CN101374528A - Therapeutic uses of RTP801 - Google Patents
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- CN101374528A CN101374528ACNA2007800027089ACN200780002708ACN101374528ACN 101374528 ACN101374528 ACN 101374528ACN A2007800027089 ACNA2007800027089 ACN A2007800027089ACN 200780002708 ACN200780002708 ACN 200780002708ACN 101374528 ACN101374528 ACN 101374528A
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Abstract
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Translated fromChinese在本申请全文中，包括美国专利的各种出版物和专利申请通过引用作者、年份和专利号来参考。这些出版物和专利的公开内容以及专利申请通过引用并入本申请，以便更全面地描述本发明所属领域的现有技术状态。Throughout this application, various publications and patent applications, including US patents, are referenced by citing author, year, and patent number. The disclosures of these publications and patents, as well as patent applications, are incorporated by reference into this application in order to more fully describe the state of the art to which this invention pertains.
技术领域technical field
本发明涉及抑制RTP801基因的新颖siRNA分子及该分子用以治疗所有类型呼吸病症(包括肺部病症)、眼病和症状、微血管病症、血管生成及细胞凋亡相关的病状及听力障碍的用途。The present invention relates to novel siRNA molecules inhibiting the RTP801 gene and the use of such molecules for the treatment of all types of respiratory disorders including pulmonary disorders, eye diseases and symptoms, microvascular disorders, angiogenesis and apoptosis related conditions and hearing impairment.
技术背景technical background
慢性阻塞性肺病(COPD)Chronic Obstructive Pulmonary Disease (COPD)
慢性阻塞性肺病(COPD)影响超过一千六百万美国人，且在美国其为第四大死亡原因。吸烟引起大部分衰竭性疾病事件，但亦不能排除其它环境因素(Petty TL.2003.Definition，epidemiology，course，and prognosis of COPD.Clin.Cornerstone，5-10)。Chronic obstructive pulmonary disease (COPD) affects more than 16 million Americans and is the fourth leading cause of death in the United States. Smoking causes most debilitating disease events, but other environmental factors cannot be ruled out (Petty TL. 2003. Definition, epidemiology, course, and prognosis of COPD. Clin. Cornerstone, 5-10).
肺气肿为COPD的主要表现。末端细支气管末梢的周边空间的永久性破坏为气肿的特征(Tuder RM等人，Oxidative stress andapoptosis interact and cause emphysema due to vascularendothelial growth factor blocade.Am J Respir Cell Mol Biol，29：88-97；2003)。气肿亦以炎性细胞(诸如细支气管及肺泡结构中的巨噬细胞及嗜中性粒细胞)的蓄积为特征(Petty，2003)。Emphysema is the main manifestation of COPD. Permanent destruction of the peripheral space distal to the terminal bronchioles is characteristic of emphysema (Tuder RM et al., Oxidative stress andapoptosis interact and cause emphysema due to vascularendothelial growth factor blocade. Am J Respir Cell Mol Biol, 29:88-97; 2003 ). Emphysema is also characterized by the accumulation of inflammatory cells such as macrophages and neutrophils in the bronchioles and alveolar structures (Petty, 2003).
气肿的发病机理是复杂且多因子的。在人类中，已显示藉由炎性细胞产生的蛋白酶抑制剂(诸如α1-抗胰蛋白酶)的不足会促成蛋白酶/抗蛋白酶不平衡，藉此促进烟(CS)诱发的气肿中肺泡细胞外基质的破坏(Eriksson，S.1964.Pulmonary Emphysema and Alpha1-Antitrypsin Deficiency.Acta Med Scand 175：197-205.Joos，L.，Pare，P.D.及Sandford，A.J.2002.Genetic risk factors ofchronic obstructive pulmonary disease.Swiss Med Wkly132：27-37)。如藉由巨噬细胞金属弹性蛋白酶基因剔除小鼠对抗由慢性吸入CS引起的气肿所证实，基质金属蛋白酶(MMP)在实验性气肿中起重要作用(Hautamaki等人：Requirement for macrophage elastasefor cigarette smoke-induced emphysema in mice.Science277：2002-2004)。此外，转基因小鼠中肺部介白素-13的过度表达导致MMP及组织蛋白酶依赖性气肿(Zheng，T等人，2000.Inducibletargeting of IL-13 to the adult lung causes matrixmetalloproteinase-and cathepsin-dependent emphysema.J ClinInvest 106：1081-1093)。近期工作描述了隔细胞细胞凋亡涉及导致气肿的肺组织损伤(RangasamiT等人，Genetic ablation of Nrf2enhances susceptibility to cigarette smoke-induced emphysemain mice.Submitted to Journal of Clinincal Investigation.；Tuder RM等人，Oxidative stress and apoptosis interact and causeemphysema due to vascular endothelial growth factor blocade.Am J Respir Cell Mol Biol，29：88-97；2003.；Yokohori N，AoshibaK，Nagai A，Increased levels of cell death and proliferationin alveolar wall cells in patients with pulmonary emphysema.Chest.2004年2月；125(2)：626-32.；Aoshiba K，Yokohori N，NagaiA.，Alveolar wall apoptosis causes lung destruction andemphysematous changes.Am J Respir Cell Mol Biol.2003年5月；28(5)：555-62)。The pathogenesis of emphysema is complex and multifactorial. In humans, deficiency of protease inhibitors such as α1-antitrypsin produced by inflammatory cells has been shown to contribute to protease/antiprotease imbalance, thereby promoting extracellular Matrix destruction (Eriksson, S.1964. Pulmonary Emphysema and Alpha1-Antitrypsin Deficiency. Acta Med Scand 175:197-205. Joos, L., Pare, P.D. and Sandford, A.J.2002. Genetic risk factors of chronic obstructive pulmonary disease. MedWkly132:27-37). Matrix metalloproteinases (MMPs) play an important role in experimental emphysema as demonstrated by macrophage metalloelastase knockout mice against emphysema induced by chronic inhalation of CS (Hautamaki et al.: Requirement for macrophage elastase for cigarette smoke-induced emphysema in mice. Science 277: 2002-2004). Furthermore, overexpression of interleukin-13 in the lungs of transgenic mice resulted in MMP and cathepsin-dependent emphysema (Zheng, T et al., 2000. Inducible targeting of IL-13 to the adult lung causes matrixmetalloproteinase-and cathepsin-dependent emphysema. J ClinInvest 106:1081-1093). Recent work has described the involvement of septal cell apoptosis in lung tissue injury leading to emphysema (RangasamiT et al., Genetic ablation of Nrf2enhances susceptibility to cigarette smoke-induced emphysemain mice. Submitted to Journal of Clinincal Investigation.; Tuder RM et al., Oxidative stress and apoptosis interact and causeemphysema due to vascular endothelial growth factor blocade. Am J Respir Cell Mol Biol, 29:88-97; 2003.; Pulmonary emphysema. Chest. 2004 February; 125(2): 626-32.; Aoshiba K, Yokohori N, Nagai A., Alveolar wall apoptosis causes lung destruction and emphysematous changes. Am J Respir Cell Mol Biol. 2003 May; 28(5):555-62).
在以气肿中肺破坏的两条路径为基础的机理中，首先应提及活性氧(ROS)的过度形成。充分确定氧化强化剂/抗氧化剂的不平衡存在于吸烟者的血液及肺组织中(Hulea SA等人：Cigarette smoking causesbiochemical changes in blood that are suggestive of oxidativestress：a case-control study.J Environ Pathol Toxicol Oncol.1995；14(3-4)：173-80.；Rahman I，MacNee W.Lung glutathioneand oxidative stress：implications in cigarette smoke-inducedairway disease.Am J Physiol.1999年12月；277(6 Pt 1)：L1067-88.；MacNee W.Oxidants/antioxidants and COPD.Chest.2000年5月；117(5 Suppl 1)：303S-17S.；Marwick JA，Kirkham P，Gilmour PS，Donaldson K，MacNEE W，Rahman I.Cigarette smoke-induced oxidative stress and TGF-betal increase p21waf1/cip1expression in alveolar epithelial cells.Ann N Y AcadSci.2002年11月；973：278-83.；Aoshiba K，Koinuma M，Yokohori N，NagaiA.Immunohistochemical evaluation of oxidative stress in murinelungs after cigarette smoke exposure.Inhal Toxicol.2003年9月；15(10)：1029-38.；Dekhuijzen PN.Antioxidant properties ofN-acetylcysteine：their relevance in relation to chronicobstructive pulmonary disease.Eur Respir J.2004年4月；23(4)：629-36.；Tuder RM，Zhen L，Cho CY，Taraseviciene-StewartL，Kasahara Y，Salvemini D，Voelkel NF及Flores SC.Oxidativestress and apoptosis interact and cause emphysema due tovascular endothelial growth factor blocade.Am J Respir CellMol Biol，29：88-97；2003)。将小鼠暴露于CS一小时后，肺泡上皮细胞中8-羟基-2′-脱氧鸟苷(8-OHdG)、尤其II型显著增加(参见上文Inhal Toxicol.2003年9月；15(10)：1029-38)。Among the mechanisms based on the two pathways of lung destruction in emphysema, the excessive formation of reactive oxygen species (ROS) should be mentioned first. It is well established that an oxidative enhancer/antioxidant imbalance exists in the blood and lung tissue of smokers (Hulea SA et al: Cigarette smoking causes biochemical changes in blood that are suggestive of oxidative stress: a case-control study. J Environ Pathol Toxicol Oncol .1995; 14(3-4):173-80.; Rahman I, MacNee W. Lung glutathione and oxidative stress: implications in cigarette smoke-induced airway disease. Am J Physiol. 1999 Dec; 277(6 Pt 1): L1067-88.; MacNee W. Oxidants/antioxidants and COPD. Chest. 2000 May;117(5 Suppl 1):303S-17S.; Marwick JA, Kirkham P, Gilmour PS, Donaldson K, MacNEE W, Rahman I . Cigarette smoke-induced oxidative stress and TGF-betal increase p21waf1/cip1 expression in alveolar epithelial cells. Ann N Y Acad Sci. 2002 Nov; 973:278-83.; Aoshiba K, Koinuma M, Yoicalkohori N, Nagais Atoimmunization of oxidative stress in murinelungs after cigarette smoke exposure. Inhal Toxicol. 2003 September; 15(10): 1029-38.; Dekhuijzen PN. Antioxidant properties of N-acetylcysteine: their relevance inur relation to J dis chronic obstructive Respulmonase pul 2004 Apr;23(4):629-36.; Tuder RM, Zhe n L, Cho CY, Taraseviciene-StewartL, Kasahara Y, Salvemini D, Voelkel NF, and Flores SC. Oxidativestress and apoptosis interact and cause emphysema due tovascular endothelial growth factor blocade. Am J Respir CellMol Biol, 209:88) . One hour after mice were exposed to CS, 8-hydroxy-2'-deoxyguanosine (8-OHdG), especially type II, was significantly increased in alveolar epithelial cells (see supra Inhal Toxicol. 2003 Sep; 15(10 ): 1029-38).
已知过度产生的活性氧具有细胞毒性活性，其细胞毒性活性源自直接DNA破坏作用及细胞凋亡信号传递路径的活化(Takahashi A，Masuda A，Sun M，Centonze VE，Herman B.Oxidative stress-inducedapoptosis is associated with alterations in mitochondrialcaspase activity and Bcl-2-dependent alterations inmitochondrial pH(pHm).Brain Res Bull.2004年2月15日；62(6)：497-504.；Taniyama Y，Griendling KK.Reactive oxygen speciesin the vasculature：molecular and cellular mechanisms.Hypertension.2003年12月；42(6)：1075-81.Epub 2003年10月27日；Higuchi Y.Chromosomal DNA fragmentation in apoptosis andnecrosis induced by oxidative stress.Biochem Pharmacol.2003年10月15日；66(8)：1527-35.；Punj V，Chakrabarty AM.Redoxprote in sinmammalian cell death：an evolutionarily conservedfunction in mitochondria and prokaryotes.Cell Microbiol.2003年4月；5(4)：225-31.；Ueda S，Masutani H，Nakamura H，Tanaka T，Ueno M，Yodoi J.Redox control of cell death.Antioxid RedoxSignal.2002年6月；4(3)：405-14)。Excessively produced reactive oxygen species are known to have cytotoxic activity derived from direct DNA damage and activation of apoptotic signaling pathways (Takahashi A, Masuda A, Sun M, Centonze VE, Herman B. Oxidative stress- induced apoptosis is associated with alterations in mitochondrial caspase activity and Bcl-2-dependent alterations inmitochondrial pH(pHm). Brain Res Bull. 2004 Feb 15;62(6):497-504.; Taniyama Y, Griendling KK. Reactive oxygen Species in the vasculature: molecular and cellular mechanisms. Hypertension. 2003 Dec; 42(6): 1075-81. Epub 2003 Oct 27; Higuchi Y. Chromosomal DNA fragmentation in apoptosis and necrosis induced by oxidative stress. Biochem Phare 2003 Oct 15;66(8):1527-35.; Punj V, Chakrabarty AM. Redoxprote in sinmammalian cell death: an evolutionarily conserved function in mitochondria and prokaryotes. Cell Microbiol. 2003 Apr;5(4): 225-31.; Ueda S, Masutani H, Nakamura H, Tanaka T, Ueno M, Yodoi J. Redox control of cell death. Antioxid Redox Signal. 2002 Jun;4(3):405-14).
ROS不仅自身具有细胞毒性，其亦为促炎症刺激物，为氧化还原敏感性转录因子NFkB及AP-1的重要活化剂(Rahman I.Oxidative stressand gene transcription in asthma and chronic obstructivepulmonary disease：antioxidant therapeutic targets.Curr DrugTargets Inflamm Allergy.2002年9月；1(3)：291-315中回顾)。而两种转录因子强烈涉及促炎性细胞因子(Renard P，Raes M.Theproinflammatory transcription factor NFkappaB：a potentialtarget for novel therapeutical strategies.Cell Biol Toxicol.1999；15(6)：341-4.；Lentsch AB，Ward PA.The NFkappaBb/IkappaBsystem in acute inflammation.Arch Immunol Ther Exp(Warsz).2000；48(2)：59-63中回顾)及基质降解蛋白酶(Andela VB，GordonAH，Zotalis G，Rosier RN，Goater JJ，Lewis GD，Schwarz EM，PuzasJE，O′Keefe RJ.NFkappaB：a pivotal transcription factor inprostate cancer metastasis to bone.Clin Orthop.2003年10月；(415 Suppl)：S75-85.；Fleenor DL，Pang IH，Clark AF.Involvementof AP-1 in interleukin-1 alpha-stimulated MMP-3 expression inhuman trabecular meshwork cells.Invest Ophthalmol Vis Sci.2003年8月；44(8)：3494-501.；Ruhul Amin AR，Senga T，Oo ML，ThantAA，Hamaguchi M.Secretion of matrixmetalloproteinase-9 by theproinflammatory cytokine，IL-1 beta：a role for the dualsignalling pathways，Akt and Erk.Genes Cells.2003年6月；8(6)：515-23)转录的刺激作用。而促炎性细胞因子充当亦分泌基质降解酶、细胞因子及活性氧的炎性细胞的引诱剂。因此，看来病理因子(例如CS)会引发其中活性氧充当肺破坏的主要介体的病理网络。ROS not only has cytotoxicity itself, but also is a pro-inflammatory stimulus and an important activator of redox-sensitive transcription factors NFkB and AP-1 (Rahman I. Oxidative stress and gene transcription in asthma and chronic obstructive pulmonary disease: antioxidant therapeutic targets. Reviewed in Curr Drug Targets Inflamm Allergy. 2002 Sep;1(3):291-315). Whereas two transcription factors are strongly involved in proinflammatory cytokines (Renard P, Raes M. The proinflammatory transcription factor NFkappaB: a potential target for novel therapeutic strategies. Cell Biol Toxicol. 1999; 15(6): 341-4.; Lentsch AB, Ward PA. The NFkappaBb/IkappaBsystem in acute inflammation. Arch Immunol Ther Exp (Warsz). 2000; 48(2): 59-63 reviewed) and matrix degrading proteases (Andela VB, Gordon AH, Zotalis G, Rosier RN, Goater JJ Fleenor DL, Pang IH, Clark AF. Involvement of AP-1 in interleukin-1 alpha-stimulated MMP-3 expression in human trabecular meshwork cells. Invest Ophthalmol Vis Sci. 2003 Aug; 44(8): 3494-501.; Ruhul Amin AR, Senga T, Oo ML, ThantAA, Hamaguchi M. Secretion of matrixmetalloproteinase-9 by the proinflammatory cytokine, IL-1 beta: a role for the dual signaling pathways, Akt and Erk. Genes Cells. 2003 Jun;8(6):515-23) Stimulation of transcription. In turn, pro-inflammatory cytokines act as attractants for inflammatory cells that also secrete matrix-degrading enzymes, cytokines, and reactive oxygen species. Thus, it appears that pathological agents such as CS trigger a pathological network in which reactive oxygen species act as the main mediators of lung destruction.
所吸入的烟的活性氧(ROS)及由炎性细胞内源形成的活性氧都会促进肺内氧化负荷增加。Reactive oxygen species (ROS) from inhaled smoke and endogenously formed ROS from inflammatory cells contribute to an increase in the oxidative load in the lungs.
关于COPD发病机理的另一病理因子为所观测到的VEGF及VEGFRII在气肿患者的肺中的表达减少(Yasunori Kasahara，Rubin M.Tuder，Carlyne D.Cool，David A.Lynch，Sonia C.Flores及Norbert F.Voelkel.Endothelial Cell Death and Decreased Expression ofVascular Endothelial Growth Factor and Vascular EndothelialGrowth Factor Receptor 2 in Emphysema.Am J Respir Crit CareMed，第163卷，第737-744页，2001)。此外，使用化学VEGFR抑制剂来抑制VEGF信号传输会导致肺泡中隔内皮细胞及接着内皮细胞细胞凋亡，此可能归因于肺泡内两种类型细胞的亲密结构/功能关系的破坏(Yasunori Kasahara，Rubin M.Tuder，LaimuteTaraseviciene-Stewart，Timothy D.Le Cras，Steven Abman，PeterK.Hirth，Johannes Waltenberger及Norbert F.Voelkel.Inhibition of VEGF receptors causes lung cell apoptosis andemphysema.J.Clin.Invest.106：1311-1319(2000)；Voelkel NF，Cool CD.Pulmonary vascular involvement in chronic obstructivepulmonary disease.Eur Respir J Suppl.2003年11月；46：28s-32s)。Another pathological factor in the pathogenesis of COPD is the observed reduced expression of VEGF and VEGFRII in the lungs of patients with emphysema (Yasunori Kasahara, Rubin M. Tuder, Carlyne D. Cool, David A. Lynch, Sonia C. Flores and Norbert F. Voelkel. Endothelial Cell Death and Decreased Expression of Vascular Endothelial Growth Factor and Vascular Endothelial
黄斑变性macular degeneration
在美国，65岁以上个体中最佳矫正视力降低的最通常原因是称为年龄相关的黄斑变性(AMD)的视网膜病症。随着AMD发展，此疾病以敏锐中心视力的损失为特征。AMD所影响的眼部区域为黄斑-视网膜中心的小区域，主要包含感光细胞。占AMD患者约85％-90％的所谓＂干式＂AMD包括归因于细胞整体萎缩的眼睛色素分布改变、感光体损失及视网膜功能衰退。所谓＂湿式＂AMD包括导致视网膜下空间中出现凝块或疤痕的异常脉络膜血管增生。因此，由于形成神经视网膜下异常脉络膜新生血管网(脉络膜新血管生成，CNV)而出现湿式AMD的发作。新形成的血管极易漏。此导致视网膜下体液及血液蓄积，从而导致视力损失。最终，在所涉及的区域中功能视网膜完全损失，成为涉及脉络膜及视网膜形式的大的椭圆形疤痕。虽然干式AMD患者可保持品质降低的视力，但湿式AMD常导致失明。(Hamdi & Kenney，Age-related Maculardegeneration-a new viewpoint，Frontiers in Bioscience，e305-314，2003年5月)。CNV不仅发生在湿式AMD中，且亦发生在其它眼部病变中，诸如眼部组织胞浆菌病综合征、血管样条纹、布鲁赫氏膜(Bruch′s membrane)破裂、近视性变性、眼部肿瘤及某些视网膜退化疾病。In the United States, the most common cause of reduced best-corrected visual acuity in individuals over the age of 65 is a retinal disorder called age-related macular degeneration (AMD). As AMD progresses, the disease is characterized by loss of acute central vision. The area of the eye affected by AMD is the macula - the small area in the center of the retina that contains mainly photoreceptor cells. The so-called "dry" form of AMD, which accounts for about 85%-90% of AMD patients, includes changes in eye pigment distribution, loss of photoreceptors, and decline in retinal function due to global atrophy of cells. So-called "wet" AMD involves abnormal choroidal vascular proliferation that leads to clots or scarring in the subretinal space. Thus, the onset of wet AMD occurs due to the formation of an abnormal choroidal neovascular network (choroidal neovascularization, CNV) under the neural retina. Newly formed blood vessels are extremely leaky. This leads to the accumulation of fluid and blood under the retina, which leads to vision loss. Eventually, there is complete loss of functional retina in the area involved, in the form of a large oval scar involving the choroid and retina. While patients with dry AMD can maintain reduced-quality vision, wet AMD often leads to blindness. (Hamdi & Kenney, Age-related Macular degeneration-a new viewpoint, Frontiers in Bioscience, e305-314, May 2003). CNV occurs not only in wet AMD but also in other ocular lesions such as ocular histoplasmosis syndrome, vascular striae, rupture of Bruch's membrane, myopic degeneration, Eye tumors and certain retinal degenerative diseases.
进行的大量研究已确定AMD的若干危险因素，诸如吸烟、年老、家族病史(Milton，Am J Ophthalmol 88，269(1979)；Mitchell等人，Ophthalmology 102，1450-1460(1995)；Smith等人，Ophthalmology108，697-704(2001))、性别(女性中可能性高七倍：Klein等人，Ophthalmology 99，933-943(1992))及种族(白种人最易受影响)。额外危险因素可包括眼睛特征(诸如远视及浅色眼)以及心血管疾病及高血压。亦已证实遗传涉及疾病的发作进程(参见上文Hamdi&Kenney)。Numerous studies conducted have identified several risk factors for AMD, such as smoking, old age, family history (Milton, Am J Ophthalmol 88, 269 (1979); Mitchell et al., Ophthalmology 102, 1450-1460 (1995); Smith et al. , Ophthalmology 108, 697-704 (2001)), gender (seven times more likely in women: Klein et al., Ophthalmology 99, 933-943 (1992)) and race (Caucasians are most susceptible). Additional risk factors may include eye characteristics such as hyperopia and light-colored eyes, as well as cardiovascular disease and high blood pressure. Genetics has also been shown to be involved in the course of the disease (see Hamdi & Kenney, supra).
Acuity Pharmaceuticals及Sirna Therapeutics两个公司最近均已申请用于治疗AMD的分别抑制VEGF及VEGF-R1(Flt-1)的siRNA分子IND。这类分子分别称为Cand5抑制剂及027抑制剂。Both Acuity Pharmaceuticals and Sirna Therapeutics have recently applied for IND for siRNA molecules that inhibit VEGF and VEGF-R1 (Flt-1), respectively, for the treatment of AMD. Such molecules are called Cand5 inhibitors and 027 inhibitors, respectively.
微血管病症Microvascular Disorders
微血管病症包含主要影响微毛细管及淋巴管的广泛病状，且因此在直接外科手术介入的范畴外。微血管疾病可广泛分成血管痉挛、脉管炎及淋巴阻塞。此外，多种已知的血管病状具有针对其的微血管元素。Microvascular disorders encompass a broad range of conditions primarily affecting microcapillaries and lymphatic vessels, and are therefore outside the realm of direct surgical intervention. Microvascular disease can be broadly divided into vasospasm, vasculitis, and lymphatic obstruction. Furthermore, various known vascular conditions have microvascular elements directed against them.
·血管痉挛性疾病-血管痉挛性疾病为一组相对普通病状，其中由于未知原因周边血管收缩反射极敏感。此导致不适当的血管收缩及组织缺血，甚至达到组织损失的程度。虽然血管痉挛症状常与温度或振动器的使用相关，但其可能继发于其它病状。• Vasospasm disorders - Vasospasm disorders are a group of relatively common conditions in which for unknown reasons the peripheral vasoconstriction reflex is extremely sensitive. This results in inappropriate vasoconstriction and tissue ischemia, even to the point of tissue loss. Although vasospasm symptoms are often associated with temperature or vibrator use, they may be secondary to other conditions.
·脉管炎疾病-脉管炎疾病是涉及微循环中的主要炎症过程的疾病。脉管炎常为自体免疫或结缔组织病症的部分，且一般不受外科处理影响，而若症状严重，则需免疫抑制处理。• Vasculitic diseases - Vasculitic diseases are diseases that involve primarily inflammatory processes in the microcirculation. Vasculitis is often part of an autoimmune or connective tissue disorder and is generally unaffected by surgical management, although severe symptoms require immunosuppressive management.
·淋巴阻塞性疾病-下肢或上肢慢性肿胀(淋巴水肿)是周边淋巴阻塞的结果。此是具有大量原因的相对罕见病状，某些是天生的，某些是后天的。治疗的主要依靠是合身压缩外衣及使用间歇性压缩装置。• Lymphatic obstructive disease - Chronic swelling of the lower or upper extremities (lymphedema) is the result of peripheral lymphatic obstruction. This is a relatively rare condition with a number of causes, some congenital and some acquired. Treatment relies primarily on well-fitting compression garments and the use of intermittent compression devices.
与糖尿病相关的微血管病理Microvascular pathology associated with diabetes
糖尿病是失明的主导原因，截肢及阳痿的第一原因，且为最常发生的慢性儿童疾病之一。在美国，糖尿病亦为晚期肾病的主导原因，与其它肾病相比发病率达31％。糖尿病亦为占所有移植手术的22％的肾移植的最常见指征。Diabetes is the leading cause of blindness, the number one cause of amputations and impotence, and is one of the most common chronic childhood diseases. Diabetes is also the leading cause of end-stage renal disease in the United States, with an incidence rate of 31% compared with other renal diseases. Diabetes is also the most common indication for kidney transplantation accounting for 22% of all transplants.
一般而言，糖尿病并发症可广泛分为微血管或大血管疾病。微血管并发症包括神经病(神经损伤)、肾病(肾脏疾病)及视力障碍(例如视网膜病、青光眼、白内障及角膜病)。在视网膜、肾小球及神经滋养血管(vasa nervorum)中，类似的病理生理学特征表征糖尿病特异性微血管疾病。In general, diabetic complications can be broadly classified as microvascular or macrovascular disease. Microvascular complications include neuropathy (nerve damage), nephropathy (kidney disease), and visual disturbances (such as retinopathy, glaucoma, cataract, and corneal disease). In the retina, glomeruli, and vasa nerves, similar pathophysiological features characterize diabetes-specific microvascular disease.
与糖尿病相关的微血管病理是定义为可能发生在(例如)长期患糖尿病的人中的最小血管(毛细管)疾病。Diabetes-associated microvascular pathology is defined as disease of the smallest blood vessels (capillaries) that can occur, for example, in persons with long-standing diabetes.
血管壁变得异常厚，但很脆弱。因此，其渗出血液、漏出蛋白且减慢血液流过身体。The blood vessel walls become unusually thick, but fragile. Thus, it seeps blood, leaks protein and slows blood flow through the body.
临床及动物模型数据表明慢性高血糖是所有类型糖尿病性微血管疾病的主要引发因素。高血糖的持续时间及严重程度均与糖尿病性微血管疾病进程的程度及速率大大相关。虽然所有糖尿病性细胞均暴露于高含量血浆葡萄糖，但高血糖破坏仅限于发展细胞内高血糖的那些细胞类型(例如内皮细胞)。内皮细胞发展细胞内高血糖，因为其不同于其它多种细胞，当内皮细胞暴露于细胞外高血糖时其不能下调葡萄糖输送。该细胞内高血糖对发展糖尿病病理而言为必需且足够的，此藉由下列事实来证实：GLUT1葡萄糖转运体在培养于正常葡萄糖环境中的肾小球系膜细胞中的过度表达模拟糖尿病表型，从而诱发胶原蛋白IV型、胶原蛋白I的相同增加且使纤维结合蛋白基因表现为糖尿病性高血糖。Data from clinical and animal models suggest that chronic hyperglycemia is a major causative factor for all types of diabetic microvascular disease. Both the duration and severity of hyperglycemia are strongly correlated with the degree and rate of progression of diabetic microvascular disease. Although all diabetic cells are exposed to high levels of plasma glucose, hyperglycemic damage is restricted to those cell types that develop intracellular hyperglycemia (eg, endothelial cells). Endothelial cells develop intracellular hyperglycemia because, unlike many other cells, they are unable to downregulate glucose delivery when exposed to extracellular hyperglycemia. This intracellular hyperglycemia is both necessary and sufficient for the development of diabetic pathology, as evidenced by the fact that overexpression of the GLUT1 glucose transporter in mesangial cells cultured in a normoglucose environment mimics the diabetic expression type, thereby inducing the same increase in collagen type IV, collagen I and expressing the fibronectin gene as diabetic hyperglycemia.
