CN1298445A - 神经纤维网素反义寡核苷酸序列及其用于调控细胞生长的方法 - Google Patents
神经纤维网素反义寡核苷酸序列及其用于调控细胞生长的方法 Download PDFInfo
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- CN1298445A CN1298445A CN99805388A CN99805388A CN1298445A CN 1298445 A CN1298445 A CN 1298445A CN 99805388 A CN99805388 A CN 99805388A CN 99805388 A CN99805388 A CN 99805388A CN 1298445 A CN1298445 A CN 1298445A
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Abstract
本发明涉及与神经纤维网素基因互补的寡核苷酸,其可调控哺乳动物的肿瘤细胞生长及血管生成。本发明也涉及在抑制哺乳动物的肿瘤细胞生长和血管生成中使用这类化合物的方法。本发明还涉及药物组合物,其包含药物上可接受的赋形剂及有效量的本发明化合物。
Description
相关申请的参考
本申请要求以美国临时申请序号60/082,791(1988年4月23日申请)作为优先权,该申请的全文并入本文作为参考。
发明背景
发明领域
本发明涉及与哺乳动物神经纤维网素(或VEGF165R)的mRNA互补的寡核苷酸,该寡核苷酸可调控哺乳动物的细胞生长。本发明也涉及在抑制哺乳动物肿瘤细胞生长及抑制哺乳动物血管生成中使用这类化合物的方法。本发明还涉及药物组合物,其包含药物上可接受的赋形剂及有效量的本发明化合物。
参考文献
下列出版物、专利申请及专利在本申请中以标示数字方式引用:
1.Tischer,E.,等人,“人血管内皮生长因子的基因。通过可变外显子剪接编码多种蛋白形式”,生物化学杂志266:11947-54,(1991).
2.Poltorak,Z.,等人,“VEGF145,可结合胞外基质的分泌的血管内皮生长因子同种型”,生物化学杂志272:7151-8,1997.
3.Terman,B.I.,等人,“作为血管内皮细胞生长因子受体的KDR酪氨酸激酶的鉴定”,生物化学生物物理研究通讯187:1579-86,1992.
4.Millauer,B.,等人,“高亲和性VEGF结合和发育表达表明Flk-1是血管发生和血管生成的主要调节物”,细胞72:835-46,1993.
5.Shibuya,M.,等人,与fms家族密切相关的新的人受体型酪氨酸激酶基因(flt)的核苷酸序列和表达”,癌基因5:519-24,1990.
6.de Vries,C.,等人,“fms样酪氨酸激酶,一种血管内皮生长因子的受体”,科学255:989-91,1992.
7.Kawakami,A.,等人,“细胞表面蛋白—神经纤维网素在小鼠神经系统中的发育调节表达”,神经生物学杂志29:1-17,1996.
8.Takagi,S.,等人,“细胞粘着分子—神经纤维网素在发育中的小鸡神经系统中的表达”,发育生物学170:207-22,1995.
9.Soker,S.,等人,“神经纤维网素由内皮细胞和肿瘤细胞作为血管内皮生长因子的同种型特异性受体来表达”,细胞92:735-45,1998.
10.Soker,S.,等人,“对应于VEGF165的外显子7-编码区的肽对血管内皮生长因子(VEGF)诱导的内皮细胞增殖的抑制作用”,生物化学杂志272:31582-8,1997.
11.He,Z.和Tessier-Lavigne,M.“神经纤维网素是轴突化学排斥物Semaphorin III的受体”,细胞90:739-51,1997.
12.Mitsuhashi,M.“设计特异性反义寡核苷酸序列的策略”,胃肠道学杂志32:282-7,1997.
13.Alama,A.,等人,“作为治疗剂的反义寡核苷酸”,药物学研究36:171-8,1997.
14.Curcio,L.D.,等人,“作为癌基因表达调制物的寡核苷酸”,药物治疗74:317-32,1997.
15.Brem,S.,等人,“通过防止玻璃体中新血管形成延长肿瘤的休眠”,癌症研究36:2807-12,1976.
16.Holmgren,L.,等人,“微量转移的休眠:在血管生成抑制存在下的平衡增殖和编程性细胞死亡”,自然医学(Nat Med.)1:149-53,1995.
17.Parangi,S.,等人,“转基因小鼠的抗血管生成治疗可再削弱肿瘤生长”,美国国家科学院院报93:2002-7,1996.
18.Choy等人,“涉及核糖核苷酸还原酶的药物抗性的分子机理:在不断增加的药物浓度存在下选择的一系列克隆相关小鼠细胞系中的羟基脲抗性”,癌症研究48:2029-2035(1988)
19.Fan等人,“核糖核苷酸还原酶R2组分是可与活化癌基因协同作用于确定转变和恶性潜力的新的恶性决定子”,美国国家科学院院报93:14036-40(1996)
20.Huang和Wright,“成纤维细胞生长因子介导的药物抗性的改变和基因扩增的迹象”,癌基因9:491-499(1994)
21.国际专利申请公开号WO99/02556,“Semaphorin受体”
22.国际专利申请公开号WO99/04263,“Semaphorin受体”
23.Remington的药物科学,宾西法尼亚费城,Mace出版公司,第17版(1985)
24.Sambrook等人,分子克隆:实验室手册,纽约,冷泉港实验室(1989,1992)
25.Ausubel等人,分子生物学的现行方案,马里兰巴尔的摩,John Wiley and Sons(1989)
26.Perbal,分子克隆实用指南,纽约,John Wiley & Sons(1988)
27.Hurta和Wright,“由H-ras引起的恶变通过转化生长因子β导致核糖核苷酸还原酶表达的异常调节”细胞生物化学杂志57:543-556(1995)
28.国际专利申请公开号WO97/21808,“修饰的VEGF反义寡核苷酸”
29.Nielsen等人;科学(1991)354:1497
30.Good和Nielsen;“靶向核糖体RNA的肽核酸对翻译和细菌生长的抑制作用”,美国国家科学院院报(1998)95:2073-2076
31.Buchardt,deceased,等人,美国专利5,766,855
32.Buchardt,deceased,等人,美国专利5,719,262
33.美国专利5,034,506
34.Altschul,等人“基本的局部对比检索工具”,分子生物学杂志(1990)215:403-10;
35.Devereux J.等人,“VAX的一整套序列分析程序”,核酸研究(1984)12:387-395;
36.Chang等人;体细胞基因治疗,CRC出版社,Ann Arbor MI(1995);
37.Vega等人;基因寻靶,CRC出版社,Ann Arbor MI(1995)
38.载体:分子克隆载体及其应用的评述,Butterworths,BostonMA(1988)
39.Sullivan,美国专利5,225,347
40.美国专利5,023,252,1991年6月11日授权
41.Felgner等人,美国专利5,580,859
