CN1703229B - 治疗前列腺和其他癌的组合物和其制备药物的用途 - Google Patents

治疗前列腺和其他癌的组合物和其制备药物的用途 Download PDF

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CN1703229B
CN1703229B CN2003801009272A CN200380100927A CN1703229B CN 1703229 B CN1703229 B CN 1703229B CN 2003801009272 A CN2003801009272 A CN 2003801009272A CN 200380100927 A CN200380100927 A CN 200380100927A CN 1703229 B CN1703229 B CN 1703229B
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M·E·格利夫
P·罗基
M·西奈夫斯基
E·贝拉尔特
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Abstract

本发明利用了靶定体内热休克蛋白(hsp)27的治疗剂来提供对患有过表达hsp27的前列腺癌和其他癌的个体，尤其是人类个体的治疗。按照本发明，治疗剂，例如对hsp27mRNA，如人hsp27mRNA具有序列特异性的反义寡核苷酸或者RNAi核苷酸抑制剂，以治疗有效量被施用于患有表达升高的hsp27水平的前列腺癌或其他癌的个体。该治疗剂适于制备成药物组合物，该药物组合物包含药学上可接受的载体，并且以剂量单位形式被包装。优选的剂量单位形式是可注射的剂量单位形式。

Description

治疗前列腺和其他癌的组合物和其制备药物的用途
本申请要求2002年10月2日提交的美国临时申请号60/415,859和2003年4月18日提交美国临时申请号60/463,952的利益，这两个申请在此处都在允许并入的权限内被并入作为参考。 
发明背景 
本申请涉及治疗前列腺和其他癌的组合物和方法，这些癌与正常组织相比，在疾病发展的至少一些阶段表达升高的hsp27水平。 
前列腺癌是影响男性的最常见的癌症，是西方世界男性中癌症死亡的第二位最主要的原因。因为前列腺癌是雄激素-敏感肿瘤，雄激素消退，例如，通过阉割，被用于晚期前列腺癌患者的一些治疗方案中。雄激素消退导致前列腺肿瘤广泛凋亡，并因此导致该疾病的退化。然而，阉割-诱导的凋亡是不完全的，并且最终发生雄激素-独立的幸存肿瘤细胞的发展。该发展是提高存活和生命质量的主要障碍，因此已经针对雄激素-独立的细胞进行了尝试。这些尝试集中在针对雄激素-独立的肿瘤细胞的非-激素治疗(Yagoda等人，Cancer71(Supp.3)：1098-1109(1993)；Oh等人，J.Urol.60：1220-1229(1998))，然而，到目前为止还没有非激素剂提高了存活。因此提出了备选方法。 
已经观察到雄激素消退后前列腺肿瘤细胞增量表达许多蛋白质。假定这些蛋白质的至少一些与雄激素消退时所观察到的凋亡性细胞死亡有关(Raffo等人，CancerRes..：4448-4445(1995)；Kra jewska等人，Am.J.Pathol.148：1567-1576(1996)；McDonnell等人，Cancer Res.52：6940-6944(1992))。 
发明概述 
本发明利用了体内靶定热休克蛋白(hsp)27的治疗剂来提供对患有前列腺癌和过表达hsp27的其他癌的个体，尤其是人类个体的治疗。按照本发明，治疗剂，例如对hsp27mRNA如人hsp27mRNA具有序列特异性的反义寡核苷酸或者RNAi核苷酸抑制剂，以治疗有效量被施用于患有表达升高的hsp27水平的前列腺癌或其他癌的个体。该治疗剂适于制备成药物组合物，该药物组合物包含药学上可接受的载体，并 且以剂量单位形式被包装。优选的剂量单位形式是可注射的剂量单位形式。 