异常内皮细胞功能：在糖尿病早期，在结构改变变得明显之前，高血糖引起血流异常及视网膜、肾小球及周边神经血管滋养血管的血管渗透性。据信血流及毛细管内压力的增加反映高血糖诱发的毛细管床的输出侧上氧化氮(NO)产生减少且可能反映对血管紧张素II的敏感性增加。由于毛细管内压力增加及内皮细胞功能障碍，故视网膜毛细管显示荧光素渗漏增加且肾小球毛细管具有增加的白蛋白排泄率(AER)。类似改变发生在周边神经的营养血管中。在糖尿病早期，增加的渗透性是可逆的；然而，随着时间发展，其变得不可逆。Abnormal endothelial function: In early diabetes, before structural changes become apparent, hyperglycemia causes abnormal blood flow and vascular permeability of the retina, glomerulus, and peripheral neurovascular vasa vasorum. Increases in blood flow and intracapillary pressure are believed to reflect hyperglycemia-induced decreased nitric oxide (NO) production on the output side of the capillary bed and possibly increased sensitivity to angiotensin II. Retinal capillaries show increased fluorescein leakage and glomerular capillaries have increased albumin excretion rate (AER) due to increased intracapillary pressure and endothelial cell dysfunction. Similar changes occur in the nutrient vessels of peripheral nerves. Early in diabetes, increased permeability is reversible; however, it becomes irreversible over time.
增加的血管壁蛋白蓄积Increased protein accumulation in blood vessel walls
糖尿病性微血管疾病的常见病理生理特征为血管内腔渐进变窄且最终阻塞，此导致患病组织的不当灌注及功能。早期高血糖诱发的微血管高血压及增加的血管渗透性藉由三个过程促成不可逆微血管阻塞：A common pathophysiological feature of diabetic microvascular disease is progressive narrowing and eventual occlusion of the vascular lumen, which results in improper perfusion and function of the diseased tissue. Early hyperglycemia-induced microvascular hypertension and increased vascular permeability contribute to irreversible microvascular occlusion through three processes:
·第一个过程是过碘酸雪夫氏(periodic acid-Schiff，PAS)阳性的含碳水化合物的血浆蛋白异常渗漏，该血浆蛋白沉积在毛细管壁中且可刺激血管周细胞(诸如周细胞及肾小球系膜细胞)以制造生长因子及细胞外基质。The first process is abnormal leakage of periodic acid-Schiff (PAS)-positive carbohydrate-containing plasma proteins that deposit in capillary walls and stimulate perivascular cells such as pericytes and glomerular mesangial cells) to produce growth factors and extracellular matrix.
·第二个过程是生长因子(诸如转化生长因子β1(TGF-β1))的外渗，此过程直接刺激细胞外基质组份的过度产生，且可诱发某些并发症相关的细胞类型的细胞凋亡。The second process is the extravasation of growth factors, such as transforming growth factor beta 1 (TGF-beta1), which directly stimulates the overproduction of extracellular matrix components and can induce certain complication-related cell types apoptosis.
·第三个过程是高血压诱发的对内皮细胞及支持细胞的病理基因表达的刺激作用，该细胞包括glut-1葡萄糖转运体、生长因子、生长因子受体、细胞外基质组份及可活化循环白细胞的黏着分子。观测到糖尿病性微血管疾病的严重程度的单方面减少另外伴随眼或肾动脉狭窄发生，与此概念一致。The third process is hypertension-induced stimulation of pathological gene expression in endothelial and Sertoli cells, which include the glut-1 glucose transporter, growth factors, growth factor receptors, extracellular matrix components, and activatable Adhesion molecules of circulating leukocytes. The observation that a unilateral reduction in the severity of diabetic microvascular disease was additionally accompanied by narrowing of the ocular or renal arteries was consistent with this concept.
微血管细胞损失及血管阻塞Microvascular cell loss and vascular occlusion
糖尿病性微血管内腔的渐进变窄及阻塞亦伴随微血管细胞损失。在视网膜中，糖尿病诱发米勒细胞(Müller cell)及神经节细胞、周细胞及内皮细胞的渐进式细胞死亡。在肾小球中，虽然衰退的肾功能与普遍毛细管阻塞及足细胞损失相关，但构成肾小球细胞损失基础的机理尚未可知。在血管滋养血管中，内皮细胞及周细胞退化发生，且这类微血管变化似乎先于糖尿病性周围神经病的发展。虽然糖尿病中神经轴突退化的多焦点分布构成微血管阻塞的病因，但藉由预防正常神经轴突修复及再生可促成高血糖诱发的神经营养素减少。The progressive narrowing and occlusion of diabetic microvascular lumen is also accompanied by the loss of microvascular cells. In the retina, diabetes induces progressive cell death of Müller cells as well as ganglion cells, pericytes and endothelial cells. In the glomerulus, the mechanisms underlying glomerular cell loss are unknown, although diminished renal function is associated with widespread capillary obstruction and loss of podocytes. In the vasa vasa, degeneration of endothelial cells and pericytes occurs, and such microvascular changes appear to precede the development of diabetic peripheral neuropathy. Although the multifocal distribution of axonal degeneration in diabetes constitutes the etiology of microvascular obstruction, hyperglycemia-induced neurotrophic depletion may be contributed by preventing normal axonal repair and regeneration.
糖尿病性微血管疾病的另一常见特征称为高血糖记忆，或正常葡萄糖稳定的后续时期内高血糖诱发的微血管改变的持续或发展。此现象的最显著实例是糖尿病狗的组织学正常眼睛中严重视网膜病的发展，其完全发生在2.5年高血糖后的2.5年正常化血糖时期内。高血糖诱发的所选基质基因转录的增加亦在活体内恢复血糖浓度正常后持续数周，且在经培养的内皮细胞中发生相对不明显但在品质上类似的高血糖诱发的所选基质基因转录的延期增加。Another common feature of diabetic microvascular disease is called hyperglycemic memory, or the persistence or development of hyperglycemia-induced microvascular changes during subsequent periods of normoglucose stabilization. The most striking example of this phenomenon is the development of severe retinopathy in the histologically normal eye of a diabetic dog, which occurs exclusively within the 2.5 year period of normalizing blood glucose after 2.5 years of hyperglycemia. Hyperglycemia-induced increases in transcription of selected matrix genes also persisted for weeks after normoglycemia in vivo, and a relatively inconspicuous but qualitatively similar hyperglycemia-induced increase in selected matrix genes occurred in cultured endothelial cells Increased delay in transcription.
其它信息参见＂Shared pathophysi ologic features ofmicrovascular complications of diabetes＂(Larsen：WilliamsTextbook of Endocrinology，第10版，Copyright 
微血管并发症不仅发生在明显糖尿病中，且亦归因于葡萄糖耐受性异常(IGT)。IGT的微血管并发症：神经病、视网膜病及肾微量蛋白尿。Microvascular complications not only occur in overt diabetes, but are also due to impaired glucose tolerance (IGT). Microvascular complications of IGT: neuropathy, retinopathy, and renal microalbuminuria.
糖尿病性神经病diabetic neuropathy
糖尿病性神经病是与糖尿病相关的神经病症(周围神经损伤)。这类病状常源自涉及支持神经(血管滋养血管)的小血管的糖尿病性微血管损伤。与糖尿病性神经病相关的相对普通病状包括第三神经麻痹(third nerve palsy)、单神经病、多发性单神经病、糖尿病性肌萎缩、疼痛性多发性神经病、自主神经病及胸腹神经病及最常见形式周围神经病(其主要影响脚及腿)。存在四个涉及糖尿病性神经病发展的因素：微血管疾病、晚期糖基化终点产物、蛋白激酶C及多元醇路径。Diabetic neuropathy is a neurological disorder (peripheral nerve damage) associated with diabetes. Such conditions often arise from diabetic microvascular damage involving the small blood vessels that support the nerves (vasa vasa). Relatively common conditions associated with diabetic neuropathy include third nerve palsy, mononeuropathy, multiple mononeuropathy, diabetic amyotrophy, painful polyneuropathy, autonomic and thoracoabdominal neuropathies and the most common forms of peripheral neuropathy Neuropathy (which primarily affects the feet and legs). There are four factors involved in the development of diabetic neuropathy: microvascular disease, advanced glycation end products, protein kinase C, and the polyol pathway.
糖尿病性神经病中的微血管疾病Microvascular disease in diabetic neuropathy
血管及神经疾病紧密相关且交织在一起。血管视正常神经功能而定，且神经视足够血流而定。微血管系统中的第一病理改变为血管收缩。当疾病发展时，神经元功能障碍与血管异常的发展(诸如毛细血管基膜增厚及内皮增生，其促成氧张力减少及低氧)紧密相关。神经元缺血为糖尿病性神经病的公认特征。血管扩张剂(例如血管紧张素转化酶抑制剂，α1拮抗剂)可导致神经元血流的实质改善以及神经传导速度的相应改善。因此，微血管功能障碍早在糖尿病中发生，与神经功能障碍的进程并行，且可足以支持糖尿病性神经病中观测到的结构、功能及临床改变的严重程度。周围神经病(腿)感觉运动神经病是糖尿病中腿部溃疡的发病机理的重要部分。Vascular and neurological diseases are closely related and intertwined. Blood vessels depend on normal nerve function, and nerves depend on adequate blood flow. The first pathological change in the microvasculature is vasoconstriction. As the disease progresses, neuronal dysfunction is closely associated with the development of vascular abnormalities such as thickening of the capillary basement membrane and endothelial hyperplasia, which contribute to decreased oxygen tension and hypoxia. Neuronal ischemia is a well-recognized feature of diabetic neuropathy. Vasodilators (eg, angiotensin-converting enzyme inhibitors,
神经病是一种随着时间发生在一半以上患有2型糖尿病的患者中的常见糖尿病并发症。神经传导研究证实神经病已存在于在糖尿病诊断期的10-18％患者中，此表明周围神经损伤发生在疾病早期且伴随较轻微血糖调节不良。神经病为糖尿病的早期临床标记的概念早在四十多年前已提出，且大部分研究报导IGT与神经病之间的联系。大部分患有IGT及相关神经病的患者具有对称的远程感觉多发性神经病，伴随显著神经痛。IGT神经病(Microvascular complications of impairedglucose tolerance-Perspectives in Diabetes，J.RobinsonSingleton，in Diabetes December 1，2003)在表型上类似于早期糖尿病性神经病，其亦引起感觉症状，包括疼痛及自主神经功能障碍。在对患有早期糖尿病性神经病的669名患者进行的调查中，感觉症状存在大于60％，阳痿存在接近40％，且其它自主神经涉及33％，但运动涉及的迹象仅为12％。这类临床发现表明早期显著涉及运载疼痛、温度及自主神经信号的小的无髓鞘神经纤维。根据皮肤活组织检查直接量化无髓鞘表皮内神经纤维，显示出患有与IGT及早期糖尿病相关的神经病的患者中存在类似纤维损失及改变的形态学。Neuropathy is a common diabetic complication that occurs over time in more than half of patients with
自主神经功能障碍、尤其勃起功能障碍及改变的心迷走神经反应是糖尿病中神经性损伤的常见早期特征。对IGT患者的工作亦表明流行性迷走神经自主神经障碍：单独研究已在多于年龄相符的血糖浓度正常对照受检者的IGT患者中发现运动后异常心率恢复、相对深呼吸而言迟钝的R-R时间间隔变化性及减少的呼出与呼入比率(迷走神经自主神经障碍的所有测量)。Autonomic dysfunction, especially erectile dysfunction, and altered cardiovagal responses are common early features of neuropathic damage in diabetes. Work with IGT patients also suggests prevalent vagal autonomic dysfunction: a separate study has found abnormal heart rate recovery after exercise, blunted R-R intervals relative to deep breathing in more IGT patients than age-matched euglycemic control subjects Variability and decreased exhalation to inhalation ratio (all measures of vagal autonomic dysfunction).
糖尿病中的神经损伤影响运动、感觉及自主神经纤维。运动神经病引起肌肉软弱、萎缩及局部麻痹。感觉神经病导致疼痛、压力及热的保护性感觉丧失。疼痛缺乏会导致脚部无感觉的多个问题，包括溃疡、未察觉的外伤及夏科氏神经性关节病。患者可能并不寻求治疗直至伤口加深。感觉及运动功能障碍的组合可引起患者置放异常压力于脚上，导致可能导致感染的外伤。自主交感神经病引起血管扩张及出汗减少，此结果导致尤其倾向于皮肤破裂以及微血管流动的功能改变的温暖、过度干燥脚。自主神经功能障碍(及真皮结构的去神经支配)亦导致皮肤完整性的损失，此为微生物入侵提供理想位点。神经病性脚并不自发溃烂；更确切言之，其为伴随神经病的特定形式外伤的组合。Nerve damage in diabetes affects motor, sensory, and autonomic fibers. Motor neuropathy causes muscle weakness, atrophy, and partial paralysis. Sensory neuropathy results in loss of protective sensation to pain, pressure, and heat. The lack of pain can lead to a variety of problems in the feet without sensation, including ulcers, unnoticed trauma, and Charcot's neuroarthropathy. Patients may not seek treatment until the wound has deepened. The combination of sensory and motor dysfunction can cause the patient to place abnormal pressure on the foot, resulting in trauma that can lead to infection. Autonomic sympathetic neuropathy causes vasodilation and decreased sweating, with the result that warm, excessively dry feet are especially prone to skin breakdown and functional changes in microvascular flow. Autonomic dysfunction (and denervation of dermal structures) also results in loss of skin integrity, which provides an ideal site for microbial invasion. Neuropathic feet do not ulcerate spontaneously; rather, they are a combination of specific forms of trauma that accompany neuropathy.
微血管功能障碍早在糖尿病中发生，与神经功能障碍的进程并行，且可足以支持糖尿病性神经病中观测到的结构、功能及临床改变的严重程度。Microvascular dysfunction occurs early in diabetes, parallels the progression of neurological dysfunction, and may be sufficient to support the severity of the structural, functional, and clinical changes observed in diabetic neuropathy.
晚期糖基化终产物-葡萄糖的细胞内含量升高会引起与蛋白的非酶共价键结合，此改变蛋白结构且破坏其功能。这类糖基化蛋白中的一些涉及糖尿病性神经病及糖尿病的其它长期并发症的病理学。Elevated intracellular levels of the end product of advanced glycation, glucose, cause non-enzymatic covalent binding to proteins, which alters protein structure and disrupts its function. Some of these glycosylated proteins are involved in the pathology of diabetic neuropathy and other long-term complications of diabetes.
蛋白激酶C(PKC)-PKC涉及糖尿病性神经病的病理学。葡萄糖的增加含量引起活化PKC的细胞内二酰甘油的增加。动物模型中的PKC抑制剂藉由增加神经元血流来增加神经传导速度。Protein kinase C (PKC) - PKC is involved in the pathology of diabetic neuropathy. Increased levels of glucose cause an increase in intracellular diacylglycerol that activates PKC. PKC inhibitors in animal models increase nerve conduction velocity by increasing neuronal blood flow.
感觉运动多发性神经病sensorimotor polyneuropathy
较长神经纤维比较短神经纤维更受影响，此是因为神经传导速度与神经长度成比例地减慢。在此综合征中，减少的感觉及反射的丧失首先发生在两侧脚趾，接着向上延伸。此常被描述为麻痹、感觉丧失、感觉迟钝及夜间疼痛的手套-袜样分布。疼痛可感觉如同燃烧、刺痛感觉、疼痛或迟钝。四肢麻木感觉是常见的。在早期罹患本体感觉(亦即腿在空间何处的感觉)丧失。当这类患者踏在异物(例如碎片)上时或当其因为不适合的鞋而形成茧时，其不能感觉到。因此，这类患者处于形成脚部及腿部溃疡及感染的危险中，此可导致截肢。类似地，这类患者可获得膝盖、踝或脚部的多处骨折，且形成夏科氏关节。运动功能的丧失导致脚趾的背屈挛缩，所谓槌状趾。这类挛缩不仅发生在脚部，亦发生在手部。Longer nerve fibers are more affected than shorter nerve fibers because nerve conduction velocity slows in proportion to nerve length. In this syndrome, decreased sensation and loss of reflexes occur first in the toes on both sides and then extend upward. This is often described as a glove-sock distribution of paralysis, sensory loss, dysesthesia, and nocturnal pain. Pain can feel like burning, tingling, aching, or dull. A numb feeling in the extremities is common. Loss of proprioception (ie the sense of where the leg is in space) is suffered early on. Such patients cannot feel when they step on foreign objects (such as debris) or when they form calluses from ill-fitting shoes. Thus, such patients are at risk of developing foot and leg ulcers and infections, which can lead to amputation. Similarly, such patients can have multiple fractures of the knee, ankle, or foot and develop Charcot joints. Loss of motor function results in a dorsiflexion contracture of the toe, a so-called hammer toe. This type of contracture occurs not only in the feet, but also in the hands.
自主神经病autonomic neuropathy
自主神经系统包含服务心脏、胃肠道及泌尿系统的神经。自主神经病可影响这类器官系统中的任一个。糖尿病中最常公认的自主神经功能障碍为体位性低血压或当患者站立时昏晕的不适感觉。在糖尿病性自主神经病的情形下，其是归因于用以适当调节心率及血管紧张性以保持血液连续且完全流过脑部的心脏及动脉的衰竭。此症状常伴随窦性呼吸变化(亦即正常呼吸下看见的心率的通常改变)的损失。当发现此两个存在时，亦存在心脏自主神经病。The autonomic nervous system includes nerves that serve the heart, gastrointestinal tract, and urinary system. Autonomic neuropathy can affect any of these organ systems. The most commonly recognized autonomic dysfunction in diabetes is orthostatic hypotension, or the uncomfortable feeling of fainting when the patient stands. In the case of diabetic autonomic neuropathy, it is due to failure of the heart and arteries that properly regulate heart rate and vascular tone to maintain a continuous and complete flow of blood through the brain. This symptom is often accompanied by a loss of sinus respiratory variability (ie, the usual changes in heart rate seen with normal breathing). When these two are found present, cardiac autonomic neuropathy is also present.
胃肠道的表现形式包括延迟的胃排空、胃瘫、恶心、气胀及腹泻。因为多名糖尿病患者口服治疗其糖尿病的药剂，所以这类药剂的吸收极大地受延迟的胃排空影响。当已存在正常或低血糖时，当饭前口服糖尿病药剂且药剂直至数小时或有时数天后方可吸收时，此可导致低血糖。小肠运动迟缓可引起细菌过度生长，在高血糖存在下变得更糟。此导致气胀、气体及腹泻。Gastrointestinal manifestations include delayed gastric emptying, gastroparesis, nausea, gas, and diarrhea. Because many diabetic patients take medications for their diabetes orally, the absorption of such medications is greatly affected by delayed gastric emptying. When normoglycemia or hypoglycemia is already present, this can lead to hypoglycemia when diabetes medications are taken orally before meals and the medication is not absorbed until hours or sometimes days later. Slow motility of the small intestine can cause bacterial overgrowth, which gets worse in the presence of high blood sugar. This results in bloating, gas and diarrhea.
泌尿系统症状包括尿频、尿急、尿失禁及尿滞留。另外，因为糖尿滞留，所以尿道感染时常发生。尿滞留可导致膀胱憩室、膀胱结石、返流性肾病。Urinary symptoms include urinary frequency, urgency, urinary incontinence, and urinary retention. In addition, urinary tract infections often occur because of the retention of diabetes. Urinary retention can lead to bladder diverticulum, bladder stones, reflux nephropathy.
脑部神经病brain neuropathy
当脑部神经受影响时，动眼神经(第三)神经病最为常见。动眼神经控制除侧直肌及上斜肌以外的移动眼睛的所有肌肉。其亦用以压缩瞳孔且打开眼睑。糖尿病性第三神经麻痹的发作常突然发生，以额骨或眼眶周疼痛起始且接着复视。除控制瞳孔大小的动眼神经外，由第三神经支配的所有动眼神经均可受到影响。虽然支配眼睛侧直肌(侧向移动眼睛)的第六神经(外展神经)亦常受到影响，但涉及第四神经滑车神经(支配上斜肌，其移动眼睛向下)却并不常见。胸或腰椎神经的单神经病可发生且导致类似心肌梗塞、胆囊炎或阑尾炎的疼痛综合征。糖尿病具有更高的压迫性神经病(诸如腕管综合征)发病率。Oculomotor (third) neuropathy is most common when nerves in the brain are affected. The oculomotor nerve controls all the muscles that move the eye except the rectus lateralis and superior oblique. It also acts to compress the pupil and open the eyelid. The onset of diabetic third nerve palsy often occurs suddenly, starting with frontal or periorbital pain and followed by diplopia. All oculomotor nerves innervated by the third nerve can be affected except the one that controls pupil size. Although the sixth nerve (abducens nerve), which innervates the lateral rectus oculi muscle (which moves the eye sideways), is also commonly affected, involvement of the fourth nerve trochlear nerve (which innervates the superior oblique muscle, which moves the eye downward) is less common. Mononeuropathy of the thoracic or lumbar nerves can occur and result in a pain syndrome resembling myocardial infarction, cholecystitis, or appendicitis. Diabetes has a higher incidence of compressive neuropathies such as carpal tunnel syndrome.
糖尿病性肢体缺血及糖尿病性足部溃疡Diabetic Limb Ischemia and Diabetic Foot Ulcer
糖尿病及压力可削弱微血管循环且导致下肢上的皮肤变化，此继而可导致溃疡形成及随后感染。微血管变化导致肢体肌肉微血管病以及发生周围组织缺血的倾向及对缺血事件的血管生成补偿反应减少。微血管病理学加剧周围血管疾病(PVD)(或周围动脉疾病(PAD)或下肢动脉疾病(LEAD)-大血管并发症-归因于动脉粥样硬化的腿中动脉变窄)。PVD较早发生在糖尿病中，其更严重且分布广泛，且常包括影响腿、眼及肾的间发微循环问题。Diabetes and stress can impair microvascular circulation and cause skin changes on the lower extremities, which in turn can lead to ulceration and subsequent infection. Microvascular changes lead to limb muscle microangiopathy as well as a predisposition to develop peripheral tissue ischemia and a reduced angiogenic compensatory response to ischemic events. Microvascular pathology is exacerbated by peripheral vascular disease (PVD) (or peripheral arterial disease (PAD) or lower extremity arterial disease (LEAD) - a macrovascular complication - narrowing of the middle leg arteries due to atherosclerosis). PVD occurs earlier in diabetes, is more severe and widespread, and often includes intermittent microcirculatory problems affecting the legs, eyes, and kidneys.
足部溃疡及坏疽为常见PAD联合病状。具有削弱感觉的并发周围神经病使脚易患外伤、溃疡及感染。糖尿病中PAD的进程中混有诸如周围神经病及脚与下肢对疼痛及外伤无感觉的共病。在削弱的循环及削弱的感觉下，溃疡及感染发生。骨髓炎及坏疽的发展可迫使截肢。Foot ulcers and gangrene are common PAD symptoms. Concurrent peripheral neuropathy with diminished sensation predisposes the foot to trauma, ulceration, and infection. The course of PAD in diabetes is mixed with comorbidities such as peripheral neuropathy and insensitivity to pain and trauma in the feet and lower extremities. With impaired circulation and impaired sensation, ulcers and infections occur. The development of osteomyelitis and gangrene can force amputation.
患有糖尿病者比非糖尿病患者高达25倍地更有可能遭受下肢截肢，此说明预防足部溃疡及随后肢体丧失的需要。糖尿病足部溃疡可能不仅与PAD联合发生，亦可能与神经病、静脉功能不全(静脉曲张)、外伤及感染联合。PAD促成产生或加速溃疡的这类其它病状。足部溃疡未必代表PAD发展，因为足部溃疡可能在适当临床周围动脉灌注存在下发生。基于患者的研究表明在患有周围神经病的糖尿病患者中足部溃疡的危险增加且足底压力高。在南部威斯康星(Wisconsin)的基于人口的研究中，足或踝上溃疡或伤口病史的发病率为15％的所有糖尿病患者。年龄小于30岁的经诊断糖尿病个体的发病率较高，男性中(16％)略高于女性中(13％)，且经胰岛素治疗的糖尿病患者中(17％)高于未接受胰岛素的患者中(10％)。发病率随年龄而增加，尤其在年龄大于30岁的经诊断糖尿病患者中。在来自欧洲的患者研究中，糖尿病患者中足部溃疡的发病率为50岁以下患者中3％、60岁以下患者中7％及80岁以下患者中14％。70岁男性中发病率比70岁女性中发病率大。People with diabetes are up to 25 times more likely to suffer lower limb amputation than non-diabetics, illustrating the need to prevent foot ulcers and subsequent limb loss. Diabetic foot ulcers may occur not only in combination with PAD, but also in combination with neuropathy, venous insufficiency (varicose veins), trauma, and infection. PAD contributes to or accelerates these other conditions that produce ulcers. Foot ulcers do not necessarily represent the development of PAD, as foot ulcers can occur in the presence of appropriate clinical peripheral arterial perfusion. Patient-based studies suggest an increased risk of foot ulcers and high plantar pressure in diabetic patients with peripheral neuropathy. In a population-based study in southern Wisconsin, a history of foot or ankle ulcers or wounds occurred in 15% of all diabetic patients. Diagnosed diabetic individuals younger than 30 years had a higher incidence, slightly higher in males (16%) than females (13%), and higher in insulin-treated diabetics (17%) than those who did not receive insulin Medium (10%). Incidence increases with age, especially among diagnosed diabetic patients older than 30 years. In a study of patients from Europe, the incidence of foot ulcers in diabetic patients was 3% of those under 50 years of age, 7% of those under 60 years of age, and 14% of those under 80 years of age. The incidence rate among 70-year-old men is higher than that among 70-year-old women.
在糖尿病患者中，足部缺血及感染为严重甚至致命的事件；然而，神经病却为最难以治疗的病状。关于糖尿病脚的临床及病理学表现的所有方面的医学及外科文献占大多数。单独或组合且严重程度不同的神经病、血管病、视网膜病及肾病可影响糖尿病脚的治疗。In diabetic patients, foot ischemia and infection are serious and even fatal events; however, neuropathy is the most difficult condition to treat. The medical and surgical literature predominates on all aspects of the clinical and pathological manifestations of the diabetic foot. Neuropathy, vascular disease, retinopathy, and nephropathy, alone or in combination and of varying severity, can affect the management of the diabetic foot.
每年，82,000例截肢术在患有糖尿病的患者中实施。这类截肢术多半在老龄人口中实施。糖尿病导致的截肢可起因于多个病源，包括足部溃疡、缺血、静脉性腿部溃疡(亦即，继发于静脉回流的溃疡)及脚跟溃疡(亦即，脚跟中未经治疗的褥疮导致的溃疡)。这类截肢多半源自溃疡。患有糖尿病的患者中，足部溃疡的发病率为12％。此外，患有1型糖尿病的患者中下肢溃疡的20年累积发生率为9.9％。糖尿病诱发的截肢导致39％至68％的五年死亡率且与增加的额外截肢的危险相关联。与无溃疡的患者相比，患有糖尿病性足部溃疡的患者的住院长度长约60％。Each year, 82,000 amputations are performed on patients with diabetes. Most of these amputations are performed in the elderly population. Amputations due to diabetes can arise from multiple etiologies, including foot ulcers, ischemia, venous leg ulcers (ie, ulcers secondary to venous return), and heel ulcers (ie, untreated pressure sores in the heel resulting in ulcers). Most of these amputations result from ulcers. Among patients with diabetes, the incidence of foot ulcers is 12%. Furthermore, the 20-year cumulative incidence of lower extremity ulcers among patients with
糖尿病性神经病削弱视健康C纤维疼痛感受器功能而定，且引起响应疼痛刺激的局部血管扩张的神经轴突反射。此病状进一步损害存在于糖尿病性神经病脚部压力病状(诸如损伤或炎症)中的血管扩张反应。此削弱可部分解释虽然下肢血管成功再形成但糖尿病性神经病脚部某些溃疡为何治愈缓慢或无法治愈的原因。Diabetic neuropathy impairs axonal reflexes dependent on healthy C fiber nociceptor function and elicits local vasodilation in response to painful stimuli. This condition further impairs the vasodilatory response present in diabetic neuropathic foot pressure conditions such as injury or inflammation. This attenuation may partly explain why some ulcers in diabetic neuropathic feet heal slowly or fail to heal despite successful revascularization of the lower extremities.
因此，糖尿病性足部溃疡的最常见病因路径可经鉴别为神经病(感觉丧失)、畸形(例如突出跖骨头)及外伤(例如，不合脚的鞋袜)的组合。Thus, the most common etiological pathway for diabetic foot ulcers can be identified as a combination of neuropathy (loss of sensation), deformity (eg, protruding metatarsal heads), and trauma (eg, ill-fitting footwear).
