然碱基,糖和糖间(骨架)连键组成的核苷酸或核苷单体的寡聚体或多聚体。该术语还包括含有非天然单体或其部分且功能相似的经修饰或取代的寡聚体。这种经修饰或取代的寡聚体优于天然寡聚体,因为前者的细胞摄入能力增强,或在核酶的存在下稳定性增强。该术语还包括含有两个或多个不同化学区域的嵌合寡核苷酸。例如,嵌合寡核苷酸可含有至少一个可赋予有利特性(如核酶抗性增加,细胞摄入能力增强)的经修饰的核苷酸区域,或者本发明的两个或多个寡核苷酸可以连接形成嵌合寡核苷酸。
本发明的反义寡核苷酸可以是核糖核酸或脱氧核糖核酸,并可含有天然或合成的单体碱基,所述碱基包括腺嘌呤,鸟嘌呤,胞嘧啶,胸腺嘧啶和尿嘧啶。寡核苷酸也可含有经修饰的碱基,如黄嘌呤,次黄嘌呤,2-氨基腺嘌呤,6-甲基,2-丙基和其它烷基腺嘌呤,5-卤尿嘧啶,5-卤胞嘧啶,6-氮尿嘧啶,6-氮胞嘧啶和6-氮胸腺嘧啶,假尿嘧啶,4-硫尿嘧啶,8-卤腺嘌呤,8-氨基腺嘌呤,8-巯基腺嘌呤,8-巯基烷基腺嘌呤,8-羟基腺嘌呤和其它8-取代腺嘌呤,8-卤鸟嘌呤,8-氨基鸟嘌呤,8-巯基鸟嘌呤,8-巯基烷基鸟嘌呤,8-羟基鸟嘌呤和其它8-取代的鸟嘌呤,其它氮和脱氮尿嘧啶,胸腺嘧啶,胞嘧啶或鸟嘌呤,5-三氟甲基尿嘧啶和5-三氟胞嘧啶。修饰也可包括将其它化学基团,如甲基,乙基,丙基结合至包括糖,碱基或骨架组分的寡核苷酸的多个部分。
本发明的反义寡核苷酸也可在磷酸酯骨架,短链烷基或环烷基糖间连键中含有磷氧杂原子或含有短链杂原子或杂环糖间连键。例如,反义寡核苷酸可含有甲基膦酸酯,硫代磷酸酯,二硫代磷酸酯,磷酸三酯和吗啉代寡聚体。反义寡核苷酸可含有连接4至6个3’末端核苷酸的硫代磷酸酯键。硫代磷酸酯键可连接所有核苷酸。硫代磷酸酯连键可以是混合的Rp和Sp对映异构体,或者是有规立构或基本上有规立构的Rp或Sp形式。
反义寡核苷酸也可具有糖模拟物。寡核苷酸可具有至少一个含经修饰碱基和/或糖的核苷酸,如2’-O-取代的核糖核苷酸。本发明意义上的术语“2’-O-取代”指的是用含有1-6个饱和或不饱和碳原子的-O-低级烷基,或用具有2-6个碳原子的-O-芳基或烯丙基取代戊糖组成成分的2’位,其中所述烷基,芳基或烯丙基可以是非取代的,或也可以被例如卤,羟基,三氟甲基,氰基,硝基,酰基,酰氧基,烷氧基,羧基,烷酯基或氨基取代。本发明的寡核苷酸可包括4或5个5’末端被2’-O-烷基化的核糖核苷酸和/或4或5个3’末端被2’-O-烷基化的(2’-O-alylated)核糖核苷酸。
本发明的反义寡核苷酸也可含有核苷酸类似物,其中核苷酸的结构发生了根本性的变化。这种寡核苷酸类似物的一个例子是肽核酸(PNA),其中DNA(或RNA)中的脱氧核糖(或核糖)磷酸酯骨架被聚酰胺骨架所取代,所述聚酰胺骨架与肽中的相类似(Nielsen等29;Good和Nielsen30;Buchardt,deceased等31;美国专利 5,766,855;Buchardt,deceased等32;美国专利5,719,262)。PNA类似物对酶降解具有抗性,体内和体外的寿命延长。相对于天然的核酸分子而言,PNA与互补DNA序列的结合能力更强,原因是PNA链和DNA链之间缺乏电荷排斥。
本发明的寡核苷酸也可包括含有多聚体骨架,环骨架或无环骨架的其它核苷酸。例如,核苷酸可含有吗啉代骨架结构(美国专利5,034,506(33))。
当本发明的寡核苷酸经修饰后对DNA和RNA核酶的降解不敏感,或者被置入本身可保护寡核苷酸免受DNA或RNA核酶降解的传递载体中时,所述寡核苷酸即为“核酶抗性”。核酶抗性的寡核苷酸包括例如甲基膦酸酯,硫代磷酸酯,二硫代磷酸酯,磷酸三酯和吗啉代寡聚体。赋予核酶抗性的适当传递载体包括例如脂质体。
本发明的寡核苷酸也可含有例如改善寡核苷酸之药物动力学或药效学特性的基团。
反义寡核苷酸优选选自与IGF-Ⅱ基因互补的序列以使序列最小可能的显示出双链体形成,发夹形成和同寡聚体/序列重复但具有高至中等的结合IGF-Ⅱ基因序列的效力。使用计算机模型设计程序OLIGO引物分析软件第5.0版(由National Biosciences,Inc.,Plymouth,MN分发)可测定这些特性。该计算机程序可定性估测这5个参数。
或者,也可在两种或多种哺乳动物之间的IGF-Ⅱ基因序列高度保守的基础上选择反义寡核苷酸。使用Wisconsin大学计算机小组(GCG)软件(Devereux J等(35))的BLASTN程序(Altschul等(34))以及国立生物技术信息中心(NCBI)数据库可测定这些特性。
反义寡核苷酸可包括突变,如取代,插入和缺失。优选低于10%的序列具有突变。
反义寡核苷酸一般含有至少约3个核苷酸或核苷酸类似物,更优选至少约为5个核苷酸,更优选至少约为7个核苷酸,更优选至少约为9个核苷酸,最优选至少约为20个核苷酸。反义寡核苷酸优选少于约100个核苷酸或核苷酸类似物,更优选少于约50个核苷酸或核苷酸类似物,最优选少于约35个核苷酸或核苷酸类似物。
优选反义寡核苷酸与胎儿IGF-Ⅱ转录物的5’非翻译区互补。“胎儿IGF-Ⅱ转录物的非翻译区”指的是在胎儿细胞中转录形成主要的IGF-Ⅱ转录物而不形成成人IGF-Ⅱ转录物(成人细胞中的主要转录物)部分的IGF-Ⅱ基因部分。优选“胎儿IGF-Ⅱ转录物的非翻译区”是IGF-Ⅱ基因的外显子4,5和6。最优选“胎儿IGF-Ⅱ转录物的非翻译区”基本上是图11A-C所示的外显子4,5和6的序列。
优选反义寡核苷酸含有下表1和2所示的序列。
表1被设计用于靶向人IGF-Ⅱ胎儿mRNA的反义序列
SEQ IDNO. | 名称 | 序列5′-3′ | Tm(℃ | ΔG(kcal/mol) |
1 | GTI4001 | GGC TCG CTG GGG CAG GAG GA | 74.6 | -46.5 |
2 | GTI4002 | GCT GGT GGG CAG AGC GCG GG | 78.0 | -48.5 |
3 | GTI4003 | TTG GTG TCT ACA GCT CAG CA | 57.8 | -35.2 |
4 | GTI4004 | CAG CGA GGC AGC GGG CGG CG | 82.7 | -52.5 |
5 | GTI4005 | TCG GGC GAA GCG GGG ATG GG | 79.0 | -50.4 |
6 | GTI4006 | CGG GCC TCG GGA GGG GGA CA | 78.2 | -49.4 |
7 | GTI4007 | GAC CGC GGG CGC CCA GCT CG | 81.7 | -51.9 |
8 | GTI4008 | ACG TCG AGG GGC CGG GGG AG | 77.4 | -49.3 |
9 | GTI4009 | CGG GAG AAA GAG CGG GGG CC | 75.1 | -48.5 |