42.Dreeley等人,科学,258:1650-1654(1992)
43.Uhlmann等人,化学评论90:534-583(1990)
44.Agrawal等人,生物技术动态10:152-158(1992)
45.Smith等人,(1994)Invest.Ophthalmol.Vis.Sci35:101-111
46.Pierce等人,(1995)美国国家科学院院报92:905-9
上列所有出版物、专利申请和专利与具体指明每一出版物、专利申请或专利全文并入作为参考一样以它们的全部内容并入本文作为参考。
目前的技术水平
新微血管的增生被称为血管生成或者新血管生成,对小的局部肿瘤扩张成大的恶性生长的转变具有决定性的影响。若排除血液供给的适当发展,肿瘤的生长则急剧减弱。
视网膜的新生血管疾病,例如糖尿病性视网膜病变,早产性视网膜病变及与年龄有关的斑点变性,在美国及世界各地均是引起眼盲的主因。在糖尿病过程中,其视网膜血管出现变化而导致血管渗漏及血管脱落最后导致视网膜缺氧症状。这种视网膜新血管生成的影响之一是导致出血和视网膜剥离。早产性视网膜病变乃是引起幼儿眼盲的常见原因。当发育中的视网膜增加代谢需求时,视网膜血管停止发育成末梢视网膜而导致局部缺血及局部缺氧的情形。缺氧的结果引发其后的视网膜新血管生成而导致永久性视力损失。眼睛新血管生成也是镰状细胞性视网膜病变,新血管性青光眼,视网膜血管闭塞及其它缺氧性疾病的基本病因。最近的实验数据显示,血管内皮生长因子的表达与视网膜新血管生成之间有高度相关性(28)。
肿瘤细胞产生的数个血管生成因子中,显示血管内皮生长因子(VEGF)为肿瘤血管生成及新血管生成的主要介体。人类VEGF单体有5种不同的同种型,其中以VEGF121和VEGF165含量最多(1,2)。VEGF活性在于其与内层有肿瘤血管分布的内皮细胞上存在的高亲和性酪氨酸激酶受体的结合作用。两种这样的受体已被分离:KDR/Flk-1(3,4),其看来是VEGF信号与Flt-1的主要转导物(5,6)。
最近克隆出的神经纤维网素或VEGF165R或血管内皮生长因子受体为由内皮细胞表达的VEGF165的新的同种型特异性受体(9),而该受体最早是以介导神经元细胞引导的脑衰蛋白/信号素(collapsin/semaphorin)的受体被分离出来的(7,8)。人类神经纤维网素的核酸序列已被报导(9,11,21,22)。神经纤维网素担当VEGF165与KDR/Flk-1结合的共同受体,并且调控接下来的生物活性,即肿瘤诱发的血管生成。在被试验的很少数中,其也在肿瘤衍生的细胞,如MDA-MB-231乳癌细胞及PC3前列腺癌细胞中高度表达(9,10)。其中也显示VEGF可结合Hela,黑素瘤及NIH 3T3细胞。
反义技术已被广泛采用,不仅是一种有用的研究工具(12),而且是获得用于治疗许多人类疾病(包括癌症)的新的治疗用化合物的合理手段(13,14)。反义寡核苷酸可与mRNA序列特异性杂交并且抑制对人类癌症的引发和/或进展具有重要性的蛋白质表达。因此,需要鉴定直接针对神经纤维网素的反义寡核苷酸,其作用是以较高特异性及较低毒性抑制神经纤维网素/VEGF165R的表达和产生。
发明概述
本发明涉及反义寡核苷酸,其可调控哺乳动物的神经纤维网素基因的表达和神经纤维网素/VEGF165R的产生,并涉及包含此类反义寡核苷酸的药物组合物。本发明也涉及利用此类反义寡核苷酸抑制哺乳动物的肿瘤生长和转移中所涉及的新毛细血管增生或血管生成或新血管生成。
因此,本发明组合物的一个方面涉及约3至约100个核苷酸的反义寡核苷酸,其包含与哺乳动物的神经纤维网素mRNA互补的核苷酸。该反义寡核苷酸可具有核酸酶抗性,且具有一个或多个硫代磷酸酯核苷酸间键。该反义寡核苷酸可能进一步包含其它不与神经纤维网素mRNA互补的核苷酸。
本发明组合物另一方面涉及约20至约100个核苷酸的反义寡核苷酸,其包括选自示于表1中SEQ ID NOs:1-30的序列,该寡核苷酸可抑制神经纤维网素表达。
本发明组合物另一方面涉及包含约20至约100个核苷酸的寡核苷酸序列的载体,该寡核苷酸包括选自示于表1中SEQ ID NOs:1-30的序列,其可抑制神经纤维网素表达。
本发明组合物另一方面涉及一种药物组合物,其包含药物上可接受的赋形剂及有效量的约20至约100个核苷酸的反义寡核苷酸,其中包括选自示于表1的SEQ ID NOs:1-30的序列,该寡核苷酸可抑制神经纤维网素表达。
本发明方法一方面涉及用于抑制哺乳动物肿瘤生长的方法,其包括对疑有肿瘤的哺乳动物施用有效量的反义寡核苷酸,该寡核苷酸的大小约3至约100个核苷酸并且包含一段在抑制肿瘤生长的条件下与哺乳动物神经纤维网素mRNA互补的序列。该反义寡核苷酸可与化疗药物共同投药。
本发明方法另一方面涉及一种抑制哺乳动物肿瘤转移的方法,其包括对疑有肿瘤的哺乳动物施用有效量的反义寡核苷酸,其大小约3至约100个核苷酸且包含在抑制肿瘤转移的条件下可与哺乳动物神经纤维网素mRNA互补的序列。该反义寡核苷酸可与化疗药物同时使用。
本发明方法另一方面涉及一种抑制哺乳动物的血管生成或新血管生成的方法,其包括对该哺乳动物施用有效量的反义寡核苷酸,其大小约3至约100个核苷酸,且可在抑制新血管生成的条件下,与哺乳动物神经纤维网素mRNA互补。
本发明方法另一方面涉及一种抑制神经纤维网素表达的方法,其包括使对神经纤维网素具有特异性的核酸与反义寡核苷酸接触,该反义寡核苷酸大小约20至约100个核苷酸并且包括一段选自示于表1中SEQ ID NOs:1-30的序列,该寡核苷酸可抑制神经纤维网素表达。
图示简述
图1A-F是经由施用指定的反义寡核苷酸抑制不同细胞系集落形成能力的百分比图。图1A为人类黑素瘤细胞系C8161的抑制百分比;图1B为人类肺癌细胞系A459的抑制百分比;图1C为人类黑素瘤细胞系A2058的抑制百分比;图1D为人类结肠癌细胞系HT-29的抑制百分比;图1E为人类前列腺癌细胞系PC-3的抑制百分比;及图1F为人类胰腺癌细胞系AsPC-1的抑制百分比。
图2A和2B是施用下列反义寡核苷酸后的人类黑素瘤癌细胞系A2058(图2B)或人类乳癌细胞系MDA-MB-231(图2A)的RNA的Northern印迹法放射自显影图:GTI3601[SEQ ID NO:1];GTI3602[SEQ ID NO:2];GTI3603[SEQ ID NO:3];GTI3604[SEQ ID NO:4];GTI3610[SEQ ID NO:10];GTI3611[SEQ ID NO:11];及GTI3612[SEQID NO:12]。
图3A在投药(反义寡核苷酸GTI3602[SEQ ID NO:2])或不投药(生理食盐水)的小鼠右胁注射人类HT-29结肠癌细胞后肿瘤体积随时间的变化图。
图3B为投药(反义寡核苷酸GTI3602[SEQ ID NO:2])或不投药(生理食盐水)的小鼠右胁注射人类HT-29结肠癌细胞后20天内的肿瘤重量变化图。
图4为经过反义寡核苷酸GTI3611[SEQ ID NO:11]或GTI3602[SEQ ID NO:2]处理或未经处理(对照组)之后人类黑素瘤细胞系C8161在每只小鼠体内肺转移的平均数量图。
图5为人类神经纤维网素cDNA的核苷酸序列。[SEQ ID NO:33]。
图6为大鼠神经纤维网素cDNA的核苷酸序列。[SEQ ID NO:34]。
图7为小鼠神经纤维网素cDNA的核苷酸序列。[SEQ ID NO:35]。
发明详述