附图简述 
图1A-G显示了暴露于Seq.ID Nos.1-81的反义寡核苷酸的细胞中的mRNA表达试验结果。 
图2显示了hsp27反义对PC3细胞中hsp27表达的影响。 
图3A和3B显示了用hsp27反义治疗后肿瘤体积和血清PSA。 
图4A和4B显示了用hsp27反义与或不与紫杉酚(taxol)治疗后肿瘤体积的变化。 
图5显示了用RNAi治疗后hsp27mRNA的减少。 
图6A和6B显示了用RNAi治疗后所表达的hsp27蛋白的量。 
图7A-7C显示了前列腺癌细胞的反义和RNAi治疗后的结果。 
图8显示了T24膀胱癌细胞中hsp27表达。 
图9显示了从NHT组织阵列中hsp27的免疫组织学评估确定的hsp27的免疫反应性。 
发明详述 
本发明涉及减少体内活性hsp27的有效量的组合物。用于本发明的示例性组合物为反义hsp27寡核苷酸或RNAi核苷酸抑制剂。本发明还涉及这些组合物在以升高的量表达hsp27的前列腺癌和其他癌的治疗中的用途。 
如用于本申请的说明书和权利要求书中的，术语“活性hsp27”指具有在胁迫时作为伴侣蛋白稳定蛋白质结构并且尤其抑制半胱天冬酶-3(一种凋亡介质)活性的活性hsp27。可通过限制hsp27的产生或者以比hsp27产生更快的速度降解hsp27减少hsp27的总量，通过将hsp27转化成非活性形式，例如，通过以非活性复合物隔离hsp27(如用抗-hsp27抗体)，可以实现活性hsp27的水平的降低。 
如在本说明书和权利要求书中所用的，可以治疗的癌是与相同组织类型的非癌性细胞相比以升高的量表达hsp27的那些癌。示例性癌包括但不限于前列腺癌、膀胱癌、肺癌、乳腺癌、骨肉瘤、胰腺癌、结肠癌、黑素瘤、睾丸癌、结直肠癌、尿道上皮癌、肾细胞癌、肝细胞癌、白血病、淋巴瘤和卵巢癌和中枢神经系统恶性肿瘤。 
如在本说明书和权利要求书中所用的，术语“序列特异性”指使 用Watson-Crick碱基配对，寡核苷酸和hsp27靶标之间的互补关系的存在足够产生细胞内条件下的特异结合。完全互补性是所希望的，但是不是绝对需要的，尤其当使用更长的寡核苷酸时。 
从例如NCBI记录号AB020027、X54079、NM-006308、NM-001540和NM-001541公知人hsp27mRNA的序列。该cDNA序列(Seq.ID No.91)形成了反义寡核苷酸和RNAi核苷酸抑制剂开发的基础。反义和RNAi的优选序列是靶定Seq.ID No.91中核苷酸131-161、241-261、361-371、551-580、661-681和744-764区域中碱基的那些序列。为了靶定这些区域内的碱基，反义或RNAi分子必须具有对包括所列碱基的至少一个，优选所列碱基的至少10个的区域的序列特异性。 
适宜的反义寡核苷酸具有12到35个核苷酸的长度并且具有对hsp27mRNA序列的序列特异性。制备了反义寡核苷酸并试验了它们减少活性hsp27mRNA的量的能力，这些反义寡核苷酸如Seq ID Nos.1到82所阐明。优选的反义寡核苷酸具有序列5′-ggggacgcggcgctcggtcat-3′(Seq.IDNo.81)或5′-gggacgcggcgctcggtcat-3′(Seq.IDNo.82)，其靶定hsp27mRNA的翻译起始位点，以及具有Seq.ID Nos.25、36、56、57、67和76的那些序列。 