10 | GTI4010 | CGA GAG GGC GGG CGT GAG GG | 77.0 | -48.4 |
11 | GTI4011 | CAG CGA GAG GCG GGC AGG CG | 78.2 | -49.0 |
12 | GTI4012 | CGG GCT GTCTTC GGG CTG GG | 74.9 | -47.0 |
13 | GTI4013 | GCG ACG GGG CAG AGC GGG GG | 80.7 | -51.4 |
14 | GTI4014 | CGC TGC CGC CCA CCT CCC TG | 77.8 | -48.5 |
15 | GTI4015 | TTG GTG TCT GGA AGC CGG C | 72.0 | -44.3 |
反义寡核苷酸选自与人IGF-ⅡmRNA互补的序列以使序列最不可能显示出双链体形成,发夹形成和同寡聚体/序列重复但可高效结合IGF-ⅡmRNA序列并含有GC夹。另外,还消除了对人和小鼠中的其它常见或重复序列的错误引发。使用计算机模型设计程序OLIGO引物分析软件第5.0版(由National Biosciences,Inc.,Plymouth,MN分发)可测定这些特性。表2具有与人IGF-ⅡmRNA的所有区域互补之序列的反义寡核苷酸
SEQNo. | 名称 | 序列5′-3′ | Tm(℃) | ΔG(kcal/mol) |
16 | GTI4016 | TTC CCC ATT GGG ATT CCC AT | 66.8 | -42.4 |
17 | GTI4017 | GTC CAC CAG CTC CCC GCC GC | 76.9 | -47.9 |
18 | GTI4018 | CGA TGC CAC GGC TGC GAC GG | 77.6 | -47.6 |
19 | GTI4019 | ACG CAG GAG GGC AGG CAG GC | 74.7 | -46.5 |
20 | GTI4020 | GCG AGC ACG TGA CCC CGG CG | 78.7 | -48.6 |
21 | GTI4021 | CGT GGG CGG GGT CTT GGG TG | 75.4 | -46.7 |
22 | GTI4022 | TGT TTC GGG GAG GCG GGG CA | 77.5 | -48 8 |
23 | GTI4023 | GCG GTA CGA GCG ACG TGC CC | 73.8 | -45.9 |
24 | GTI4024 | CAA ATG CCG CCG GCC GCA CA | 79.7 | -49.8 |
25 | GTI4025 | CGC ATC AGT GCA CGG CCC CC | 76.5 | -46.9 |
SEQ IDNO. | 名称 | 序列5′-3′ | Tm(℃) | △G(kcal/mol) |
26 | GTI4026 | GTG CGG AAG GCG GCC ACC CT | 76.4 | -48.2 |
27 | GTI4027 | CAG GGT GCT GAG GGG CGG GC | 76.9 | -48.0 |
28 | GTI4028 | GCT CCG GGG CCC AAG CAA CC | 75.9 | -48.3 |
29 | GTI4029 | CCC TAG GCG CCG CGG TGG TG | 77.6 | -49.3 |
30 | GTI4030 | TGG CAT GGA CGA CCC CCG GG | 77.7 | -48.1 |
31 | GTI4031 | GGG CCG CAA GGT GGA CCG AG | 74.8 | -46.7 |
反义寡核苷酸选自与人IGF-ⅡmRNA互补的序列以使序列最不可能显示出双链体形成,发夹形成和同寡聚体/序列重复但可高效结合IGF-ⅡmRNA序列并含有GC夹。另外,还消除了对人和小鼠中的其它常见或重复序列的错误引发。使用计算机模型设计程序OLIGO引物分析软件第5.0版(由National Biosciences,Inc.,Plymouth,MN分发)可测定这些特性。
在表1和2中,“Tm”是根据毗邻热动力学值计算出的寡核苷酸双链体解链温度。在此温度下,50%核酸分子为双链体,50%已变性。“ΔG”是寡核苷酸的自由能,它测定的是寡核苷酸双链体的稳定性。
术语“烷基”指的是单价烷基,优选其具有1至20个碳原子,更优选其具有1至6个碳原子。该术语所包括的基团的例子如下:甲基,乙基,正-丙基,异-丙基,正-丁基,异-丁基,正-己基等。
术语“芳基”指的是具有单个环(如苯基)或多个缩合(融合)环(如萘基或蒽基)的6至14个碳原子的不饱和芳香族碳环基团。优选芳基包括苯基,萘基等。
术语“卤”或“卤素”指的是氟,氯,溴和碘,优选为氟或氯。有关含有一个或多个取代基的上述任一基团,应理解所述基团不含空间上无法实施和/或无法合成的任何取代或取代模式。另外,本发明的化合物包括由这些化合物中的取代所致的所有立体化学异构体。
术语“药物可接受的”指的是不会干扰活性成分之生物活性效力的无毒物质。该物质与如细胞,细胞培养物,组织或生物体的生物系统相容。
术语“药物可接受的盐”指的是可保留本发明反义寡核苷酸的生物效力和特性而本身是或不是生物所需要的盐。在很多情况下,本发明的反义寡核苷酸能利用氨基和/或羧基或其类似基团的存在形成酸和/或碱加成盐。
可由无机和有机碱制备药物可接受的碱加成盐。衍生自无机碱的盐包括例如钠,钾,锂,铵,钙和镁盐。衍生自有机碱的盐包括但不限于伯胺,叔胺和仲胺的盐,所述胺如烷基胺,二烷基胺,三烷基胺,取代烷基胺,二(取代烷基)胺,三(取代烷基)胺,链烯基胺,二链烯基胺,三链烯基胺,取代链烯基胺,二(取代链烯基)胺,三(取代链烯基)胺,环烷基胺,二(环烷基)胺,三(环烷基)胺,取代环烷基胺,二取代环烷基胺,三取代环烷基胺,环链烯基胺,二(环链烯基)胺,三(环链烯基)胺,取代环链烯基胺,二取代环链烯基胺,三取代环链烯基胺,芳基胺,二芳基胺,三芳基胺,杂芳基胺,二杂芳基胺,三杂芳基胺,杂环胺,二杂环胺,三杂环胺,混合的二-和三-胺,其中胺上的至少两个取代基有所不同并选自烷基,取代烷基,链烯基,取代链烯基,环烷基,取代环烷基,环链烯基,取代环链烯基,芳基,杂芳基,杂环等。还包括其中的两个或三个取代基与氨基氮一起形成杂环或杂芳基的胺。
适当的胺的例子包括例如异丙基胺,三甲基胺,二乙基胺,三(异丙基)胺,三(正丙基)胺,乙醇胺,2-二甲基氨基乙醇,氨基丁三醇,赖氨酸,精氨酸,组氨酸,咖啡因,普鲁卡因,哈胺,胆碱,甜菜碱,乙二胺,葡糖胺,N-烷基葡糖胺,可可碱,嘌呤,哌嗪,哌啶,吗啉,N-乙基哌啶等。应理解其它羧酸衍生物也可用于本发明实践,例如,羧酸酰胺,包括氨甲酰,低级烷基氨甲酰,二烷基氨甲酰等。