本发明涉及与哺乳动物神经纤维网素mRNA互补的寡核苷酸,该寡核苷酸可调控细胞生长。
神经纤维网素为血管内皮生长因子或VEGF的受体。已发现VEGF可调控肿瘤诱发的血管生成。神经纤维网素也可在肿瘤衍生的细胞中高度表达,如MDA-MD-231乳癌细胞和组织培养细胞如Hela和NIH 3T3细胞。此点表示,除了其在刺激血管生成中的作用之外,神经纤维网素可能以一种自体分泌的形式来增强肿瘤细胞存活,分化或游动性而作为肿瘤细胞本身的VEGF活性的唯一信号转导物。另一种可能性则是神经纤维网素可能具有储存或螯合的功能。定义:
本文中采用下列名词的定义如下:
本文使用的“反义寡核苷酸”一词是指与所要的mRNA互补的核苷酸序列。优选的是,该反义寡核苷酸与哺乳动物神经纤维网素mRNA或VEGF165R mRNA中可有效地作为抑制神经纤维网素表达的靶的部分互补。该反义寡核苷酸应可与mRNA任何5′非翻译区域,该mRNA的编码区域或3′非翻译区域互补。最优选的是,该反义寡核苷酸与述于图5的核苷酸序列互补。
在不受限于任何理论或反应机理的情形下,一般认为反义寡核苷酸的活性取决于该寡核苷酸与其靶核酸的结合(例如,与至少一段基因组区域,基因或其mRNA转录本结合),从而以杂交扣留的方式或以使用RNA酶H破坏靶RNA的方式破坏靶的功能(当与RNA杂交时活化RNA酶H的能力),进而抑制神经纤维网素表达。
“寡核苷酸”一词是指由天然碱基、糖,和糖间(主链)键组成的核苷酸的寡聚物或聚合物或核苷单体。该名词也包括经修饰或经取代的寡聚物,其包含功能类似的非天然单体或其部分。此类经修饰或经取代的寡聚物在如增强细胞吸收,或在核酸酶存在下增加稳定性等性质上可能优于天然存在的形式。本名词也包括嵌合型寡核苷酸,具包含两个或多个化学性不同的区域。例如,嵌合型寡核苷酸可包含至少一个可提供有益性质(例如,增加核酸酶抗性,增加细胞吸收)的经修饰的核苷酸的区域,或者两个或多个本发明的寡核苷酸可连接形成嵌合型寡核苷酸。
本发明的反义寡核苷酸可以是核糖核酸或脱氧核糖核酸,而且可包含天然或合成的碱基单体,其包括腺嘌呤、鸟嘌呤、胞嘧啶、胸腺嘧啶及尿嘧啶。寡核苷酸也可包含经修饰的碱基如黄嘌呤,次黄嘌呤,2-氨基腺嘌呤,6-甲基,2-丙基及其它烷基的腺嘌呤,5-卤代尿嘧啶,5-卤代胞嘧啶,6-氮杂尿嘧啶,6-氮杂胞嘧啶及6-氮杂胸腺嘧啶,假尿嘧啶,4-硫尿嘧啶,8-卤代腺嘌呤,8-氨基腺嘌呤,8-巯基腺嘌呤,8-巯基烷基腺嘌呤,8-羟基腺嘌呤及其它8-取代的腺嘌呤、8-卤代鸟嘌呤、8-氨基鸟嘌呤,8-巯基鸟嘌呤,8-硫烷基鸟嘌呤,8-羟基鸟嘌呤及其它8-取代的鸟嘌呤,其它氮杂和去氮杂尿嘧啶,胸腺嘧啶,胞嘧啶或鸟嘌呤,5-三氟甲基尿嘧啶及5-三氟胞嘧啶。该修饰作用也可包括附接其它化学基团,如甲基,乙基,丙基至该寡核苷酸不同部位,包括糖,碱基或主链成分。
本发明反义寡核苷酸也可在磷酸主链,短链烷基或环烷基糖间键或短链杂原子或杂环糖间键中包含经修饰的磷氧杂原子。例如,该反义寡核苷酸可包含膦酸甲酯,硫代磷酸酯,二硫代磷酸酯,磷酸三酯,和吗啉代寡聚物。该反义寡核苷酸可在连接4至6个3′-末端核苷酸之间包含硫代磷酸酯键。该硫代磷酸酯键可连接所有核苷酸。硫代磷酸酯键可为混合的RP及SP对映异构体,或可为呈RP或SP型的立体规整型或几近立体规整型。
反义寡核苷酸也可具有糖拟似物。该寡核苷酸可具有至少一个核苷酸,其包含经修饰的碱基和/或糖,如2′-O-取代的核糖核苷酸。为了本发明的目的,“2′-O-取代的”一词意指戊糖部分的2′位置经一个含1-6个饱和或未饱和碳原子的-O-低级烷基取代,或经一个含有2-6个碳原子的-O-芳基或烯丙基取代,其中该烷基,芳基或烯丙基可未取代或经取代,例如经卤素,羟基,三氟甲基,氰基,硝基,酰基,酰氧基,烷氧基,羧基,烷氧羰基,或氨基取代。本发明的寡核苷酸可在其5′末端含4或5个2′-O-烷基化的核糖核苷酸和/或于其3′末端包含4或5个2′-O-烷基化的核糖核苷酸。
本发明的反义寡核苷酸也可包含核苷酸类似物,其中核苷酸的架构发生了根本上的改变。此类核苷酸类似物的实例为肽核酸(PNA),其中该DNA的脱氧核糖(或RNA的核糖)磷酸主链改换为类似肽中主链的聚酰胺主链(Nielsen等29;Good和Nielsen30;Buchardt,已故,等31,美国专利5,766,855;Buchardt,已故,等32,美国专利5,719,262)。PNA类似物已证实对酶的降解具有抗性,可于活体内及活体外延长其寿命。由于PNA股与DNA股之间缺少电荷相斥力,因此PNAs与互补DNA序列的结合力比与天然核酸分子的结合力强。
本发明寡核苷酸也可包括其它含有聚合物主链,环状主链,或者无环主链的核苷酸。例如,该核苷酸可包含吗啉代主链架构(美国专利5,034,50633)。
本发明寡核苷酸当经过修饰而不致被DNA及RNA核酸酶降解,或置入一种运输媒体中以保护寡核苷酸免于受到DNA或RNA核酸酶降解时,则具有“核酸酶抗性”。核酸酶抗性寡核苷酸包括例如膦酸甲酯,硫代磷酸酯,二硫代磷酸酯,磷酸三酯,和吗啉代寡聚物。赋予核酸酶抗性的适当运输媒体包括例如脂质体。
本发明寡核苷酸也可包含化学基团,如用于改进寡核苷酸药物代谢动力学性质的基团,或用于改进寡核苷酸药物动力学性质的基团。
该反义寡核苷酸选自与神经纤维网素基因互补的序列。优选的是,该序列在形成双螺旋体,形成发夹结构和均寡聚物/序列重复段等方面具有最小的可能性,但对结合神经纤维网素基因序列则具有高至中度的潜力。这些性质可使用计算机模仿程序OLIGO引物分析软件,第5版[由国家生物科学公司(National Biosciences,Inc.,普里茅斯,麻塞诸塞州)发行]测定。此计算机程序可定性估测该5项参数。
或者,该反义寡核苷酸也可根据对于两种或多种哺乳动物之间的神经纤维网素基因来说该序列高度保守来选择。这些性质可使用威斯康辛大学计算机组(GCG)软件(Devereux J.等人35)的BLASTN程序(Altschul,等人34)以及国家生物科学信息中心(NCBI)的数据库来确定。
该反义寡核苷酸可包括突变,例如取代,插入和缺乏。优选的是具有突变的序列少于10%。
该反义寡核苷酸一般包含至少约3个核苷酸或核苷酸类似物,优选包含至少约5个核苷酸,更优选至少约7个核苷酸,更优选至少约9个核苷酸,最优选至少约20个核苷酸。该反义寡核苷酸少于约100个核苷酸或核苷酸类似物较佳,少于约50个核苷酸或核苷酸类似物更佳,少于约35个核苷酸或核苷酸类似物最佳。
优选的是,该反义寡核苷酸包含下表1所示的序列。
表1
具有与人类神经纤维网素mRNA互补的序列的反义寡核苷酸
序列辨别号 | 名称 | 序列5′-3′ | Tm(℃) | ΔG(千卡/摩尔) |
1 | GTI3601 | GAG CGG CAG CCC CCT CTC CA | 74.6 | -46.5 |
2 | GTI3602 | CGA GCA CGG CGC AGA GGA GC | 74.2 | -45.7 |
3 | GTI3603 | GGA CGA GGG CGA GCA CGG CG | 78.0 | -48.6 |
4 | GTI3604 | TGG GTC CGG AGC CTG AAT CA | 69.0 | -42.2 |
5 | GTI3605 | TTT TTC AGG GAA TCC GGG GG | 69.1 | -44.6 |
6 | GTI3606 | GGG TAG TTC AGG CGG GAG CG | 69.9 | -44.3 |