RNA干涉或者“RNAi”最初由Fire和同事提出用于描述当双链RNA(dsRNA)被导入蠕虫时能够阻碍基因表达这一现象(Fire等人(1998)Nature 391，806-811，此处并入作为参考)。dsRNA指导包括脊椎动物的许多生物中基因特异的、转录后沉默，并已经提供了研究基因功能的新的工具。RNAi与mRNA降解有关，但是该干涉的生化机理还未知。在Carthew等人(2001)Current Opinions in CellBiology 13，244-248，和Elbashir等人(2001)Nature 411，494-498中还描述了RNAi的用途，这两篇文献在此处都被并入作为参考。本发明的RNAi分子是约21到约23个核苷酸的双链或单链RNA，其调节介导RNA抑制。即，本发明的分离的RNAi介导hsp27基因的mRNA的降解。 
术语RNA、RNA分子、RNA碎片(segment)和RNA片段(fragment)可以互换使用以表示介导RNA干涉的RNA。这些术语包括双链RNA、单链RNA、分离的RNA(部分纯化的RNA、基本纯的RNA、合成的RNA、重 组产生的RNA)，以及被修饰的RNA，其通过一个或多个核苷酸的加入、缺失、置换和/或修饰而与天然发生的RNA不同。这些修饰可以包括将非核苷酸物质，如加到RNA的末端或内部(RNA的一个或多个核苷酸处)。本发明的RNA分子中的核苷酸还可以含有非标准核苷酸，包括非天然发生的核苷酸或者脱氧核糖核苷酸。总之，所有这些修饰的RNAi化合物被称为类似物或者天然发生的RNA的类似物。本发明的RNA仅需要具有与天然RNA的足够类似性从而其能够介导RNAi。如此处所用的短语“介导RNAi”表示并指出辨别哪些mRNA将被RNAi机器或方法影响的能力。介导RNAi的RNA与RNAi机器相互作用从而其指导该机器降解特定mRNAs或者降低靶蛋白的表达。在一个实施方案中，本发明涉及RNA分子，这些RNA分子指导它们的序列所对应的特定mRNA的切割。不必有序列的完全对应，但是该对应必须足够以便能够通过切割或者靶mRNA的表达的缺乏使得该RNA指导RNAi抑制。 
如上面所指出的，本发明的RNA分子通常含有一个RNA部分和某一些额外部分，例如，脱氧核糖核苷酸部分。该RNA分子中核苷酸的总数适宜地小于49以便成为RNAi的有效介导剂。在优选的RNA分子中，核苷酸的数目为16到29，更优选18到23，最优选21-23。 
适宜的RNAi分子的RNA部分在Seq.IDNos.83-90中给出。这些序列是有义RNA链。它们可以与相应的反义链组合用于RNAi治疗中。 
可以修饰用作反义或者RNAi分子的寡核苷酸以增加体内寡核苷酸的稳定性。例如，寡核苷酸可用作硫代磷酸衍生物(用硫原子置换非-成桥磷酰基氧原子)，其具有对核酸酶消化的增强的抗性。MOE修饰(ISIS骨架)也是有效的。 
可以使用本领域中公知的各种机理实施反义寡核苷酸的施用，包括裸露施用和在药学上可接受的脂质载体中施用。例如，在美国专利号5,855,911和5,417,978中公开了反义递送的脂质载体，这些专利在此被并入作为参考。通常，通过静脉内、腹膜内、皮下或口服途径，或者直接局部肿瘤注射施用反义(寡核苷酸)。 
所施用的反义寡核苷酸或其他治疗剂的量是有效减少活性hsp27的量的量。应该明白该量将随着所用的反义核苷酸或其他治疗剂的有效性，和所用的载体的性质而变。对于任何给定组合物的适宜量的确 定在本领域技术中，通过所设计的标准试验系列可以评估适宜的治疗水平。 
本发明的RNAi分子用于治疗包括人类患者的患者的疗法中，这些患者患有癌症或者一种其他疾病，其中通过所靶定蛋白的表达的抑制得到治疗益处。通过口服、每天一次或多次注射(静脉内、皮下、膀胱内、或鞘内)或者连续静脉内或鞘内施用一个或多个治疗周期，对患者施用本发明的siRNA分子以达到适于所靶定的mRNA和蛋白质的调节的血浆和组织浓度。 
前列腺癌是在晚期癌，尤其在变成雄激素独立的癌中过表达hsp27的一种癌。图9显示了从NHT组织阵列中hsp27的免疫组织学评估确定的hsp27的免疫反应性。在良性样品中，免疫反应性限于基底层。随着新辅助治疗时间的增加，免疫反应性增加，雄激素独立的肿瘤表现出非常强的反应性。对于前列腺癌的治疗，本发明的治疗组合物适于在雄激素消退起始后施用。可通过手术(除去两个睾丸)或者内科(药物诱导的睾酮抑制)去雄完成雄激素消退的引发，这两种去雄现今被指示用于前列腺癌的治疗。可通过各种方案，包括LHRH剂或者抗雄激素实现内科去雄(Gleave等人，CMAJ160：225-232(1999))。在Gleave等人，Eur.Urol.34(Supp.3)：37-41(1998)中描述了间歇疗法，其中实现了可逆的雄激素消退。 