可由无机和有机酸制备药物可接受的酸加成盐。衍生自无机酸的盐包括盐酸,氢溴酸,硫酸,硝酸,磷酸等。衍生自有机酸的盐包括醋酸,丙炔酸,乙醇酸,丙酮酸,草酸,苹果酸,丙二酸,琥珀酸,马来酸,富马酸,酒石酸,柠檬酸,苯甲酸,肉桂酸,扁桃酸,甲磺酸,乙烷磺酸,对甲苯磺酸,水杨酸等。
术语“IGF-Ⅱ基因”或“胰岛素样生长因子Ⅱ”指的是编码能结合IGF-Ⅰ或IGF-Ⅱ受体的蛋白质的任何基因。优选IGF-Ⅱ基因之一个或多个区域的核苷酸序列与图11A-D所示外显子4,5,6或7-9的序列基本上相似。
术语“互补”指的是反义寡核苷酸序列能结合靶序列,即IGF-Ⅱ基因(或mRNA)。优选反义寡核苷酸在生理条件下,通过例如Watson-Crick碱基配对(寡核苷酸和单链核酸之间的相互作用)或Hoogsteen碱基配对(寡核苷酸和双链核酸之间的相互作用)或通过任何其它方式(包括在寡核苷酸与RNA结合的情况下导致假结形成)与核酸序列结合。通过观察对核酸序列之功能的干扰来测定生理条件下通过Watson-Crick或Hoogsteen碱基配对的结合是可行的。
优选反义寡核苷酸序列与靶序列之间的同一性至少约为75%,优选至少约为90%,最优选与靶序列之间的同一性至少约为95%以允许几个碱基的缺口或错配存在。通过使用例如Wisconsin大学计算机小组(GCG)软件的BLASTN程序可测定同一性。优选反义寡核苷酸序列与IGF-ⅡmRNA杂交,通过本文所述的OLIGO引物分析软件第5.0版测定其解链温度至少为45℃,更优选至少约为50℃,最优选至少约为55℃。
术语“抑制生长”指的是将至少一种肿瘤细胞类型的生长降低或抑制优选至少10%,更优选至少50%,最优选至少75%。通过测定裸鼠肿瘤的大小或肿瘤细胞在体外不能形成集落这一特性可测定对肿瘤生长的抑制作用。
术语“抑制转移”指的是将所产生的转移肿瘤的数目减少或抑制优选至少10%,更优选至少50%。通过实施例中所述的方法和本领域已知的其它方法可测定该抑制作用。
术语“抑制IGF-Ⅱ表达”指的是当将反义寡核苷酸施用于细胞时,该寡核苷酸降低了细胞产生的IGF-ⅡmRNA水平或IGF-Ⅱ蛋白质水平。
术语“哺乳动物”指的是所有哺乳动物,包括人,绵羊,牛,马,猪,狗,猫和小鼠等,优选哺乳动物指的是人。
“被怀疑患有肿瘤的哺乳动物”指的是哺乳动物患有增殖性病症或肿瘤或被诊断为增殖性病症或肿瘤或以前曾被诊断为增殖性病症或肿瘤,肿瘤已被外科切除,哺乳动物仍被怀疑具有一些残留的肿瘤细胞。反义寡核苷酸的制备
通过常规的和众所周知的技术可制备本发明的反义寡核苷酸。例如,使用固相合成,尤其是使用商购仪器,如可购自Applied BiosystemsCanada公司,Mississauga,加拿大的仪器可制备寡核苷酸。通过用本领域已知的方法酶促消化天然IGF-Ⅱ基因也可制备寡核苷酸。
通过本领域公知的方法,如氨基磷酸酯或H-磷酸酯化学可制备这些寡核苷酸,所述方法可以手工操作或按Uhlmann等(21)和Agrawal等(22)所述通过自动化的合成仪进行。分离和纯化反义寡核苷酸
必要时,可通过任何适当的分离或纯化方法分离和纯化本文所述的反义寡核苷酸,所述方法如过滤,提取,结晶,柱层析,薄层层析,厚层层析,制备性低或高压液相层析或上述方法的组合。然而,也可使用其它功能等同的分离方法。
考虑到寡核苷酸的序列并使用本领域已知的方法,可构建含有反义寡核苷酸序列的表达载体。
本领域技术人员可构建载体,所述载体含有获得反义寡核苷酸序列之适当转录所需的所有表达元件。因此,本发明提供了含有与编码反义寡核苷酸之序列可操作相连的转录控制序列的载体。适当的转录和翻译元件有多个来源,包括细菌,真菌,病毒,哺乳动物或昆虫基因。适当元件的选择取决于所选的宿主细胞。
载体中可包括报道基因。适当的报道基因包括β-半乳糖苷酶(如lacZ),氯霉素,乙酰基转移酶,萤火虫萤光素酶或免疫球蛋白或其部分。通过监测报道基因的表达可监测反义寡核苷酸的转录。
通过本领域的多种已知方法中的任一种可将载体导入细胞或组织。所述方法通常描述于Sambrook等24;Ausubel等25;Chang等36;Vega等37和载体:分子克隆载体及其用途概述38并包括例如稳定或瞬时转染,脂转染法,电穿孔和用重组病毒载体感染。
通过感染导入核酸有几个优点,可获得较高的效力和组织类型的特异性。病毒一般感染特定的细胞类型并在其中繁殖,因此,可使用病毒的特异性将载体靶向体内或组织内或混合的细胞培养物中的特定细胞类型。也可用特定的受体或配体修饰病毒载体以通过受体介导的事件改变靶特异性。
希望本发明的寡核苷酸是切割mRNA的核酶。优选核酶具有与本发明的寡核苷酸序列和切割mRNA所必需的催化中心同源的序列。例如,可选择破坏IGF-ⅡmRNA的同源核酶序列。本发明所用的核酶类型可选自本领域已知的类型。已鉴定出几个核酶结构家族,包括Ⅰ组内含子,RNA酶P,丁型肝炎病毒核酶,锤头状核酶和最初得自烟草环斑病毒卫星RNA(sTRSV)的发夹核酶(Sullivan 1994,美国专利5,225,34739)。锤头状核酶和发夹核酶基元最常适用于反式切割mRNA以用于基因治疗(Sullivan 1994)。本发明优选使用发夹核酶。通常,核酶的长度为30至100个核苷酸。
本发明的寡核苷酸可以是不溶解的,例如,寡核苷酸可结合于适当的载体上。适当载体的例子是琼脂糖,纤维素,葡聚糖,Sephadex,Sepharose,羧甲基纤维素,聚苯乙烯,滤纸,离子交换树脂,塑料薄膜,塑料管,玻璃珠,多胺-甲基乙烯基-醚-马来酸共聚物,氨基酸共聚物,乙烯-马来酸共聚物,尼龙,丝织物等。载体的外形可以是管,检测板,珠盘,球形等。
通过使用已知的化学或物理方法,如溴化氰偶联法使寡核苷酸与适当的不溶性载体反应即可制备不溶解的寡核苷酸。药物配制
当用作药物时,通常以药物组合物的形式施用反义寡核苷酸。可通过包括口服,直肠,经皮,皮下,静脉内,肌内和鼻内的多种途径施用这些化合物。这些化合物作为可注射的和口服的组合物都是有效的。可按药学领域众所周知的方式制备所述组合物,该组合物中可含有至少一种活性化合物。例如,可以静脉内注射药物组合物。希望能将药物组合物直接注射至需治疗的肿瘤中。
本发明还包括含有一种或多种反义寡核苷酸作为活性成分并同时含有药物可接受的载体或赋形剂的药物组合物。在制备本发明的组合物时,通常将活性成分与赋形剂混合,用赋形剂稀释活性成分,或将活性成分包裹在胶囊,药囊,纸或其它容器形式的载体内。当赋形剂用作稀释剂时,它可以是用作活性成分之载体或介质的固体,半固体或液体物质。因此,组合物的形式可以是片剂,丸剂,粉末,锭剂,药囊,扁囊剂,池剂,悬浮液,乳剂,溶液,糖浆,气雾剂(作为固体或溶于液体介质中),含有例如占重量10%的活性化合物的软膏剂,软和硬的明胶胶囊,栓剂,无菌的注射溶液和无菌的包装粉末。