7 | GTI3607 | AAT GGC GCC CTG TGT CCC GA | 73.4 | -45.4 |
8 | GTI3608 | GTG CCC AGC CAG AGC GAC TG | 69.5 | -42.0 |
9 | GTI3609 | TGA GGT GCG GGT GGA AGT GC | 69.6 | -42.0 |
10 | GTI3610 | GTG CCG ACG TGG GAC CCA GA | 71.6 | -43.1 |
11 | GTI3611 | GAC CCC CAG GGC ACT CAT GG | 70.1 | -42.9 |
12 | GTI3612 | CGA CCC CAC AGA CAG CCC CC | 72.4 | -44.4 |
13 | GTI3613 | TCT CTG TCC TCC AAA TCG AA | 58.6 | -36.5 |
14 | GTI3614 | TGC TTC CCA CCC TGA ATG AT | 63.3 | -39.2 |
15 | GTI3615 | TGG GAA TAG ATG AAG TTG CC | 58.4 | -37.1 |
16 | GTI3617 | TCC TCT GGC TTC TGG TAG CG | 63.8 | -39.9 |
17 | GTI3618 | AGG TTT CCT TTT CCG ATT TC | 59.0 | -38.6 |
18 | GTI3619 | GTG CTC CCT GTT TCA TCA AT | 58.0 | -36.2 |
19 | GTI3620 | CAT TGC CTG GCT TCC TGG AG | 66.2 | -41.1 |
20 | GTI3621 | CCC AGG GCA CTC ATG GCT AT | 65.5 | -41.0 |
21 | GTI3622 | GCT GAG AAA CCT TCT TTT GC | 57.9 | -37.0 |
22 | GTI3623 | AAC ATC TGT GGG GTT GGT GT | 60.3 | -36.9 |
23 | GTI3624 | TCG GAC AAA TCG AGT TAT CA | 57.1 | -36.0 |
24 | GTI3625 | CAA CAT TCC AGA GCA AGG AT | 58.2 | -36.5 |
25 | GTI3626 | CGA TCT TGA ACT TCC TCA TG | 56.0 | -35.2 |
26 | GTI3627 | CCT GTG AGC TGG AAG TCA TC | 58.2 | -35.7 |
27 | GTI3628 | CAT GTG ATA CCA GAA GGT CA | 53.9 | -33.5 |
28 | GTI3629 | CCA ACA GGC ACA GTA CAG CA | 60.8 | -36.7 |
29 | GTI3630 | ACC ATC CAC AAG TTC AAA GT | 54.8 | -34.5 |
30 | GTI3631 | ACC ACA GGG CTC ACC AGG CG | 71.0 | -43.2 |
表1的反义寡核苷酸选自与人类神经纤维网素/VEGF165R mRNA互补的序列,使该序列形成双螺旋体,发夹结构和均寡聚物/序列重复段的可能性最小,但与神经纤维网素/VEGF165R mRNA序列结合的潜力高。此外,可避免错误导向人类及小鼠中其它经常出现或重复的序列。这些性质是采用计算机模仿程序OLIGO引物分析软件,第5版(由国际生物科学公司,普里茅斯,麻塞诸塞州发行)测定。
表1的“Tm”是指根据其最近邻热力学值计算的寡核苷酸双螺旋的熔点。在此温度下50%的核酸分子维持双螺旋,50%的核酸分子变性。该“ΔG”是寡核苷酸的自由能,其为寡核苷酸双螺旋稳定性的测定值。
“烷基”一词指碳原子数优选为1至20个,更优选为1至6个的单价烷基。此名词的实例如甲基,乙基,正丙基,异丙基,正丁基,异丁基,正己基,及其类似物。
“芳基”一词是指6至14个碳原子的不饱和芳香族碳环基,具有单环(例如苯基)或多重缩合(稠合)环(例如,萘基或蒽基)。优选的芳基包括苯基,萘基及其类似物。
“卤”或“卤素”一词指氟、氯、溴和碘,优选氟或氯。
关于上列包含一个或多个取代基的任何基团,应该理解此类基团当然不包含立体结构上不实际和/或不可能合成的任何取代或取代形式。此外,本发明化合物包括所有因此类化合物取代所产生的立体化学异构体。
“药物上可接受的”一词指一种无毒材料,其不干扰该活性成分的生物活性的有效性。该材料可与生物系统如细胞、细胞培养物、组织或生物体相容。
术语“药物上可接受的盐”指可维持本发明反义寡核苷酸的生物有效性及性质而且不是生物学上或其它方面不需要的盐。在许多情况下,本发明反义寡核苷酸可因含有氨基和/或羧基或其它类似基团而形成酸式盐和/或碱式盐。
药物上可接受的碱加成盐可由无机和有机碱制备。衍生自无机碱的盐包括例如:钠、钾、锂、铵,钙及镁盐。衍生自有机碱的盐包括(但不限于):伯、仲和叔胺类的盐,例如烷基胺,二烷基胺,三烷基胺,取代的烷基胺,二(取代的烷基)胺,三(取代的烷基)胺,链烯基胺,二链烯基胺,三链烯基胺,取代的链烯基胺,二(取代的链烯基)胺,三(取代的链烯基)胺,环烷基胺,二(环烷基)胺,三(环烷基)胺,取代的环烷基胺,二取代环烷基胺,三取代环烷基胺,环烯基胺,二(环烯基)胺,三(环烯基)胺,取代的环烯基胺,二取代环烯基胺,三取代环烯基胺,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,杂环基胺,二杂环基胺,三杂环基胺,混合的二胺及三胺类,其中该胺类至少有两个选自下列的不同取代基:烷基,取代烷基,烯基,取代的烯基,环烷基,取代环烷基,环烯基,取代环烯基,芳基,杂芳基,杂环基,及其类似物。其也包括胺类,其中2或3个取代基与氨基氮一起形成杂环或杂芳基。
适宜的胺类实例包括例如:异丙胺,三甲胺,二乙胺,三(异丙基)胺,三(正丙基)胺,乙醇胺,2-二甲氨基乙醇,三甲醇氨基甲烷,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,及其类似物。也应该理解,其它羧酸衍生物将适用于本发明,例如,羧酸酰胺,包括甲酰胺,低级烷基甲酰胺,二烷基甲酰胺,及其类似物。
药物上可接受的酸加成盐可由无机及有机酸制备。衍生自无机酸的盐类包括盐酸,氢溴酸,硫酸,硝酸,磷酸,及其类似物的盐类。衍生自有机酸的盐类包括乙酸,丙酸,羟基乙酸,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,乙磺酸,对-甲苯磺酸,水杨酸,及其类似物的盐类。
“神经纤维网素基因”一词指可编码能够作为信号素或者VEGF受体的蛋白质的任何基因。优选的是,此神经纤维网素mRNA的序列基本上类似于图5,6或7所示的序列。
“互补”一词意指该反义寡核苷酸序列可与其靶序列,即该神经纤维网素基因(或mRNA)结合。优选的是,该反义寡核苷酸在生理条件下与该核酸序列结合,例如,经由华生-克里克(Watson-Crick)碱基配对法(寡核苷酸与单股核酸间的相互作用)或经由胡斯丁(Hoogsteen)碱基配对法(寡核苷酸及双股核酸间的相互作用)或者经由任何其它方法,包括若寡核苷酸与RNA结合,则形成假结。经由华生-克里克或胡斯丁碱基配对法,在生理条件下的结合实际是通过观察核酸序列功能的干扰情形来测定的。