Hsp27表达的抑制可以是暂时的，并且对于前列腺癌的治疗，理想地应该与雄激素消退一致地发生。在人类中，抑制表达的该方法应该在雄激素消退的一天或两天内(之前或之后)有效开始并且延长约3到6个月。这可能需要多次剂量来完成。然而，应该明白时间期限可以进一步延长，在阉割前开始并进行足够的时间而不背离本发明的范围。 
按照本发明的治疗癌症，包括前列腺癌的方法可以还包括施用化学治疗剂和/或针对不同靶标的额另外的反义寡核苷酸。其他治疗剂的实例包括，但不限于，紫杉醇(paclitaxel或docetaxel)、盐酸米托蒽酯(mitoxanthrone)和针对Bc1-2、Bc1-xl或c-myc的反义(寡核苷酸)。使用反义或RNAi对hsp27的抑制可以用于增强如紫杉醇或吉西他滨(gemcitabine)，以及用于治疗前列腺、乳腺、肺、尿道上皮和其他癌的生物剂的活性。
现在将关于下面的非限制性实施例进一步描述本发明。 
实施例1 
制备如Seq.ID Nos.1-81中定义的许多反义化合物，并将人前列腺癌PC3细胞暴露于Oligofectamine载体中的50nM特异反义寡核苷酸后通过RNA印迹检验人前列腺癌PC3细胞中Hsp27 mRNA表达水平。在图中显示了关于Seq.ID Nos.1-81的这些试验的结果(作为只有Oligofectamine的对照的百分比)。如所示，尽管不是所有反义序列都有效，但在hsp27 mRNA的全部长度中发现了有效反义序列。 
实施例2 
用6种不同hsp27-反义寡核苷酸的三种浓度(10、30和50nM)，在10cm盘中，用Oligofectamine载体转染40％汇合的PC3前列腺癌细胞。第一次处理后48小时提取RNA并通过RNA印迹分析。所检查的反义寡核苷酸是具有Seq.ID Nos.67、57、25、76、56和36的那些反义寡核苷酸。作为对照，实施了杂乱寡核苷酸和仅Oligofectamine的实验。所有试验的寡核苷酸表现出在至少一种浓度下关于对照的hsp27的下调。Seqs.ID71和74表现出最有效，在10nM具有最显著的下调。在图2中图示性描绘了相对于GAPDH对照的结果。 
实施例3 
将LNCaP前列腺癌细胞的异种移植物导入小鼠，评价阉割使雄激素消退后腹膜内施用10mg/kg hsp27-反义寡核苷酸(Seq.ID No.82)每天一次，持续4周后的效果。如图3A和3B所示，用杂乱对照治疗后的几周中肿瘤体积和血清PSA增加，表明发展到雄激素独立，从而失去了阉割疗法的效能。相反，当给予hsp27反义寡核苷酸治疗时在相同时限中没有观察到雄激素独立。 
实施例4 
将PC3前列腺癌细胞的异种移植物导入小鼠，评价阉割使雄激素消退后腹膜内施用10mg/kg hsp27-反义寡核苷酸(Seq.ID No.82)每天一次，持续4周，用和不用紫杉酚后的效果。如图4A和4B中所示，与杂乱寡核苷酸相比，使用hsp27-反义治疗显著减小肿瘤体积。当紫杉酚治疗与反义治疗组合时，该效果被增强。图4A图解单剂抗肿瘤活性而图4B图解hsp27反义的施用可以体内使细胞对紫杉醇敏感 化。4B中的对照是杂乱(寡核苷酸)加紫杉酚。 
实施例5 
在PC3细胞中试验具有依照Seq.ID Nos.84、85、87、88和90的序列的RNAi分子。将这些PC细胞用各种量的hsp27 siRNA或杂乱对照转染。转染后2天，提取总RNA并通过RNA印迹分析hsp27和28S水平。仅用Oligofectimine处理的细胞用作额外对照。图5显示了对28S mRNA对照标准化后hsp27mRNA的密度计测量。如所示，与杂乱对照相比，Seq.ID Nos.84、85、87、88和90对于显著降低hsp27表达都有效。 