制备制剂时,必需研磨活性化合物以在与其它成分混合之前提供适当的颗粒大小。如果活性化合物基本上不溶解,一般将其研磨至颗粒大小小于200目。如果活性化合物基本上是水溶性的,一般通过研磨来调节颗粒大小以使制剂中的颗粒分布基本上均匀,如研磨至40目左右。
适当赋形剂的一些例子包括乳糖,葡萄糖,蔗糖,山梨糖醇,甘露糖醇,淀粉,金合欢胶,磷酸钙,藻酸盐,黄蓍胶,明胶,硅酸钙,微晶纤维素,聚乙烯吡咯烷酮,纤维素,无菌水,糖浆和甲基纤维素。制剂中还可包括:润滑剂,如滑石粉,硬脂酸镁和矿物油;湿润剂;乳化剂和悬浮剂;防腐剂,如甲基-和丙基羟基-苯甲酸盐;甜味剂;和调味剂。也可使用本领域已知的方法配制本发明的组合物,使得给患者施用之后,能快速控释或缓释活性成分。
优选将组合物配制成单位剂量形式,每个剂量含有约1%至约95%,更优选约5%至约90%的活性成分。术语“单位剂量形式”指的是适于用作人受试者和其它哺乳动物的单一剂量的物理分离单位,每个单位含有经计算可产生所需治疗效果的预定量的活性物质以及适当的药物赋形剂。
反义寡核苷酸在宽的剂量范围内都有效,一般以药物有效量给药。有效量是给药时可减轻症状的量。优选有效量是能抑制肿瘤细胞生长的量,优选有效量是约0.1mg/kg体重至约20mg/kg体重。然而,应理解实际施用的反义寡核苷酸的量由医生根据有关情况决定,所述情况包括治疗的疾病,选定的给药途径,实际施用的化合物,年龄,体重和个体患者的反应,患者症状的严重程度等。疗程从几天至几个月不等或直至减轻疾病为止。反义寡核苷酸可以与其它已知的疗法一起使用。当与一种或多种其它的疗法一起使用时,可同时或依次施用寡核苷酸和其它治疗。如果是依次施用,护理医生将决定施用寡核苷酸与其它疗法的适当次序。
为了制备固体组合物,如片剂,可将主要的活性成分/反义寡核苷酸与药物赋形剂混合以形成含有本发明化合物之均质混合物的固体预配制组合物。当将所述预配制的组合物称为均质时,意味着活性成分均匀分散在整个组合物中,使得组合物很容易地被次分为同样有效的单位剂量形式,如片剂,丸剂和胶囊。
可包被或要不然复合本发明的片剂或丸剂以提供具有作用时间延长之优点的剂量形式。例如,片剂或丸剂可含有内部剂量组分和外部剂量组分,后者是前者的外膜形式。这两种组分可由肠层分隔开,所述肠层可以防止在胃中崩解并可使内部组分完整地进入十二指肠或延缓释放。多种物质可用作所述肠层或外被,所述物质包括多种聚合酸和聚合酸与如虫胶,鲸蜡醇和乙酸纤维素之物质的混合物。
其中掺入本发明的新组合物以口服或注射途径给药的液体形式包括水溶液,适当调味的糖浆,含水或油的悬浮液和用诸如玉米油,棉子油,芝麻油,椰子油或花生油的食用油调味的乳剂,以及酏剂和类似的药物载体。
用于吸入或吹入的组合物包括溶于药物可接受的含水或有机溶剂或其混合物中的溶液和悬浮液和粉末。液体或固体组合物可含有本文所述的适当的药物可接受的赋形剂。优选通过口服或鼻呼吸途径施用组合物以产生局部或全身疗效。可使用惰性气体喷雾优选溶于药物可接受的溶剂中的组合物,可直接由喷雾装置中吸入喷雾溶液,或者使喷雾装置贴附在面罩或间歇正压呼吸器上。优选由以适当方式传递制剂的装置口服或鼻内施用溶液,悬浮液或粉末组合物。
本发明的药物组合物可以是脂质体的形式,其中除了其它药物可接受的载体外,寡核苷酸还与以聚集体形式存在于水溶液中的两亲性试剂(如脂质)混合,所述聚集体形式如微团,不溶性单层,液晶或片层。脂质体制剂中所用的适当脂质包括但不限于甘油单酯,甘油二酯,硫脑苷脂,溶血卵磷脂,磷脂,皂苷,胆汁酸等。一个特别有用的脂质载体是lipofectin。制备所述脂质体制剂的方法是本领域技术人员众所周知的,例见国际专利号WO97/21808(28)。药物组合物可进一步包括能使寡核苷酸至细胞的传递增强的化合物,如环化糊精等或缓释聚合物。本发明方法中使用的另一种优选制剂使用经皮传递器具(“贴片”)。可使用这种经皮补片控量连续或不连续注入本发明的反义寡核苷酸。传递药剂所用的经皮补片的制备和使用是本领域众所周知的,例见美国专利5,023,25240(列入本文作为参考)。可制备这种补片以连续,脉动或按需传递药剂。
另一种优选的传递方法包括穿过真皮层“散弹枪法”传递裸露的反义寡核苷酸。“裸露”反义寡核苷酸的传递是本领域众所周知的,例见Felgner等,美国专利5,580,85941。希望在“散弹枪法”传递反义寡核苷酸之前将反义寡核苷酸包装于脂质载体中。
下列配方的例子阐明了本发明代表性的药物组合物。
配方实施例1
制备了含有下列成分的硬明胶胶囊:
成分 含量(mg/胶囊)
活性成分 30.0
淀粉 305.0
硬脂酸镁 5.0
混合上述成分并以340mg的量填入硬明胶胶囊。
配方实施例2
使用下列成分制备片剂:
成分 含量(mg/片)
活性成分 25.0
纤维素,微晶 200.0
胶态二氧化硅 10.0
硬胇酸 5. 0混合上述组分并压制成片剂,每片重为240mg。配方实施例3制备含有下列组分的干粉吸入制剂:
成分 重量%
活性成分 5
乳糖 95将活性成分与乳糖混合,并将混合物加入干粉吸入器中。配方实施例4按下述制备各含有30mg活性成分的片剂:
成分 含量(mg/片)
活性成分 30.0mg
淀粉 45.0mg
微晶纤维素 35.0mg聚乙烯吡咯烷酮(无菌水中的10%溶液) 4.0mg
羧甲基淀粉钠 4.5mg
硬脂酸镁 0.5mg
滑石粉 1.0mg
总重量 120mg将活性成分,淀粉和纤维素穿过20目的U.S.筛并充分混合。将聚乙烯吡咯烷酮溶液与所得粉末混合,然后穿过16目的U.S.筛。于50至60℃干燥所产生的颗粒并穿过16目的U.S.筛。然后在颗粒中加入预先穿过30目的U.S.筛的羧甲基淀粉钠,硬脂酸镁和滑石粉,混合后用片剂机压制产生每片重120mg的片剂。
配方实施例5
按下述制备各含有40mg药物的胶囊:
成分 含量(mg/胶囊)
活性成分 40.0mg
淀粉 109.0mg
硬脂酸镁 1.0mg
总重量 150.0mg
混合活性成分,淀粉和硬脂酸镁,穿过20目的U.S.筛并以150mg的量填入硬明胶胶囊。
配方实施例6
按下述制备各含有25mg活性成分的栓剂:
成分 含量
活性成分 25mg
饱和脂肪酸甘油酯 至2000mg
将活性成分穿过60目的U.S.筛并悬浮于预先用必需的最低温度融化的饱和脂肪酸甘油酯中。然后将混合物注入容量名义上为2.0g的栓剂模具中并使其冷却。
配方实施例7
按下述制备每5.0ml剂量含有50mg药物的悬浮液:
成分 含量
活性成分 50.0mg
黄原胶 4.0mg
羧甲基纤维素钠(11%)
微晶纤维素(89%) 50.0mg
蔗糖 1.75g
苯甲酸钠 10.0mg
调味剂和着色剂 q.v.