该反义寡核苷酸序列与靶序列具有至少75%同一性较佳,以至少90%同一性更佳,以与靶序列具有至少95%同一性最佳,其仅容许少数碱基缺口或碱基错配。同一性的测定可以使用例如威斯康辛大学计算机组(GCG)软件的BLASTN程序。该反义寡核苷酸序列与神经纤维网素mRNA杂交的熔点优选为至少45℃,更优选为至少约50℃,最优选为至少约55℃,此熔点是使用本文所述的OLIGO引物分析软件程序第5版所确定的。
“抑制生长”一词指使至少一种肿瘤细胞型态的生长受到至少10%的减少或抑制,以至少50%更佳,以至少75%最佳。肿瘤细胞生长减少的测定可以测量该肿瘤于裸鼠中的大小或者该肿瘤细胞于活体外无法形成集落的能力的方式进行。
“抑制血管生成”一词指减少或抑制新血管生成。此可使用本领域已知的方法测定。一种氧诱发视网膜新血管生成的小鼠模型已经确立,处理组动物的发生率为100%,且可以定量(45,46)。使用此模型可测定神经纤维网素的抑制与视网膜新血管生成的抑制之间的相关性。也可由Northern印迹法及原位杂交分析法测定神经纤维网素表达水平的变化来确认此结果。
“抑制转移”一词指减少或抑制发展成转移性肿瘤的数目,以至少10%较佳,以至少50%更佳。其可采用述于实施例中的方法和本领域中已知的其它方法测定。
“抑制神经纤维网素的表达”一词指当对细胞施用反义寡核苷酸时,其可降低神经纤维网素mRNA的水平或由细胞产生的神经纤维网素蛋白质的水平。
“哺乳动物”一词意指所有哺乳动物,包括人类,羊,牛,马,猪、狗、猫、及小鼠等,优选的是指人类。
“疑有肿瘤的哺乳动物”意指该哺乳动物可能具有增生病变或肿瘤,或者已被诊断具有增生病变或肿瘤,或过去曾被诊断患有增生病变或肿瘤,但该肿瘤已手术割除而该哺乳动物仍疑似潜藏一些残存的肿瘤细胞。反义寡核苷酸的制备
本发明反义寡核苷酸可利用传统及熟知的技术制备。例如,该寡核苷酸可使用固相合成方法制备,特别是使用可购得的仪器如由加拿大应用生物系统公司(Mississauga,加拿大)所销售的仪器。该寡核苷酸也可使用本领域中已知的方法,以酶解天然存在的神经纤维网素基因而制备。
这些寡核苷酸可使用本领域已知的方法制备,如氨基磷酸酯或者氢膦酸酯化学制备,其可以人工进行或以自动合成仪器执行(如乌曼(Uhlmann)等人(43)及阿格瓦(Agrawal)等人(44)所述)。反义寡核苷酸的分离及纯化
本文所述的反义寡核苷酸的分离及纯化若需要时可以任何合适的分离或纯化方法进行,如,例如过滤,萃取、结晶,柱层析,薄层层析,厚层层析,制备型低压或高压液相层析,或者上述方法的组合。然而,其它等同的分离方法当然也可采用。
可根据该反义寡核苷酸的序列并使用本领域中已知的步骤构建包含该反义寡核苷酸序列的表达载体。
本领域的技术人员可构建载体,使包含达到该反义寡核苷酸序列所需要的转录作用所需的所有表达元件。因此,本发明提供的载体包括一段转录调控序列,其与一段编码反义寡核苷酸的序列操纵性地连接。适宜的转录及翻译元件可来自不同来源,包括细菌,真菌,病毒,哺乳动物或昆虫基因。适当元件的选择乃是根据所选用的宿主细胞而定。
报道基因可包括于此载体中。适宜的报道基因包括β-半乳糖苷酶(例如lacZ),氯霉素、乙酰转移酶、萤火虫萤光素酶,或免疫球蛋白或其部分。可以追踪该报道基因的表达来监控该反义寡核苷酸的转录。
该载体可使用本领域内已知的众多方法中的任一种方法导入细胞或组织中。此类方法一般可见于Sambrook等人24;Ausubel等人25;张等人36;Vega等人37;以及“载体:分子克隆载体及其应用”38,并包括,例如,稳定转染或瞬时转染,脂转染法,电穿孔法,及以重组病毒载体感染。
以感染方式导入核酸的作法提供几个优点。可得到较高效率及对组织型态的特异性。病毒主要于特定的细胞型态中感染及繁衍。因此,该病毒的特异性可使载体于活体内或组织中或细胞的混合培养物中针对特定的细胞型态。病毒载体也可经由特异性受体或配位体修饰,通过受体介导的事件改变靶特异性。
本发明寡核苷酸可为一种可裂解mRNA的核糖酶。该核糖酶最好具有与本发明寡核苷酸序列具同源性且具有裂解mRNA所必需的催化中心的序列。例如,可选用可破坏神经纤维网素mRNA的同源性核糖酶序列。本发明采用的核糖酶种类可选自本领域内已知的种类。已鉴定出数种核糖酶结构族群,包括第I组内含子,RNA酶P,肝炎δ病毒核酶,锤头核酶及源自烟叶环斑病病毒卫星RNA(sTRSV)负链的发夹核酶(Sullivan 1994,美国专利5,225,34739)。锤头核酶和发夹核酶基元最常用于基因疗法中mRNA的反式切割(Sullivan 1994)。发夹核酶优选用于本发明。一般而言,该核酶长度为30至100个核苷酸。
本发明寡核苷酸可以不溶解。例如,该寡核苷酸可结合于适宜的载体。适宜的载体实例为琼脂糖,纤维素,葡聚糖,交联葡聚糖(Sephadex),琼脂糖(Sepharose),羧甲基纤维素聚苯乙烯,滤纸,离子交换树脂,塑胶膜,塑胶管,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,丝等。该载体可呈例如:管状,试验板状,珠状物圆盘状,球状等。
该不溶的寡核苷酸的制法可采用已知的化学或物理方法,由该物质与适宜的不溶性载剂反应,例如,进行溴化氰偶合法。药物调配物
当反义寡核苷酸作为药物使用时,该反义寡核苷酸通常以药物组合物的形式进行投药。此类化合物可经由数种途径投药,包括口服,直肠,透皮,皮下,静脉内,肌内及鼻内。这些化合物以注射组合物或口服组合物形式皆有效。此类组合物乃依据药物领域中熟知的方式制备,并且包含至少一种活性化合物。例如,该药物组合物是经静脉内投药。该药物组合物应可直接投药至欲治疗的肿瘤中。
本发明也包括药物组合物,其包含作为活性成分的一种或多种反义寡核苷酸与药物上可接受的载体或赋形剂结合。制造本发明组合物时,活性成分通常与赋形剂混合,以赋形剂稀释或封装于此种载体中,此种载体可呈胶囊,小药包,纸包或其它容器的形式。当该赋形剂作为稀释剂时,其可为固体,半固体或液体物质,该物质充当活性成分的媒介物,载体或介质。因此,该组合物可呈片剂,丸粒,粉剂,扁囊锭,小药包,扁囊剂,酏剂,悬浮液,乳液,溶液,糖浆,气雾剂(呈固体或含于液体介质中),软膏(其包含例如至多10%(重量)的活性化合物),软及硬明胶胶囊,栓剂,无菌注射液,及无菌包装粉剂。
在制备调配物时,可能需要研磨该活性化合物,以便在与其它成分混合以前提供适当颗粒大小。若该活性化合物基本上不溶,通常研磨至小于200筛目的颗粒大小。如果活性化合物基本上是水溶性的,则一般通过研磨调节颗粒大小以提供于调配物中基本上均匀的分布,例如约40筛目。
一些适宜的赋形剂的实例包括乳糖,葡萄糖,蔗糖,山梨糖醇,甘露糖醇,淀粉,阿拉伯树胶,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,糖浆及甲基纤维素。该调配物可另外包括:润滑剂如:滑石,硬脂酸镁,及矿物油;润温剂;乳化剂和悬浮剂;防腐剂如羟基苯甲酸甲酯及丙酯;甜味剂;及调味剂。可使用本领域中已知的方法配制本发明的组合物,使之给患者服用后可提供迅速持久的或延迟的释放。