大部分外科医生偏爱实施腘动脉或胫动脉旁路手术，因为与更近端程序相比保肢及肢体开放率较差。若腘动脉或胫动脉旁路手术不能恢复明显脚脉搏，则据报导脚旁路手术为患有糖尿病及缺血性脚伤的患者提供更耐久且更有效的保肢程序。患有糖尿病的患者中，甚至大面积多段阻塞疾病不会对保肢造成妨碍。虽然严重伤口并发症可具有严重结果，但其在脚旁路移植后并不常见。适当控制预先存在的足部感染及谨慎移植开道已显示出有效避免其它并发症。下肢中血管成形术日渐得到利用。然而，必须强调为使血管成形术有效，若欲使更近端血管成形术成功，则末端血管或供养血管必须张开。Most surgeons prefer popliteal or tibial artery bypass because of poorer limb salvage and limb access rates compared with more proximal procedures. If popliteal or tibial artery bypass surgery fails to restore a significant foot pulse, foot bypass surgery has been reported to provide a more durable and effective limb-salvage procedure for patients with diabetes and ischemic foot injuries. In patients with diabetes, even extensive multisegmental obstructive disease does not impede limb salvage. Although serious wound complications can have serious consequences, they are uncommon after foot bypass grafts. Proper control of pre-existing foot infections and careful grafting have been shown to be effective in avoiding other complications. Angioplasty in the lower extremities is increasingly utilized. However, it must be emphasized that for angioplasty to be effective, the distal or feeding vessels must be dilated if more proximal angioplasty is to be successful.
虽然可在某些患者中处理糖尿病性溃疡/肢体病理(藉由清创手术、抗生素治疗、使用刺激粒状组织的制剂(新颖胶原蛋白及血管生成)及减少伤口中细菌负荷)，但可更优选地治疗这类病状和/或减轻症状的药物组合物将为有益的。While diabetic ulcer/limb pathology can be managed in some patients (by debridement surgery, antibiotic therapy, use of agents that stimulate granular tissue (novel collagen and angiogenesis) and reduce bacterial load in the wound), it may be more preferred Pharmaceutical compositions that effectively treat and/or alleviate symptoms of such conditions would be beneficial.
其它信息参见American Journal of Surgery，第187卷，第5期第1增补版，2004年5月1日，Copyright
糖尿病中冠状动脉微血管功能障碍Coronary microvascular dysfunction in diabetes
根据先前实验研究及尸体解剖已熟知糖尿病中的组织病理学与微循环功能障碍之间的相互关系，其中常发现基膜增厚、血管周围纤维化、血管变薄及毛细管出血。虽然一篇近期论文证实了病理学与眼部微血管功能障碍之间的相互关系(Am J Physiol 2003；285)，但仍难以在活体内证实这类资料。然而，大量临床研究表明不仅显性糖尿病可影响冠状动脉微循环，削弱的代谢控制亦可影响冠状动脉微循环(Hypert Res 2002；25：893)。Werner提到Sambuceti等人的重要论文(Circulation 2001；104：1129)，该论文说明在成功地重新打开梗死相关动脉后患者的微血管功能障碍持续存在，且可解释这类患者中增加的心脏血管发病率及死亡率。大量急性再灌注研究不断证实发病率及死亡率与梗死相关动脉自身的重新打开无关，而更取决于TIMI流量+/-心肌呈色(myocardial blush)(Stone 2002；FeldmannCirculation 2003)。Herrmann表明冠状动脉微循环的完整性可能为此文中最重要的临床及预后因子(Circulation 2001)。保护装置的中性影响(TIMI流量、ST分辨率或MACE并无相关变化)可表明微循环的功能削弱为预后的主要决定因素。不断增加的证据亦表明冠状动脉微血管功能障碍在非阻塞性CAD中起主要作用。冠状动脉内皮细胞功能障碍仍为这类患者中的强预后因子。The correlation between histopathology and microcirculatory dysfunction in diabetes is well known from previous experimental studies and autopsies, where basement membrane thickening, perivascular fibrosis, vascular thinning, and capillary hemorrhage are often found. Although a recent paper demonstrated a correlation between pathology and ocular microvascular dysfunction (
糖尿病性肾病(糖尿病患者的肾功能障碍)Diabetic nephropathy (kidney dysfunction in people with diabetes)
糖尿病性肾病包含微量白蛋白尿(微血管疾病影响)、蛋白尿及ESRD。糖尿病为肾衰竭的最常见病因，占新病例的40百分比以上。甚至当药物及饮食能够控制糖尿病时，糖尿病亦可导致肾病及肾衰竭。大部分患有糖尿病的人不会形成足够严重而引起肾衰竭的肾病。在美国，约一千六百万人患有糖尿病，且约100,000人患有作为糖尿病的结果的肾衰竭。Diabetic nephropathy includes microalbuminuria (affected by microvascular disease), proteinuria, and ESRD. Diabetes is the most common cause of kidney failure, accounting for more than 40 percent of new cases. Even when drugs and diet can control diabetes, diabetes can lead to kidney disease and kidney failure. Most people with diabetes do not develop kidney disease severe enough to cause kidney failure. In the United States, about 16 million people have diabetes, and about 100,000 people have kidney failure as a result of diabetes.
糖尿病性视网膜病diabetic retinopathy
在糖尿病状态下，高血糖导致视网膜血流量减少、视网膜高渗透性、感光体神经传导的延迟及视网膜神经元细胞死亡。在短期糖尿病中，已于视网膜内核层内鉴别神经元细胞死亡。具体地说，细胞凋亡被限定于神经胶质细胞(诸如米勒细胞及星形胶质细胞)且显示出其发生在STZ诱发的糖尿病大鼠模型中的一个月糖尿病内。这类事件的病因是多因子的，包括二酰甘油/PKC路径的活化、氧化应力及非酶糖基化。这类事件的组合使得视网膜含氧量低，且最终导致糖尿病性视网膜病的形成。视网膜缺血与糖尿病性视网膜的早期变化之间的一种可能关系为低氧诱发的生长因子(诸如VEGF)的产生。低氧反应的主要调节因子已鉴别为低氧诱发性因子-1(HIF-1)，其控制调节细胞增殖及血管生成的基因。先前研究已证实HIF-1泛素化的抑制导致与低氧反应性组件(HRE)结合及VEGF mRNA产生。In the diabetic state, hyperglycemia leads to decreased retinal blood flow, retinal hyperpermeability, delayed photoreceptor nerve conduction, and retinal neuronal cell death. In short-term diabetes, neuronal cell death has been identified within the inner retinal layer. Specifically, apoptosis was restricted to glial cells such as Müller cells and astrocytes and was shown to occur within one month of diabetes in the STZ-induced diabetic rat model. The etiology of such events is multifactorial and includes activation of the diacylglycerol/PKC pathway, oxidative stress, and nonenzymatic glycosylation. The combination of these events makes the retina hypoxic and ultimately leads to the development of diabetic retinopathy. One possible relationship between retinal ischemia and early changes in the diabetic retina is the production of hypoxia-induced growth factors such as VEGF. A master regulator of the hypoxic response has been identified as hypoxia-inducible factor-1 (HIF-1), which controls genes that regulate cell proliferation and angiogenesis. Previous studies have demonstrated that inhibition of HIF-1 ubiquitination leads to association with the hypoxia-responsive element (HRE) and VEGF mRNA production.
糖尿病性视网膜病是定义为藉由慢性高血糖引起的渐进式视网膜脉管结构功能障碍。糖尿病性视网膜病的主要特征包括脉管动脉瘤、视网膜出血、视网膜脂质渗出物、棉絮斑、毛细管无灌注、黄斑水肿及新血管生成。相关特征包括玻璃体出血、视网膜脱离、新生血管性青光眼、早期白内障及脑神经麻痹。Diabetic retinopathy is defined as progressive retinal vasculature dysfunction caused by chronic hyperglycemia. The main features of diabetic retinopathy include vascular aneurysms, retinal hemorrhages, retinal lipid exudates, cotton wool spots, capillary non-perfusion, macular edema, and neovascularization. Associated features include vitreous hemorrhage, retinal detachment, neovascular glaucoma, early cataract, and cranial nerve palsy.
在美国，一千六百万人患有1型及2型糖尿病。80％的1型患者在15年内形成糖尿病性视网膜病，而84％的2型糖尿病患者在19年内形成视网膜病。这类数字为针对眼部新血管结构疾病的治疗剂建立了重要市场。糖尿病性视网膜病的发展具有时间依赖性。虽然最佳应控制血糖，但可预期长年患有疾病的患者最终将形成某一形式的视网膜病。国家预防失明组织(The National Society to Prevent Blindness)估计，在美国有四百万至六百万糖尿病患者患有糖尿病性视网膜病。经评估的增生性糖尿病性视网膜病及糖尿病性黄斑水肿新病例的年发生率分别为65,000及75,000，而发病率分别为700,000及500,000。在美国，糖尿病性视网膜病每年引起12,000至24,000个失明新病例。视网膜病应藉由外科手术方法来治疗，此方法有效减少严重视力损失，但视网膜的经激光部分不可逆地遭到破坏。现无可采用的药物治疗。In the United States, 16 million people have
主要影响毛细管的微血管疾病(糖尿病)藉由破坏结膜、视网膜及中枢神经系统中的脉管结构来影响眼睛。患者可存在长期球结膜注射的病史以及虽然食欲比正常食欲大(多食)、异常口渴(剧渴症)及异常频繁排尿(多尿)但重量减轻的全身性疾病。Microvascular disease (diabetes) that primarily affects capillaries affects the eye by destroying vascular structures in the conjunctiva, retina, and central nervous system. Patients may have a history of long-term bulbar conjunctival injections and systemic disease with weight loss despite a greater than normal appetite (hyperphagia), abnormal thirst (hyperdipsia), and abnormally frequent urination (polyuria).
继发自不稳定血糖的波动视力为常见眼部标记。晶状体内的肿胀导致大的突然折射改变以及早期白内障形成。视力变化将视疾病的严重程度及阶段而定。Fluctuating vision secondary to unstable blood sugar is a common ocular sign. Swelling within the lens leads to large sudden refractive changes and early cataract formation. Vision changes will depend on the severity and stage of the disease.
在视网膜中，小动脉及毛细管变弱可导致视网膜内点及斑点出血、渗出物、视网膜内微血管异常(IRMA)、脉管动脉瘤、水肿及棉絮梗死的特征表像。增生性糖尿病性视网膜病为血管严重损害的结果且可视作神经盘新血管生成(NVD)、神经盘外新血管生成(NVE)及虹膜新血管生成(NVI或虹膜红变)。神经学并发症包括第三、第四及第六脑神经麻痹以及糖尿病性视乳头炎及面神经麻痹。In the retina, weakened arterioles and capillaries can lead to the characteristic appearance of intraretinal point and spot hemorrhages, exudates, intraretinal microvascular abnormalities (IRMA), vascular aneurysms, edema, and cotton wool infarction. Proliferative diabetic retinopathy is the result of severe damage to blood vessels and can be seen as neural disc neovascularization (NVD), neural extradiscal neovascularization (NVE), and iris neovascularization (NVI or iridosis). Neurological complications included third, fourth and sixth cranial nerve palsies as well as diabetic papillitis and facial nerve palsies.
糖尿病为共享葡萄糖失耐之一组遗传影响的疾病。其特征为作为胰岛素缺乏或功能障碍或细胞胰岛素受体缺乏或功能障碍结果的代谢调节病症。Diabetes is a group of diseases that share a genetic influence of glucose intolerance. It is characterized as a disorder of metabolic regulation as a result of insulin deficiency or dysfunction or cellular insulin receptor deficiency or dysfunction.
涉及山梨糖醇形成的生物化学在周细胞的破坏中起作用，而周细胞为支撑血管内皮的细胞。当支撑性周细胞毁坏时，毛细管内皮遭到损害，导致血管血液、蛋白及脂质渗漏。与葡萄糖负荷的增浓血液组合，此产生血管功能不全、毛细管无灌注、视网膜低氧、改变的结构及减少的功能。对在视网膜新血管生成起源中起作用的血管增生因子的形成及释放了解不足。Biochemistry involving sorbitol formation plays a role in the destruction of pericytes, the cells that support the vascular endothelium. When the supporting pericytes are destroyed, the capillary endothelium is damaged, resulting in blood, protein, and lipid leakage from the vessel. Combined with glucose-loaded thickened blood, this produces vascular insufficiency, capillary non-perfusion, retinal hypoxia, altered structure, and reduced function. The formation and release of angiogenic factors that play a role in the origin of retinal neovascularization are poorly understood.
在药物控制后，经数周至数月的过程，糖尿病的大部分非视力危险续发症减轻。在存在大的折射变化的情形下，患者可需要暂时眼镜处方直至折射稳定。当视网膜病威胁黄斑或当新血管增生时，患者可求助于激光凝固法。糖尿病性视网膜病研究(DRS)已最终证实全视网膜光凝术成功地减小了高危患者罹患严重视力损失的危险。其将高危特征定义为：(1)大小为神经盘直径的四分之一至三分之一的视神经盘新血管生成(NVD)及(2)具有任何玻璃体出血的视神经盘外新血管生成(NVE)。After drug control, most of the non-visual risk complications of diabetes are alleviated over the course of weeks to months. In situations where there are large changes in refraction, the patient may require a temporary eyeglass prescription until the refraction stabilizes. When retinopathy threatens the macula or when new blood vessels grow, patients may turn to laser photocoagulation. The Diabetic Retinopathy Study (DRS) has conclusively demonstrated that panretinal photocoagulation successfully reduces the risk of severe vision loss in high-risk patients. It defines high-risk features as: (1) optic disc neovascularization (NVD) that is one-quarter to one-third the size of the disc diameter and (2) extra-optic disc neovascularization with any vitreous hemorrhage ( NVE).
糖尿病性黄斑水肿(DME)Diabetic Macular Edema (DME)
DME为糖尿病性视网膜病的并发症，其为影响视网膜血管的疾病。糖尿病性视网膜病导致视网膜中的多种异常，包括视网膜增厚及水肿、出血、血流受阻、体液自血管过度渗透及最终阶段中的异常血管生长。此血管生长可导致大出血及严重视网膜破坏。当糖尿病性视网膜病的血管渗漏引起黄斑肿胀时，将其称为DME。DME的主要症状为中心视力的损失。与DME相关的危险因素包括控制不足的血糖含量、高血压、引起体液滞留的异常肾功能、高胆固醇含量及其它一般全身性因素。DME is a complication of diabetic retinopathy, a disease that affects blood vessels in the retina. Diabetic retinopathy results in a variety of abnormalities in the retina, including retinal thickening and edema, hemorrhage, obstruction of blood flow, hyperinfiltration of fluid from blood vessels and, in the final stage, abnormal blood vessel growth. This blood vessel growth can lead to massive hemorrhage and severe retinal damage. When the leaky blood vessels in diabetic retinopathy cause swelling in the macula, it's called DME. The main symptom of DME is loss of central vision. Risk factors associated with DME include poorly controlled blood glucose levels, high blood pressure, abnormal kidney function causing fluid retention, high cholesterol levels, and other general systemic factors.
根据世界卫生组织(the World Health Organization)，糖尿病性视网膜病为工作年龄成人失明的主要病因及糖尿病患者视力损失的主要病因。美国糖尿病协会(The American Diabetes Ass ociation)报导美国存在约一千八百万糖尿病患者且美国每年新诊断出的糖尿病病例为约一百三十万个。美国防盲协会(Prevent Blindness America)及美国眼科机构(the National Eye Institute)估计在美国有超过五百三十万18岁或18岁以上的人患有糖尿病性视网膜病，包括约500,000个患有DME的人。CDC估计每年美国DME新病例为约75,000个。According to the World Health Organization, diabetic retinopathy is the leading cause of blindness in working-age adults and the leading cause of vision loss in people with diabetes. The American Diabetes Association reports that there are approximately 18 million diabetics in the United States and that approximately 1.3 million new cases of diabetes are diagnosed each year in the United States. Prevent Blindness America and the National Eye Institute estimate that more than 5.3 million
其它神经病other neuropathy
除糖尿病外，神经病的常见病因为带状疱疹感染、慢性或急性外伤(包括外科手术)及各种神经毒素。作为周围神经癌症的直接结果(例如被肿瘤压缩)及多种化学疗法药物的副作用，神经痛在癌症中为常见的。In addition to diabetes, common causes of neuropathy are herpes zoster infection, chronic or acute trauma (including surgery), and various neurotoxins. Neuralgia is common in cancer as a direct result of peripheral nerve cancer (eg, compression by tumors) and as a side effect of many chemotherapy drugs.
微血管疾病-血管及神经疾病紧密相关且交织在一起。血管视正常神经功能而定，且神经视足够血流而定。微血管系统中的第一病理改变为血管收缩。当疾病发展时，神经元功能障碍与血管异常的发展(诸如毛细管基膜增厚及内皮增生，其促成氧张力减少及低氧)紧密相关。血管扩张剂(例如血管紧张素转化酶抑制剂，α1拮抗剂)可导致神经元血流量的实质改善以及神经传导速度的相应改善。Microvascular Disease - Vascular and neurological diseases are closely related and intertwined. Blood vessels depend on normal nerve function, and nerves depend on adequate blood flow. The first pathological change in the microvasculature is vasoconstriction. As the disease progresses, neuronal dysfunction is closely associated with the development of vascular abnormalities such as thickening of the capillary basement membrane and endothelial hyperplasia, which contribute to decreased oxygen tension and hypoxia. Vasodilators (eg, angiotensin-converting enzyme inhibitors,
临床表现形式clinical manifestations
神经病影响所有周围神经：疼痛纤维、运动神经元、自主神经。因为所有器官及系统均受到支配，所以神经病必定可影响所有器官及系统。虽然存在基于受影响的器官系统及成员的若干不同综合征，但这类综合征决非唯一的。患者可患有感觉运动及自主神经病或任何其它组合。Neuropathy affects all peripheral nerves: pain fibers, motor neurons, autonomic nerves. Since all organs and systems are innervated, neuroses must affect all organs and systems. Although several different syndromes exist based on the organ system and members affected, this class is by no means unique. Patients may suffer from sensorimotor and autonomic neuropathy or any other combination.
虽然预先了解神经病的代谢病因，但以中断这类病理进程为目标的治疗受副作用及功效缺乏限制。因此，治疗具有症状性且不解决根本问题。针对藉由感觉运动神经病引起的疼痛的药剂包括三环抗抑制剂(TCA)、血清素再吸收抑制剂(SSRI)及抗癫痫药物(AED)。这类药剂均无法逆转导致糖尿病性神经病的病理进程且亦无法改变疾病的残酷过程。因此，能更优选地治疗这类病状和/或减轻症状的药物组合物为适用的。Despite advance knowledge of the metabolic etiology of neuropathy, treatments aimed at interrupting such pathological processes are limited by side effects and lack of efficacy. Therefore, treatment is symptomatic and does not address the underlying problem. Agents targeting pain caused by sensorimotor neuropathy include tricyclic antiinhibitors (TCAs), serotonin reuptake inhibitors (SSRIs) and antiepileptic drugs (AEDs). None of these agents can reverse the pathological process leading to diabetic neuropathy nor alter the brutal course of the disease. Accordingly, pharmaceutical compositions that more preferably treat and/or alleviate symptoms of such conditions are suitable.
其它视网膜病other retinopathy
视网膜微血管病(AIDS视网膜病)Retinal microvascular disease (AIDS retinopathy)
视网膜微血管病在100％AIDS患者中可见到。其特征在于视网膜内出血、脉管动脉瘤、罗斯氏斑点(Roth spot)、棉絮斑(神经纤维层的微梗死)及血管周覆盖。虽然据信视网膜病归因于循环免疫复合物、细胞毒性物质局部释放、异常血液流变学及内皮细胞感染HIV，但其病因未知。AIDS视网膜病现为常见的，使得无糖尿病或高血压但处于HIV危险中的患者应提示医师考虑病毒测试。虽然对AIDS视网膜病并无特定治疗，但其持续存在可促使医师复查HIV疗法的功效及患者顺应性。Retinal microangiopathy is seen in 100% of AIDS patients. It is characterized by intraretinal hemorrhages, vascular aneurysms, Roth spots, cotton wool spots (microinfarcts of the nerve fiber layer), and perivascular coverage. Although retinopathy is believed to be due to circulating immune complexes, local release of cytotoxic substances, abnormal hemorheology, and HIV infection of endothelial cells, its etiology is unknown. AIDS retinopathy is now common enough that patients without diabetes or hypertension but at risk for HIV should prompt physicians to consider viral testing. Although there is no specific treatment for AIDS retinopathy, its persistence may prompt physicians to review the efficacy of HIV therapy and patient compliance.
骨髓移植(BMT)视网膜病Bone Marrow Transplantation (BMT) Retinopathy
骨髓移植视网膜病首先在1983年报导。虽然其通常发生在六个月内，但其亦可发生在BMT后迟至62个月。诸如糖尿病及高血压的危险因素可藉由加剧缺血性微血管病来推进BMT视网膜病的发展。BMT视网膜病的发展对年龄、性别或种族的偏好未知。患者呈现视力减退和/或视场缺陷。后段发现通常为两侧且对称的。临床表现形式包括多个棉絮斑、毛细管扩张、脉管动脉瘤、黄斑水肿、硬渗出物及视网膜出血。荧光素血管摄影术证实毛细管无灌注且脱落、视网膜内微血管异常、脉管动脉瘤及黄斑水肿。虽然BMT视网膜病的精确病因尚未说明，但其似乎受若干因素影响：环孢霉素毒性、全身放射(TBI)及化学治疗剂。环孢霉素为一种抑制移植物与宿主的免疫反应的强免疫调节剂。其可导致内皮细胞损害及神经副作用，且结果，已提出其为BMT视网膜病的病因。然而，BMT视网膜病可在缺乏环孢霉素使用下形成，且环孢霉素尚未显示出在自体或同源骨髓接受者中引起BMT视网膜病。因此，环孢霉素似乎并非BMT视网膜病的唯一病因。亦暗示全身放射(TBI)为BMT视网膜病的病因。放射会损害视网膜微血管系统且导致缺血性异种移植血管病。诸如放射总剂量及放射与骨髓切除之间的时间间隔的变量似乎为重要的。然而，BMT视网膜病可发生在未接受TBI的患者中，且BMT视网膜病在接收类似剂量放射的实体器官移植接受者中并未观测到。因此，虽然TBI并非唯一病因，但其在BMT视网膜病发生中为另一起作用因素。已提出化学治疗剂为BMT视网膜病中潜在起作用的因素。诸如顺铂(cisplatin)、卡莫司汀(carmustine)及环磷酰胺的药物可引起眼部副作用，包括视乳头水肿、视神经炎、视场缺陷及皮质盲。已表明这类化学治疗剂可使患者易遭受放射引起的视网膜破坏且增强放射的不利影响。一般而言，患有BMT视网膜病的患者具有好的预后性。视网膜病常在停止或减少环孢霉素剂量后二至四个月内减轻。在一报导中，69％患者经历视网膜探测的完全消退，且46％患者完全恢复其基本视力。因为BMT视网膜病具有良好预后性及相对无渐进性性质，所以常无需主动干预。Bone marrow transplant retinopathy was first reported in 1983. Although it usually occurs within six months, it can occur as late as 62 months after BMT. Risk factors such as diabetes and hypertension can promote the development of BMT retinopathy by exacerbating ischemic microangiopathy. The age, gender, or race preference for the development of BMT retinopathy is unknown. Patients present with decreased vision and/or visual field defects. Posterior findings are usually bilateral and symmetrical. Clinical manifestations include multiple cotton wool spots, telangiectasia, vascular aneurysms, macular edema, hard exudates, and retinal hemorrhages. Fluorescein angiography demonstrated capillary nonperfusion and detachment, intraretinal microvascular abnormalities, vascular aneurysms, and macular edema. Although the precise etiology of BMT retinopathy has not been elucidated, it appears to be influenced by several factors: cyclosporine toxicity, total body radiation (TBI), and chemotherapeutic agents. Cyclosporine is a potent immunomodulator that suppresses the immune response of the graft and the host. It can cause endothelial cell damage and neurological side effects, and as a result, it has been proposed as the cause of BMT retinopathy. However, BMT retinopathy can develop in the absence of cyclosporine use, and cyclosporine has not been shown to cause BMT retinopathy in autologous or syngeneic bone marrow recipients. Thus, cyclosporine does not appear to be the only cause of BMT retinopathy. Total body radiation (TBI) has also been implicated as the etiology of BMT retinopathy. Radiation damages the retinal microvasculature and leads to ischemic xenograft vasculopathy. Variables such as total radiation dose and time interval between radiation and myelectomy appear to be important. However, BMT retinopathy can occur in patients who have not received TBI, and BMT retinopathy has not been observed in solid organ transplant recipients who received similar doses of radiation. Therefore, although TBI is not the only etiology, it is another contributing factor in the development of BMT retinopathy. Chemotherapeutic agents have been proposed as potentially contributing factors in BMT retinopathy. Drugs such as cisplatin, carmustine, and cyclophosphamide can cause ocular side effects, including papilledema, optic neuritis, visual field defects, and cortical blindness. Such chemotherapeutic agents have been shown to predispose patients to radiation-induced retinal damage and enhance the adverse effects of radiation. In general, patients with BMT retinopathy have a good prognosis. Retinopathy usually resolves within two to four months after stopping or reducing the dose of cyclosporine. In one report, 69% of patients experienced complete regression of retinal detection, and 46% of patients fully recovered their basal vision. Because of the good prognosis and relatively nonprogressive nature of BMT retinopathy, active intervention is often not required.
缺血性病状ischemic condition
缺血可分成两类：第一类包括常发生在患有糖尿病的患者中(亦即在股动脉、腘动脉及胫后动脉中)的加速性动脉粥样硬化。这类血管的直径常仅为1或2cm，其可形成严重减少血流量的动脉粥样硬化斑。大的血管完全阻塞后，可发生中风、心肌梗塞、缺血及难愈性糖尿病性足部溃疡。此种形式的缺血基本上为大血管疾病。Ischemia can be divided into two categories: The first category consists of accelerated atherosclerosis that often occurs in patients with diabetes, ie in the femoral, popliteal and posterior tibial arteries. These blood vessels, often only 1 or 2 cm in diameter, can form atherosclerotic plaques that severely reduce blood flow. After complete occlusion of large blood vessels, stroke, myocardial infarction, ischemia, and refractory diabetic foot ulcers can occur. This form of ischemia is essentially macrovascular disease.
中风后痴呆dementia after stroke
中风后25％的人患有痴呆，而其它多人在后来5至10年内发展成痴呆。此外，多个体亦经历其高等脑功能(诸如计划能力及处理信息的速度)的更微妙损害且处于极高的随后发展成痴呆的危险中。在此过程(称为微血管疾病)中，脑深部中极小的中风似乎在该过程中为基本的，导致中风后痴呆的经鉴别的特定脑萎缩类型。Twenty-five percent of people have dementia after a stroke, and many others develop dementia within the next 5 to 10 years. In addition, many individuals also experience more subtle impairments of their higher brain functions, such as planning ability and speed of processing information, and are at very high risk of subsequently developing dementia. Minimal strokes in the deep brain appear to be fundamental in this process, called microvascular disease, leading to the identified specific type of brain atrophy of post-stroke dementia.
眼部缺血综合征ocular ischemic syndrome
罹患眼部缺血综合征(OIS)的患者一般为老人，年龄介于50与80之间。患病男性一般为女性两倍。患者很少为无症状的。减少的视力即刻发生在90％的病例中，且40％的患者伴随眼痛。亦可伴随短暂缺血发作或阵发性黑蒙或存在此类先前病史。患者在呈现的时间亦具有显著已知或未知的全身性疾病。最常遇到的全身性疾病为高血压、糖尿病、缺血性心脏病、中风及周围血管疾病。在更少程度上，患者由于巨细胞动脉炎(GCA)而显示OSI。Patients with ocular ischemic syndrome (OIS) are generally elderly, between the ages of 50 and 80. Males are generally twice as affected as females. Patients are rarely asymptomatic. Reduced vision occurs immediately in 90% of cases and is accompanied by eye pain in 40% of patients. It may also be accompanied by transient ischemic attacks or paroxysmal amaurosis or a prior history of such. Patients also had significant known or unknown systemic disease at the time of presentation. The most commonly encountered systemic diseases were hypertension, diabetes mellitus, ischemic heart disease, stroke, and peripheral vascular disease. To a lesser extent, patients exhibit OSI due to giant cell arteritis (GCA).
单侧研究结果存在于80％的病例中。通常研究结果可包括晚期单侧白内障、前段炎症、无症状性前房反应、黄斑水肿、扩张但未弯曲的视网膜静脉、周围点中央(mid-peripheral dot)及斑点出血、棉絮斑、渗出物及神经盘与视网膜的新血管生成。亦可存在自发的动脉跳动、增加的眼内压及伴随新生血管性青光眼(NVG)的虹膜及角膜的新血管生成。虽然患者可显示前段新血管生成，但归因于对睫状体的低动脉灌注，可出现眼部张力衰弱。有时，存在可见视网膜栓塞(Hollenhorst斑)。Unilateral findings were present in 80% of cases. Common findings may include advanced unilateral cataract, anterior segment inflammation, asymptomatic anterior chamber reaction, macular edema, dilated but not tortuous retinal veins, mid-peripheral dot and spot hemorrhages, cotton wool spots, exudates and neovascularization of the neural disc and retina. There may also be spontaneous arterial beating, increased intraocular pressure, and neovascularization of the iris and cornea with neovascular glaucoma (NVG). Although the patient may show anterior segment neovascularization, there may be attenuation of the eye due to hypoarterial perfusion to the ciliary body. Occasionally, a visible retinal emboli (Hollenhorst spots) is present.