纯水 至5.0ml
混合活性成分,蔗糖和黄原胶,穿过10目的U.S.筛,然后与预先制备的微晶纤维素和羧甲基纤维素钠水溶液混合。用一些水稀释苯甲酸钠,调味剂和着色剂,边搅拌边加入,再加入足够量的水以产生所需的体积。
配方实施例8
成分 含量(mg/胶囊)
活性成分 15.0mg
淀粉 407.0mg
硬脂酸镁 3.0mg
总重量 425.0mg
混合活性成分,淀粉和硬脂酸镁,穿过20目的U.S.筛并以425.0mg的量填入硬明胶胶囊。
配方实施例9
按下述制备制剂:
成分 含量
活性成分 5.0mg
玉米油 1.0ml
配方实施例10
按下述制备局部制剂:
成分 含量
活性成分 1-10g
乳化蜡 30g
液体石蜡 20g
白色软石蜡 至100g
加热白色的软石蜡直至融化,将液体石蜡和乳化蜡混合在一起,搅拌直至溶解。加入活性成分,持续搅拌直至均匀分散,然后将混合物冷却为固体。
本发明所用的其它适当制剂见于Remington’s PharmaceuticalSciences(23)。
可将反义寡核苷酸或含有反义寡核苷酸的药物组合物包装于方便使用的试剂盒中,该试剂盒将必需的物质包装于适当容器中。
治疗制剂形式的本发明的反义寡核苷酸可用于治疗与肿瘤生长相关的疾病和病症。在该方法中,将能有效抑制IGF-Ⅱ胎儿转录物之表达的治疗量的寡核苷酸施用于细胞。该细胞可以是细胞培养物,组织培养物的一部分,或是如人的哺乳动物的整个身体的一部分。
本发明的寡核苷酸和核酶调节肿瘤细胞生长,因此,本发明提供了干扰或抑制哺乳动物肿瘤细胞生长的方法,所述方法包括将肿瘤或肿瘤细胞与本发明的反义寡核苷酸接触。
术语“接触”指的是在细胞悬浮液或组织样品中加入寡核苷酸,核酶等,或直接或间接给动物体内的细胞或组织施用寡核苷酸等。
可使用该方法治疗包括多种形式的癌症或肿瘤的增殖性病症,如白血病,(Hodgkins和非-Hodgkins)淋巴瘤,肉瘤,黑素瘤,腺瘤,实体组织癌,含氧量低的肿瘤,口腔,咽,喉和肺的鳞状细胞癌,泌尿生殖道癌症,如子宫颈和膀胱癌,造血癌症,结肠癌,乳腺癌,胰腺癌,肾癌,脑癌,皮肤癌,肝癌,头和颈癌,和神经系统的癌症以及良性损害,如乳头状瘤。也包括其它增殖性病症,如牛皮癣和涉及关节硬化的病症。
也可使用本发明的寡核苷酸治疗对药物具有抗性的肿瘤。药物抗性肿瘤的例子包括对诸如5-氟尿嘧啶,丝裂霉素C,氨甲蝶呤或羟基脲的化学治疗剂具有抗性的肿瘤和表达高水平P-糖蛋白的肿瘤,已知P-糖蛋白可赋予对诸如秋水仙素,长春花碱和阿霉素的多种抗癌药物的抗性;或表达多个-药物抗性蛋白质(如Dreeley等(28)所述)的肿瘤。因此,希望本发明的寡核苷酸与已知的抗癌化合物或化学治疗剂一起使用。化学治疗剂是可抑制肿瘤生长的化合物,该药剂包括但不限于5-氟尿嘧啶,丝裂霉素C,氨甲蝶呤和羟基脲。希望化学治疗剂的量为有效量,即足以抑制肿瘤生长的量或低于有效量。
已发现本发明的寡核苷酸可降低转移肿瘤,如C8161黑素瘤细胞的生长。在本发明的一个实施方案中,提供了降低哺乳动物转移肿瘤生长的方法,所述方法包括施用有效量的约3个至约100个核苷酸长的寡核苷酸,其含有与哺乳动物胎儿IGF-ⅡmRNA之5’非翻译区互补的序列,该序列可选自表1所示的寡核苷酸。在另一个实施方案中,提供了降低哺乳动物转移肿瘤生长的方法,所述方法包括施用有效量的约20个至约100个核苷酸长的寡核苷酸,其含有选自表2所示SEQ IDNO:17-31的序列。
使用病毒或非病毒载体可传递寡核苷酸。可将序列掺入盒或构建体中以使本发明的寡核苷酸能在细胞中表达。优选构建体含有适当的转录控制区域以使寡核苷酸能在细胞中转录。
因此,本发明提供了载体,其含有与编码本发明的寡核苷酸的序列可操作相连的转录控制序列。本发明还提供了被上述载体转化的宿主细胞,其选自适当的真核和原核细胞。
适当的载体是已知的,优选其含有获得序列的必要转录所必需的所有表达元件。噬粒是这种有利载体的特例,因为它们既可用作质粒也可用作噬菌体载体。载体的例子包括如噬菌体,杆状病毒,逆转录病毒,DNA病毒的病毒,脂质体和其它重组载体。载体也可含有用于原核或真核宿主系统的元件。本领域技术人员知道何种宿主系统与特定载体相容。
可通过稳定或瞬时转染,脂转染法,电穿孔和用重组病毒载体感染将载体导入细胞。
可在载体中添加其它特征以确保其安全性和/或增强其治疗效力,所述特征包括例如可用于阴性选择被重组病毒感染之细胞的标记。这种阴性选择标记的例子是赋予对抗病毒药丙氧鸟苷的敏感性的TK基因。也可包括将表达局限于特定细胞类型的特征。这种特征包括例如特异于所需细胞类型的启动子和调节元件。
逆转录病毒载体是另一例可用于体内导入所需核酸的载体,因为它们具有诸如侧向感染和靶向特异性的优点。侧向感染是单个感染细胞产生很多可感染相邻细胞的后代毒粒的过程,其结果是大面积细胞被快速感染。
可根据需要靶向的细胞类型选择本发明方法中所用的载体。例如,如果需治疗乳腺癌,即可使用特异于上皮细胞的载体。类似地,如果需治疗造血系统的细胞,则优选特异于血细胞的病毒载体。实用性
本发明的反义寡核苷酸具有多种用途。它们可用于抑制哺乳动物细胞中的IGF-Ⅱ基因的表达,导致该细胞的生长受抑制。所述寡核苷酸也可用作杂交探针以检测哺乳动物细胞中IGF-ⅡmRNA的存在。当如此使用时,可用适当的可测基团(如放射性同位素,配体,特定结合对的另一个成员,如生物素)标记寡核苷酸。最后,将寡核苷酸用作分子量标记。
为了进一步阐明本发明及其优点,给出了下列特定的实施例,但这些实施例不以任何方式限制权利要求书的范围。实施例
在下列实施例中,所有温度以摄氏度表示(除非另有说明),所有百分比皆为重量百分比(除非另有说明)。
在下列实施例中,下列缩写具有下列含义。如果未定义缩写,其具有其公知的含义:
AS=反义
cDNA=互补的脱氧核糖核酸
ODN=寡脱氧核苷酸
μM=微摩尔
mM=毫摩尔
M=摩尔
ml=毫升
μl=微升
mg=毫克
μg=微克
PAGE=聚丙烯酰胺凝胶电泳
rpm=每分钟的转数
ΔG=自由能,寡核苷酸双链体稳定性的测定结果
kcal=千卡
FBS=胎牛血清
DTT=二硫苏糖醇
SDS=十二烷基硫酸钠
PBS=磷酸缓冲盐水
PMSF=苯甲基磺酰氟
GAPDH=甘油醛-3-磷酸脱氢酶
IgG=免疫球蛋白G
kDa=千道尔顿
PCR=聚合酶链反应
Tris-HCl=三(羟甲基)氨基甲烷-盐酸
TRIzol=总RNA分离试剂
ECL=Western印迹检测试剂
IGF-Ⅰ=胰岛素样生长因子Ⅰ
IGF-Ⅱ=胰岛素样生长因子Ⅱ
UTR=非翻译区分子生物学的一般方法
本领域已知的,本文中未具体描述的标准分子生物学技术一般参照Sambrook等24;Ausubel等25和Perbal26。寡核苷酸
反义寡核苷酸选自与IGF-ⅡmRNA互补的序列以使序列最不可能显示出双链体形成,发夹形成和同寡聚体/序列重复但可高效结合IGF-ⅡmRNA序列。另外,还消除了对人和小鼠中的其它常见或重复序列的错误引发。使用计算机模型设计程序OLIGO引物分析软件第5.0版(由International Biosciences,Inc.,Plymouth,MN分发)可测定这些特性。根据此分析结果,设计硫代磷酸反义寡核苷酸,然后通过本领域众所周知的方法制备该寡核苷酸。细胞系
包括胚胎横纹肌肉瘤(RD),成神经细胞瘤(SK-N-AS),维尔姆斯瘤(G401),黑素瘤(C8161),人前列腺腺癌(PC-3),转移性胰腺腺癌(AsPC-1)的5个不同的人癌细胞系得自美国典型培养物保藏中心(ATCC)。将细胞系维持在添加有10%胎牛血清(FBS)的α-MEM培养基(Gibco BRL,Gaithersburg,MD)中。实施例1.通过与IGF-Ⅱ互补的反义寡核苷酸抑制癌症细胞系的生长