该组合物最好配制成单位剂型,每一剂量包含约1%至约95%,更常用约5%至约90%的活性成分。“单位剂型”一词指作为单一剂量适于人类对象和其它哺乳动物的物理性不连续单位,每一单位包含预先计算确定可产生所需疗效的用量的活性物质,与适宜的药物赋形剂组合。
该反义寡核苷酸的有效剂量范围相当广,而且一般以药物有效量服用。有效量即当施药时可缓解症状的量。该有效量优选为可抑制肿瘤细胞生长的量。该有效量以约0.1毫克/公斤体重至约20毫克/公斤体重为宜。然而应该理解,实际上反义寡核苷酸的投药剂量将由医师根据有关条件决定,包括欲治疗的病症,选择的投药途径,实际投药的化合物,个别患者的年龄,重量,及反应,患者症状的严重性,等等。疗程可能持续数天至数个月或直至达到减轻该疾病的目的。该反义寡核苷酸可与其它已知疗法合并投药。当与一种或多种其它疗法共同投药时,该寡核苷酸可与其它治疗药物同时投药,也可依次投药。若依次投药,负责的医师将决定施用该寡核苷酸与其它疗法并用的适当顺序。
制备固体组合物如片剂时,由主要活性成分/反义寡核苷酸与药物赋形剂混合,形成固体预配制组合物,其包含本发明化合物的均匀混合物。当提及这些预配制组合物为均质时,其意指该活性成分乃均匀分布于该组合物中,以致可以将该组合物容易地再分成同等有效的剂型例如片剂,丸粒及胶囊。
本发明片剂或丸粒可涂覆包衣或另外复合成可提供延长作用的优点的剂型。例如,该片剂或丸粒可包含内部剂量及外部剂量成分,后者为前者的包膜形式。此二种成分可以肠溶性涂层分隔,该肠溶性涂层用于抵抗在胃中崩解并且容许该内部成分完整地通过直达十二指肠或得以延后释出。多种物质皆可用于此类肠溶性涂层或包衣,此类物质包括数种聚合性酸和聚合性酸与如虫胶,鲸蜡醇,和纤维素乙酸酯的混合物。
包含本发明新颖组合物的可供口服或注射用的液体剂型包括水溶液,适当调味的糖浆,水性或油性悬浮液,及用食用油如玉米油,棉籽油,芝麻油,椰子油或花生油,以及酏剂和类似的药物媒介物的加味乳液。
用于吸入或吹入的组合物包括含在药物上可接受的水性或有机溶剂中或其混合物中的溶液和悬浮液,及粉剂。该液体或固体组合物可能包含本文所述的适当的药物上可接受的赋形剂。该组合物优选经口或鼻呼吸管道投药,以达到局部或全身效果。最好含在药物上可接受的溶剂中的组合物可使用惰性气体制成喷雾剂。喷雾溶液可由喷雾器直接吸入或可将该喷雾器附接于面罩蓬或间歇性正压力呼吸机上。溶液,悬浮液或粉剂组合物可使用于适当方式传送调配物的装置投药,优选的是经由口腔或鼻腔投药。
本发明药物组合物可呈脂质体形式,其中寡核苷酸除了与其它的药物上可接受的载体组合外,还与亲两性剂如脂质组合,该脂质以聚集形式如胶束,不溶性单层,液晶或于水溶液中的薄片层存在。适合脂质体调配物的脂质包括(但不限于):单酸甘油酯,二酸甘油酯,硫酸脑苷脂,脱脂酸卵磷脂,磷脂,皂角苷,胆汁酸及其类似物。一种特别有用的脂质载体为脂转染试剂。此类脂质体调配物的制法在本领域技艺范围内,例如,国际专利WO97/21808(28)。该药物组合物可进一步包括化合物如环糊精及其类似物,其可加强寡核苷酸传送至细胞中或缓释的聚合物中。
另一种适用于本发明方法的优选调配物采用透皮传送装置(“贴剂”)。此类透皮贴剂可用于以控制量持续或间歇注入本发明的反义寡核苷酸。用于输送药剂的透皮贴剂的构成及用法是本领域中熟知的。请参阅,例如,美国专利5,023,25240,该专利是以提及的方式并于本文。此类贴剂可构成用于持续性传送,脉冲式传送或依需求传送药剂。
另一种优选的传送方法涉及以“短枪”传送未包覆的反义寡核苷酸通过皮肤表层。“未包覆”的反义寡核苷酸的传送是本领域中熟知的。请参阅,例如,Felgner等人,美国专利5,580,85941。在以“短枪”方式传送该反义寡核苷酸以前,可将该反义寡核苷酸包装在脂小泡中。
下列制剂实施例说明本发明代表性药物组合物。
制剂实施例1
制备包含下列成分的硬明胶胶囊:成分
用量(毫克/粒胶囊)活性成分 30.0淀粉 305.0硬脂酸镁 5.0
混合上述成分,并以每颗胶囊340毫克的量装填至硬明胶胶囊中。
制剂实施例2
使用下列成分制备片剂调配物:成分
用量(毫克/药片)活性成分 25.0微晶纤维素 200.0胶体二氧化硅 10.0硬脂酸 5.0
掺合上列成分并压缩成片剂,每片重240毫克。
制剂实施例3
制备包含下列成分的干粉吸入性调配物:成分
重量百分比(%)活性成分 5乳糖 95
混合该活性成分及乳糖,并将混合物装填至于粉吸入装置中。
制剂实施例4
依下列方法制备各含30毫克活性成分的片剂:成分
用量(毫克/药片)活性成分 30.0毫克淀粉 45.0毫克微晶纤维素 35.0毫克聚乙烯吡咯烷酮 4.0毫克(含在无菌水中的10%水溶液)羟甲基淀粉钠 4.5毫克硬脂酸镁 0.5毫克滑石
1.0毫克总共 120毫克
使活性成分,淀粉及纤维素通过20号筛目美国滤网过筛并均匀混合。将聚乙烯吡咯烷酮溶液和所形成的粉末混合,然后通过16号筛目美国滤网过筛。将所产生的颗粒置于50℃至60℃间干燥,通过16号筛目美国滤网过筛。将羧甲基淀粉钠,硬脂酸镁及滑石预先通过30号筛目美国滤网过筛,然后加入至前述颗粒中,混合后,以制片机压缩成片剂,每片重120毫克。
制剂实施例5
依下列方式制备每颗含40毫克药物的胶囊:成分
用量(毫克/胶囊)活性成分 40.0毫克淀粉 109.0毫克硬脂酸镁
1.0毫克总共 150毫克
掺合该活性成分,淀粉及硬脂酸镁,通过20号筛目的美国滤网过筛,以每颗150毫克的量装填至硬明胶胶囊中。
制剂实施例6
依下列方式制备每颗包含25毫克活性成分的栓剂:成分
数量活性成分 25毫克饱和脂肪酸甘油酯,加至 2,000毫克
该活性成分通过60号筛目美国滤网过筛并使其悬浮于预先使用最低温熔化的饱和脂肪酸甘油酯中。然后将该混合物倒入公称2.0克容量的栓剂模具中,并使之冷却。
制剂实施例7
依下列方法制备每5.0毫升包含50毫克药物的悬浮液:成分
用量活性成分 50.0毫克黄原胶 4.0毫克羧甲基纤维素钠(11%) -微晶纤维素(89%) 50.0毫克蔗糖 1.75克苯甲酸钠 10.0毫克香料及色素 适量纯水加至 5.0毫升
掺合该活性成分,蔗糖及黄原胶,通过10号筛目的美国滤网过筛,然后与由微晶纤维素和羧甲基纤维素钠预先制成的水溶液混合。使用部分水稀释苯甲酸钠,香料及色素并且一边搅拌一边添加。最后添加足量水,达到所需体积。
制剂实施例8 成分
用量(毫克/粒胶囊)活性成分 15.0毫克淀粉 407.0毫克硬脂酸镁 3.0毫克总共 425.0毫克
掺合该活性成分、淀粉及硬脂酸镁,通过20号筛目的美国滤网过筛,并以每颗425.0毫克的量装填至硬明胶胶囊中。
制剂实施例9依下列方法制备调配物:成分
用量活性成分 5.0毫克玉米油 1.0毫升
制剂实施例10依下列方法制备局部调配物:成分
用量活性成分 1-10克乳化用蜡 30克液体石蜡 20克白色软石蜡 加至100克
加热白色软石蜡至熔化。将该液体石蜡与乳化用蜡合并并搅拌直至溶解。添加该活性成分并且持续搅拌直至分散。冷却该混合物直至固化。
其它适用于本发明的调配物可见于“雷氏药物科学”23一书(Remington’s Pharmaceutical Sciences23)。
该反义寡核苷酸或包含该反义寡核苷酸的药物组合物可包装至便利的试剂盒,此试剂盒提供装填至适宜的容器所需要的材料。
呈医疗调配物形式的本发明的反义寡核苷酸适用于治疗与血管生成和新血管生成相关的疾病和病变及病症,其包括(但不限于)视网膜新血管生成及肿瘤生长。此类方法中,将抑制神经纤维网素表达的有效治疗剂量的寡核苷酸投药给细胞。此细胞可为细胞培养物或组织培养物的一部分,或是哺乳动物,如人类的整个躯体。