OIS中的研究结果是藉由常见颈动脉的分叉处的内颈动脉粥样硬化溃疡及狭窄引起的。5％的患有内动脉狭窄的患者发展成OIS。然而，若狭窄度超过90％，则仅OIS发生。颈动脉狭窄减少了对眼睛的灌注压力，导致上述缺血现象。狭窄达到90％后，视网膜中央动脉(CRA)的灌注压力仅下降至50％。减少的动脉压力常表现为CRA的自发跳动。研究结果可变且可包括任一或所有上文研究结果。The findings in OIS were driven by internal carotid atherosclerotic ulceration and stenosis at the bifurcations of the common carotid arteries. Five percent of patients with internal artery stenosis develop OIS. However, only OIS occurs if the stenosis exceeds 90%. Narrowing of the carotid arteries reduces the perfusion pressure on the eye, leading to the ischemia described above. After the stenosis reaches 90%, the perfusion pressure of the central retinal artery (CRA) only drops to 50%. Reduced arterial pressure often manifests as spontaneous beating of the CRA. Study results are variable and may include any or all of the above study results.
OIS患者患有必须分析的显著全身性疾病。心脏死亡为OIS患者中死亡率的主要原因-五年死亡率为40％。由于此原因，针对完全血清学、EKG、ECG及颈动脉评估，OIS患者必须求助于心脏病专家。Patients with OIS have significant systemic disease that must be analyzed. Cardiac death is the leading cause of mortality in OIS patients - 40% at five years. For this reason, patients with OIS must refer to a cardiologist for complete serology, EKG, ECG, and carotid artery evaluation.
肾的微血管疾病renal microvascular disease
肾涉及影响全身性及肾微血管系统的大量谨慎临床病理病状。这类病状中的某些以主要针对内皮细胞的损伤为特征，诸如：The kidney is involved in a number of discreet clinicopathological conditions affecting the systemic and renal microvasculature. Some of these conditions are characterized by damage primarily to endothelial cells, such as:
·溶血性尿毒综合征(HUS)及栓塞性血小板减少性紫斑(TTP)HUS及TTP是以微小血管内溶血性贫血及可变器官损害为特征的紧密相关疾病。传统上，当肾衰竭为HUS综合征的显著特征时，诊断出HUS，其常见于儿童。成人中，神经损害常占优势，且继而将该综合征称为TTP。栓塞性微血管病为两种综合征中的根本病理损害，且在患有HUS或TTP的患者中的临床及实验室研究结果在很大程度上重复。此促使若干研究者将两种综合征视作单一疾病实体的连续体。发病机理：实验资料强烈表明内皮细胞损伤为HUS/TTP的发病机理中的主要事件。内皮损害引发一连串事件，包括局部血管内凝结、纤维蛋白沉积及血小板活化及凝聚。最终结果为不同形式HUS/TTP综合征的常见栓塞性微血管病的组织病理学研究结果。若HUS/TTP未经治疗，则死亡率达90％。辅助治疗(包括透析、抗高血压药物、输血及神经并发症的处理)有助于改善HUS/TTP患者的存活。适当体液平衡及肠休息在与腹泻相关的典型HUS的治疗中是重要的。Hemolytic uremic syndrome (HUS) and thrombotic thrombocytopenic purpura (TTP) HUS and TTP are closely related diseases characterized by microvascular hemolytic anemia and variable organ damage. Traditionally, HUS is diagnosed when renal failure is a prominent feature of the HUS syndrome, which is common in children. In adults, neurological damage often predominates, and the syndrome has since been termed TTP. Embolic microangiopathy is the underlying pathologic lesion in both syndromes, and clinical and laboratory findings have largely replicated in patients with HUS or TTP. This has prompted several investigators to view both syndromes as a continuum of a single disease entity. Pathogenesis: Experimental data strongly suggest that endothelial cell injury is a major event in the pathogenesis of HUS/TTP. Endothelial damage triggers a cascade of events including localized intravascular coagulation, fibrin deposition, and platelet activation and aggregation. The final results are the results of a histopathological study of common embolic microangiopathy in different forms of HUS/TTP syndrome. If HUS/TTP is untreated, the mortality rate is as high as 90%. Adjuvant therapy (including dialysis, antihypertensive drugs, blood transfusion, and management of neurological complications) can help improve survival in patients with HUS/TTP. Proper fluid balance and bowel rest are important in the management of typical HUS associated with diarrhea.
·放射性肾炎-超过2500rad的肾辐射的长期结果可分成五种临床综合征：· Radiation nephritis - long-term results of renal radiation exceeding 2500 rad can be divided into five clinical syndromes:
(i)6至13个月潜伏期后，急性放射性肾炎发生在约40％患者中。在大部分导致晚期肾的病例中，其临床特征在于高血压、蛋白尿、水肿及渐进式肾衰竭的突然发作。(i) After an incubation period of 6 to 13 months, acute radiation nephritis occurs in approximately 40% of patients. In most cases leading to end stage renal failure, the clinical features are sudden onset of hypertension, proteinuria, edema, and progressive renal failure.
(ii)相反地，慢性放射性肾炎具有在最初损伤后18个月与14年之间变化的潜伏期。其隐伏发作且特征在于高血压、蛋白尿及肾功能逐渐丧失。(ii) In contrast, chronic radiation nephritis has a latency period that varies between 18 months and 14 years after the initial injury. It has an insidious onset and is characterized by hypertension, proteinuria, and gradual loss of renal function.
(iii)在曝露于放射后5至19年，第三种综合征表现为良性蛋白尿以及正常肾功能。(iii) The third syndrome presents with benign proteinuria and normal
(iv)第四组患者在2至5年后仅显示良性高血压且可具有可变蛋白尿。晚期恶性高血压在患有慢性放射性肾炎或良性高血压的患者经受辐射后18个月至11年发生。移除患病肾，从而逆转高血压。已报导放射引起的对肾动脉的损害以及后续肾血管性高血压。(iv) A fourth group of patients shows only benign hypertension after 2 to 5 years and may have variable proteinuria. Late malignant hypertension occurs 18 months to 11 years after exposure to radiation in patients with chronic radiation nephritis or benign hypertension. Remove the diseased kidney, thereby reversing high blood pressure. Radiation-induced damage to the renal arteries and subsequent renovascular hypertension have been reported.
(v)类似于急性放射性肾炎的肾功能不全综合征已在经全身放射(TBI)治疗的骨髓移植(BMT)患者中观测到。(v) Renal insufficiency syndrome similar to acute radiation nephritis has been observed in bone marrow transplant (BMT) patients treated with total body irradiation (TBI).
已报导放射会引起内皮细胞功能障碍，但在放射后早期并不伤害血管平滑肌细胞。放射可直接损害DNA，导致这类细胞的再生减少及肾小球毛细管及小管中的基膜脱落。不清楚此最初损害最终如何导致肾小球硬化、小管萎缩及间质性纤维化。假定内皮细胞层退化可导致毛细管及更细小动脉中的血管内血栓。接着，此肾内血管病将解释表征放射性肾炎的渐进式肾纤维化及高血压。近期对经辐射的小鼠肾的研究显示出肾皮质中的白细胞具有剂量依赖性增加，此表明炎症过程在放射诱发的肾炎中的作用。Radiation has been reported to cause endothelial dysfunction but not to damage vascular smooth muscle cells in the early post-irradiation period. Radiation can directly damage DNA, resulting in reduced regeneration of these cells and detachment of the basement membrane in glomerular capillaries and tubules. It is unclear how this initial lesion eventually leads to glomerulosclerosis, tubular atrophy, and interstitial fibrosis. It is postulated that degeneration of the endothelial cell layer can lead to intravascular thrombus in capillaries and smaller arteries. This intrarenal vascular disease will then explain the progressive renal fibrosis and hypertension that characterize radiation nephritis. A recent study of irradiated mouse kidneys showed a dose-dependent increase in leukocytes in the renal cortex, suggesting a role for inflammatory processes in radiation-induced nephritis.
在其它肾病中，肾的微血管结构涉及自体免疫病症，诸如全身性硬化症(硬皮病)。肾涉及性全身性硬化症表现为缓慢渐进式慢性肾病或硬皮病肾危症(SRC)，其特征在于恶性高血压及急性氮血症。假定SRC是由肾中类雷诺(Raynaud-like)现象引起的。严重血管痉挛导致皮层缺血及肾素及血管紧张素II的产生增强，继而维持肾血管收缩。激素变化(怀孕)、身体及情感压力或冷的温度可引发类雷诺动脉血管痉挛。ACE抑制剂治疗SRC的显著益处强调了肾素-血管紧张素系统在维持肾缺血中的作用。在虽然进行抗高血压治疗但仍发展成严重肾功能不全的SRC患者中，必须进行透析。已使用腹膜透析及血液透析。末期肾病(ESRD)网对患有全身性硬化症诱发的ESDR且在1983年与1985年之间透析的患者进行了报导，揭示三年存活率为33％。In other kidney diseases, the microvascular structure of the kidney is involved in autoimmune disorders such as systemic sclerosis (scleroderma). Renal-related systemic sclerosis manifests as slowly progressive chronic kidney disease or scleroderma renal crisis (SRC), characterized by malignant hypertension and acute azotemia. SRC is postulated to be caused by a Raynaud-like phenomenon in the kidney. Severe vasospasm leads to cortical ischemia and enhanced production of renin and angiotensin II, which in turn maintain renal vasoconstriction. Hormonal changes (pregnancy), physical and emotional stress, or cold temperatures can trigger Raynaud-like artery vasospasms. The marked benefit of ACE inhibitors in SRC underscores the role of the renin-angiotensin system in maintaining renal ischemia. Dialysis is mandatory in SRC patients who develop severe renal insufficiency despite antihypertensive therapy. Peritoneal dialysis and hemodialysis have been used. The End Stage Renal Disease (ESRD) Network reported on patients with systemic sclerosis-induced ESDR who were on dialysis between 1983 and 1985, revealing a three-year survival rate of 33%.
在镰状细胞疾病中，肾微循环亦可受到影响，由于氧随直管反向移动而在肾髓质的深脉管中获得低氧张力，因此肾尤其患有镰状细胞疾病。更小的肾动脉及小动脉亦可为自大脉管壁去除的含胆固醇物质损伤血栓栓塞的位点。Renal microcirculation can also be affected in sickle cell disease, the kidney in particular suffers from sickle cell disease due to the acquisition of low oxygen tension in the deep vessels of the renal medulla due to the reverse movement of oxygen with the straight ducts. Smaller renal arteries and arterioles can also be sites of thromboembolic injury from removal of cholesterol-containing material from the walls of large vessels.
作为一组引起肾微血管结构暂时或永久阻塞的疾病，该疾病一致导致肾小球灌注受到破坏，且因此使肾小球滤过率受到破坏，藉此对系统稳定构成严重威胁。As a group of diseases that cause temporary or permanent obstruction of renal microvascular structures, the diseases collectively result in impaired glomerular perfusion, and thus glomerular filtration rate, thereby posing a serious threat to systemic stability.
急性肾衰竭(ARF)acute renal failure (ARF)
ARF可藉由微血管或大血管疾病(主要肾动脉阻塞或严重腹部大动脉疾病)引起。典型微血管疾病常呈现因为肾小球毛细管血栓或阻塞而发生的微血管病变性溶血及急性肾衰竭，常伴随血小板减少。这类疾病的典型实例包括：ARF can be caused by microvascular or macrovascular disease (major renal artery occlusion or severe abdominal aortic disease). Typical microvascular disease presents with microangiopathic hemolysis and acute renal failure due to glomerular capillary thrombosis or obstruction, often accompanied by thrombocytopenia. Typical examples of such disorders include:
a)栓塞性血小板减少性紫斑-栓塞性血小板减少性紫斑中的典型五个症状包括发烧、神经变化、肾衰竭、微血管病变性溶血性贫血及血小板减少。a) Thrombocytopenic Purpura - The typical five symptoms in thrombotic thrombocytopenic purpura include fever, neurological changes, renal failure, microangiopathic hemolytic anemia, and thrombocytopenia.
b)溶血性尿毒综合征-溶血性尿毒综合征类似于栓塞性血小板减少性紫斑，但不呈现神经变化。b) Hemolytic uremic syndrome - Hemolytic uremic syndrome resembles thrombotic thrombocytopenic purpura, but does not exhibit neurological changes.
c)HELLP综合征(溶血、上升的肝酶及低血小板)。HELLP综合征为一种发生在怀孕女性中的溶血性尿毒综合征，此外亦伴随转胺酶上升。c) HELLP syndrome (hemolysis, elevated liver enzymes and low platelets). HELLP syndrome is a hemolytic uremic syndrome that occurs in pregnant women and is also associated with elevated transaminases.
虽然急性肾衰竭可呈现在所有医学装置中，但其主要在医院中获得。该病状在5％的就医患者中发展，且约0.5％的就医患者需要透析。在过去40年期间，急性肾衰竭的存活率并未改善，主要因为患病患者现已变老且具有更多共病。在患有急性肾衰竭的患者中，感染占死亡的75％，且心肺并发症为第二最常见死亡原因。视肾衰竭的严重程度而定，死亡率可介于7％与高达80％之间。急性肾衰竭可分成三类：肾前性、肾因性及肾后性ARF。肾因性ARF再分成四类：肾小管病、肾小球病、血管病(包括微血管)及间质病。Although acute renal failure can present in all medical settings, it is primarily acquired in hospitals. The condition develops in 5% of hospital visits, and approximately 0.5% of hospital visits require dialysis. Survival rates for acute renal failure have not improved over the past 40 years, mainly because affected patients are now older and have more comorbidities. In patients with acute renal failure, infections account for 75% of deaths, with cardiopulmonary complications being the second most common cause of death. Depending on the severity of kidney failure, the mortality rate can range from 7% to as high as 80%. Acute renal failure can be divided into three categories: prerenal, renal and postrenal ARF. Nephrogenic ARF is subdivided into four categories: tubular disease, glomerular disease, vascular disease (including microvascular) and interstitial disease.
渐进式肾病progressive kidney disease
有迹象表明渐进式肾病以微血管结构的渐进式损失为特征。微血管结构的损失与肾小球及肾小管间质性疤痕的发展直接相关。机制藉由内皮细胞增生反应的减少来介导，且毛细管修复中的此损害藉由肾中血管生成因子(血管内皮细胞生长因子)及抗血管生成因子(凝血栓蛋白1)的局部表现的改变来介导。血管生成生长因子平衡的变化藉由巨噬细胞相关的细胞因子(介白素-1β)及血管活性介体来介导。最终，引起兴趣的证据表明血管生成和/或毛细管修复的刺激作用可稳定肾功能且减缓进程，且此益处的出现独立于对BP或蛋白尿的影响。There are indications that progressive renal disease is characterized by progressive loss of microvascular structure. Loss of microvascular architecture is directly related to the development of glomerular and tubulointerstitial scarring. The mechanism is mediated by a decrease in the proliferative response of endothelial cells, and this impairment in capillary repair is mediated by changes in the local expression of angiogenic (vascular endothelial growth factor) and antiangiogenic (thrombin 1) factors in the kidney to mediate. Changes in the balance of angiogenic growth factors are mediated by macrophage-associated cytokines (interleukin-1β) and vasoactive mediators. Finally, intriguing evidence suggests that stimulation of angiogenesis and/or capillary repair can stabilize renal function and slow progression, and that this benefit occurs independently of effects on BP or proteinuria.
其它信息参见Brenner & Rector′s The Kidney，第七版，Copyright
听力障碍hearing impairment
化学药品诱发的耳毒性Chemical-Induced Ototoxicity
各种耳毒性治疗药物对听细胞及螺旋神经节神经元的毒性作用常为限制其治疗有效性的因素。主要耳毒性药物包括广泛使用的化学治疗剂顺铂及其类似物、常用于治疗藉由革兰氏阴性细菌引起的感染的氨基糖苷抗生素(例如庆大霉素(gentamicin))、奎宁(quinine)及其类似物、水杨酸盐及其类似物及髓袢利尿剂。The toxic effects of various ototoxic therapeutic drugs on auditory cells and spiral ganglion neurons are often factors that limit their therapeutic effectiveness. Major ototoxic drugs include the widely used chemotherapeutic agent cisplatin and its analogs, aminoglycoside antibiotics (such as gentamicin) commonly used to treat infections caused by Gram-negative bacteria, quinine ) and its analogs, salicylates and their analogs, and loop diuretics.
举例而言，已知抗细菌氨基糖苷(诸如庆大霉素、链霉素、卡那霉素(kanamycin)、妥布霉素(tobramycin)及其类似物)具有严重毒性、尤其耳毒性及肾脏危害性，此性质会减少该抗菌剂的有效性(参见Goodman及Gilman，The Pharmacological Basis of Therapeutics，第6版，A.Goodman Gilman等人编辑；Macmillan Publishing Co.，Inc.，New York，第1169-71页(1980))。明显地，耳毒性为抗生素给药的剂量限制性副作用。历时一周以上每日接收1公克的4％至15％患者发展成可测量的听力损失，若治疗继续，则听力损失慢慢变严重且可导致完全永久耳聋。For example, antibacterial aminoglycosides such as gentamicin, streptomycin, kanamycin, tobramycin and their analogs are known to have severe toxicity, especially ototoxicity and renal Hazardousness, a property that reduces the effectiveness of the antimicrobial (see Goodman and Gilman, The Pharmacological Basis of Therapeutics, 6th ed., edited by A. Goodman Gilman et al.; Macmillan Publishing Co., Inc., New York, pp. 1169 -71 pages (1980)). Clearly, ototoxicity is a dose-limiting side effect of antibiotic administration. Between 4% and 15% of patients receiving 1 gram daily for over a week develop measurable hearing loss, which becomes progressively severe and can lead to complete permanent deafness if treatment continues.
耳毒性亦为严重的剂量限制性顺铂副作用，顺铂为铂的配位错合物，已证实其对多种人类癌症(包括睾丸癌、卵巢癌、膀胱癌及头部及颈部癌)有效。顺铂()破坏听觉及前庭系统。由于消炎、止痛、解热及抗血栓作用，故水杨酸盐(诸如阿司匹林(aspirin))为最常用的治疗药物。不幸地，其亦具有耳毒性副作用。其常导致耳鸣(＂耳朵里鸣响＂)及暂时听力损失。此外，若该药物以高剂量使用长时间，则听力障碍可持续且不可逆。Ototoxicity is also a serious dose-limiting side effect of cisplatin, a coordination complex of platinum that has been shown to be effective in a variety of human cancers (including testicular, ovarian, bladder, and head and neck cancers). efficient. Cisplatin ( ) destroys the auditory and vestibular systems. Salicylates (such as aspirin) are the most commonly used therapeutic drugs due to their anti-inflammatory, analgesic, antipyretic and antithrombotic effects. Unfortunately, it also has ototoxic side effects. It often causes tinnitus ("ringing in the ears") and temporary hearing loss. In addition, hearing impairment can be persistent and irreversible if this drug is used at high doses for an extended period of time.
因此，需要预防、减小或治疗涉及内耳组织、尤其内耳毛细胞的内耳病症及听力障碍的发病率和/或严重程度的方式。尤其关注作为耳毒性治疗药物(包括顺铂及其类似物、氨基糖苷抗生素、水杨酸盐及其类似物或髓袢利尿剂)的不良副作用出现的那些病状。此外，需要以更高剂量且因此以更有效剂量使用这类诱发耳毒性的医药药物并伴随预防或减少藉由这类药物引起的耳毒作用的方法。需要一种为预防或治疗与内耳组织损伤、损失或退化相关、尤其由耳毒素诱发且尤其涉及内耳毛细胞的听力障碍提供安全、有效及长时期方式的方法。此外，亦需要用于治疗源自内耳外伤(声创伤)的损害及耳聋的方法及组合物。Accordingly, there is a need for ways to prevent, reduce or treat the incidence and/or severity of inner ear disorders and hearing impairments involving inner ear tissue, particularly inner ear hair cells. Of particular concern are those conditions that occur as adverse side effects of ototoxic treatment drugs, including cisplatin and its analogs, aminoglycoside antibiotics, salicylates and their analogs, or loop diuretics. Furthermore, there is a need for using such ototoxicity-inducing pharmaceutical drugs at higher doses and thus more effective doses with concomitant methods of preventing or reducing the ototoxic effects caused by such drugs. There is a need for a method that provides a safe, effective and long-term means of preventing or treating hearing impairment associated with damage, loss or degeneration of inner ear tissue, especially induced by ototoxins and especially involving inner ear hair cells. Additionally, there is a need for methods and compositions for treating damage and deafness resulting from inner ear trauma (acoustic trauma).
不受理论限制，相信诱发耳毒性及声创伤的顺铂药物及其它药物(诸如氨基糖苷抗生素)可经由内耳组织、尤其内耳毛细胞中的渐进式细胞死亡或细胞凋亡而诱发耳毒作用(Zhang等人，Neuroscience 120(2003)191-205；Wang等人，J.Neuroscience 23((24)：8596-8607))。在哺乳动物中，听觉毛细胞仅在胚胎形成期间产生且若在出生后生命期间丧失则不再生，因此，毛细胞丧失导致完全且不可逆耳聋。不幸地，目前并无治疗耳蜗及逆转此病状的有效疗法。因此，预防听觉毛细胞的细胞死亡的有效疗法具有重要治疗价值。Without being bound by theory, it is believed that cisplatin and other drugs that induce ototoxicity and acoustic trauma, such as aminoglycoside antibiotics, may induce ototoxic effects via progressive cell death or apoptosis in inner ear tissues, especially inner ear hair cells ( Zhang et al., Neuroscience 120 (2003) 191-205; Wang et al., J. Neuroscience 23 ((24):8596-8607)). In mammals, auditory hair cells are generated only during embryogenesis and do not regenerate if lost during postnatal life; thus, hair cell loss results in complete and irreversible deafness. Unfortunately, there is currently no effective therapy to treat the cochlea and reverse the condition. Effective therapies to prevent cell death of auditory hair cells are therefore of great therapeutic value.
褥疮bedsore
褥疮为受损皮肤及组织的区域，当持续压力(通常来自床或轮椅)切断身体的脆弱部分、尤其腚部、臀部及脚后跟皮肤的循环时，则形成褥疮。缺乏适当血流量会导致患病组织的缺血性坏死及溃疡。褥疮最常发生在感觉减少或缺乏的患者或虚弱、消瘦、瘫痪或长期卧床不起的患者中。骶骨、坐骨、大转子、外踝及脚后跟上的组织尤其易患病；视患者的位置而定，可涉及其它位点。Bed sores are areas of damaged skin and tissue that develop when sustained pressure (usually from a bed or wheelchair) cuts off circulation to the skin in vulnerable parts of the body, especially the buttocks, buttocks, and heels. Lack of proper blood flow can lead to ischemic necrosis and ulceration of diseased tissue. Bed sores most commonly occur in patients with reduced or absent sensation or in patients who are weak, emaciated, paralyzed, or chronically bedridden. Tissues on the sacrum, ischium, greater trochanter, lateral malleolus, and heel are particularly susceptible; depending on patient location, other sites may be involved.
褥疮为通常仅极缓慢治愈的创伤，且尤其在该情况下改善及更快速治愈对患者极具重要性。此外，当治愈性得到改善且更快速发生时，治疗罹患该创伤的患者所涉及的费用显著减少。Bed sores are wounds that usually only heal very slowly, and especially in this case improved and faster healing is of great importance to the patient. Furthermore, when healing is improved and occurs more rapidly, the costs involved in treating patients suffering from this trauma are significantly reduced.
缺血性病状ischemic condition
缺血性损伤为因氧剥夺而使细胞损伤的最通常临床表现。研究缺血性损伤的最适用模型包括完全阻塞末端动脉之一者至器官(例如冠状动脉)及检查由动脉供应的区域中的组织(例如心肌)。在缺血期间，复杂病理改变发生在不同细胞系统中。高达某一程度，针对在不同类型细胞中变化的持续时间，若藉由血流恢复再次获得氧及代谢基质，则损伤可进行修补且患病细胞亦可恢复。随着缺血持续时间的进一步扩大，归因于正在进行的损伤机制的持续进程，细胞结构继续损坏。随着时间过去，细胞的能量机构-粒线体氧化动力室及糖分解路径-受到无法恢复的破坏，且血流恢复(再灌注)不能救助受到破坏的细胞。即使细胞能量机构保持完整，对基因组或细胞膜的无法恢复的破坏确保致命结果，而与再灌注无关。此不可逆损伤常表现为坏死，但细胞凋亡亦可起作用。在某些情形下，当血流量恢复至先前已缺血但尚未死亡的细胞时，损伤常反常地恶化且加速进行-此为再灌注损伤。Ischemic injury is the most common clinical manifestation of cellular damage due to oxygen deprivation. The most useful model to study ischemic injury involves complete occlusion of one of the terminal arteries to an organ (such as a coronary artery) and examining tissue in the area supplied by the artery (such as the heart muscle). During ischemia, complex pathological changes occur in different cellular systems. Up to a certain extent, for varying durations in different types of cells, if oxygen and metabolic substrates are again available through restoration of blood flow, damage can be repaired and diseased cells can also recover. With further extension of the duration of ischemia, cellular structures continue to be damaged due to the continued progression of the ongoing injury mechanism. Over time, the energy machinery of the cell - the mitochondrial oxidative powerhouse and the glycolysis pathway - is irreparably damaged, and restoration of blood flow (reperfusion) cannot rescue the damaged cell. Even if the cellular energy machinery remains intact, irreversible damage to the genome or cell membrane ensures lethal outcomes independent of reperfusion. This irreversible damage often manifests as necrosis, but apoptosis may also play a role. In certain instances, when blood flow is restored to cells that were previously ischemic but not dead, the injury is paradoxically worsened and accelerated - this is reperfusion injury.
再灌注损伤可发生在多种病状中，尤其在医学介入期间，包括(但不限于)血管成形术、心脏外科手术或溶栓；器官移植；由于整形手术；在严重间室综合征期间；在加重肢体的再连接期间；由于多器官衰竭综合征；在脑中由于中风或脑外伤；与慢性创伤(诸如褥疮、静脉溃疡及糖尿病性溃疡)有关；骨骼肌缺血或肢体移植期间；由于肠系膜缺血或急性缺血性肠病；由于下躯体缺血而造成的呼吸衰竭(导致肺循环血压过高、血氧不足及非心脏性肺水肿)；在肾移植；主要外科手术、外伤及及脓毒性休克以及失血性休克后观测到的急性肾衰竭；脓毒病；由于急性血管阻塞而发生的视网膜缺血(在大量眼病(诸如急性青光眼、糖尿病性视网膜病、高血压性视网膜病及视网膜血管阻塞)中导致视力损失)；耳蜗缺血；针对头部及颈部缺陷的微血管外科手术中的瓣衰竭；硬皮病中的雷诺现象及相关数字缺血损害；脊髓损伤；血管外科手术；外伤横纹肌溶解(挤压综合征)；及肌红蛋白尿。Reperfusion injury can occur in a variety of conditions, especially during medical interventions including, but not limited to, angioplasty, cardiac surgery, or thrombolysis; organ transplantation; due to orthopedic surgery; during severe compartment syndrome; During reattachment of exacerbated limbs; due to multiple organ failure syndrome; in the brain due to stroke or traumatic brain injury; associated with chronic trauma (such as decubitus ulcers, venous ulcers, and diabetic ulcers); during skeletal muscle ischemia or limb transplantation; due to mesentery Ischemia or acute ischemic enteropathy; respiratory failure due to lower body ischemia (resulting in pulmonary circulation hypertension, hypoxemia, and noncardiac pulmonary edema); in kidney transplantation; major surgery, trauma, and abscess Acute renal failure observed after toxic shock and hemorrhagic shock; sepsis; retinal ischemia due to acute vascular occlusion (in a number of eye diseases such as acute glaucoma, diabetic retinopathy, hypertensive retinopathy, and retinal vascular cochlear ischemia; valve failure in microvascular surgery for head and neck defects; Raynaud's phenomenon and associated digital ischemic damage in scleroderma; spinal cord injury; vascular surgery; trauma rhabdomyolysis (crush syndrome); and myoglobinuria.
此外，缺血/再灌注可涉及下列病状：高血压、高血压性脑血管疾病、动脉瘤破裂、血管收缩或阻塞-如在血栓或栓子、血管瘤、血质不调、任何形式的受损心脏功能(包括心跳骤停或衰竭、全身性低血压、心跳骤停、心脏性休克、败血性休克、脊髓外伤、头外伤、肿瘤发作、出血)及诸如中风、帕金森氏病(Parkinson′s disease)、癫痫症、抑郁、ALS、阿兹海默氏病(Alzheimer′s disease)、亨廷顿氏舞蹈病(Huntington′s disease)及任何疾病诱发的痴呆(诸如HIV诱发的痴呆)的疾病的情形下发生。In addition, ischemia/reperfusion can be involved in the following conditions: hypertension, hypertensive cerebrovascular disease, ruptured aneurysm, vasoconstriction or occlusion - as in thrombus or embolus, hemangioma, dyscrasia, any form of Impairment of cardiac function (including cardiac arrest or failure, systemic hypotension, cardiac arrest, cardiac shock, septic shock, spinal cord injury, head injury, tumor attack, hemorrhage) and conditions such as stroke, Parkinson's disease (Parkinson's disease) s disease), epilepsy, depression, ALS, Alzheimer's disease, Huntington's disease, and any disease-induced dementia (such as HIV-induced dementia) situation occurs.