使用前述方法(Choy等18)估计用不同硫代磷酸反义寡核苷酸处理的癌症细胞系的集落形成能力。具体地说,以约1×104的密度将肿瘤细胞悬浮液的等分试样接种于60mm组织培养皿中,于37℃,在添加有10%FBS的α-MEM培养基中保温过夜。用5mlPBS将细胞洗涤1次,再在阳离子脂质的存在下(Lipofectin试剂,终浓度为5μg/ml,Gibco-BRL,Gaithers-burg,MD),用0.2μM所示反义寡核苷酸将细胞处理4小时。通过用PBS洗涤细胞1次以除去反义寡核苷酸,于37℃,在添加有10%FBS的α-MEM生长培养基中将细胞培养7至10天。用亚甲蓝染色集落,通过按choy等18以及Huang和Wright20所述直接计数以进行评分。通过与缺乏反义寡核苷酸的培养物中存在的集落数目相比较,计算出抑制作用的百分比。所有实验都重复进行4次。
反义寡核苷酸对人肿瘤细胞系的集落形成能力具有抑制作用。图2A-D分别是各个反义寡核苷酸对横纹肌肉瘤(RD);人前列腺癌细胞系(PC-3);人胰腺癌细胞系(AsPC-1);和人成神经细胞瘤(SK-N-AS)的抑制作用百分比。实施例2.用与IGF-Ⅱ互补的反义寡核苷酸处理之后降低的mRNA水平
将人成神经细胞瘤(SK-N-AS)或横纹肌肉瘤细胞(RD)培养至将近铺满(70-80%),然后在阳离子脂质(Lipofectin试剂,终浓度为5μg/ml,Gibco-BRL)和Opti-MEM(Gibco-BRL)的存在下,用0.2μM与IGF-Ⅱ互补的硫代磷酸反义寡核苷酸将细胞处理4小时。用PBS洗涤细胞1次并在含有10%FBS的α-MEM培养基(Gibco-BRL)中将细胞保温16小时。在TRIzol试剂(Gibco-BRL)中制备总RNA,按Hurta和Wright(27)所述,稍加改动后进行Northern印迹分析。使用正向引物(5’-TAC CGC CCCAGT GAG ACC CT-3’)[SEQ ID NO:32],反向引物(5’-TGA CGT TTG GCCTCC CTG AA-3’)[SEQ ID NO:33]并以人结肠腺癌5’端的一段序列加cDNA文库(Clonetech,Palo Alto CA)为模板合成经32P-标记的389bp PCR片断,将上述印迹与此片断杂交。人IGF-ⅡmRNA被表达为约6kb的核苷酸转录物(Werner等6)。通过在杂交之前用亚甲蓝染色可阐明相等的RNA上样量。
图3显示出相对于对照细胞而言,反义寡核苷酸可将IGF-ⅡmRNA水平至少降低50%。实施例3.用与IGF-Ⅱ互补的反义寡核苷酸处理之后降低的IGF-Ⅱ蛋白质水平
将人成神经细胞瘤(SK-N-AS)或横纹肌肉瘤细胞(RD)培养至将近铺满(70-80%),然后在阳离子脂质(Lipofectin试剂,终浓度为5μg/ml,Gibco-BRL)和Opti-MEM(Gibco-BRL)的存在下,用0.2μM与IGF-Ⅱ互补的硫代磷酸反义寡核苷酸将细胞处理4小时。用PBS洗涤细胞1次并在含有10%FBS的α-MEM培养基(Gibco-BRL)中将细胞保温20小时。将处理和保温过程再重复1次,然后在2×样品上样缓冲液(100mM Tris-HCl,pH6.8,0.2M DTT,4%SDS,20%甘油和0.015%溴酚蓝)中制备全细胞蛋白质提取物。
按Choy等(18)和Fan等(19)所述,稍加改动后进行Western印迹分析。用抗IGF-Ⅱ抗体(1-2μg/ml)(Research Diagnostics公司,Flanders NJ),接着用稀释度为1∶7000的辣根过氧化物酶-缀合的抗山羊IgG(Sigma,St.Loius MO)检测IGF-Ⅱ的表达。通过ECL(Amersham,Arlington heights,IL)肉眼观察到约7.5kDa的蛋白质。
图4显示出用多种反义寡核苷酸处理之后,人成神经细胞瘤细胞中IGF-Ⅱ蛋白的减少。
图5显示出用多种反义寡核苷酸处理之后,人横纹肌肉瘤细胞中IGF-Ⅱ蛋白的减少。实施例4.通过用与IGF-Ⅱ互补的反义寡核苷酸静脉内治疗来抑制小鼠体内的人肿瘤细胞生长
CD-1无胸腺裸鼠购自Charles River实验室(Montreal加拿大)。将SK-N-AS人成神经细胞瘤细胞(一般为3×106个细胞/100μl PBS)皮下注入6-7周龄的CD-1无胸腺雌性裸鼠的右肋。每个实验组包括5只小鼠。当肿瘤大小达到约100mm3的体积(一般在注射肿瘤细胞6天后)之后,每隔一天以10mg/kg的剂量通过大药丸灌注将多种反义寡核苷酸施用于尾静脉。同时给对照动物仅施用盐水。随后,一般持续治疗14天。
图6A显示出多种反义寡核苷酸对CD-1裸鼠中的人成神经细胞瘤生长的影响。在治疗的14天里,平均每隔2天用测径器测量肿瘤体积,由对肿瘤体积的抑制作用估计抗肿瘤活性。图中的每个点表示由每个实验组的5只动物计算出的平均肿瘤体积。使用相关变异分析比较每个治疗组小鼠在治疗时间内的回归曲线。由该分析得出特别的假说,即斜率相同,或当斜率相同时截距也相同。所有分析都使用了SAS(统计学分析系统)6.12版。当与盐水对照相比时,施用反义寡核苷酸可抑制肿瘤生长,p值≤0.0001。
治疗结束时(通常为最后一次治疗后24小时),处死动物,测定肿瘤重量。图6B显示出肿瘤的平均重量,反义寡核苷酸对肿瘤生长具有显著的抑制作用。使用方差单向分析比较治疗组的平均值。当整个组的效果显著时,从事先使用最小二乘方平均值获得的多个比较结果中可发现显著不同的治疗组对。当比较肿瘤重量时,与盐水对照相比,反义寡核苷酸也显示出统计学上显著的抑制作用。实施例5.通过用与IGF-Ⅱ互补的反义寡核苷酸静脉内治疗来抑制小鼠体内的人肿瘤细胞生长
CD-1无胸腺裸鼠购自Charles River实验室(Montreal加拿大)。将G401人维尔姆斯瘤细胞(一般为3×106个细胞/100μl PBS)皮下注入6-7周龄的CD-1无胸腺雌性裸鼠的右肋。每个实验组包括5只小鼠。当肿瘤大小达到约100mm3的体积(一般在注射肿瘤细胞8天后)之后,每隔一天以10mg/kg的剂量通过大药丸灌注将多种反义寡核苷酸施用于尾静脉。同时给对照动物仅施用盐水。随后,一般持续治疗18天。
图7A显示出多种反义寡核苷酸对CD-1裸鼠中的人维尔姆斯瘤生长的影响。在治疗的18天里,平均每隔2天用测径器测量肿瘤体积,由对肿瘤体积的抑制作用估计抗肿瘤活性。图中的每个点表示由每个实验组的5只动物计算出的平均肿瘤体积。使用相关变异分析比较每个治疗组小鼠在治疗时间内的回归曲线。由该分析得出特别的假说,即斜率相同,或当斜率相同时截距也相同。所有分析都使用了SAS(统计学分析系统)6.12版。当与盐水对照相比时,施用反义寡核苷酸可抑制肿瘤生长,p值≤0.0002。
治疗结束时(通常为最后一次治疗后24小时),处死动物,测定肿瘤重量。图7B显示出肿瘤的平均重量,反义寡核苷酸对肿瘤生长具有显著的抑制作用。使用方差单向分析比较治疗组的平均值。当整个组的效果显著时,从事先使用最小二乘方平均值获得的多个比较结果中可发现显著不同的治疗组对。当比较肿瘤重量时与盐水对照相比,反义寡核苷酸也显示出统计学上显著的抑制作用。实施例6.通过用与IGF-Ⅱ互补的反义寡核苷酸静脉内治疗来降低小鼠体内的人肿瘤的IGF-ⅡmRNA水平