实施例6 
将具有依照Seq ID.No.84的序列的RNAi转染到PC3细胞中，并确定与Vinculin表达相比所表达的hsp27蛋白的量。结果在图6A和6B中显示。如所示，用RNAi分子治疗后观察到hsp27表达的剂量依赖的降低。 
实施例7 
将具有依照Seq ID.No.84的序列的1nM RNAi体外转染LNCaP细胞(104个细胞/孔，培养于12孔板中)。用Crustal Violet测定法监视细胞生长。如图7A所示，RNAi处理导致与仅Oligofectamine或者杂乱对照的处理相比细胞生长的降低。用PC3细胞重复该实验。图7B显示了转染后3天的活细胞％。图7C显示了用hsp27反义Seq.ID NO.82体外处理后PC3细胞的生长抑制。 
实施例8 
检测用hsp27反义(Seq.ID No82)或RNAi(Seq.ID No.84)转染的人膀胱癌T24细胞的hsp27表达。如图8所示，RNAi和反义对于降低这些细胞中表达的hsp27的量都有效。
Figure IYZ000004150144200041
Figure IYZ000004150144200091
Figure IYZ000004150144200101
Figure IYZ000004150144200121
Figure IYZ000004150144200131
Figure IYZ000004150144200181

Claims (11)

1.一种组合物，其含有治疗剂，所述治疗剂能有效降低暴露于该治疗剂的hsp27表达细胞中活性hsp27的量，其中所述组合物含有寡核苷酸作为治疗剂，该寡核苷酸具有与Seq.ID NO.91的一部分互补的序列；并且其中所述寡核苷酸是
(a)反义寡核苷酸，其具有12到35个核苷酸的长度，并且具有整体上与Seq.ID NO.91互补且靶定Seq.ID NO.91的一部分的序列，所述Seq.ID NO.91的一部分与Seq.ID No.s 25、36、56、57、67、71、74、76、81和82中的任何一个序列互补；或
(b)靶定Seq.ID NO.91，并且包含Seq.ID Nos.84、85、87、88和90之一所示序列的siRNA。
2.权利要求1的组合物，其中该寡核苷酸是反义寡核苷酸。
3.权利要求2的组合物，其中反义寡核苷酸具有骨架修饰以提供针对体内核酸酶消化的抗性。
4.权利要求3的组合物，其中反义寡核苷酸如Seq.ID No.81或82所示。
5.权利要求2的组合物，其中反义寡核苷酸如Seq.ID No.81或82所示。
6.权利要求1的组合物，其中该寡核苷酸是siRNA。
7.权利要求6的组合物，其中siRNA具有骨架修饰以提供针对体内核酸酶消化的抗性。
8.权利要求7的组合物，其中siRNA含有Seq.ID No.84。
9.权利要求6的组合物，其中siRNA含有Seq.ID No.84。
10.根据权利要求1-9任一项的组合物在制备治疗前列腺癌的药物组合物的制剂中的用途。
11.权利要求10的用途，其中药物组合物以可注射的剂量单位形式包装。
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Families Citing this family (21)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
ES2345330T3 (es) 2002-10-02 2010-09-21 The University Of British Columbia Oligonucleotidos para el tratamiento del cancer de prostata y otros canceres.