此外，缺血事件可由对中枢神经系统的机械损伤引起，诸如源自对头或脊骨的重击。外伤可包括诸如擦伤(abrasion)、切口(incision)、挫伤(contusion)、刺破(puncture)、压伤(compression)等组织损害，诸如可源于外部对象与头、颈或脊柱的任一部位或其从属物的外伤性接触。外伤的其它形式可源于藉由体液不当累积(例如正常脑脊髓或玻璃体液产生的阻塞或功能障碍、翻转或体积调节或硬膜下或硬膜内血肿或水肿)而造成的CNS组织的收缩或压缩。类似地，外伤收缩或压缩可源于大量异常组织(诸如转移性或原发性肿瘤)的存在。In addition, ischemic events can result from mechanical injury to the central nervous system, such as from a blow to the head or spine. Trauma can include tissue damage such as abrasions, incisions, contusions, punctures, compressions, etc., such as can originate from external objects and any Traumatic contact of the site or its appendages. Other forms of trauma may result from constriction of CNS tissue by inappropriate accumulation of fluid such as obstruction or dysfunction of normal cerebrospinal or vitreous humor, inversion or volume adjustment, or subdural or intradural hematoma or edema or compressed. Similarly, traumatic constriction or compression may result from the presence of a large amount of abnormal tissue, such as a metastatic or primary tumor.
总的，用于预防和/或治疗COPD、黄斑变性微血管疾病及耳毒性病状的疗法的当前模式并不令人满意，且因此需要发展用于获得此目的的新颖化合物。亦需要发展可治疗当前与某些药物及病状相关、尤其与顺铂化学疗法及某些抗生素相关的耳毒性作用而不牺牲该药物的有效性的疗法及药剂。此外，亦需要发展可治疗与内耳中的声创伤或机械外伤相关的耳毒性作用的疗法及药剂。此外，亦需要发展用于治疗褥疮、缺血及缺血-再灌注相关病状的疗法及药剂。上文所揭示的所有疾病及适应症以及本文所述的其它疾病及病状(诸如MI)亦可藉由本发明的新颖化合物来治疗。Overall, the current modality of therapy for the prevention and/or treatment of COPD, macular degeneration microvascular disease and ototoxic conditions is unsatisfactory and therefore there is a need to develop novel compounds for this purpose. There is also a need to develop therapies and agents that can treat the ototoxic effects currently associated with certain drugs and conditions, especially cisplatin chemotherapy and certain antibiotics, without sacrificing the effectiveness of the drugs. Furthermore, there is also a need to develop therapies and agents that can treat the ototoxic effects associated with acoustic trauma or mechanical trauma in the inner ear. In addition, there is a need to develop therapies and agents for the treatment of decubitus ulcers, ischemia and ischemia-reperfusion related conditions. All diseases and indications disclosed above, as well as other diseases and conditions described herein, such as MI, can also be treated by the novel compounds of the present invention.
RTP801RTP801
基因RTP801首先由本申请案的共同受让人报导。美国专利第6455674、6555667及6740738号(均转让于本申请案的共同受让人之一)自身揭示且主张RTP801聚核苷酸及多肽及针对多肽的抗体。RTP801表示靶向可调节低氧诱发的发病机理(与诸如VEGF的生长因子无关)的低氧诱发因子-1(HIF-1)的独特基因。The gene RTP801 was first reported by the common assignee of the present application. US Patent Nos. 6,455,674, 6,555,667, and 6,740,738 (all assigned to one of the common assignees of the present application) themselves disclose and claim RTP801 polynucleotides and polypeptides and antibodies against the polypeptides. RTP801 represents a unique gene targeting hypoxia-inducible factor-1 (HIF-1) that can regulate hypoxia-induced pathogenesis independent of growth factors such as VEGF.
下列专利申请案及公开案给出背景信息的状态。The following patent applications and publications give the status of background information.
WO 2001070979涉及在卵巢癌细胞中过度表达的核酸标记。WO 2001070979 relates to nucleic acid markers overexpressed in ovarian cancer cells.
US 6673549揭示包含为回应甾类治疗而差异表达的cDNA的组合。US 6673549 discloses combinations comprising cDNAs that are differentially expressed in response to steroid treatment.
美国申请案2003165864涉及在经DNA去甲基化剂处理的细胞中差异表达的cDNA。US application 2003165864 relates to cDNAs differentially expressed in cells treated with DNA demethylating agents.
美国申请案2003108871涉及包含在经治疗人类C3A肝细胞培养物中差异表达的若干cDNA的组合物。US application 2003108871 relates to a composition comprising several cDNAs differentially expressed in cultures of treated human C3A hepatocytes.
美国申请案2002119463揭示一种新颖组合物，其适用于治疗及诊断前列腺癌，该组合物包含在前列腺癌中差异表达的人类cDNA。US application 2002119463 discloses a novel composition suitable for the treatment and diagnosis of prostate cancer, the composition comprising human cDNA differentially expressed in prostate cancer.
WO 2004018999揭示一种用于分析、表征、监视、预防及治疗子宫颈癌的方法。WO 2004018999 discloses a method for analysis, characterization, monitoring, prevention and treatment of cervical cancer.
EP 1394274涉及一种用于测试支气管哮喘或慢性阻塞性肺病的方法，该方法藉由将来自受检者的生物样品中的标记基因的表达量与来自健康受检者的样品中的基因的表达量相比较来进行。EP 1394274 relates to a method for testing bronchial asthma or chronic obstructive pulmonary disease by comparing the expression levels of marker genes in biological samples from subjects with the expression levels of genes in samples from healthy subjects Quantitatively compared.
WO 2002101075涉及适用于检测、表征、预防及治疗人类子宫颈癌的经分离核酸分子。WO 2002101075 relates to isolated nucleic acid molecules suitable for detection, characterization, prevention and treatment of human cervical cancer.
WO 2003010205涉及用于治疗伤口愈合、视网膜病、缺血、炎症、微血管病、骨愈合及皮肤炎症的血管生成抑制。WO 2003010205 relates to angiogenesis inhibition for the treatment of wound healing, retinopathy, ischemia, inflammation, microangiopathy, bone healing and skin inflammation.
WO 2002046465涉及鉴别涉及疾病的基因以治疗低氧调节的病状。WO 2002046465 relates to the identification of genes involved in disease for the treatment of hypoxia regulated pathologies.
WO 2002031111涉及多肽及其编码的蛋白，且因此提供多种用途。WO 2002031111 relates to polypeptides and proteins encoded by them, and thus provides a variety of uses.
WO 2001012659涉及适用于重组DNA方法的核酸。WO 2001012659 relates to nucleic acids suitable for use in recombinant DNA methods.
WO 2001077289揭示衍生自多种人类组织来源的623种聚核苷酸。WO 2001077289 discloses 623 polynucleotides derived from various human tissue sources.
WO 2003101283涉及包含多种cDNA及蛋白的组合。WO 2003101283 relates to combinations comprising various cDNAs and proteins.
JP 2003259877涉及多种肝纤维化疾病标记。JP 2003259877 relates to various liver fibrosis disease markers.
Tzipora Shoshani等人，Identification of a NovelHypoxia-Inducible Factor 1-Responsive Gene，RTP801，Involvedin Apoptosis.MOLECULAR AND CELLULAR BIOLOGY，2002年4月，第2283-2293页；此论文由本发明的发明者共同创作，其详细描述了RTP801基因(接着，新颖HIF-1依赖性基因)的发现。Tzipora Shoshani et al., Identification of a NovelHypoxia-Inducible Factor 1-Responsive Gene, RTP801, Involvedin Apoptosis.MOLECULAR AND CELLULAR BIOLOGY, April 2002, pages 2283-2293; The discovery of the RTP801 gene (and subsequently, a novel HIF-1-dependent gene) is described.
Anat Brafman等人，Inhibition of Oxygen-Induced Retinopathyin RTP801-Deficient Mice.Invest Ophthalmol Vis Sci.2004年10月；45(10)：3796-805)；此论文亦由本发明的发明者共同创作，其证实了在RTP801基因剔除小鼠中氧过多不会引起视网膜毛细管网络的退化。Anat Brafman et al., Inhibition of Oxygen-Induced Retinopathy RTP801-Deficient Mice. Invest Ophthalmol Vis Sci. 2004 October; 45(10): 3796-805); this paper, also co-authored by the inventors of the present invention, demonstrates Hyperoxia does not cause degeneration of the retinal capillary network in RTP801 knockout mice
Leif W.Ellisen等人，REDD1，a Developmentally RegulatedTranscriptional Target of p63 and p53，Links p63 to Regulationof Reactive Oxygen Species.Molecular Cell，第10卷，995-1005，2002年11月；此论文证实了RTP801(其中称为REDD1)的过度表达会导致活性氧的产生增加。Leif W. Ellisen et al., REDD1, a Developmentally Regulated Transcriptional Target of p63 and p53, Links p63 to Regulation of Reactive Oxygen Species. Molecular Cell, Vol. 10, 995-1005, Nov. 2002; this paper confirms that RTP801 (which states Overexpression of REDD1) leads to increased production of reactive oxygen species.
Richard DR，Berra E及Pouyssegur J.Non-hypoxic pathwaymediates the induction of hypoxia-inducible factor 1 alpha invascular smooth muscle cells.J Biol.Chem.2000年9月1日；275(35)：26765-71。此论文证实了HIF-1依赖性转录可藉由活性氧的过度产生来诱发。Richard DR, Berra E, and Pouyssegur J. Non-hypoxic pathway mediates the induction of hypoxia-
Rangasami T等人，Genetic ablation of Nrf2 enhancessusceptibility to cigarette smoke-induced emphysema in mice。提交给Journal of Clinical Investigation。此作品涉及具有受损抗氧化剂防御的小鼠。Rangasami T et al., Genetic ablation of Nrf2 enhanceusceptibility to cigarette smoke-induced emphysema in mice. Submitted to Journal of Clinical Investigation. This work concerns mice with impaired antioxidant defenses.
发明内容Contents of the invention
本发明提供用于治疗微血管病症、黄斑变性、呼吸病症及脊髓损伤或疾病的新颖方法及组合物。The present invention provides novel methods and compositions for treating microvascular disorders, macular degeneration, respiratory disorders, and spinal cord injuries or diseases.
在一实施方式中，提供抑制RTP801且可用以治疗各种疾病及适应症的新颖分子。In one embodiment, novel molecules are provided that inhibit RTP801 and can be used to treat various diseases and indications.
在另一实施方式中，本发明提供一种用于治疗罹患微血管病症、黄斑变性或呼吸病症的患者的方法，该方法包括将包含RTP801抑制剂的药物组合物给药于患者。In another embodiment, the present invention provides a method for treating a patient suffering from a microvascular disorder, macular degeneration or a respiratory disorder, the method comprising administering to the patient a pharmaceutical composition comprising an RTP801 inhibitor.
本发明的另一实施例涉及一种用于治疗罹患COPD的患者的方法，该方法包括将包含治疗有效量的RTP801抑制剂的药物组合物给药于患者。在一实施方式中，抑制剂为siRNA分子、反义分子、抗体(诸如中和抗体)、显性阴性肽或核糖核酸酶。Another embodiment of the present invention relates to a method for treating a patient suffering from COPD, the method comprising administering to the patient a pharmaceutical composition comprising a therapeutically effective amount of an RTP801 inhibitor. In one embodiment, the inhibitor is an siRNA molecule, an antisense molecule, an antibody (such as a neutralizing antibody), a dominant negative peptide, or a ribonuclease.
本发明的另一实施例涉及一种用于治疗罹患黄斑变性的患者的方法，该方法包括将包含治疗有效量的RTP801抑制剂的药物组合物给药于患者。在一实施方式中，抑制剂为siRNA分子、反义分子、抗体(诸如中和抗体)、显性阴性肽或核糖核酸酶。Another embodiment of the present invention relates to a method for treating a patient suffering from macular degeneration, the method comprising administering to the patient a pharmaceutical composition comprising a therapeutically effective amount of an RTP801 inhibitor. In one embodiment, the inhibitor is an siRNA molecule, an antisense molecule, an antibody (such as a neutralizing antibody), a dominant negative peptide, or a ribonuclease.
本发明的另一实施例涉及一种用于治疗罹患微血管病症的患者的方法，该方法包括将包含治疗有效量的RTP801抑制剂的药物组合物给药于患者。在一实施方式中，抑制剂为siRNA分子、反义分子、抗体(诸如中和抗体)、显性阴性肽或核糖核酸酶。Another embodiment of the present invention is directed to a method for treating a patient suffering from a microvascular disorder, the method comprising administering to the patient a pharmaceutical composition comprising a therapeutically effective amount of an RTP801 inhibitor. In one embodiment, the inhibitor is an siRNA molecule, an antisense molecule, an antibody (such as a neutralizing antibody), a dominant negative peptide, or a ribonuclease.
本发明的另一实施例提供治疗有效量的RTP801抑制剂用于制备用于促进罹患呼吸病症的患者恢复的药剂的用途。在一实施方式中，呼吸病症为COPD且抑制剂优选为siRNA。Another embodiment of the present invention provides the use of a therapeutically effective amount of an RTP801 inhibitor for the manufacture of a medicament for promoting recovery in a patient suffering from a respiratory disorder. In one embodiment, the respiratory disorder is COPD and the inhibitor is preferably siRNA.
本发明的另一实施例提供治疗有效剂量的RTP801抑制剂用于制备用于促进罹患黄斑变性的患者恢复的药剂的用途。在一实施方式中，黄斑变性为AMD且抑制剂优选为siRNA。Another embodiment of the present invention provides the use of a therapeutically effective dose of an RTP801 inhibitor for the manufacture of a medicament for promoting recovery in a patient suffering from macular degeneration. In one embodiment, the macular degeneration is AMD and the inhibitor is preferably siRNA.
本发明的另一实施例提供治疗有效量的RTP801抑制剂用于制备用于促进罹患微血管病症的患者恢复的药剂的用途。在一实施方式中，微血管病症为糖尿病性视网膜病且抑制剂优选为siRNA。Another embodiment of the present invention provides the use of a therapeutically effective amount of an RTP801 inhibitor for the manufacture of a medicament for promoting recovery in a patient suffering from a microvascular disorder. In one embodiment, the microvascular disorder is diabetic retinopathy and the inhibitor is preferably siRNA.
本发明一般涉及用于治疗或预防听力障碍(或平衡障碍)、尤其与内耳毛细胞的细胞死亡相关的听力障碍的发生率或严重程度的方法及组合物。该方法及组合物包括将预防或治疗有效量的一种或多种下调RTP801基因表达的化合物、尤其新颖小干扰RNA(siRNA)给药于需要该治疗的哺乳动物。The present invention generally relates to methods and compositions for treating or preventing the incidence or severity of hearing impairment (or balance impairment), especially hearing impairment associated with cell death of inner ear hair cells. The methods and compositions include administering a prophylactically or therapeutically effective amount of one or more compounds that down-regulate RTP801 gene expression, especially novel small interfering RNA (siRNA), to a mammal in need of such treatment.
更具体地说，本发明提供用于治疗罹患听力障碍或与内耳毛细胞的细胞死亡相关的其它耳病理的患者的方法及组合物。该耳病理可为声创伤、机械外伤或耳毒素诱发的听力损失的结果。本发明的方法包括将一或多种下调RTP801基因表达的化合物、尤其抑制RTP801的siRNA通常作为药物组合物以治疗有效剂量给药于患者以藉此治疗患者。More specifically, the present invention provides methods and compositions for treating patients suffering from hearing impairment or other otic pathologies associated with cell death of inner ear hair cells. This ear pathology can be the result of acoustic trauma, mechanical trauma, or ototoxin-induced hearing loss. The method of the present invention includes administering one or more compounds that down-regulate the expression of RTP801 gene, especially siRNA that inhibits RTP801, to a patient, usually as a pharmaceutical composition, in a therapeutically effective dose, thereby treating the patient.
在一实施方式中，本发明提供用于治疗的经改良组合物及方法，其需要给药具有耳毒性、听力障碍副作用的药物，该药物与治疗有效量的一种或多种抑制RTP801的siRNA分子组合以治疗或预防由该药物诱发的耳毒性。本发明的组合物可在诱发内耳细胞凋亡组织损伤的耳毒性、听力损害性药物给药之前、之后以适当间隔给药或大体上同时给药。类似地，此治疗可有效作为在引起声创伤的病状前或与其结合的预防性治疗。In one embodiment, the present invention provides improved compositions and methods for treatment requiring administration of a drug with side effects of ototoxicity, hearing impairment, and a therapeutically effective amount of one or more siRNAs that inhibit RTP801 Combination of molecules to treat or prevent ototoxicity induced by this drug. The composition of the present invention can be administered at appropriate intervals or substantially simultaneously before, after, or at appropriate intervals before or after administration of ototoxic or hearing-impairing drugs that induce apoptotic tissue damage in inner ear cells. Similarly, this treatment may be effective as a prophylactic treatment prior to or in conjunction with a condition causing acoustic trauma.
因此，本发明之一目标是提供含有与耳毒性、听力损害性药物组合的治疗有效量的一种或多种抑制RTP801的siRNA分子的经改良组合物以将其给药于哺乳动物。该组合药物可分开给药；在全身性给药耳毒性、听力损害性药物时可局部给药抑制RTP801的siRNA分子。siRNA分子可在耳毒性药物之前、同时或之后给药。该组合组合物可另外含有可药用载体。该药物组合物应具有比单独耳毒性药物低的耳毒性，且优选应具有比通常使用的剂量高的耳毒性药物剂量。该经改良组合物的实例包括与治疗有效量的一种或多种抑制RTP801的siRNA分子组合的顺铂或其它耳毒性赘生性药剂或氨基糖苷抗生素。Accordingly, it is an object of the present invention to provide improved compositions comprising a therapeutically effective amount of one or more siRNA molecules inhibiting RTP801 in combination with ototoxic, hearing-impairing drugs for administration to mammals. The combined drug can be administered separately; when ototoxic and hearing-impairing drugs are administered systemically, the siRNA molecules inhibiting RTP801 can be administered locally. The siRNA molecule can be administered before, simultaneously with or after the ototoxic drug. The combination composition may additionally contain a pharmaceutically acceptable carrier. The pharmaceutical composition should have lower ototoxicity than the ototoxic drug alone, and preferably should have a higher dose of the ototoxic drug than is usually used. Examples of such improved compositions include cisplatin or other ototoxic neoplastic agents or aminoglycoside antibiotics in combination with a therapeutically effective amount of one or more siRNA molecules that inhibit RTP801.
此外，本发明亦涉及本发明的组合物在需要利尿剂的情形下的用途。本发明提供长期寻求可治疗与某些利尿剂普遍相关且尤其与更普遍及通用的髓袢利尿剂相关的耳毒作用而不牺牲利尿剂的利尿效能的疗法及药剂的本领域的解决方法。Furthermore, the invention also relates to the use of the composition according to the invention in situations where a diuretic is required. The present invention provides a solution in the art to the long search for therapies and agents that can treat the ototoxic effects commonly associated with certain diuretics, and particularly with the more common and common loop diuretics, without sacrificing the diuretic efficacy of the diuretics.
此外，本发明亦涉及本发明的组合物在需要奎宁或类奎宁化合物的情形下的用途。本发明提供长期寻求可治疗与某些奎宁相关的耳毒作用而不牺牲奎宁的效能的疗法及药剂的本领域的解决方法。Furthermore, the invention also relates to the use of the compositions of the invention in situations where quinine or quinine-like compounds are required. The present invention provides a solution in the art to the long-standing quest for therapies and agents that can treat certain quinine-related ototoxic effects without sacrificing the efficacy of quinine.
本发明另外涉及用于治疗或预防褥疮的发生率或严重程度的方法及组合物。该方法及组合物包括将预防或治疗有效量的一种或多种下调RTP801基因表达的化合物、尤其新颖小干扰RNA(siRNA)给药于需要该治疗的哺乳动物。The present invention additionally relates to methods and compositions for treating or preventing the incidence or severity of decubitus ulcers. The methods and compositions include administering a prophylactically or therapeutically effective amount of one or more compounds that down-regulate RTP801 gene expression, especially novel small interfering RNA (siRNA), to a mammal in need of such treatment.
此外，本发明亦涉及用于治疗如本文所述的任何缺血或缺血-再灌注损伤或病状的方法及组合物。该方法及组合物包括将预防或治疗有效量的一种或多种下调RTP801基因表达的化合物、尤其新颖小干扰RNA(siRNA)给药于需要该治疗的哺乳动物。Furthermore, the present invention also relates to methods and compositions for treating any ischemia or ischemia-reperfusion injury or condition as described herein. The methods and compositions include administering a prophylactically or therapeutically effective amount of one or more compounds that down-regulate RTP801 gene expression, especially novel small interfering RNA (siRNA), to a mammal in need of such treatment.
本发明的详细描述Detailed description of the invention
在本发明的某些实施例中，本发明涉及RTP801基因或多肽的抑制以治疗眼病、呼吸病症、微血管病症、听力障碍及缺血性病状。如本文所述，本发明所用的优选抑制剂为生物分子。In certain embodiments of the invention, the invention relates to inhibition of the RTP801 gene or polypeptide for the treatment of ophthalmic diseases, respiratory disorders, microvascular disorders, hearing impairment and ischemic conditions. As described herein, preferred inhibitors for use in the invention are biomolecules.
不受理论限制，本发明的发明者发现RTP801涉及多种疾病病况，包括微血管病症、眼病、呼吸病症、听力障碍、褥疮、缺血性病状及脊髓损伤及疾病，且应有益地抑制RTP801以治疗该疾病或病症中的任一个。本文详细讨论抑制RTP801的方法、分子及组合物，且在治疗罹患该病状中的任一个的患者中可有益地使用该分子和/或组合物中的任一个。Without being limited by theory, the inventors of the present invention have discovered that RTP801 is involved in a variety of disease conditions, including microvascular disorders, eye diseases, respiratory disorders, hearing impairment, decubitus ulcers, ischemic conditions, and spinal cord injuries and diseases, and that inhibition of RTP801 should be beneficial for the treatment of Any of the diseases or conditions. Methods, molecules and compositions for inhibiting RTP801 are discussed in detail herein, and any of the molecules and/or compositions may be beneficially used in the treatment of patients suffering from any of these conditions.
本发明提供用于抑制RTP801基因活体内表达的方法及组合物。一般而言，该方法包括以足以藉由RNA干扰机制下调靶向基因表达的量给药寡核糖核苷酸(诸如靶向特定mRNA且与其杂交的小干扰RNA(亦即siRNA))或可在细胞中产生siRNA的核酸物质。具体说，该方法可用以抑制RTP801基因的表达以治疗呼吸病症、微血管病症、眼病及听力障碍。The invention provides a method and composition for inhibiting the expression of RTP801 gene in vivo. In general, the method includes administering an oligoribonucleotide (such as a small interfering RNA (ie, siRNA) that targets a specific mRNA and hybridizes thereto) in an amount sufficient to down-regulate the expression of a targeted gene by an RNA interference mechanism or can be expressed at A nucleic acid substance in a cell that produces siRNA. Specifically, the method can be used to inhibit the expression of RTP801 gene to treat respiratory diseases, microvascular diseases, eye diseases and hearing impairment.
因此，在一实施方式中，本发明提供一种用于治疗罹患选自微血管病症、眼病、呼吸病症、听力障碍、褥疮或脊髓损伤或其它创伤的病状的患者的方法，该方法包括将包含RTP801抑制剂的药物组合物以治疗有效量给药于患者以藉此治疗患者。本发明另外提供用于治疗罹患微血管病症、眼病、呼吸病症、听力障碍、褥疮或脊髓损伤或其它创伤的患者的方法，该方法包括将包含RTP801抑制剂的药物组合物以足以促进恢复的剂量及历经足以促进恢复的时间给药于患者。眼病可为黄斑变性，尤其诸如年龄相关的黄斑变性(AMD)。微血管病症可尤其为糖尿病性视网膜病或急性肾衰竭。呼吸病症可尤其为慢性阻塞性肺病(COPD)、气肿、慢性支气管炎、哮喘及肺癌。听力障碍可为声创伤或顺铂诱发的耳聋。RTP801抑制剂可选自大量分子，包括(但不限于)诸如聚核苷酸的化合物、反义片段、靶向RTP801基因mRNA的RNA分子(诸如核糖核酸酶或siRNA(诸如表A-D的siRNA且具体地说表A的siRNA No：14、22、23、25、27、39、41、42、49及50或表D的siRNA No：257、260-262及264-268))或包含其的表达载体、诸如显性阴性肽的多肽、抗体(诸如特异性结合存在于包含连续氨基酸的多肽(多肽序列在图2(SEQ ID NO：2)中示出)内的抗原决定簇的抗体)或在某些情形下为酶。此外，RTP801抑制剂可为诸如小分子(例如，具有低分子量(例如低于2000道尔顿的分子量)的化学分子)的化学抑制剂。特异性RTP801抑制剂在下文中给出。Accordingly, in one embodiment, the present invention provides a method for treating a patient suffering from a condition selected from a microvascular disorder, an eye disease, a respiratory disorder, a hearing impairment, a decubitus ulcer or a spinal cord injury or other trauma, the method comprising comprising RTP801 The pharmaceutical composition of the inhibitor is administered to a patient in a therapeutically effective amount to thereby treat the patient. The present invention further provides a method for treating a patient suffering from a microvascular disorder, eye disease, respiratory disorder, hearing impairment, decubitus ulcer or spinal cord injury or other trauma, the method comprising administering a pharmaceutical composition comprising an RTP801 inhibitor in a dose sufficient to promote recovery and Administer to the patient for a time sufficient to promote recovery. The eye disease may be macular degeneration, such as age-related macular degeneration (AMD), among others. The microvascular disorder may especially be diabetic retinopathy or acute renal failure. Respiratory disorders may be chronic obstructive pulmonary disease (COPD), emphysema, chronic bronchitis, asthma and lung cancer, among others. Hearing impairment can be acoustic trauma or cisplatin-induced deafness. RTP801 inhibitors can be selected from a large number of molecules, including (but not limited to) compounds such as polynucleotides, antisense fragments, RNA molecules targeting RTP801 gene mRNA (such as ribonucleases or siRNAs (such as the siRNAs of Tables A-D and specifically Said siRNA Nos of Table A: 14, 22, 23, 25, 27, 39, 41, 42, 49 and 50 or siRNA Nos of Table D: 257, 260-262 and 264-268)) or expressions comprising the same A carrier, a polypeptide such as a dominant negative peptide, an antibody (such as an antibody that specifically binds to an epitope present in a polypeptide comprising contiguous amino acids (the polypeptide sequence is shown in Figure 2 (SEQ ID NO: 2)) or in In some cases enzymes. Furthermore, the RTP801 inhibitor can be a chemical inhibitor such as a small molecule (eg, a chemical molecule having a low molecular weight, eg, a molecular weight below 2000 Daltons). Specific RTP801 inhibitors are given below.
本发明另外提供一种用于治疗罹患黄斑变性、COPD、糖尿病性视网膜病或声创伤诱发的耳聋的患者的方法，该方法包括将包含治疗有效量的包含聚核苷酸(其特异性杂交自RTP801基因转录的mRNA且下调RTP801基因表达以藉此治疗患者)的RTP801抑制剂的药物组合物给药于患者。聚核苷酸可为包含具有与表A-D中所陈述的任一序列(SEQ IDNO：3-536)一致的序列的连续核苷酸的siRNA且具体地说表A的siRNANo：14、22、23、25、27、39、41、42、49及50或表D的siRNA No：257、260-262及264-268。The present invention additionally provides a method for treating a patient suffering from macular degeneration, COPD, diabetic retinopathy or acoustic trauma-induced deafness, the method comprising treating a therapeutically effective amount of a polynucleotide (which is specifically hybridized from mRNA of RTP801 gene transcription and down-regulation of RTP801 gene expression to thereby treat patients) pharmaceutical compositions of RTP801 inhibitors are administered to patients. The polynucleotide may be an siRNA comprising contiguous nucleotides having a sequence consistent with any of the sequences set forth in Tables A-D (SEQ ID NO: 3-536) and specifically siRNA Nos of Table A: 14, 22, 23 , 25, 27, 39, 41, 42, 49 and 50 or siRNA Nos of Table D: 257, 260-262 and 264-268.
此外，本发明的另一实施例涉及一种用于治疗罹患微血管病症、眼病、呼吸病症、听力障碍、褥疮或脊髓损伤或其它创伤的患者的方法，该方法包括将包含治疗有效量的包含siRNA分子(任选为详述于表A-D的任一个中的siRNA分子)的RTP801抑制剂的药物组合物以一定剂量及历经一段时间给药于患者以藉此治疗患者。In addition, another embodiment of the present invention relates to a method for treating a patient suffering from a microvascular disorder, eye disease, respiratory disorder, hearing impairment, decubitus ulcer or spinal cord injury or other trauma, the method comprising administering a therapeutically effective amount of A pharmaceutical composition of an RTP801 inhibitor of a molecule (optionally an siRNA molecule as detailed in any one of Tables A-D) is administered to a patient at a dose and over a period of time to thereby treat the patient.