CD-1无胸腺裸鼠购自Charles River实验室(Montreal加拿大)。将SK-N-AS人成神经细胞瘤细胞(一般为3×106个细胞/100μl PBS)皮下注入6-7周龄的CD-1无胸腺雌性裸鼠的右肋。每个实验组包括5只小鼠。当肿瘤大小达到约100mm3的体积(一般在注射肿瘤细胞6天后)之后,每隔一天以10mg/kg的剂量通过大药丸灌注将多种反义寡核苷酸施用于尾静脉。同时给对照动物仅施用盐水。7次注射后处死小鼠,立即将切下的大小相似的肿瘤片断收集至TRIzol试剂(GIBCO BRL)中,并快速匀浆以制备mRNA。
为了测定反义寡核苷酸对IGF-ⅡmRNA水平的影响,按Hurta和Wright(27)所述,稍加改动后进行Northern印迹分析。使用正向引物(5’-TAC CGC CCC AGT GAG ACC CT-3’)[SEQ ID NO:32],反向引物(5’-TGACGT TTG GCC TCC CTG AA-3’)[SEQ ID NO:33]并以人结肠腺癌5’端的一段序列加cDNA文库(Clonetech,Palo Alto CA)为模板合成经32p-标记的389bp PCR片断,将上述印迹与此片断杂交。人IGF-ⅡmRNA被表达为约6kb的核苷酸转录物(Werner等6),按Hurta和Wright(27)所述,将其水平与甘油醛-3-磷酸脱氢酶(GADPH)mRNA相比较。
图8显示出被反义寡核苷酸GTI4006[SEQ ID NO:6]治疗的肿瘤中IGF-ⅡmRNA水平的降低。实施例7.通过用与IGF-Ⅱ互补的反义寡核苷酸静脉内治疗来降低小鼠体内的人肿瘤的IGF-Ⅱ蛋白质水平
CD-1无胸腺裸鼠购自Charles River实验室(Montreal加拿大)。将SK-N-AS人成神经细胞瘤细胞(一般为3×106个细胞/100μl PBS)皮下注入6-7周龄的CD-1无胸腺雌性裸鼠的右肋。每个实验组包括5只小鼠。当肿瘤大小达到约100mm3的体积(一般在注射肿瘤细胞6天后)之后,每隔一天以10mg/kg的剂量通过大药丸灌注将多种反义寡核苷酸施用于尾静脉。同时给对照动物仅施用盐水。7次注射后处死小鼠,立即将切下的大小相似的肿瘤片断收集至RIPA提取缓冲液(50mM Tris-HCl,pH7.5,150mM亮抑蛋白酶肽)中,并快速匀浆以制备蛋白质。
为了测定反义寡核苷酸对IGF-Ⅱ蛋白质水平的影响,按Choy等(18)和Fan等(19)所述,稍加改动后进行Western印迹分析。在15%SDS-PAGE凝胶上分级分离蛋白质提取物(10-20μg),然后转移至硝酸纤维素膜上,通过用印度墨汁染色肉眼观察。用抗IGF-Ⅱ抗体(1-2μg/ml(Research Diagnostics公司,Flanders NJ),接着用稀释度为1∶7000的辣根过氧化物酶-缀合的抗山羊IgG(Sigma,St.Loius MO)检测IGF-Ⅱ的表达。通过ECL(Amersham,Arlington heights,IL)肉眼观察到约7.5kDa的蛋白质。
图9显示出提取自肿瘤细胞的蛋白质的Western印迹。检测的每种反义寡核苷酸都能降低肿瘤中IGF-Ⅱ蛋白质的水平。下方显示的用印度墨汁染色的一部分印迹可阐明每个泳道相等的上样量。实施例8.通过反义寡核苷酸抑制实验转移
按Fan等,199619所述估计用不同反义寡核苷酸处理的C8161人黑素瘤细胞的实验转移。以2×106的密度将细胞悬浮液的等分试样接种于100mm组织培养皿中,于37℃,在添加有10%FBS的α-MEM培养基中保温过夜。用10ml PBS将细胞洗涤1次,再在阳离子脂质的存在下(Lipofectin试剂,终浓度为5μg/ml,Gibco-BRL),用0.2μM寡核苷酸将细胞处理4小时。通过用PBS洗涤细胞1次以除去反义寡核苷酸,并用胰蛋白酶消化细胞。离心收集细胞,将悬浮于0.1ml PBS中的约1×105个细胞注射至6-8周龄的CD-1无胸腺雌性裸鼠的尾静脉中,5周后估计肺部肿瘤的数目,用苦味酸染料溶液(75%苦味酸,20%甲醛,5%冰醋酸)染色从各个小鼠体内切下的肺。
图10显示出用多种反义寡核苷酸处理肿瘤细胞之后,雌性裸鼠中肺部肿瘤数目的降低。
Claims (26)
1.反义寡核苷酸,包括约3至约100个核苷酸,其中的寡核苷酸含有与哺乳动物胎儿IGF-ⅡmRNA之5’非翻译区互补的序列。
2.权利要求1的反义寡核苷酸,其进一步含有一个或多个硫代磷酸酯核苷酸间连键。
3.权利要求1的反义寡核苷酸,其进一步含有不与IGF-ⅡmRNA互补的其它核苷酸。
4.权利要求1的反义寡核苷酸,其中序列选自表1的SEQ IDNO:1-15。
5.反义寡核苷酸,包括约20至约100个核苷酸,其中的寡核苷酸含有选自表2的SEQ ID NO:17-31的序列。
6.权利要求5的反义寡核苷酸,其进一步含有一个或多个硫代磷酸酯核苷酸间连键。
7.权利要求5的反义寡核苷酸,其进一步含有不与IGF-ⅡmRNA互补的其它核苷酸。
8.含有反义寡核苷酸序列的载体,其中所述寡核苷酸序列长度为约3至约100个核苷酸,并含有与哺乳动物胎儿IGF-ⅡmRNA之5’非翻译区互补的序列。
9.含有反义寡核苷酸序列的载体,其中所述寡核苷酸序列长度为约20至约100个核苷酸,并含有选自表2的SEQ ID NO:17-31的序列。
10.药物组合物,其含有药物可接受的赋形剂和有效量的反义寡核苷酸,其中所述寡核苷酸序列长度为约3至约100个核苷酸,并含有与哺乳动物胎儿IGF-ⅡmRNA之5’非翻译区互补的序列。
11.权利要求10的药物组合物,其中序列选自表1的SEQ IDNO:1-15。
12.药物组合物,其含有药物可接受的赋形剂和有效量的反义寡核苷酸,其中所述寡核苷酸序列长度为约20至约100个核苷酸,并含有选自表2的SEQ ID NO:17-31的序列。
13.抑制哺乳动物肿瘤生长的方法,所述方法包括:在能抑制肿瘤生长的条件下,给怀疑患有肿瘤的哺乳动物施用有效量的反义寡核苷酸,该寡核苷酸长度约为3至100个核苷酸,并含有与哺乳动物胎儿IGF-ⅡmRNA之5’非翻译区互补的序列。
14.权利要求13的方法,进一步包括给哺乳动物施用化学治疗剂的步骤。
15.权利要求13的方法,其中寡核苷酸可含有选自表1之SEQ IDNO:1至15的序列。
16.权利要求13的方法,其中寡核苷酸为核酶抗性。
17.抑制哺乳动物肿瘤生长的方法,所述方法包括:在能抑制肿瘤生长的条件下,给怀疑患有肿瘤的哺乳动物施用有效量的反义寡核苷酸,该寡核苷酸长度约为20至100个核苷酸,并含有选自表2之SEQ IDNO:17至31的序列。
18.权利要求17的方法,其中寡核苷酸为核酶抗性。
19.权利要求17的方法,进一步包括给哺乳动物施用化学治疗剂的步骤。
20.抑制哺乳动物肿瘤转移的方法,所述方法包括:在能抑制肿瘤转移的条件下,给怀疑患有转移性肿瘤的哺乳动物施用有效量的反义寡核苷酸,该寡核苷酸长度约为3至100个核苷酸,并含有与哺乳动物胎儿IGF-ⅡmRNA之5’非翻译区互补的序列。
21.权利要求20的方法,进一步包括给哺乳动物施用化学治疗剂的步骤。
22.权利要求20的方法,其中寡核苷酸为核酶抗性。
23.权利要求20的方法,其中寡核苷酸含有选自表1之SEQ ID NO:1至15的序列。
24.抑制哺乳动物肿瘤转移的方法,所述方法包括:在能抑制肿瘤转移的条件下,给怀疑患有转移性肿瘤的哺乳动物施用有效量的反义寡核苷酸,该寡核苷酸长度约为20至100个核苷酸,并含有选自表2之SEQ ID NO:17至31的序列。
25.权利要求24的方法,进一步包括给哺乳动物施用化学治疗剂的步骤。
26.权利要求25的方法,其中寡核苷酸为核酶抗性。