本发明寡核苷酸及核酶可调控肿瘤细胞生长。因此,所提供用于干扰或抑制哺乳动物肿瘤细胞生长的方法包括利用本发明反义寡核苷酸与肿瘤或肿瘤细胞接触。在未受于理论或反应机理限制下,据信该反义寡核苷酸可以两种方式抑制肿瘤生长。其可以自体分泌的方式直接作用于肿瘤细胞而抑制肿瘤生长。第二种方式或另外一种方式即该反义寡核苷酸可抑制与肿瘤生长有关的新血管生成,从而减少对肿瘤的血液供给。
“接触”一词指添加寡核苷酸,核酶等至细胞悬浮液或组织样本,或直接或间接施用寡核苷酸等至动物体内的细胞或组织。
这些方法可用于治疗增生性病变,包括许多种不同型态的癌或肿瘤,如肉瘤,黑素瘤,腺瘤,实体组织癌瘤,缺氧性肿瘤,口腔,咽,喉及肺的鳞状细胞癌瘤,泌尿生殖器癌,如子宫颈和膀胱癌,血癌,结肠癌,乳癌,胰腺癌,肾脏癌,脑癌,皮肤癌,肝癌,头和颈癌,神经系统癌,以及良性病变如乳头状瘤。
这些方法可用于治疗新血管病变,如糖尿病引起的视网膜病变,早产性视网膜病变和与年龄有关的斑点性退化。
本发明寡核苷酸也可用于治疗抗药性肿瘤。抗药性肿瘤实例为可抵抗如5-氟尿嘧啶,丝裂霉素C,氨甲蝶呤或羟脲的肿瘤和表达高含量P-糖蛋白的肿瘤,已知其对多种抗癌药物如秋水仙素,长春花碱和阿霉素具有抗性;或如Dreeley等人42文中所述可表达多重抗药性蛋白质的肿瘤。因此,本发明寡核苷酸可并用或另外增加使用已知的抗癌化合物或化疗剂。化疗剂是可抑制肿瘤生长的化合物。此类药剂包括(但不限于)5-氟尿嘧啶,丝裂霉素C,胺甲蝶呤及羟脲。化疗剂的投药量可为有效量,即足以抑制肿瘤生长的量,或少于有效量。
已发现,本发明寡核苷酸可减少转移性肿瘤生长,如MDA-MB-231乳腺癌,HT-29结肠腺癌,A549肺癌,和A2058黑素瘤癌细胞。本发明的实施方案提供一种减少哺乳动物转移性肿瘤生长的方法,包括施用一定量的与神经纤维网素mRNA互补的寡核苷酸,或示于表1的寡核苷酸。
本发明寡核苷酸可减少血管生成。本发明的一个实施方案提供治疗新血管病变的方法。
该寡核苷酸可采用病毒载体或非病毒载体传送。序列可并入盒或构建体中,使本发明寡核苷酸在细胞中表达。优选该构建体包含适宜的转录控制区,以使该寡核苷酸可于此细胞中转录。
因此,本发明提供一种载体,其包含一段与编码本发明寡核苷酸的序列操纵性连接的转录控制序列。本发明进一步提供选自适当真核及原核细胞的宿主细胞,使用这些载体进行转化。
合适载体是已知的并且优选包含达到序列所需的转录作用所必需的表达元件。噬菌粒为此类有益载体的具体实例,因为其可用作为质粒载体或噬菌体载体。载体的实例包括病毒如噬菌体,杆状病毒,逆转录病毒,DNA病毒,脂质体和其它重组载体。该载体也可包含用于原核宿主系统或真核宿主系统的元件。本领域的普通技术人员知道何种宿主系统可与特定载体相容。
该载体可经由稳定转染或瞬时转染,脂转染法,电穿孔法及以重组病毒载体感染等方式导入细胞。
可在该载体中增加其它特征,以确保其安全性和/或增强其疗效。此类特征包括,例如,能用于负选择经重组病毒感染的细胞的标记物。此类负选择标记物的实例为TK基因,其对抗病毒性9-[1,3-二羟-2-丙氧甲基]鸟嘌呤具有敏感性。也包括限定于特定细胞型态中表达的特征。此类特征包括,例如,对所需细胞型态具有特异性的启动子和调节元件。
逆转录病毒载体为适用于活体内导入所需核酸的另一个载体实例,因其提供如侧感染和寻靶特异性等优点。侧感染为单感染细胞可藉以产生许多子代病毒粒子以感染毗邻细胞的过程。其结果是大面积被迅速感染。
本发明方法所用的载体可根据寻靶所需的细胞型态选择。例如,若治疗乳癌,则可使用对表皮细胞具有特异性的载体。同样地,若治疗造血系统细胞,则对血液细胞具有特异性的病毒载体是优选的。用途
本发明反义寡核苷酸适用于多种用途。其可用于抑制哺乳动物细胞中神经纤维网素基因的表达,其结果是抑制细胞生长。其可用于抑制肿瘤细胞生长和/或新血管生成。该寡核苷酸可用作为杂交探针,以检测哺乳动物细胞中神经纤维网素mRNA存在与否。当采用该寡核苷酸作为探针时,可用合适的检测基团(如放射性同位素,配位体,特异性结合配对的另一成员,如,生物素)进行标记。最后,该寡核苷酸可用作为分子量标记物。
为了进一步阐述本发明及其优点,给出下列具体实施例,但它们并不以任何方式限制本发明权利要求书的范围。
实施例
在下列实施例中,所有温度均以摄氏温度表示(除非另外指明)且所有百分比均为重量百分比(除非另外指明)。
在下列实施例中,下列缩写的含义如下述。倘若某一缩写未被定义,则是其普遍接受的含义。
AS = 反义
cDNA = 互补脱氧核糖核酸
ODN = 寡核苷酸
μM = 微摩尔浓度
mM = 毫摩尔浓度
M = 摩尔浓度
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,寡核苷酸双螺旋稳定性的量度
kcal = 千卡
FBS = 胎牛血清
DTT = 二硫苏糖醇
SDS = 十二烷基硫酸钠
PBS = 磷酸盐缓冲生理食盐水
PMSF = 苯甲基磺酰氟
GAPDH = 3-磷酸甘油醛脱氢酶
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
VEGF = 血管内皮生长因子分子生物学一般方法
本领域中已知的标准分子生物技术和未明确说明的分子生物技术一般是依照Sambrook等人24;Ausubel等人25;和Perbal26等人著作的说明进行的。寡核苷酸
该反义寡核苷酸选自与神经纤维网素mRNA互补的序列,此类序列形成双螺旋,形成发夹结构和均寡聚物/序列重复的可能性最小,但具有高潜力可结合神经纤维网素mRNA序列。此外,也避免错误导向人类和小鼠中其它常常出现的或重复的序列。此类性质是使用计算机模仿程序OLIGO引物分析软件,第5版(国际生物科学公司,普里茅斯,麻塞诸塞州)测定的。根据此分析法设计了硫代磷酸酯反义寡核苷酸,然后依据本领域已知的方法制造。细胞系
自美国典型培养物保藏中心(ATCC)取得七种不同的人类癌细胞系包括肺癌(A549),黑素瘤(C8161),乳细胞腺癌(MDA-MB-231),转移性胰腺癌(AsPC-1),结肠腺癌(HT-29),人类黑素瘤细胞系A2058,人类胰腺癌PC3。这些细胞系维持在补充了10%胎牛血清(FBS)的α-MEM培养基(Bibco BRL,盖瑟斯堡,马里兰州)中。实施例1 与神经纤维网素互补的反义寡核苷酸对癌细胞系生长的抑 制
采用过去曾说明的方法(Choy等人18)估测经过不同反义寡核苷酸处理后的癌细胞系的集落形成能力。具体地说,取等分的肿瘤细胞悬浮液接种至60毫米组织培养皿中,密度约1×104个,并且在补充了10%FBS的α-MEM培养基中,于37℃温育过夜。细胞以5ml PBS洗涤一次,并且于阳离子性脂质(脂转染试剂,终浓度,5μg/ml,Gibco-BRL,盖瑟斯堡,马里兰州)存在下,以0.2μM指定的反义寡核苷酸处理4小时。以PBS清洗该细胞一次清除该反义寡核苷酸,而后将该细胞置于生长培养基(补充了10% FBS的α-MEM培养基)中于37℃下培养7至10天。细胞集落以亚甲基蓝染色并通过直接计数评分(如Choy等人18以及Huang和Wright20所述)。抑制百分比的计算法是与在不含反义寡核苷酸下生长的细胞培养物中存在的集落数比较而得。所有的实验均重复4次。