提供另一种用于治疗罹患微血管病症、眼病、呼吸病症、听力障碍、褥疮或脊髓损伤或其它创伤的患者的方法，该方法包括将包含治疗有效剂量的靶向RTP801基因mRNA的RNA分子的药物组合物以一定剂量及历经一段时间给药于患者以藉此治疗患者。RNA分子可为siRNA分子(诸如表A-D中详述的siRNA分子且具体地说表A的siRNA No：14、22、23、25、27、39、41、42、49及50或表D的siRNA No：257、260-262及264-268)或核糖核酸酶。Provided is another method for treating a patient suffering from a microvascular disorder, eye disease, respiratory disorder, hearing impairment, decubitus or spinal cord injury or other trauma, the method comprising administering a therapeutically effective dose of a medicament comprising an RNA molecule targeting RTP801 gene mRNA Compositions are administered to a patient in dosages and over a period of time to thereby treat the patient. The RNA molecule can be an siRNA molecule such as the siRNA molecules detailed in Tables A-D and specifically the siRNA Nos: 14, 22, 23, 25, 27, 39, 41, 42, 49 and 50 of Table A or the siRNA of Table D No: 257, 260-262 and 264-268) or ribonuclease.
本发明另外提供一种用于治疗罹患微血管病症、眼病、呼吸病症、听力障碍、褥疮或脊髓损伤或其它创伤或本文所揭示的任一病状的患者的方法，该方法包括将包含治疗有效剂量的靶向RTP801基因mRNA的siRNA分子(任选为表A-D的任一个中所详述的siRNA分子)的药物组合物以一定剂量及历经一段时间给药于患者以藉此治疗患者。此外，眼病可为黄斑变性，诸如年龄相关的黄斑变性(AMD)；微血管病症可为糖尿病性视网膜病或急性肾衰竭；呼吸病症可为COPD且所治疗COPD的方面可包含(但不限于)气肿、慢性支气管炎或两者；且听力障碍可为声创伤诱发的耳聋。The present invention additionally provides a method for treating a patient suffering from a microvascular disorder, eye disease, respiratory disorder, hearing impairment, decubitus or spinal cord injury or other trauma, or any of the conditions disclosed herein, comprising a therapeutically effective dose of A pharmaceutical composition of siRNA molecules targeting RTP801 gene mRNA (optionally siRNA molecules detailed in any one of Tables A-D) is administered to a patient at a dose and over a period of time to thereby treat the patient. Additionally, the eye disease can be macular degeneration, such as age-related macular degeneration (AMD); the microvascular disorder can be diabetic retinopathy or acute renal failure; the respiratory disorder can be COPD and aspects of COPD treated can include, but are not limited to, respiratory disorders. edema, chronic bronchitis, or both; and the hearing impairment may be acoustic trauma-induced deafness.
此外，本发明亦涉及本文所揭示的新颖siRNA在治疗其中抑制RTP801表达有益的听力障碍中的用途。在一实施方式中，本发明构成一种用于治疗患有或倾向于患有听力(或平衡)障碍的哺乳动物或预防性治疗患有其中抑制RTP801表达有益的病状的哺乳动物的方法。本发明的此实施例的方法应预防或减少源自尤其由声创伤或耳毒性药剂引起的内耳细胞损伤、损失或退化的听力(平衡)障碍的发生率或严重程度。在此实施例中，本发明的方法包括给药治疗有效量的一种或多种下调RTP801基因表达的化合物、尤其本发明的新颖siRNA。Furthermore, the present invention also relates to the use of the novel siRNAs disclosed herein in the treatment of hearing disorders in which inhibition of RTP801 expression is beneficial. In one embodiment, the invention constitutes a method for treating a mammal suffering from or prone to suffering hearing (or balance) impairment or prophylactically treating a mammal suffering from a condition in which inhibition of RTP801 expression is beneficial. The method of this embodiment of the invention should prevent or reduce the incidence or severity of hearing (balance) impairment resulting from inner ear cell damage, loss or degeneration caused, inter alia, by acoustic trauma or ototoxic agents. In this embodiment, the methods of the invention comprise administering a therapeutically effective amount of one or more compounds that down-regulate RTP801 gene expression, particularly the novel siRNAs of the invention.
在一实施方式中，本发明的目标是提供一种用于治疗哺乳动物以预防、减少或治疗听力障碍、病症或不平衡、优选声创伤或耳毒素诱发的听力病状的方法，该方法藉由将本发明的组合物给药于需要该治疗的哺乳动物来进行。一实施例是一种用于治疗其中声创伤源自曾经暴露于极大声音或连续暴露于经长时间可引起耳聋的噪声环境的听力障碍的方法。听力障碍亦可藉由耳的物理创伤引起。另一实施例是一种用于治疗其中耳毒性源自治疗有效量的耳毒性药物的给药的听力障碍的方法。典型耳毒性药物为化学治疗剂，例如抗赘生剂及抗生素。其它可能候选者包括髓袢利尿剂、奎宁或类奎宁化合物及水杨酸盐及类水杨酸盐化合物。当耳毒性化合物为抗生素、优选氨基糖苷抗生素时，这类方法尤其有效。耳毒性氨基糖苷抗生素包括(但不限于)新霉素、巴龙霉素、核糖霉素、利维霉素、卡那霉素、阿米卡星、妥布霉素、紫霉素、庆大霉素、西索米星、奈替米星、链霉素、地贝卡星、复提霉素及双氢链霉素或其组合。特定抗生素包括新霉素B、卡那霉素A、卡那霉素B、庆大霉素C1、庆大霉素C1a及庆大霉素C2。当耳毒性化合物为赘生性药剂(诸如长春新碱、长春碱、顺铂及类顺铂化合物及紫杉醇及类紫杉醇化合物)时，本发明的方法亦有效。In one embodiment, the object of the present invention is to provide a method for treating a mammal to prevent, reduce or treat a hearing impairment, disorder or imbalance, preferably acoustic trauma or ototoxin-induced hearing pathology, by This is done by administering the composition of the present invention to a mammal in need of such treatment. One embodiment is a method for treating hearing impairment in which the acoustic trauma results from previous exposure to loud sounds or continuous exposure to a deafening environment over a prolonged period of time. Hearing impairment can also be caused by physical trauma to the ear. Another embodiment is a method for treating hearing impairment wherein ototoxicity results from administration of a therapeutically effective amount of an ototoxic drug. Typical ototoxic drugs are chemotherapeutics such as antineoplastic agents and antibiotics. Other possible candidates include loop diuretics, quinine or quinine-like compounds, and salicylates and salicylate-like compounds. Such methods are especially effective when the ototoxic compound is an antibiotic, preferably an aminoglycoside antibiotic. Ototoxic aminoglycoside antibiotics include (but are not limited to) neomycin, paromomycin, ribomycin, rivamycin, kanamycin, amikacin, tobramycin, puromycin, genta Mycin, sisomicin, netilmicin, streptomycin, dibekacin, formomycin and dihydrostreptomycin or combinations thereof. Specific antibiotics include neomycin B, kanamycin A, kanamycin B, gentamicin C1, gentamicin C1a, and gentamicin C2. The methods of the invention are also effective when the ototoxic compound is a neoplastic agent such as vincristine, vinblastine, cisplatin and cisplatin-like compounds, and paclitaxel and paclitaxel-like compounds.
在以治疗或预防听力障碍为目标的某些实施例中，本发明的组合物应与耳毒素共给药。举例而言，提供一种藉由给药氨基糖苷抗生素来治疗哺乳动物感染的经改良方法，其改良之处包含将治疗有效量的一种或多种下调RTP801基因表达的化合物、尤其新颖siRNA给药于需要该治疗的患者以减少或预防与抗生素相关的耳毒素诱发的听力障碍。减少或预防耳毒素诱发的听力障碍的化合物、尤其新颖siRNA优选应局部给药于内耳中，任选经鼓膜给药。In certain embodiments where the treatment or prevention of hearing impairment is an objective, the compositions of the present invention will be co-administered with ototoxins. For example, there is provided an improved method of treating infection in a mammal by administering an aminoglycoside antibiotic, the improvement comprising administering to It is prescribed to reduce or prevent antibiotic-associated ototoxin-induced hearing impairment in patients in need of such treatment. Compounds that reduce or prevent ototoxin-induced hearing impairment, especially the novel siRNAs, should preferably be administered topically in the inner ear, optionally transtympanically.
在另一实施方式中，提供一种藉由给药化学治疗化合物来治疗哺乳动物癌症的经改良方法，其改良之处包含将治疗有效量的本发明组合物给药于需要该治疗的患者以减少或预防与化学治疗药物相关的耳毒素诱发的听力障碍。在另一实施方式中，该治疗方法应用于源自化学治疗剂给药的听力障碍以治疗其耳毒性副作用。应用于本发明方法的耳毒性化学治疗剂包括(但不限于)抗赘生剂，包括顺铂或类顺铂化合物、紫杉醇或类紫杉醇化合物及相信引起耳毒素诱发的听力障碍的其它化学治疗剂(例如长春新碱，一种用以治疗恶性血液病及肉瘤的抗赘生性药物)。类顺铂化合物包括卡铂(
在另一实施方式中，本发明的方法应用于治疗或预防褥疮的发生率或严重程度。该方法及组合物包括将预防或治疗有效量的一种或多种下调RTP801基因表达的化合物、尤其新颖小干扰RNA(siRNA)给药于需要该治疗的哺乳动物。治疗或预防褥疮发生率或严重程度的化合物、尤其新颖siRNA优选应在受损区域内局部给药。当持续压力(通常来自床或轮椅)切断身体的脆弱部分的循环时，本发明的方法及组合物有效治疗且预防所发展的褥疮。该方法及组合物有效用于感觉减少或缺乏的患者或虚弱、消瘦、瘫痪或长期卧床不起的患者。使用本发明的组合物，该组合物亦有效地改善褥疮的治愈。组合物可在治愈过程中的任何特定阶段使用，包括任何治愈起始前的阶段或甚至特定疮形成前的阶段(预防性治疗)。In another embodiment, the methods of the invention are used to treat or prevent the incidence or severity of decubitus ulcers. The methods and compositions include administering a prophylactically or therapeutically effective amount of one or more compounds that down-regulate RTP801 gene expression, especially novel small interfering RNA (siRNA), to a mammal in need of such treatment. Compounds, especially the novel siRNAs, that treat or prevent the incidence or severity of decubitus ulcers should preferably be administered topically in the affected area. The methods and compositions of the invention are effective in treating and preventing bedsores that develop when constant pressure, usually from a bed or wheelchair, cuts off circulation to vulnerable parts of the body. The methods and compositions are useful for patients with reduced or absent sensation or who are weak, emaciated, paralyzed or chronically bedridden. Using the composition of the present invention, the composition is also effective in improving the healing of bedsores. The composition may be used at any specific stage in the healing process, including any stage prior to the onset of healing or even the stage prior to the development of a particular sore (prophylactic treatment).
本发明的其它种类的待治疗创伤亦包括i)一般创伤，诸如外科伤口、外伤、感染创伤、缺血创伤、热创伤、化学创伤及大泡性创伤；ii)口腔特异性创伤，诸如拔牙后创伤、尤其与包囊及脓肿治疗有关的牙髓创伤、细菌、病毒或自体免疫器官的溃疡及损害、机械创伤、化学创伤、热创伤、感染创伤及苔癣样创伤、疱疹溃疡、阿夫他性口腔炎(stomatitis aphthosa)、急性坏死溃疡性牙龈炎及口腔灼烧综合征；及iii)皮肤上的创伤，诸如赘瘤、灼伤(例如化学、热)、损害(细菌、病毒、自体免疫)、咬伤及外科手术切口。Other types of wounds to be treated according to the present invention also include i) general wounds, such as surgical wounds, trauma, infected wounds, ischemic wounds, thermal wounds, chemical wounds, and bullous wounds; ii) oral cavity-specific wounds, such as after tooth extraction Trauma, especially pulp trauma related to the treatment of cysts and abscesses, ulcers and lesions of bacterial, viral or autoimmune organs, mechanical trauma, chemical trauma, thermal trauma, infected wounds and lichenoid wounds, herpetic ulcers, aphthous ulcers stomatitis aphthosa, acute necrotizing ulcerative gingivitis and burning mouth syndrome; and iii) wounds on the skin such as neoplasms, burns (e.g. chemical, thermal), lesions (bacterial, viral, autoimmune) , bites and surgical incisions.
本发明的方法及组合物亦有效治疗且预防包括褥疮、静脉溃疡及糖尿病性溃疡的任何慢性创伤。在所有这类慢性创伤类型中，潜在加速事件为缺血时期、继而再灌注时期。这类缺血-再灌注事件常重复，此意谓缺血-再灌注的不利影响加强且最终足以引起溃疡。对褥疮及糖尿病性足部溃疡而言，缺血事件为足以防止组织灌注的压力延长的结果，且当压力最终解除时，再灌注损伤发生。本发明的组合物有效抑制由慢性创伤中的缺血-再灌注引起的损害。The methods and compositions of the invention are also effective in the treatment and prevention of any chronic wound including decubitus ulcers, venous ulcers and diabetic ulcers. In all of these types of chronic trauma, the potentially accelerating event is a period of ischemia followed by a period of reperfusion. Such ischemia-reperfusion events are often repeated, which means that the adverse effects of ischemia-reperfusion are intensified and eventually sufficient to cause ulceration. For decubitus ulcers and diabetic foot ulcers, ischemic events are the result of prolonged pressure sufficient to prevent tissue perfusion, and when the pressure is finally relieved, reperfusion injury occurs. The composition of the present invention effectively inhibits damage caused by ischemia-reperfusion in chronic wounds.
本发明的组合物亦有效用于与缺血-再灌注相关的其它病状，诸如(但不限于)器官移植、小肠及结肠吻合术、大血管手术、缝合分离肢体、气囊血管成形术或任何心脏外科手术、中风或脑外伤、肢体移植、肺循环血压过高、血氧不足及非心脏性肺水肿、急性肾衰竭、急性青光眼、糖尿病性视网膜病、高血压性视网膜病、视网膜血管阻塞、耳蜗缺血、微血管外科手术及硬皮病中的缺血损害。The compositions of the present invention are also effective in other conditions associated with ischemia-reperfusion, such as, but not limited to, organ transplantation, small bowel and colon anastomosis, major vessel surgery, suture separation of limbs, balloon angioplasty, or any cardiac Surgery, stroke or traumatic brain injury, limb transplantation, pulmonary hypertension, hypoxemia and noncardiac pulmonary edema, acute renal failure, acute glaucoma, diabetic retinopathy, hypertensive retinopathy, retinal vascular occlusion, cochlear defect Ischemic damage in blood, microvascular surgery, and scleroderma.
本发明的方法及组合物亦有效治疗声创伤或机械外伤，优选地有效治疗导致内耳毛细胞损失的声创伤或机械外伤。本发明中待治疗的声创伤可藉由单一暴露于极大声音或在长期每日暴露于85分贝以上的大声音之后而引起。本发明中待治疗的机械内耳外伤为(例如)将电子装置插入内耳的手术后的内耳外伤。本发明的组合物预防或最小化与手术相关的对内耳毛细胞造成的损害。减少或预防耳毒素诱发的听力障碍的化合物、尤其新颖siRNA优选应局部给药于内耳中。The methods and compositions of the present invention are also effective in treating acoustic or mechanical trauma, preferably acoustic or mechanical trauma resulting in loss of inner ear hair cells. The acoustic trauma to be treated in the present invention may be caused by a single exposure to loud sounds or after long-term daily exposure to loud sounds above 85 decibels. The mechanical inner ear trauma to be treated in the present invention is, for example, post-operative inner ear trauma in which an electronic device is inserted into the inner ear. The compositions of the present invention prevent or minimize surgery-related damage to inner ear hair cells. Compounds, especially the novel siRNAs, that reduce or prevent ototoxin-induced hearing impairment should preferably be administered topically in the inner ear.
此外，本发明的化合物亦可用以治疗其中涉及缺血、任选缺血-再灌注的任何病状。该病状包括源自下列情形的缺血或缺血-再灌注：血管成形术、心脏外科手术或溶栓、器官移植、由于整形手术、严重间室综合征期间、分离肢体的再连接期间、由于多器官衰竭综合征、脑中由于中风或脑外伤、与慢性创伤(诸如褥疮、静脉溃疡及糖尿病性溃疡)有关、骨骼肌缺血或肢体移植期间、由于肠系膜缺血或急性缺血性肠病、由于下躯体缺血造成的呼吸衰竭(导致肺循环血压过高、血氧不足及非心脏性气肿)、肾移植、大外科手术、外伤及脓毒性休克以及失血性休克后观测到的急性肾衰竭、脓毒病、由于急性血管阻塞而发生的视网膜缺血(在大量眼病(诸如急性青光眼、糖尿病性视网膜病、高血压性视网膜病及视网膜血管阻塞)中导致视力损失)、耳蜗缺血、针对头部及颈部缺陷的微血管外科手术中的瓣衰竭、硬皮病中的雷诺现象及相关数字缺血损害、脊髓损伤、血管外科手术、外伤横纹肌溶解(挤压综合征)及肌红蛋白尿。此外，缺血/再灌注可涉及下列病状：高血压、高血压性脑血管疾病、动脉瘤破裂、血管收缩或阻塞-如在血栓或栓子、血管瘤、血质不调、任何形式的受损心脏功能(包括心跳骤停或衰竭、全身性低血压、心跳骤停、心脏性休克、败血性休克、脊髓外伤、头外伤、癫痫发作、出血)及诸如中风、帕金森氏病、癫痫症、抑郁、ALS、阿兹海默氏病、亨廷顿氏舞蹈病及任何疾病诱发的痴呆(诸如HIV诱发的痴呆)的疾病的情形下发生。此外，缺血事件可由对中枢神经系统的机械损伤引起，诸如源自对头或脊骨的重击。外伤可包括诸如擦伤、切口、挫伤、刺破、压伤等组织损害，诸如可源于外部对象与头、颈或脊柱的任一部位或其从属物的外伤性接触。外伤的其它形式可源于藉由体液不当累积(例如正常脑脊髓或玻璃体液产生的阻塞或功能障碍、翻转或体积调节或硬膜下或硬膜内血肿或水肿)而造成的CNS组织的收缩或压缩。类似地，外伤收缩或压缩可源于大量异常组织(诸如转移性或原发性肿瘤)的存在。Furthermore, the compounds of the present invention may also be used to treat any condition in which ischemia, optionally ischemia-reperfusion is involved. The condition includes ischemia or ischemia-reperfusion resulting from angioplasty, cardiac surgery or thrombolysis, organ transplantation, due to plastic surgery, during severe compartment syndrome, during reattachment of a separated limb, due to Multiple organ failure syndrome, in the brain due to stroke or traumatic brain injury, associated with chronic trauma (such as decubitus ulcers, venous ulcers, and diabetic ulcers), during skeletal muscle ischemia or limb transplantation, due to mesenteric ischemia or acute ischemic bowel disease , respiratory failure due to lower body ischemia (resulting in pulmonary hypertension, hypoxemia, and noncardiac emphysema), renal transplantation, major surgery, trauma and septic shock, and acute renal failure observed after hemorrhagic shock Failure, sepsis, retinal ischemia due to acute vascular occlusion (resulting in vision loss in a number of eye diseases such as acute glaucoma, diabetic retinopathy, hypertensive retinopathy, and retinal vascular occlusion), cochlear ischemia, Valve failure in microvascular surgery for head and neck defects, Raynaud's phenomenon and associated digital ischemic damage in scleroderma, spinal cord injury, vascular surgery, traumatic rhabdomyolysis (crush syndrome) and myoglobin Urine. In addition, ischemia/reperfusion can be involved in the following conditions: hypertension, hypertensive cerebrovascular disease, ruptured aneurysm, vasoconstriction or occlusion - as in thrombus or embolus, hemangioma, dyscrasia, any form of Impairment of cardiac function (including cardiac arrest or failure, systemic hypotension, cardiac arrest, cardiac shock, septic shock, spinal cord trauma, head trauma, seizures, hemorrhage) and conditions such as stroke, Parkinson's disease, epilepsy , depression, ALS, Alzheimer's disease, Huntington's disease, and any disease-induced dementia such as HIV-induced dementia. In addition, ischemic events can result from mechanical injury to the central nervous system, such as from a blow to the head or spine. Trauma may include tissue damage such as abrasions, cuts, contusions, punctures, crush wounds, etc., such as may result from traumatic contact of an external object with any part of the head, neck, or spine, or its appendages. Other forms of trauma may result from constriction of CNS tissue by inappropriate accumulation of fluid such as obstruction or dysfunction of normal cerebrospinal or vitreous humor, inversion or volume adjustment, or subdural or intradural hematoma or edema or compressed. Similarly, traumatic constriction or compression may result from the presence of a large amount of abnormal tissue, such as a metastatic or primary tumor.
＂治疗疾病＂是指给药有效改善与疾病相关的症状的治疗物质以减轻严重程度或治愈疾病或预防疾病发生。＂治疗＂是指治疗性治疗及预防性方法，其中目标是预防或减缓(减轻)疾病或病症。"Treating a disease" refers to the administration of a therapeutic substance effective to ameliorate symptoms associated with a disease in order to lessen the severity or cure the disease or prevent the disease from occurring. "Treatment"refers to both therapeutic treatment and prophylactic methods, wherein the object is to prevent or slow down (lessen) a disease or condition.
＂治疗有效量＂是指有效获得患者或其生理系统的改良的医药化合物或组合物的量，其改良之处包括(但不限于)改善存活率、更快恢复或改善或消除症状及由本领域技术人员选择的适当测定方法的其它指示。"Therapeutically effective amount" means an amount of a pharmaceutical compound or composition effective to obtain an improvement in the patient or its physiological system, including, but not limited to, improved survival, faster recovery, or amelioration or elimination of symptoms as well as those known in the art. Other indications of appropriate assay methods at the choice of the skilled artisan.
治疗本文所揭示的疾病且包括于本发明中的方法可包括给药RTP801抑制剂，结合另一RTP801抑制剂(一种改良如下详述的活性成份的药理性质的物质)或已知有效治疗待治疗疾病(诸如黄斑变性、COPD、ARF、DR、顺铂或声创伤诱发的耳聋)的另一化合物。＂结合......＂意谓之前、同时或之后。关于例示性结合疗法的进一步详述在下文中给出。Methods of treating the diseases disclosed herein and encompassed by the present invention may comprise administering an RTP801 inhibitor in combination with another RTP801 inhibitor (a substance that modifies the pharmacological properties of the active ingredient as detailed below) or known effective treatment Another compound for the treatment of diseases such as macular degeneration, COPD, ARF, DR, cisplatin, or acoustic trauma-induced deafness. "In conjunction with..." means before, while or after. Further details on exemplary combination therapies are given below.
在另一实施方式中，本发明提供治疗有效剂量的RTP801抑制剂用于制备用于促进罹患如上文详述的黄斑变性、COPD、ARF、DR、顺铂或声创伤诱发的耳聋或任何其它眼病、微血管或呼吸病状或听力障碍的患者恢复的药剂的用途，及治疗有效量的RTP801抑制剂用于制备用于治疗该疾病及病状的药剂的用途。在此实施例中，RTP801抑制剂可包含聚核苷酸，该聚核苷酸包含具有包含图1中所陈述的序列(SEQ IDNO：1)的反义序列的序列的连续核苷酸。此外，RTP801抑制剂可为包含具有作为图1中所陈述的序列(SEQ ID NO：1)的反义序列的序列的聚核苷酸的表达载体。根据该用途，RTP801抑制剂亦可为抗体，诸如特异性结合存在于包含连续氨基酸的多肽(其序列显示于图2中(SEQ IDNo：2))中的抗原决定簇的中和抗体。此外，RTP801抑制剂可为靶向RTP801基因mRNA的RNA分子(任选为siRNA，任选为包含具有与表A-D中所陈述的任何序列(SEQ ID NO：3-536)一致的序列的连续核苷酸的siRNA且具体地说表A的siRNA No：14、22、23、25、27、39、41、42、49及50或表D的siRNA No：257、260-262及264-268)或核糖核酸酶。In another embodiment, the present invention provides a therapeutically effective dose of an RTP801 inhibitor for use in the manufacture of a drug for promoting macular degeneration, COPD, ARF, DR, cisplatin or acoustic trauma-induced deafness or any other ocular disease as detailed above. , the use of a medicament for the recovery of patients with microvascular or respiratory conditions or hearing impairment, and the use of a therapeutically effective amount of an RTP801 inhibitor for the preparation of a medicament for the treatment of such diseases and conditions. In this embodiment, the RTP801 inhibitor may comprise a polynucleotide comprising contiguous nucleotides having a sequence comprising an antisense sequence to the sequence set forth in Figure 1 (SEQ ID NO: 1). In addition, the RTP801 inhibitor may be an expression vector comprising a polynucleotide having a sequence that is an antisense sequence to the sequence set forth in Figure 1 (SEQ ID NO: 1). According to this use, the RTP801 inhibitor may also be an antibody, such as a neutralizing antibody that specifically binds to an epitope present in a polypeptide comprising contiguous amino acids, the sequence of which is shown in Figure 2 (SEQ ID No: 2). In addition, the RTP801 inhibitor may be an RNA molecule (optionally siRNA, optionally comprising a contiguous core having a sequence consistent with any of the sequences set forth in Tables A-D (SEQ ID NO: 3-536) targeting RTP801 gene mRNA. nucleotides and specifically siRNA Nos of Table A: 14, 22, 23, 25, 27, 39, 41, 42, 49 and 50 or siRNA Nos of Table D: 257, 260-262 and 264-268) or ribonuclease.
因此，根据本文所揭示的信息，本文所揭示的任一方法、本文所揭示的任一用途及本文所揭示的任一药物组合物中待使用的RTP801抑制剂均可选自由下列各物组成的组：siRNA、包含siRNA的载体、表达siRNA的载体及内源性加工成siRNA中的任一分子。如本文详述，该siRNA优选为包含具有与表A-D中所陈述的任一序列(SEQ ID NO：3-536)一致的序列的连续核苷酸的siRNA且具体地说表A的siRNA No：14、22、23、25、27、39、41、42、49及50或表D的siRNA No：257、260-262及264-268。Therefore, according to the information disclosed herein, the RTP801 inhibitor to be used in any of the methods disclosed herein, in any of the uses disclosed herein, and in any of the pharmaceutical compositions disclosed herein can be selected from the following: Group: any molecule of siRNA, vector containing siRNA, vector expressing siRNA, and endogenously processed siRNA. As detailed herein, the siRNA is preferably an siRNA comprising contiguous nucleotides having a sequence consistent with any of the sequences set forth in Tables A-D (SEQ ID NO: 3-536) and specifically the siRNA No of Table A: siRNA Nos. 14, 22, 23, 25, 27, 39, 41, 42, 49 and 50 or Table D: 257, 260-262 and 264-268.
＂呼吸病症＂是指呼吸系统的病状、疾病或综合征，包括(但不限于)所有类型的肺部病症，尤其包括慢性阻塞性肺病(COPD)、气肿、慢性支气管炎、哮喘及肺癌。气肿及慢性支气管炎可作为COPD的部分发生或独立发生。"Respiratory disorder" means a condition, disease or syndrome of the respiratory system, including, but not limited to, all types of pulmonary disorders including, among others, chronic obstructive pulmonary disease (COPD), emphysema, chronic bronchitis, asthma and lung cancer. Emphysema and chronic bronchitis can occur as part of COPD or independently.