WO2006089209A2 (en) * 2005-02-17 2006-08-24 The Board Of Trustees Of The Leland Stanford Junior University Txr1 and enhanced taxane sensitivity based on the modulation of a pathway mediated thereby
WO2007025229A2 (en) * 2005-08-25 2007-03-01 Isis Pharmaceuticals, Inc. Compositions and their uses directed to hsp27
EP1984399A4 (en) * 2006-02-10 2010-03-03 Univ California TRANSDUCIBLABLE DELIVERY OF SIRANA BY MEANS OF DSRNA BINDEDOMADE FUSIONS TO PTD / CPPS
US8242258B2 (en) * 2006-12-03 2012-08-14 Agilent Technologies, Inc. Protecting groups for RNA synthesis
US9381209B2 (en) * 2007-03-05 2016-07-05 The University Of British Columbia Treatment of squamous cell carcinoma with HSP27 antisense oligonucleotides and radiotherapy
JP2010521460A (ja) 2007-03-12 2010-06-24 アンティジェン・エクスプレス・インコーポレーテッド 癌免疫療法におけるＩｉ−ＲＮＡｉ関与Ｉｉ抑制
CA2656577A1 (en) * 2008-04-14 2009-10-14 The University Of British Columbia Method for evaluation of a cancer
AU2009265194A1 (en) 2008-07-02 2010-01-07 Enzon Pharmaceuticals Inc RNA antagonists targeting Hsp27
NZ589262A (en) * 2008-07-18 2012-05-25 Oncogenex Technologies Inc Antisense formulation
WO2011054811A1 (en) 2009-11-03 2011-05-12 Santaris Pharma A/S Rna antagonists targeting hsp27 combination therapy
KR101217866B1 (ko) * 2010-08-18 2013-01-03 한국원자력의학원 혈관신생 억제용 약학 조성물 및 혈관신생 억제용 활성물질을 스크리닝하는 방법
DE102010056610A1 (de) * 2010-12-31 2012-07-05 Volker A. Erdmann Pharmazeutische Zusammensetzung enthaltend L-DNA
JP5658696B2 (ja) * 2011-02-25 2015-01-28 株式会社ファンケル 皮膚のストレス蓄積度の評価方法
CA2747509C (en) 2011-05-12 2017-07-18 Oncogenex Technologies Inc. Treatment of pulmonary and pleural fibrosis using hsp27 inhibitors
EP2809325A4 (en) * 2012-02-02 2015-04-01 Univ British Columbia CANCER POLYTHERAPY USES HSP27 INHIBITORS AND EGFR TYROSINE KINASE INHIBITORS OR ANTIFOLATES
GB201204785D0 (en) 2012-03-19 2012-05-02 Queen Mary & Westfield College Method for determining prognosis of prostate cancer in a subject
KR101374585B1 (ko) * 2012-05-02 2014-03-17 연세대학교 산학협력단 ＨＳＰ２７ 발현을 억제하는 ｓｈＲＮＡ
CN104781271B (zh) 2012-08-20 2018-07-06 加利福尼亚大学董事会 具有生物可逆的基团的多核苷酸
WO2016016267A1 (de) 2014-07-28 2016-02-04 Technische Universität Dresden Effiziente hemmung von hsp27
BR112018011045A2 (pt) 2015-11-30 2018-11-21 Univ British Columbia inibidores de oligonucleotídeo anti-sentido (aso) de transportador de monocarboxilato 4 (mct4) para uso como terapêutica no tratamento de câncer

Family Cites Families (19)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH08504766A (ja) * 1992-12-14 1996-05-21 スタート テクノロジー パートナーシップ ドーパミン作動性神経系の病態の診断および治療に関するドーパミン受容体ｍＲＮＡに対してアンチセンスのオリゴヌクレオチドの投与
IL108367A0 (en) * 1993-01-27 1994-04-12 Hektoen Inst For Medical Resea Antisense polynzcleotide inhibition of human growth factor-sensitive cancer cells