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US5766855A (en) | 1991-05-24 | 1998-06-16 | Buchardt, Deceased; Ole | Peptide nucleic acids having enhanced binding affinity and sequence specificity |
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WO1993024655A1 (en) * | 1992-05-27 | 1993-12-09 | Amersham International Plc | Rna analysis |
EP0668782B1 (en) | 1992-10-21 | 2001-04-11 | Temple University - Of The Commonwealth System Of Higher Education | Combination of antineoplastic agent and antisense oligonucleotides for treatment of cancer |
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WO1999029729A2 (en) * | 1997-12-09 | 1999-06-17 | Children's Medical Center Corporation | Antagonists of neuropilin receptor function and use thereof |
-
1999
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- 1999-04-23 IL IL13897699A patent/IL138976A0/xx unknown
- 1999-04-23 NZ NZ508353A patent/NZ508353A/en unknown
- 1999-04-23 KR KR1020007011618A patent/KR20010042849A/ko not_active Application Discontinuation
- 1999-04-23 EP EP99915403A patent/EP1073728A2/en not_active Withdrawn
- 1999-04-23 CA CA002326825A patent/CA2326825A1/en not_active Abandoned
- 1999-04-23 CN CN99805388A patent/CN1298445A/zh active Pending
- 1999-04-23 AU AU34021/99A patent/AU749802B2/en not_active Ceased
- 1999-04-23 BR BR9909809-1A patent/BR9909809A/pt not_active IP Right Cessation
- 1999-04-23 JP JP2000545998A patent/JP2002512792A/ja not_active Withdrawn
- 1999-04-23 AU AU34022/99A patent/AU747639C/en not_active Ceased
- 1999-04-23 WO PCT/CA1999/000324 patent/WO1999055855A2/en not_active Application Discontinuation
- 1999-04-23 CN CN99805384A patent/CN1298444A/zh active Pending
- 1999-04-23 NZ NZ508354A patent/NZ508354A/en unknown
- 1999-04-23 EP EP99915404A patent/EP1073729A2/en not_active Withdrawn
- 1999-04-23 CA CA002326824A patent/CA2326824A1/en not_active Abandoned
- 1999-04-23 KR KR1020007011617A patent/KR20010042848A/ko not_active Application Discontinuation
- 1999-04-23 WO PCT/CA1999/000323 patent/WO1999055854A2/en not_active Application Discontinuation
- 1999-04-23 JP JP2000545999A patent/JP2002512793A/ja not_active Withdrawn
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- 1999-04-24 IL IL13897799A patent/IL138977A0/xx unknown
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2002
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Also Published As
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WO1999055855A3 (en) | 2000-01-06 |
KR20010042849A (ko) | 2001-05-25 |
CA2326824A1 (en) | 1999-11-04 |
CN1298445A (zh) | 2001-06-06 |
WO1999055854A3 (en) | 2000-06-29 |
WO1999055854A2 (en) | 1999-11-04 |
EP1073729A2 (en) | 2001-02-07 |
EP1073728A2 (en) | 2001-02-07 |
NZ508354A (en) | 2005-01-28 |
BR9909809A (pt) | 2000-12-19 |
BR9909859A (pt) | 2000-12-19 |
KR20010042848A (ko) | 2001-05-25 |
IL138976A0 (en) | 2001-11-25 |
AU3402199A (en) | 1999-11-16 |
US20020187954A1 (en) | 2002-12-12 |
JP2002512793A (ja) | 2002-05-08 |
AU749802B2 (en) | 2002-07-04 |
NZ508353A (en) | 2005-01-28 |
WO1999055855A2 (en) | 1999-11-04 |
AU3402299A (en) | 1999-11-16 |
CA2326825A1 (en) | 1999-11-04 |
AU747639B2 (en) | 2002-05-16 |
JP2002512792A (ja) | 2002-05-08 |
AU747639C (en) | 2005-01-13 |
US6417169B1 (en) | 2002-07-09 |
IL138977A0 (en) | 2001-11-25 |
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