＂微血管病症＂是指任何影响微毛细管及微淋巴管的病状，尤其血管痉挛性疾病、脉管炎疾病及淋巴阻塞性疾病。微血管病症的实例尤其包括：眼病，诸如阵发性黑蒙(栓塞或继发于SLE)、抗磷脂抗体综合征、Prot CS及ATIII不足、因使用IV药物而引起的微血管病理、异常蛋白血症、颞动脉炎、前部缺血性视神经病、视神经炎(原发或继发于自体免疫疾病)、青光眼、逢希柏林道综合征(von hippel lindausyndrome)、角膜病、角膜移植排斥反应白内障、伊尔斯病(Eales′disease)、霜样树枝状视网膜脉管炎、环扎术、包括睫状体平坦部炎(pars planitis)的葡萄膜炎、脉络膜黑素瘤、脉络膜血管瘤、视神经发育不全、视网膜病状(诸如视网膜动脉阻塞、视网膜静脉阻塞、早产儿视网膜病、HIV视网膜病、外伤性视网膜病、全身性脉管炎及自体免疫疾病的视网膜病、糖尿病性视网膜病、高血压性视网膜病、放射性视网膜病、树枝状视网膜动脉或静脉阻塞、特发性视网膜脉管炎、动脉瘤、视神经网膜炎、视网膜栓塞、急性视网膜坏死、乌枪弹样视网膜脉络膜病变、长期视网膜脱离)；全身性病状，诸如糖尿病、糖尿病性视网膜病(DR)、糖尿病相关的微血管病理(如本文详述)、高血黏稠度综合征(hyperviscosity syndrome)、主动脉弓综合征及眼部缺血综合征、颈动脉海绵状瘘管、多发性硬化症、全身性红斑性狼疮症、SS-A自体抗体的小动脉炎、急性多发性出血性脉管炎、源自感染的脉管炎、源自白塞病(Behcet′s disease)的脉管炎、肉状瘤病、凝血病、神经病、肾病、肾微血管病及缺血性微血管病状。"Microvascular disorder" means any condition affecting microcapillaries and microlymphatic vessels, especially vasospastic diseases, vasculitic diseases and lymphatic obstructive diseases. Examples of microvascular disorders include, inter alia: eye diseases such as amaurosis (embolism or secondary to SLE), antiphospholipid antibody syndrome, Prot CS and ATIII insufficiency, microvascular pathology due to IV drug use, dysproteinemia , temporal arteritis, anterior ischemic optic neuropathy, optic neuritis (primary or secondary to autoimmune disease), glaucoma, von hippel lindausyndrome, corneal disease, corneal graft rejection cataract, Eales' disease, frost dendritic retinal vasculitis, cerclage, uveitis including pars planitis, choroidal melanoma, choroidal hemangioma, optic nerve development Insufficiency, retinopathy (such as retinal artery occlusion, retinal vein occlusion, retinopathy of prematurity, HIV retinopathy, traumatic retinopathy, retinopathy of systemic vasculitis and autoimmune disease, diabetic retinopathy, hypertensive retinal disease, radiation retinopathy, dendritic retinal artery or vein occlusion, idiopathic retinal vasculitis, aneurysm, optic retinitis, retinal embolism, acute retinal necrosis, black bullet-like retinochoroidopathy, long-term retinal detachment); systemic STD conditions such as diabetes mellitus, diabetic retinopathy (DR), diabetes-associated microvascular pathology (as detailed herein), hyperviscosity syndrome, aortic arch syndrome and ocular ischemic syndrome, carotid artery Cavernous fistulas, multiple sclerosis, systemic lupus erythematosus, arterioarteritis with SS-A autoantibodies, acute polyhemorrhagic vasculitis, vasculitis from infection, Behcet's disease ’s disease), vasculitis, sarcoidosis, coagulopathy, neuropathy, nephropathy, renal microangiopathy, and ischemic microvascular pathology.
微血管病症可包含新生血管性元素。术语＂新生血管性病症＂是指其中血管形成(新血管生成)对患者有害的那些病状。眼部新血管生成的实例包括：视网膜病(糖尿病性视网膜病、糖尿病性黄斑水肿、慢性青光眼、视网膜脱离及镰状细胞性视网膜病)、虹膜红变、增生性玻璃体视网膜病、炎症性疾病、慢性葡萄膜炎、赘瘤(眼癌、假神经胶质瘤及黑素瘤)、富克斯异色性虹膜睫状体炎(Fuchs′heterochromiciridocyclitis)、新生血管性青光眼、角膜新血管生成(炎症、移植及虹膜发育不全)、组合的玻璃体切除术及晶状体切除术后的新血管生成、血管疾病(视网膜缺血、脉络膜血管不足、脉络膜血栓及颈动脉缺血)、视神经新血管生成及归因于眼睛穿透或撞伤性眼部损伤的新血管生成。使用本发明的化合物及药物组合物可治疗所有这类新生血管性病状。Microvascular disorders may include neovascular elements. The term "neovascular disorder" refers to those conditions in which blood vessel formation (neovascularization) is detrimental to the patient. Examples of ocular neovascularization include: retinopathy (diabetic retinopathy, diabetic macular edema, chronic glaucoma, retinal detachment, and sickle cell retinopathy), iridosis, proliferative vitreoretinopathy, inflammatory diseases, Chronic uveitis, neoplasm (eye cancer, pseudoglioma, and melanoma), Fuchs'heterochromiciridocyclitis, neovascular glaucoma, corneal neovascularization (inflammation , transplantation, and iris hypoplasia), neovascularization after combined vitrectomy and lensectomy, vascular disease (retinal ischemia, choroidal vascular insufficiency, choroidal thrombosis, and carotid ischemia), optic nerve neovascularization and attribution Neovascularization in penetrating or contusive ocular injuries. All such neovascular conditions can be treated using the compounds and pharmaceutical compositions of the present invention.
＂眼病＂是指眼部病状、疾病或综合征，包括(但不限于)包含脉络膜新生血管(CNV)、湿式及干式AMD、眼部组织浆菌病综合征、血管样条纹、布鲁赫氏膜破裂、近视性变性、眼部肿瘤、视网膜退化疾病及视网膜静脉阻塞(RVO)的任何病状。在本文所陈述的定义下，本发明方法可治疗的本文所揭示的某些病状(诸如DR)已视作微血管病症及眼病或两者。"Eye disease" means an ocular condition, disease or syndrome including, but not limited to, choroidal neovascularization (CNV), wet and dry AMD, ocular histoplasmosis syndrome, vascular striae, Bruch Ruptured membrane, myopic degeneration, ocular tumors, retinal degenerative diseases and any condition of retinal vein occlusion (RVO). Certain conditions disclosed herein, such as DR, that are treatable by the methods of the present invention have been considered microvascular disorders and ocular diseases, or both, under the definitions set forth herein.
与本发明相关的听力障碍可归因于涉及内耳毛细胞的末梢器官损害，例如声创伤、病毒性内淋巴迷路炎、美尼尔氏病(Meniere′sdisease)。听力障碍包括耳鸣，耳鸣是在声刺激缺乏下声音的感知且可为间歇或连续的，其中诊断出感觉神经损失。听力损失可归因于细菌或病毒感染，诸如耳带状疱疹、源于急性中耳炎的化脓性迷路炎、化脓性脑膜炎、慢性中耳炎、特发性耳聋(包括病毒来源的特发性耳聋，例如由病毒引起的病毒性内淋巴迷路炎，包括腮腺炎、麻疹、流行性感冒、水痘、单核细胞增多症及腺病毒)中。听力损伤可为先天的，诸如由风疹、出生期间缺氧、血液渗至内耳(归因于分娩期间的外伤)、给药于母亲的耳毒性药物、胎儿红血球母细胞增多症及遗传性病状(包括瓦登伯格氏综合征(Waardenburg′s syndrome)及贺勒氏症(Hurler′s syndrome))引起的听力损失。听力损失可由噪声诱发，一般归因于大于约85分贝(db)的破坏内耳的噪声。听力损失优选应由影响内耳的听力部分(尤其内耳毛细胞)的声创伤或耳毒性药物引起。TheMerck Manual of Diagnosis and Therapy，第14版，(1982)，MerckSharp & Dome Research Laboratories，N.J.的196、197、198及199章及最近第16版的相应章节(包括207及210章)涉及听力及平衡障碍的描述及诊断，其以引用的方式并入本文。The hearing impairments associated with the present invention may be due to peripheral organ damage involving inner ear hair cells, eg, acoustic trauma, viral endolymphatic labyrinthitis, Meniere's disease. Hearing impairment includes tinnitus, which is the perception of sound in the absence of acoustic stimulation and can be intermittent or continuous, where sensory nerve loss is diagnosed. Hearing loss can be attributable to bacterial or viral infections such as herpes zoster oticus, suppurative labyrinthitis from acute otitis media, suppurative meningitis, chronic otitis media, idiopathic deafness (including idiopathic deafness of viral origin, e.g. Viral endolymphatic labyrinthitis caused by viruses, including mumps, measles, influenza, chickenpox, mononucleosis, and adenovirus). Hearing impairment can be congenital, such as from rubella, hypoxia during birth, leakage of blood into the inner ear (due to trauma during childbirth), ototoxic drugs administered to the mother, fetal erythroblastosis, and hereditary conditions ( Includes hearing loss caused by Waardenburg's syndrome and Hurler's syndrome). Hearing loss can be induced by noise, generally due to noise greater than about 85 decibels (db) damaging the inner ear. The hearing loss should preferably be caused by acoustic trauma or ototoxic drugs affecting the hearing part of the inner ear, especially the inner ear hair cells. TheMerck Manual of Diagnosis and Therapy, 14th Edition, (1982), MerckSharp & Dome Research Laboratories, N.J. Chapters 196, 197, 198, and 199 and the corresponding chapters of the most recent 16th Edition (including Chapters 207 and 210) deal with hearing and balance Description and diagnosis of the disorder, which is incorporated herein by reference.
不受理论限制，本发明内容中待治疗或预防的听力障碍(或平衡障碍)优选为耳毒素或外伤诱发的对内耳毛细胞造成的细胞凋亡损害。需治疗的患者包括已经历听力障碍的人、倾向于患有障碍的人及待预防障碍的人。不受理论限制，听力障碍可归因于细胞凋亡性内耳毛细胞损害或损失，其中由感染、机械损伤、大的声音、年老或化学药品诱发的耳毒性引起损害或损失。耳毒素包括治疗药物，包括抗赘生剂、水杨酸盐、奎宁及氨基糖苷抗生素、食品或药物污染物及环境或工业污染物。通常应实施治疗以预防或减少尤其源自或期望源自治疗药物的给药或暴露于包括诱发声创伤的环境的耳毒性或声创伤。治疗有效组合物优选应在暴露后立即给予以预防或减少耳毒作用或外伤作用。更优选应藉由在给药耳毒性药物或暴露于耳毒素或诱发声创伤的环境之前或同时给药组合物来预防治疗。Without being limited by theory, the hearing impairment (or balance disorder) to be treated or prevented in the present invention is preferably the apoptotic damage to inner ear hair cells induced by ototoxin or trauma. Those in need of treatment include those who have already experienced a hearing impairment, those who are prone to have the disorder and those in whom the disorder is to be prevented. Without being limited by theory, the hearing impairment may be due to apoptotic inner ear hair cell damage or loss caused by infection, mechanical injury, loud sound, aging or chemical-induced ototoxicity. Ototoxins include therapeutic drugs, including antineoplastic agents, salicylates, quinine, and aminoglycoside antibiotics, food or drug contaminants, and environmental or industrial contaminants. Treatment should generally be performed to prevent or reduce ototoxicity or acoustic trauma, particularly resulting or expected from administration of a therapeutic drug or exposure to circumstances including those that induce acoustic trauma. Therapeutically effective compositions should preferably be administered immediately after exposure to prevent or reduce ototoxic or traumatic effects. More preferably the prophylactic treatment should be by administering the composition prior to or simultaneously with the administration of ototoxic drugs or exposure to ototoxic or acoustic trauma inducing environments.
听力障碍可由化学疗法诱发。具体说，听力障碍可由诸如依托泊苷、5-FU(5-氟尿嘧啶)、顺铂、阿霉素、长春花属生物碱、长春新碱、长春碱、长春瑞宾、紫杉醇、环磷酸胺、异环磷酰胺、苯丁酸氮芥、白消安、氮芥、丝裂霉素、达卡巴嗪、卡铂、塞替派、柔红霉素、伊达比星、米托蒽醌、博来霉素、埃斯波霉素Al、放线菌素D、普卡霉素、卡莫司汀、洛莫司汀、牛磺莫司汀、链佐星、美法仑、放线菌素D、丙卡巴肼、地塞米松、泼尼松、2-氯脱氧腺苷、阿糖胞苷、多烯紫杉醇、氟达拉滨、吉西他滨、赫赛汀、羟基尿、伊立替康、甲胺喋呤、奥沙利铂、瑞土星(rituxin)、司莫司汀、表柔比星、依托泊苷、雷替曲塞及拓朴替康的化学治疗剂或这类化学治疗剂之一的化学类似物引起。最可能引起听力障碍的化学治疗剂为顺铂及类顺铂化合物。Hearing impairment can be induced by chemotherapy. Specifically, hearing impairment can be caused by factors such as etoposide, 5-FU (5-fluorouracil), cisplatin, doxorubicin, vinca alkaloids, vincristine, vinblastine, vinorelbine, paclitaxel, cyclophosphamide, Ifosfamide, chlorambucil, busulfan, nitrogen mustard, mitomycin, dacarbazine, carboplatin, thiotepa, daunorubicin, idarubicin, mitoxantrone, Leymycin, Espermycin Al, Actinomycin D, Pleucamycin, Carmustine, Lomustine, Tauromustine, Streptozocin, Melphalan, Actinomycin D , procarbazine, dexamethasone, prednisone, 2-chlorodeoxyadenosine, cytarabine, docetaxel, fludarabine, gemcitabine, Herceptin, hydroxyuria, irinotecan, methotrexate Chemotherapeutic agents of phosphatine, oxaliplatin, rituxin, semustine, epirubicin, etoposide, raltitrexed, and topotecan, or a chemical of one of such chemotherapeutic agents Analogs cause. The chemotherapeutic agents most likely to cause hearing impairment are cisplatin and cisplatin-like compounds.
在本发明内容中，＂耳毒素＂意谓经由化学作用而损伤、损害或抑制与听力相关的神经系统的声音接受器，继而损害听力(和/或平衡)的活性的物质。在本发明的内容中，耳毒性包括对内耳毛细胞的不利影响。引起听力障碍的耳毒性药剂包括(但不限于)赘生性药剂(诸如长春新碱、长春碱、顺铂及类顺铂化合物、紫杉醇及类紫杉醇化合物、双脱氧化合物(例如双脱氧肌苷))、醇类、金属、涉及工作或环境暴露的工业毒素、食品或药物污染物及过多剂量的维生素或治疗药物(例如抗生素，诸如盘尼西林或氯霉素)及大剂量的维生素A、D或B6、水杨酸盐、奎宁及髓袢利尿剂。＂暴露于耳毒性药剂＂意谓耳毒性药剂可为哺乳动物所得或接触哺乳动物。暴露于耳毒性药剂可藉由直接给药而发生，例如藉由摄取或给药食品、药物或治疗剂(例如化学治疗剂)、藉由意外污染或藉由环境暴露(例如空气或水暴露)而发生。In the context of the present invention, "ototoxin" means a substance that chemically damages, damages or inhibits the sound receptors of the hearing-related nervous system, thereby impairing the activity of hearing (and/or balance). In the context of the present invention, ototoxicity includes adverse effects on the hair cells of the inner ear. Ototoxic agents that cause hearing impairment include, but are not limited to, neoplastic agents (such as vincristine, vinblastine, cisplatin and cisplatin-like compounds, paclitaxel and paclitaxel-like compounds, dideoxy compounds (such as dideoxyinosine)) , alcohols, metals, industrial toxins involving work or environmental exposure, food or drug contaminants, and excessive doses of vitamins or therapeutic drugs (eg, antibiotics such as penicillin or chloramphenicol) and large doses of vitamins A, D, or B6 , salicylates, quinine, and loop diuretics. "Exposure to an ototoxic agent" means that the ototoxic agent is available to or exposed to a mammal. Exposure to ototoxic agents can occur by direct administration, such as by ingestion or administration of food, drugs, or therapeutic agents (such as chemotherapeutic agents), by accidental contamination, or by environmental exposure (such as air or water exposure) And happened.
＂RTP801基因＂是指编码如图1中所示的序列开放阅读框架(SEQ IDNO：1)或优选具有至少70％同一性、更优选80％同一性、甚至更优选90％或95％同一性的其任一同源序列的RTP801。此包含衍生自SEQ ID NO：1的任何序列，该序列已经历如本文所述的突变、变化或修饰。因此，在一优选实施例中，RTP801藉由根据SEQ ID NO：1的核酸序列编码。亦在本发明内，本发明的核酸仅互补且分别与编码RTP801的核酸部分一致，因为第一链段及第一链通常优选应短于本发明的核酸。亦应了解基于RTP801的氨基酸序列，编码该氨基酸序列的任何核酸序列均可藉由本领域技术人员基于遗传密码来了解。然而，归因于本发明核酸的假定作用模式，编码RTP801的核酸、优选地其mRNA最佳应为分别存在于有机体、组织和/或细胞中者，其中RTP801的表达将减少。"RTP801 gene" refers to an open reading frame encoding a sequence as shown in Figure 1 (SEQ ID NO: 1) or preferably has at least 70% identity, more preferably 80% identity, even more preferably 90% or 95% identity RTP801 of any of its homologous sequences. This includes any sequence derived from SEQ ID NO: 1 that has been mutated, altered or modified as described herein. Therefore, in a preferred embodiment, RTP801 is encoded by the nucleic acid sequence according to SEQ ID NO:1. Also within the present invention, the nucleic acid according to the invention is only complementary and in each case partially identical to the nucleic acid encoding RTP801, since the first strand and the first strand should generally preferably be shorter than the nucleic acid according to the invention. It should also be understood that based on the amino acid sequence of RTP801, any nucleic acid sequence encoding the amino acid sequence can be understood by those skilled in the art based on the genetic code. However, due to the postulated mode of action of the nucleic acids of the invention, the nucleic acid encoding RTP801, preferably its mRNA, should optimally be present in an organism, tissue and/or cell, respectively, in which the expression of RTP801 will be reduced.
＂RTP801多肽＂是指RTP801基因的多肽，且应了解为获得本发明的目的，其包括术语＂RTP779＂、＂REDD1＂、＂Ddit4＂、＂FLJ20500＂、＂Dig2＂及＂PRF1＂，其衍生自任一有机体(任选为人)、保持生物活性的剪接变异体及其片段及优选具有至少70％、更优选至少80％、甚至更优选至少90％或95％同源性的其同源物。此外，应了解此术语包含源自RTP801编码序列的较小改变的多肽，诸如可引起所得多肽与天然产生的RTP801之间的若干氨基酸不同的点突变、取代、缺失及插入。此术语亦包含藉由在高严格杂交条件下结合RTP801编码序列或基因组序列的核苷酸序列编码的多肽，该多肽已为本领域技术人员所熟知(例如Ausubel等人，Current Protocols in Molecular Biology，John Wiley and Sons，Baltimore，Maryland(1988)，1995及1998修正)。只要保留生物活性，则经化学修饰的RTP801或经化学修饰的RTP801片段亦包括在此术语中。RTP801优选应具有或包含根据SEQ.ID.NO.2的氨基酸序列。应了解，在有机体的不同组织中及一种物种的不同有机体中或在本发明的多个实施例中可应用本发明核酸的不同物种中，氨基酸序列可能存在差异。然而，当设计本发明的任何核酸时，基于本文所提供的技术教导，因此可考虑个别序列。RTP801的特定片段包括图2中所示的氨基酸1-50、51-100、101-150、151-200及201-232序列。RTP801的其它特定片段包括图2中所示的氨基酸25-74、75-124、125-174、175-224及225-232序列。如本文所用的RTP801为WO 99/09046中所述的蛋白。亦称为RTP801的RTP801已由ShoshaniT等人描述为HIF-1α的转录目标(Shoshani等人，2002，Mol Cell Biol，22，2283-93)。此外，Ellisen等人的研究(Ellisen等人，Mol Cell，10，995-1005)已鉴别出RTP801为p53依赖性DNA损害回应基因且为涉及上皮细胞分化的p63依赖性基因。又，RTP801反映了组织特异性类型的p53家族成员p63，有效类似于TP63或除TP63外亦为活体外分化的抑制剂，且涉及活性氧的调节。此外，RTP801响应低氧反应转录因子低氧诱发因子1(HIF-1)且通常在缺血性中风的动物模型中于活体外及活体内低氧期间经上调。RTP801似乎在活性氧(ROS)的调节中起作用，且ROS含量及减少的对氧化应力的敏感性均随着异常表达RTP801而增加(前述的Ellisen等人，2002；前述的Soshani等人，2002)。RTP801优选为生物活性RTP801蛋白，其优选显示那些特征中的至少之一，优选两种或两种以上且最佳每一个及这类特征中的任一个。"RTP801 polypeptide" refers to a polypeptide of the RTP801 gene, and it is understood that for the purposes of the present invention, it includes the terms "RTP779", "REDD1", "Ddit4", "FLJ20500", "Dig2" and "PRF1", derivatives thereof From any organism, optionally human, splice variants and fragments thereof that retain biological activity and homologues thereof preferably have at least 70%, more preferably at least 80%, even more preferably at least 90% or 95% homology. Furthermore, it is to be understood that this term encompasses polypeptides derived from minor alterations of the RTP801 coding sequence, such as point mutations, substitutions, deletions and insertions which may result in several amino acid differences between the resulting polypeptide and naturally occurring RTP801. The term also includes polypeptides encoded by nucleotide sequences that bind to the RTP801 coding sequence or genomic sequence under highly stringent hybridization conditions, which polypeptides are well known to those skilled in the art (e.g. Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, Maryland (1988), 1995 and 1998 revision). Chemically modified RTP801 or chemically modified fragments of RTP801 are also included in this term as long as the biological activity is retained. RTP801 should preferably have or comprise the amino acid sequence according to SEQ.ID.NO.2. It is understood that amino acid sequences may vary in different tissues of an organism and in different organisms of a species or in different species to which nucleic acids of the invention are applicable in various embodiments of the invention. However, when designing any nucleic acid of the invention, individual sequences may therefore be considered based on the technical teachings provided herein. Specific fragments of RTP801 include the amino acid 1-50, 51-100, 101-150, 151-200, and 201-232 sequences shown in Figure 2 . Other specific fragments of RTP801 include the amino acid 25-74, 75-124, 125-174, 175-224 and 225-232 sequences shown in Figure 2 . RTP801 as used herein is the protein described in WO 99/09046. RTP801, also known as RTP801, has been described by Shoshani T et al. as a transcriptional target of HIF-1α (Shoshani et al., 2002, Mol Cell Biol, 22, 2283-93). Furthermore, studies by Ellisen et al. (Ellisen et al., Mol Cell, 10, 995-1005) have identified RTP801 as a p53-dependent DNA damage response gene and as a p63-dependent gene involved in epithelial cell differentiation. In addition, RTP801 reflects the tissue-specific type of p53 family member p63, which is effectively similar to or in addition to TP63 and is also an inhibitor of in vitro differentiation, and is involved in the regulation of reactive oxygen species. Furthermore, RTP801 responds to the hypoxia-responsive transcription factor hypoxia-inducible factor 1 (HIF-1) and is often upregulated during in vitro and in vivo hypoxia in animal models of ischemic stroke. RTP801 appears to play a role in the regulation of reactive oxygen species (ROS), and both ROS levels and reduced sensitivity to oxidative stress are increased with aberrant expression of RTP801 (Ellisen et al., 2002, supra; Soshani et al., 2002, supra. ). RTP801 is preferably a biologically active RTP801 protein which preferably exhibits at least one of those characteristics, preferably two or more and optimally each and any of such characteristics.
不受理论限制，应力诱发蛋白RTP801(回应低氧、氧化应力、热应力、ER应力)是在细胞回应能量不平衡的微调中起作用的因子。同样，目标为适于治疗应从归因于应力条件的细胞凋亡救出细胞的任何疾病(例如伴随正常细胞死亡的疾病)或适应归因于RTP801表达的改变的应力条件的细胞(例如癌细胞)应被杀死的任何疾病。在后一情形下，RTP801可视作癌细胞的存活因子，且其抑制剂可作为单独疗法或作为与化学疗法或放射疗法组合的敏化药物来治疗癌症。Without being bound by theory, the stress-induced protein RTP801 (Response to Hypoxia, Oxidative Stress, Heat Stress, ER Stress) is a factor that plays a role in the fine-tuning of cellular responses to energy imbalances. Likewise, the aim is to be suitable for the treatment of any disease in which cells should be rescued from apoptosis due to stress conditions (e.g. diseases accompanied by normal cell death) or cells adapted to stress conditions due to altered expression of RTP801 (e.g. cancer cells) Any disease that should be killed. In the latter case, RTP801 can be considered as a survival factor for cancer cells, and its inhibitors can be used to treat cancer as a monotherapy or as a sensitizing drug in combination with chemotherapy or radiotherapy.
术语＂聚核苷酸＂是指包含DNA核苷酸、RNA核苷酸或两种类型的组合的任何分子，亦即包含碱基胍、胞嘧啶、胸苷、腺嘌呤、尿嘧啶或肌苷中的两者或两者以上的分子。聚核苷酸可包括天然核苷酸、经化学修饰的核苷酸及合成核苷酸或其化学类似物。术语包括＂寡核苷酸＂且包含＂核酸＂。The term "polynucleotide" refers to any molecule comprising DNA nucleotides, RNA nucleotides or a combination of both types, i.e. comprising the bases guanidine, cytosine, thymidine, adenine, uracil or inosine Two or more molecules in . Polynucleotides can include natural nucleotides, chemically modified nucleotides, and synthetic nucleotides or chemical analogs thereof. The term includes "oligonucleotide" and includes "nucleic acid."
术语＂氨基酸＂是指由20个天然产生的氨基酸、经化学修饰的氨基酸(参见下文)或合成氨基酸的任一个组成的分子。The term "amino acid" refers to a molecule consisting of any of the 20 naturally occurring amino acids, chemically modified amino acids (see below), or synthetic amino acids.
术语＂多肽＂是指包含两个或两个以上氨基酸残基的分子。该术语包括肽、多肽、蛋白及肽模拟物。The term "polypeptide" refers to a molecule comprising two or more amino acid residues. The term includes peptides, polypeptides, proteins and peptidomimetics.
＂肽模拟物＂为含有非肽结构元素的化合物，其能够模拟天然亲本肽的生物作用。某些经典肽特征(诸如酶裂解肽键)通常并不存在于肽模拟物中。A "peptidomimetic" is a compound containing non-peptide structural elements that is capable of mimicking the biological actions of the native parent peptide. Certain classical peptide features, such as enzymatic cleavage of peptide bonds, are generally not present in peptidomimetics.
术语＂显性阴性肽＂意谓藉由编码一部分蛋白的cDNA片段编码的多肽(参见Herskowitz I.：Functional inactivation of genes bydominant negative mutations.Nature.1987年9月17-23；329(6136)：219-22.Review；Roninson IB等人，Genetic suppressorelements：new tools for molecular oncology--thirteenthCornelius P.Rhoads Memorial Award Lecture.Cancer Res.1995年9月15日；55(18)：4023)。此肽可具有不同于衍生其的蛋白的功能。其可与完整蛋白相互作用且抑制蛋白活性，或其可与其它蛋白相互作用且抑制蛋白活性以响应全长(亲本)蛋白。显性阴性意谓肽能对抗天然亲本蛋白且抑制其活性以给予细胞不同特征，诸如对死亡或所关注的任何细胞表型的对抗或敏化。针对治疗介入，肽自身可作为药物组合物的活性成份来传递，或可利用已知方法将cDNA传递至细胞。The term "dominant negative peptide" means a polypeptide encoded by a cDNA fragment encoding a part of a protein (see Herskowitz I.: Functional inactivation of genes by dominant negative mutations. Nature. 1987 September 17-23; 329(6136): 219 -22. Review; Roninson IB et al., Genetic suppressor elements: new tools for molecular oncology--thirteenth Cornelius P. Rhoads Memorial Award Lecture. Cancer Res. 1995
肽及多肽的制备Preparation of peptides and polypeptides
多肽可经由若干方法而产生，例如：Polypeptides can be produced by several methods, such as:
1)合成：1) Synthesis:
合成多肽可使用市售机器使用RTP801或其部分的已知序列来制成。Synthetic polypeptides can be made using commercially available machines using the known sequence of RTP801 or a portion thereof.
2)重组方法：2) Recombination method:
制成RTP801多肽或其片段的优选方法是将包含RTP801基因的cDNA的聚核苷酸克隆至表达载体中且培养具有该载体的细胞以表达经编码的多肽，且接着纯化所得多肽，其中所有步骤均使用本领域已知的方法执行，举例而言，如Marshak等人，＂Strategies for ProteinPurification and Characterization.A laboratory course manual.＂CSHL Press(1996)(另外参见Bibl Haematol.1965；23：1165-74Appl Microbiol.1967年7月；15(4)：851-6；Can J Biochem.1968年5月；46(5)：441-4；Biochemistry.1968年7月；7(7)：2574-80；ArchBiochem Biophys.1968年9月10日；126(3)：746-72；Biochem BiophysRes Commun.1970年2月20日；38(4)：825-30)中所述的方法。A preferred method of making an RTP801 polypeptide or fragment thereof is to clone a polynucleotide comprising the cDNA of the RTP801 gene into an expression vector and to culture cells bearing the vector to express the encoded polypeptide, and then to purify the resulting polypeptide, wherein all steps All are performed using methods known in the art, for example, as in Marshak et al., "Strategies for Protein Purification and Characterization. A laboratory course manual." CSHL Press (1996) (see also Bibl Haematol. 1965; 23: 1165-74 Appl Microbiol. 1967 July; 15(4): 851-6; Can J Biochem. 1968 May; 46(5): 441-4; Biochemistry. 1968 July; 7(7): 2574-80; Arch Biochem Biophys. 1968
表达载体可包括用于控制异源物质转录的启动子，且可为构成或诱发启动子以允许选择性转录。任选可包括可要求用以获得必要转录程度的增强子。表达载体(expression vehicle)亦可包括选择基因。Expression vectors may include a promoter for controlling transcription of the heterologous material, and may be a constitutive or inducible promoter to allow selective transcription. Optionally, enhancers may be included that may be required to achieve the necessary degree of transcription. An expression vehicle may also include a selection gene.