US5417978A (en) 1993-07-29 1995-05-23 Board Of Regents, The University Of Texas System Liposomal antisense methyl phosphonate oligonucleotides and methods for their preparation and use
US5801154A (en) * 1993-10-18 1998-09-01 Isis Pharmaceuticals, Inc. Antisense oligonucleotide modulation of multidrug resistance-associated protein
US5855911A (en) 1995-08-29 1999-01-05 Board Of Regents, The University Of Texas System Liposomal phosphodiester, phosphorothioate, and P-ethoxy oligonucleotides
JPH09176011A (ja) * 1995-12-27 1997-07-08 Kureha Chem Ind Co Ltd Ｈｓｐ２７ファミリーに属するタンパク質のフラボノイド含有合成抑制剤
US20060008448A1 (en) * 1996-06-11 2006-01-12 Minzhen Xu Inhibition of li expression in mammalian cells
JPH107559A (ja) 1996-06-18 1998-01-13 Kureha Chem Ind Co Ltd Ｈｓｐ２７ファミリーに属するタンパク質のベルベリン誘導体含有合成抑制剤
US5962262A (en) * 1997-07-25 1999-10-05 Incyte Pharmaceuticals, Inc. Human heat shock 27 like protein
US6703228B1 (en) 1998-09-25 2004-03-09 Massachusetts Institute Of Technology Methods and products related to genotyping and DNA analysis
US5998148A (en) * 1999-04-08 1999-12-07 Isis Pharmaceuticals Inc. Antisense modulation of microtubule-associated protein 4 expression
CA2375467C (en) 1999-07-19 2013-10-29 The University Of British Columbia Antisense therapy for hormone-regulated tumors
AU2001249317A1 (en) * 2000-03-21 2001-10-03 Corixa Corporation Compositions and methods for the therapy and diagnosis of ovarian and endometrial cancer
US6905827B2 (en) * 2001-06-08 2005-06-14 Expression Diagnostics, Inc. Methods and compositions for diagnosing or monitoring auto immune and chronic inflammatory diseases
ES2432225T3 (es) * 2001-08-23 2013-12-02 Arizona Board Of Regents Reactivos y procedimientos para terapias del músculo liso
ES2345330T3 (es) 2002-10-02 2010-09-21 The University Of British Columbia Oligonucleotidos para el tratamiento del cancer de prostata y otros canceres.
US7250496B2 (en) * 2002-11-14 2007-07-31 Rosetta Genomics Ltd. Bioinformatically detectable group of novel regulatory genes and uses thereof
WO2007025229A2 (en) * 2005-08-25 2007-03-01 Isis Pharmaceuticals, Inc. Compositions and their uses directed to hsp27
US9381209B2 (en) 2007-03-05 2016-07-05 The University Of British Columbia Treatment of squamous cell carcinoma with HSP27 antisense oligonucleotides and radiotherapy

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
Sandrine Horman et al.anti-sense inhibition ofsmall-heat-shock-protein(HSP27)expression in MCF-7mammary-carcinoma cells induces theirspontaneousacquisition of a secretory phenotype.Int.J.Cancer82 4.1999,82(4),574-582.
Sandrine Horman et al.anti-sense inhibition ofsmall-heat-shock-protein(HSP27)expression in MCF-7mammary-carcinoma cells induces theirspontaneousacquisition of a secretory phenotype.Int.J.Cancer82 4.1999,82(4),574-582. *

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