该反义寡核苷酸对人类肿瘤细胞系的集落形成能力产生抑制效应。每种反义寡核苷酸的抑制百分比示于图1A为人类黑素瘤细胞系C8161;图1B为人类肺癌细胞系A549;图1C为人类黑素瘤细胞系A2058;图1D为人类结肠癌细胞系HT-29;图1E为人类前列腺癌细胞系PC-3;及图1F为人类胰腺癌细胞系AsPC-1。实施例2 经与神经纤维网素互补的反义寡核苷酸处理后降低的mRNA 含量
将人类黑素瘤癌细胞(A2058)或乳癌细胞(MDA-MB-231)培养至70%-80%的次融合程度,而后于阳离子性脂质(脂转染试剂,终浓度5μg/ml,Gibco-BRL)和Opti-MEM(Gibco-BRL)的存在下,以0.2μM与神经纤维网素互补的硫代磷酸酯反义寡核苷酸处理4小时。以PBS清洗细胞一次,并置于含10%FBS的α-MEM培养基(Gibco-BRL)中温育16小时。使用TRIzol试剂(Gibco-BRL)制备总RNA并依Hurta和Wright(27)文中所述的方法经过少许修改,进行Northern印迹分析法。该印迹与正向引物(5′-CGC TCC CGC CTG AAC TAC CC-3′)[SEQID NO:31],反向引物(5′-TCC CAC CCT GAA TGA TGA TG-3′)[SEQID NO:32],并使用人类直肠结肠腺癌5′-延伸段加上cDNA文库(Clonetech,帕洛阿图,加州)作为模板合成32P标记的598个碱基对的PCR片段杂交。人类神经纤维网素/VEGF165R mRNA表达为约7kb核苷酸转录本(Soker等人9)。在杂交前,利用亚甲基蓝染色印迹证实等量RNA荷载。
图2A和2B显示该反义寡核苷酸降低神经纤维网素mRNA含量至对照组细胞的至少50%。实施例3 以与神经纤维网素互补的反义寡核苷酸静脉处理对小鼠体 内人类肿瘤细胞生长的抑制作用
CD-1无胸腺裸鼠购自查理士河实验室(蒙特晏,加拿大)。将HT-29人类结肠癌细胞(一般为3×106个细胞含于100μl PBS中)皮下注射至6-7周大的CD-1无胸腺裸鼠的右肋中。每一实验组包括5只小鼠。在肿瘤大小达到体积约100立方毫米后(一般为注射肿瘤细胞5日后),采用大丸剂输注施用该反义寡核苷酸GTI3602[SEQ ID NO:2]至其尾静脉,每隔一天10mg/kg剂量。对照组动物在相同时段仅接受生理食盐水。此后一般持续处理14天。
图3A显示该反义寡核苷酸GTI3602对CD-1裸鼠中HT-29肿瘤生长的影响。以肿瘤体积受抑制程度估算抗肿瘤活性,肿瘤体积是在14天的期间内平均每隔两天以测径器测量的。图中每一点代表每个实验组中5只小鼠计算得到的平均肿瘤体积。采用协方差分析来比较各处理组内小鼠于一段时间内的回归曲线。当斜率均相等时,由该分析衍生出同等斜率或同等截距的特定假说。所有分析均采用SAS(统计分析系统)第6.12版。当与生理食盐水对照组比较时,施用该反义寡核苷酸抑制肿瘤生长的p值≤0.0001。
处理结束时(通常是在最后一次处理后24小时)处死该实验动物,并测量肿瘤重量。图3B出示肿瘤平均重量。该反义寡核苷酸显示出对肿瘤生长的显著抑制作用。采用单向方差分析比较处理组的平均值。若此组整体效应非常显著,则使用最小平方平均值进行演绎多重比较,以发现具显著差异的处理组对。当比较肿瘤重量时,该反义寡核苷酸在统计学上也比生理食盐水对照展现出显著的抑制作用。实施例4 反义寡核苷酸对实验性转移的抑制作用
依前述的方法(Fan等人,199610)估算经过不同反义寡核苷酸处理的C8161人类黑素瘤细胞的实验性转移。取等分的细胞悬浮液以2×106的密度接种至100毫米的组织培养皿中并于补充了10% FBS的α-MEM培养基中,于37℃下温育过夜。以10ml PBS洗涤细胞一次,并于阳离子性脂质(脂转染试剂,终浓度5μg/ml,Gibco-BRL)存在下处理4小时。用PBS洗涤细胞一次以除去该反义寡核苷酸,然后以胰蛋白酶处理细胞。离心收集细胞,将悬浮于0.1ml PBS中的约1×105个细胞注射到6-8周大的CD-1无胸腺雌性裸鼠的尾静脉中。5周后估算肺肿瘤数目,切除各小鼠的肺后,以苦味酸染料溶液(75%苦味酸,20%甲醛,5%冰醋酸)染色。
图4出示在以各种不同反义寡核苷酸处理肿瘤细胞后,于该雌性裸鼠体内所减少的肺肿瘤数目。
Claims (16)
1.一种反义寡核苷酸,其约20个至约100个核苷酸,并且包括选自示于表1的SEQ ID NOs:1-30的序列,该寡核苷酸可抑制神经纤维网素表达。
2.权利要求1的反义寡核苷酸,其进一步包含一个或多个硫代磷酸酯核苷酸间键。
3.权利要求1的反义寡核苷酸,其进一步包含其它不与神经纤维网素mRNA互补的核苷酸。
4.一种载体,其包含约20个至约100个核苷酸的寡核苷酸序列,该核苷酸序列包含选自示于表1的SEQ ID NOs:1-30的序列,该寡核苷酸可抑制神经纤维网素表达。
5.一种药物组合物,其包括药物上可接受的赋形剂及有效量的约20个至约100个核苷酸的反义寡核苷酸,该反义寡核苷酸包含选自于表1的SEQ ID NOs:1-30的序列,该寡核苷酸可抑制神经纤维网素表达。
6.一种抑制哺乳动物肿瘤生长的方法,包括对疑有肿瘤的哺乳动物施用有效量的反义寡核苷酸,该反义寡核苷酸的大小约3个至约100个核苷酸并且包含在抑制肿瘤生长的条件下与哺乳动物神经纤维网素mRNA互补的序列。
7.权利要求6的方法,其进一步包括对哺乳动物施用化疗剂的步骤。
8.权利要求6的方法,其中该寡核苷酸包含选自示于表1的SEQID NOs:1-30的序列。
9.权利要求6的方法,其中该寡核苷酸具有核酸酶抗性。
10.一种抑制哺乳动物肿瘤转移的方法,包括对疑有转移性肿瘤的哺乳动物施用有效量的反义寡核苷酸,该反义寡核苷酸的大小约3个至约100个核苷酸并且包含在抑制肿瘤转移的条件下可与哺乳动物神经纤维网素基因互补的序列。
11.权利要求10的方法,其进一步包括对哺乳动物施用化疗剂的步骤。
12.权利要求10的方法,其中该寡核苷酸具有核酸酶抗性。
13.权利要求10的方法,其中该寡核苷酸包括选自示于表1的SEQ ID NOs:1-30的序列。
14.一种抑制新血管生成的方法,其包括对哺乳动物施用有效量的反义寡核苷酸,该反义寡核苷酸的大小约3个至约100个核苷酸并且包含在抑制新血管生成的条件下与哺乳动物神经纤维网素基因互补的序列。
15.权利要求14的方法,其中该寡核苷酸具有核酸酶抗性。
16.权利要求14的方法,其中该寡核苷酸包括选自示于表1的SEQ ID NOs:1-30的序列。
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US6653467B1 (en) * | 2000-04-26 | 2003-11-25 | Jcr Pharmaceutical Co., Ltd. | Medicament for treatment of Duchenne muscular dystrophy |
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US20060193772A1 (en) * | 2003-09-24 | 2006-08-31 | Atsushi Ochiai | Drugs for treating cancer |
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