载体可藉由本领域已知的多种方法中的任一种而引入细胞或组织中。可发现该方法一般描述于Sambrook等人，Molecular Cloning：ALaboratory Manual，Cold Springs Harbor Laboratory，New York(1989，1992)；Ausubel等人，Current Protocols in MolecularBiology，John Wiley and Sons，Baltimore，Maryland(1989)；Vega等人，Gene Targeting，CRC Press，Ann Arbor，MI(1995)，Vectors：A Surveyo f Molecular Cloning Vectors and Their Uses，Butterworths，Boston MA(1988)及Gilboa等人(1986)中。Vectors can be introduced into cells or tissues by any of a variety of methods known in the art. This method can be found generally described in Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Springs Harbor Laboratory, New York (1989, 1992); Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, Maryland (1989) in Vega et al., Gene Targeting, CRC Press, Ann Arbor, MI (1995), Vectors: A Survey of Molecular Cloning Vectors and Their Uses, Butterworths, Boston MA (1988) and Gilboa et al. (1986).
3)自天然来源纯化：3) Purified from natural sources:
RTP801多肽或其天然产生片段可使用本领域技术人员已知的多种方法自天然来源(诸如组织)纯化，该方法诸如：用抗RTP801抗体免疫沉淀或用已知结合RTP801的任一分子进行基质结合的亲和层析。RTP801 polypeptides, or naturally occurring fragments thereof, can be purified from natural sources, such as tissues, using a variety of methods known to those skilled in the art, such as: immunoprecipitation with anti-RTP801 antibodies or matrix processing with any molecule known to bind RTP801. Binding affinity chromatography.
如本领域已知来实施蛋白纯化，举例而言，如Marshak等人，＂Strategies for Protein Purification and Characterization.Alaboratory course manual.＂CSHL Press(1996)所述。Protein purification is performed as known in the art, for example, as described in Marshak et al., "Strategies for Protein Purification and Characterization. Alaboratory course manual." CSHL Press (1996).
＂RTP801的生物效应＂或＂RTP801生物活性＂意谓RTP801在呼吸病症中的作用，该作用可为直接或间接的，且不受理论限制，其包括RTP801对由低氧或高氧条件诱发的肺泡细胞的细胞凋亡的作用。间接作用包括(但不限于)RTP801结合涉及导致细胞凋亡的信号传递级联的若干分子之一或作用于其。"Biological effects of RTP801" or "RTP801 biological activity" means the effects of RTP801 in respiratory disorders, which may be direct or indirect, and without being bound by theory, include the effect of RTP801 on the effects of hypoxic or hyperoxic conditions. Apoptotic role in alveolar cells. Indirect effects include, but are not limited to, RTP801 binding or acting on one of several molecules involved in the signaling cascade leading to apoptosis.
＂细胞凋亡＂是指生理学类型的细胞死亡，其源自某些细胞机制的活化，亦即藉由细胞机构控制的死亡。细胞凋亡可为(例如)藉由导致细胞死亡之外部触发(例如细胞因子或抗FAS抗体)或藉由内部信号活化细胞机构的结果。术语＂渐进式细胞死亡＂亦可与＂细胞凋亡＂交替使用。"Apoptosis" refers to a physiological type of cell death resulting from the activation of certain cellular mechanisms, ie death controlled by cellular machinery. Apoptosis can be the result, for example, of activation of cellular machinery by external triggers leading to cell death, such as cytokines or anti-FAS antibodies, or by internal signals. The term "progressive cell death" is also used interchangeably with "apoptosis".
＂细胞凋亡相关的疾病＂是指病源学与细胞凋亡过程完全或部分相关的疾病。该疾病可由细胞凋亡过程的功能障碍(诸如在癌症或自体免疫疾病中)或由细胞凋亡过程的过度活性引起(诸如在某些神经退化疾病中)。RTP801涉及的多种疾病为细胞凋亡相关的疾病。举例而言，细胞凋亡为干式AMD的重要机制，主要在视网膜的中心(黄斑)区域中的感光体及色素上皮细胞发生缓慢萎缩。神经视网膜细胞凋亡亦为糖尿病性视网膜病的重要机制。"Apoptosis-associated disease" refers to a disease whose etiology is wholly or partially related to the process of apoptosis. The disease can be caused by a dysfunction of the apoptotic process, such as in cancer or autoimmune diseases, or by an overactivity of the apoptotic process, such as in certain neurodegenerative diseases. Many diseases involved in RTP801 are apoptosis-related diseases. For example, apoptosis is an important mechanism in dry AMD, with slow atrophy of photoreceptors and pigment epithelium mainly in the central (macular) region of the retina. Neuroretinal cell apoptosis is also an important mechanism of diabetic retinopathy.
＂抑制剂＂为一种能够抑制基因或该基因产物的活性至足以获得所需生物或生理作用程度的化合物。＂RTP801抑制剂＂为一种能够抑制RTP801基因或RTP801基因产物、尤其人类RTP801基因或基因产物活性的化合物。该抑制剂包括影响基因转录或转译的物质以及影响基因产物活性的物质。RTP801抑制剂亦可为RTP801启动子的抑制剂。该抑制剂的实例可包括聚核苷酸(诸如AS片段、siRNA或包含其的载体)、多肽(诸如显性阴性肽、抗体及酶)、催化性RNAs(诸如核糖核酸酶)及低分子量(例如低于2000道尔顿的分子量)的化学分子。特异性RTP801抑制剂述于下文。An "inhibitor" is a compound capable of inhibiting the activity of a gene or the gene product to an extent sufficient to obtain a desired biological or physiological effect. An "RTP801 inhibitor" is a compound capable of inhibiting the activity of the RTP801 gene or RTP801 gene product, especially the human RTP801 gene or gene product. Such inhibitors include substances that affect the transcription or translation of genes as well as substances that affect the activity of gene products. The RTP801 inhibitor can also be an inhibitor of the RTP801 promoter. Examples of such inhibitors may include polynucleotides (such as AS fragments, siRNA or vectors containing them), polypeptides (such as dominant-negative peptides, antibodies, and enzymes), catalytic RNAs (such as ribonucleases), and low molecular weight ( Chemical molecules such as molecular weights below 2000 Daltons). Specific RTP801 inhibitors are described below.
＂表达载体＂是指能够并入且表达外来细胞中的异源DNA片段的载体。多种原核及真核表达载体为已知和/或可购得。适当表达载体的选择是在本领域技术人员的知识内。"Expression vector" refers to a vector capable of incorporating and expressing a heterologous DNA segment in a foreign cell. A variety of prokaryotic and eukaryotic expression vectors are known and/or commercially available. Selection of appropriate expression vectors is within the knowledge of those skilled in the art.
术语＂抗体＂尤其是指IgG、IgM、IgD、IgA及IgE抗体。该定义包括多克隆抗体或单克隆抗体。此术语是指全抗体或包含抗原结合域的抗体片段(例如无Fc部分的抗体、单链抗体、微小抗体、基本上仅由抗体的可变抗原结合域组成的片段等)。术语＂抗体＂亦可指对抗cDNA疫苗接种所获得的聚核苷酸序列的抗体。该术语亦包含保留选择性结合抗原或受体的能力的抗体片段，例示如下：The term "antibody" especially refers to IgG, IgM, IgD, IgA and IgE antibodies. This definition includes polyclonal or monoclonal antibodies. The term refers to a whole antibody or an antibody fragment comprising an antigen binding domain (eg, an antibody without an Fc portion, a single chain antibody, a minibody, a fragment consisting essentially only of the variable antigen binding domain of an antibody, etc.). The term "antibody" may also refer to antibodies raised against polynucleotide sequences obtained by cDNA vaccination. The term also encompasses antibody fragments that retain the ability to selectively bind an antigen or receptor, as exemplified below:
(1)Fab，含有可藉由用木瓜蛋白酶消化全抗体以产生轻链及一部分重链而产生的抗体分子的单价抗原结合片段的片段；(1) Fab, a fragment comprising a monovalent antigen-binding fragment of an antibody molecule that can be produced by papain digestion of a whole antibody to produce a light chain and a portion of a heavy chain;
(2)(Fab′)2，可藉由用胃蛋白酶处理全抗体而无后续还原所获得的抗体片段；F(ab′2)为藉由两个双硫键固持于一起的两个Fab片段的二聚体；(2) (Fab')2 , an antibody fragment obtainable by treating a whole antibody with pepsin without subsequent reduction; F(ab'2 ) is two Fab fragments held together by two disulfide bonds the dimer;
(3)Fv，定义为含有表达为两条链的轻链可变区及重链可变区的基因工程片段；及(3) Fv, defined as a genetically engineered fragment containing a light chain variable region and a heavy chain variable region expressed as two chains; and
(4)单链抗体(SCA)，定义为含有藉由适合多肽连接体键联的轻链可变区及重链可变区的作为基因融合单链分子的基因工程分子。(4) Single-chain antibody (SCA), defined as a genetically engineered molecule containing a light chain variable region and a heavy chain variable region linked by a suitable polypeptide linker as a genetically fused single-chain molecule.
微型抗体及微体亦包括于此术语中。微体为具有假节结构的极小(通常约28-45个氨基酸)蛋白，该微体具有高度选择性且稳定。因此，本文所揭示的包括抗体的任一应用及新颖化合物均可包括微型抗体或微体以替代作为分子的抗体部分。Minibodies and microbodies are also included in this term. Microbodies are extremely small (typically about 28-45 amino acids) proteins with a pseudoknot structure that are highly selective and stable. Accordingly, any of the uses and novel compounds disclosed herein that include antibodies may include minibodies or microbodies in place of the antibody portion of the molecule.
如本发明所用的术语＂抗原决定簇＂意谓抗体结合的抗原上的抗原决定子。抗原决定性决定子常由化学活性表面分子的组(诸如氨基酸或糖侧链)组成且常具有特异性三维结构特征以及特定电荷特征。The term "antigenic determinant" as used in the present invention means an antigenic determinant on an antigen to which an antibody binds. Antigenic determinants often consist of groups of chemically active surface molecules such as amino acids or sugar side chains and often have specific three-dimensional structural characteristics as well as specific charge characteristics.
抗RTP801抗体的制备Preparation of anti-RTP801 antibody
结合RTP801的抗体或衍生自其的片段可使用完整多肽或含有更小多肽作为免疫抗原的片段来制备。举例而言，可需要产生特异性结合N末端或C末端或RTP801的任何其它适合域的抗体。用以免疫动物的多肽可衍生自经转译的cDNA或化学合成，且必要时可结合载体蛋白。该与多肽化学偶联的通用载体包括钥孔虫戚血蓝蛋白(KLH)、甲状腺球蛋白、牛血清白蛋白(BSA)及破伤风类毒素。接着，将经偶联的多肽用于免疫动物。Antibodies that bind RTP801, or fragments derived therefrom, can be prepared using the entire polypeptide or fragments containing smaller polypeptides as immunizing antigens. For example, it may be desirable to generate antibodies that specifically bind the N- or C-terminus or any other suitable domain of RTP801. Polypeptides used to immunize animals can be derived from translated cDNA or chemically synthesized and, if necessary, bound to a carrier protein. The universal carrier chemically coupled with the polypeptide includes keyhole limpet hemocyanin (KLH), thyroglobulin, bovine serum albumin (BSA) and tetanus toxoid. Next, the conjugated polypeptide is used to immunize animals.
举例而言，必要时可藉由结合到基质并从基质洗脱来进一步纯化多克隆或单克隆抗体，所述基质上结合有产生所述抗体的多肽或肽。本领域技术人员应了解用于纯化和/或浓缩多克隆抗体以及单克隆抗体的免疫学中常见的各种技术(Coligan等人，第9单元，CurrentProtocols in Immunology，Wiley Interscience，1994)。For example, polyclonal or monoclonal antibodies can be further purified, if desired, by binding to and eluting from the matrix to which the polypeptide or peptide from which the antibody is produced is bound. Those skilled in the art will be aware of the various techniques common in immunology for purifying and/or concentrating polyclonal antibodies as well as monoclonal antibodies (Coligan et al.,
用于产生所有类型的抗体(包括片段)的方法在本领域已知(例如，参见Harlow及Lane，Antibodies：A Laboratory Manual，Cold SpringHarbor Laboratory，New York(1988))。包括在适合佐剂中制备免疫原、确定抗体结合、分离抗体、用于获得单克隆抗体的方法及人源化单克隆抗体的所有必要步骤的免疫方法均为熟练技工所知。Methods for producing antibodies of all types, including fragments, are known in the art (see, e.g., Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, New York (1988)). Immunization methods including all necessary steps for preparation of immunogens in suitable adjuvants, determination of antibody binding, isolation of antibodies, methods for obtaining monoclonal antibodies, and humanization of monoclonal antibodies are known to the skilled artisan.
抗体可为人源化抗体或人类抗体。可使用本领域已知的多种技术来人源化抗体，包括CDR-移植(EP239,400：PCT公开案WO.91/09967；美国专利第5,225,539、5,530,101及5,585,089号)、镶饰(veneering)或重塑(resurfacing)(EP 592,106；EP 519,596；Padlan，MolecularImmunology 28(4/5)：489-498(1991)；Studnicka等人，ProteinEngineering 7(6)：805-814(1994)；Roguska等人，PNAS 91：之内-973(1994))及链改组(美国专利第5,565,332号)。Antibodies can be humanized antibodies or human antibodies. Antibodies can be humanized using a variety of techniques known in the art, including CDR-grafting (EP239,400: PCT Publication WO.91/09967; US Patent Nos. 5,225,539, 5,530,101 and 5,585,089), veneering, or resurfacing (EP 592,106; EP 519,596; Padlan, Molecular Immunology 28(4/5): 489-498 (1991); Studnicka et al., Protein Engineering 7(6): 805-814 (1994); Roguska et al. , PNAS 91: Inner-973 (1994)) and chain shuffling (US Patent No. 5,565,332).
所定义的单克隆抗体包括衍生自一种物种(诸如鼠科动物、兔、山羊、大鼠、人类等)的抗体以及衍生自两种(或两种以上)物种的抗体，诸如嵌合及人源化抗体。Monoclonal antibodies as defined include antibodies derived from one species (such as murine, rabbit, goat, rat, human, etc.) as well as antibodies derived from two (or more) species, such as chimeric and human derivatized antibodies.
具体地说，完全人类抗体来治疗人类患者是特别理想的。人类抗体可藉由本领域已知的多种方法而产生，该方法包括使用衍生自人类免疫球蛋白序列的抗体文库的噬菌体展示技术。亦参见美国专利第4,444,887号及第4,716,111号及PCT公开案WO 98/46645、WO98/50433、WO 98/24893、WO 98/16654、WO 96/34096、WO 96/33735及WO 91/10741，其各自均以全文引用的方式并入本文。In particular, fully human antibodies are particularly desirable for the treatment of human patients. Human antibodies can be produced by a variety of methods known in the art, including phage display technology using antibody libraries derived from human immunoglobulin sequences. See also U.S. Patent Nos. 4,444,887 and 4,716,111 and PCT Publications WO 98/46645, WO 98/50433, WO 98/24893, WO 98/16654, WO 96/34096, WO 96/33735, and WO 91/10741, which Each is incorporated herein by reference in its entirety.
关于所有类型抗体(包括人源化抗体、人类抗体及抗体片段)的额外信息可见于WO 01/05998，其以全文引用的方式并入本文。Additional information on antibodies of all types, including humanized antibodies, human antibodies, and antibody fragments, can be found in WO 01/05998, which is incorporated herein by reference in its entirety.
中和抗体可藉由上文所讨论的方法来制备，可能存在藉由(例如)存活检定来筛检中和活性的额外步骤。Neutralizing antibodies can be prepared by the methods discussed above, possibly with the additional step of screening for neutralizing activity by, for example, a survival assay.
术语＂化合物＂、＂小分子＂、＂化学分子＂、＂小化学分子＂及＂小化合物＂在本文中可交替使用，且应了解其是指可合成产生或自天然来源获得且通常具有少于2000道尔顿、少于1000道尔顿或甚至少于600道尔顿的分子量的任何特定类型的化学部分。The terms "compound", "small molecule", "chemical molecule", "small chemical molecule" and "small compound" are used interchangeably herein, and it is understood that they refer to compounds that are synthetically produced or obtained from natural sources and generally have little Any particular type of chemical moiety with a molecular weight of less than 2000 Daltons, less than 1000 Daltons, or even less than 600 Daltons.
本发明亦涉及包含双链结构的功能核酸、其用于制造药剂的用途、包含该功能核酸的药物组合物及用于治疗患者的方法。The present invention also relates to a functional nucleic acid comprising a double-stranded structure, its use for the manufacture of a medicament, a pharmaceutical composition comprising the functional nucleic acid and a method for treating a patient.
低氧已被视为大量疾病的病理机制的关键元素，诸如与次佳氧可用性及响应低氧条件的组织损伤相关的中风、气肿及梗塞。在包括肿瘤的快速生长组织中，次佳氧可用性藉由不良新血管生成补偿。因此，至少在癌症的情形下，并不需要脉管结构的生长。Hypoxia has been recognized as a key element in the pathology of a number of diseases such as stroke, emphysema and infarction associated with suboptimal oxygen availability and tissue damage in response to hypoxic conditions. In rapidly growing tissues, including tumors, suboptimal oxygen availability is compensated by poor neovascularization. Thus, at least in the case of cancer, growth of vasculature is not required.
因此，血管生成及血管生长的抑制分别经受密集型研究。目前，已可获得某些抑制不良血管生成及血管生长的化合物。某些更显著的化合物是抑制VEGF及VEGF受体的化合物。在两种情形下，VEGF的作用应藉由阻断VEGF(例如藉由使用对抗诸如由Genentech的阿伐司汀(AVASTIN，特异性针对VEGF的单株AB)购买的VEGF的抗体)(Ferrara N；Endocr Rev.2004年8月；25(4)：581-611)或藉由阻断相应受体(亦即VEGF受体)(Traxler P，Cancer Res.2004年7月15日；64(14)：4931-41或Stadler WM等人，Clin Cancer Res.2004年5月15日；10(10)：3365-70)来避免。Therefore, inhibition of angiogenesis and vascular growth, respectively, has been subject to intensive research. Currently, certain compounds that inhibit undesirable angiogenesis and growth of blood vessels are available. Some of the more notable compounds are compounds that inhibit VEGF and VEGF receptors. In both cases, the action of VEGF should be achieved by blocking VEGF (e.g. by using antibodies against VEGF such as commercially available from Genentech's AVASTIN (monoclonal AB specific for VEGF)) (Ferrara N ; Endocr Rev.2004 August; 25(4):581-611) or by blocking the corresponding receptor (ie VEGF receptor) (Traxler P, Cancer Res.2004 July 15th; 64(14 ): 4931-41 or Stadler WM et al., Clin Cancer Res. 2004 May 15; 10(10): 3365-70) to avoid.
然而，因为血管生成及脉管结构生长为任何动物及人类中极其基本且重要的过程，所以必须关注此种化合物在实际上并不需要血管生成及血管生长的特定位点的作用，此给予适当的目标靶向或传递(与此种治疗方法相关的关键性问题)。However, because angiogenesis and the growth of vascular structures are extremely fundamental and important processes in any animal and human, attention must be paid to the action of such compounds at specific sites where angiogenesis and vascular growth are not actually required, given appropriate target targeting or delivery (a critical issue associated with this approach to therapy).
因此，本发明的目标是提供用于治疗分别涉及不良脉管结构生长及血管生成的疾病的其它方法。It is therefore an object of the present invention to provide additional methods for the treatment of diseases involving undesirable vascular structure growth and angiogenesis respectively.
＂小干扰RNA＂(siRNA)意谓一种RNA分子，其减少其内源性细胞对应物的基因/mRNA表达或使其沉默(预防)。应了解该术语包含＂RNA干扰＂(RNAi)。RNA干扰(RNAi)是指藉由小干扰RNA(siRNA)介导的哺乳动物中序列特异性转录后基因沉默的过程(Fire等人，1998，Nature391.806)。植物中的相应过程通常称为特异性转录后基因沉默或RNA沉默，且亦称为压制真菌。RNA干扰反应可表征含有siRNA的核酸内切酶复合物，该复合物通常称为RNA诱发的沉默复合物(RISC)，其介导具有siRNA双链体反义链的互补序列的单链RNA的裂解。靶向RNA的裂解可发生在siRNA双链体反义链的互补区域的中部(Elbashir等人，2001，GenesDev.，15，188)。关于这类术语及所陈述的机制的近期信息，参见Bernstein E.，Denli AM.，Hannon GJ：The rest is silence.RNA.2001年11月；7(11)：1509-21及Nishikura K.：A short primer onRNAi：RNA-directed RNA polymerase acts as a key catalyst.Cell.2001年11月16日；107(4)：415-8。可用于本发明的siRNA分子的实例在表A-D中给出。"Small interfering RNA" (siRNA) means an RNA molecule that reduces or silences (prevents) the gene/mRNA expression of its endogenous cellular counterpart. It is to be understood that the term encompasses "RNA interference" (RNAi). RNA interference (RNAi) refers to the process of sequence-specific post-transcriptional gene silencing in mammals mediated by small interfering RNA (siRNA) (Fire et al., 1998, Nature391.806 ). The corresponding process in plants is often referred to as specific post-transcriptional gene silencing or RNA silencing, and is also known as fungal suppression. The RNA interference response characterizes the siRNA-containing endonuclease complex, commonly referred to as the RNA-induced silencing complex (RISC), that mediates the cleavage of single-stranded RNA with the complementary sequence to the antisense strand of the siRNA duplex. crack. Cleavage of the targeted RNA can occur in the middle of the complementary region of the antisense strand of the siRNA duplex (Elbashir et al., 2001, Genes Dev.,15 , 188). For recent information on such terms and the mechanisms stated, see Bernstein E., Denli AM., Hannon GJ: The rest is silence. RNA. 2001 Nov;7(11):1509-21 and Nishikura K.: A short primer on RNAi: RNA-directed RNA polymerase acts as a key catalyst. Cell. 2001
近年间，RNAi已呈现为用于基因失活的最有效方法之一(NatureReviews，2002，第3卷，第737-47页；Nature，2002，第418页，第244-51页)。作为一种方法，RNAi是基于dsRNA物质进入特定蛋白复合物的能力，其在该蛋白复合物中继而靶向互补细胞RNA且特异性使其降解。具体说，藉由III型RNAse(DICER、Drosha等)将dsRNA消化成短(17-29bp)的抑制性RNA(siRNA)(Nature，2001，409卷，第363-6页；Nature，2003，425卷，第415-9页)。这类片段及互补mRNA应藉由特定RISC蛋白复合物来识别。整个过程是藉由靶向mRNA的核酸内切酶裂解来结束(Nature Reviews，2002，第3卷，第737-47页；Curr Opin MolTher.2003年6月；5(3)：217-24)。In recent years,RNAi has emerged as one of the most effective methods for gene inactivation (Nature Reviews, 2002, Vol. 3, pp. 737-47; Nature, 2002, pp. 418, pp. 244-51). As one approach, RNAi is based on the ability of dsRNA species to enter specific protein complexes where they in turn target complementary cellular RNAs and specifically cause them to be degraded. Specifically, dsRNA is digested into short (17-29bp) inhibitory RNA (siRNA) by type III RNAse (DICER, Drosha, etc.) (Nature, 2001, volume 409, pages 363-6; Nature, 2003, 425 vol., pp. 415-9). Such fragments and complementary mRNA should be recognized by specific RISC protein complexes. The whole process ends by endonuclease cleavage of targeted mRNA (Nature Reviews, 2002, Vol. 3, pp. 737-47; Curr Opin MolTher. 2003 June; 5(3): 217-24) .
关于如何设计及制备已知基因的siRNA的揭示内容，参见(例如)Pei & Tuschl On the Art of Identifying Effective and SpecificsiRNAs Nature Methods 3 No.9，2006年9月；Chalk AM，WahlestedtC，Sonnhammer EL.Improved and automated prediction ofeffective siRNA Biochem.Biophys.Res.Commun.2004年6月18日；319(1)：264-74；Sioud M，Leirdal M.，Potential design rules andenzymatic synthesis of siRNAs，Methods MolBiol.2004；252：457-69；Levenkova N，Gu Q，Rux JJ.：Gene specificsiRNA selector Bioinformatics.2004年2月12日；20(3)：430-2；及Ui-Tei K，Naito Y，Takahashi F，Haraguchi T，Ohki-Hamazaki H，Juni A，Ueda R，Saigo K.，Guidelines for the se l ection of highlyeffective siRNA sequences for mammalian and chick RNAinterference Nucleic Acids Res.2004年2月9日；32(3)：936-48。亦参见Liu Y，Braasch DA，Nulf CJ，Corey DR.Efficient andisoform-selective inhibition of cellular gene expression bypeptide nucleic acids Biochemistry，2004年2月24日；43(7)：1921-7。亦参见用于产生经修饰/更稳定siRNA的PCT公开案WO2004/015107(Atugen)及WO02/44321(Tuschl等人)以及Chiu YL，RanaTM.siRNA function in RNAi：a chemical modification analysis，RNA2003年9月；9(9)：1034-48及美国专利第5898031号及第6107094号(Crooke)。For disclosures on how to design and prepare siRNAs for known genes, see (eg) Pei & Tuschl On the Art of Identifying Effective and Specific
已发展能够在细胞内产生siRNA的以DNA为主的载体。该方法一般包括转录经有效加工以在细胞内形成siRNA的短的发夹型RNA。Paddison等人，PNAS2002，99：1443-1448；Paddison等人，Genes &Dev 2002，16：948-958；Sui等人，PNAS 2002，8：5515-5520及Brummelkamp等人，Science 2002，296：550-553。这类报导描述了产生能够特异性靶向经内源性及外源性表达的基因的siRNA的方法。DNA-based vectors capable of producing siRNA in cells have been developed. The method generally involves the transcription of short hairpin RNAs that are efficiently processed to form siRNAs within the cell. Paddison et al., PNAS2002, 99:1443-1448; Paddison et al., Genes & Dev 2002, 16:948-958; Sui et al., PNAS 2002, 8:5515-5520 and Brummelkamp et al., Science 2002, 296:550- 553. Such reports describe methods for generating siRNAs capable of specifically targeting endogenously and exogenously expressed genes.
关于siRNA的传递，参见(例如)Shen等人(FEBS letters 539：111-114(2003))；Xia等人，Nature Biotechnology 20：1006-1010(2002)；Reich等人，Molecular Vision 9：210-216(2003)；Sorensen等人(J.Mol.Biol.327：761-766(2003))；Lewis等人，NatureGenetics 32：107-108(2002)；及Simeoni等人，Nucleic AcidsResearch 31，11：2717-2724(2003)。近来，siRNA已成功用于灵长类动物中的抑制作用；进一步详述参见Tolentino等人，Retina 24(1)，2004年2月，第132-138页。Regarding delivery of siRNA, see, for example, Shen et al. (FEBS letters 539:111-114 (2003)); Xia et al., Nature Biotechnology 20:1006-1010 (2002); Reich et al., Molecular Vision 9:210- 216 (2003); Sorensen et al. (J. Mol. Biol. 327:761-766 (2003)); Lewis et al., Nature Genetics 32:107-108 (2002); and Simeoni et al.,
本发明的siRNAsiRNA of the present invention
本发明siRNA的总体说明General Description of siRNAs of the Invention
一般而言，本发明所用的siRNA包含核糖核酸，该核糖核酸包含双链结构，其中双链结构包含第一链及第二链，其中第一链包含第一连续核苷酸链段且其中该第一链段至少部分互补于靶向核酸，且第二链包含第二连续核苷酸链段且其中该第二链段至少部分与靶向核酸一致，其中该第一链和/或该第二链包含复数个经修饰核苷酸的组，该核苷酸在2′位上具有修饰，其中在该链内各组经修饰的核苷酸通过核苷酸的侧翼基团侧接于核苷酸的一或两侧，其中形成该组侧接核苷酸的侧接核苷酸为未经修饰的核苷酸或具有不同于经修饰核苷酸的修饰的核苷酸。此外，该第一链和/或该第二链可包含该复数个经修饰核苷酸且可包含该复数个组的经修饰核苷酸群。Generally speaking, the siRNA used in the present invention comprises ribonucleic acid comprising a double-stranded structure, wherein the double-stranded structure comprises a first strand and a second strand, wherein the first strand comprises a first continuous nucleotide segment and wherein the The first stretch is at least partially complementary to the target nucleic acid, and the second strand comprises a second contiguous nucleotide stretch and wherein the second stretch is at least partially identical to the target nucleic acid, wherein the first strand and/or the second strand The second strand contains a plurality of groups of modified nucleotides having a modification at the 2' position, wherein within the strand each group of modified nucleotides is flanked by the nucleotide's flanking group to the core One or both sides of a nucleotide, wherein the flanking nucleotides forming the set of flanking nucleotides are unmodified nucleotides or nucleotides with modifications other than modified nucleotides. Furthermore, the first strand and/or the second strand may comprise the plurality of modified nucleotides and may comprise the plurality of groups of modified nucleotides.
经修饰核苷酸的组和/或侧接核苷酸的组可包含大量核苷酸，其中数目是选自包含1个核苷酸至10个核苷酸的组。结合本文所说明的任何范围，应了解各范围揭示在用以界定包括界定该范围的该两个数字的范围的个别数字之间的任一个别整数。因此，在本发明情形下，该组包含一个核苷酸、两个核苷酸、三个核苷酸、四个核苷酸、五个核苷酸、六个核苷酸、七个核苷酸、八个核苷酸、九个核苷酸及十个核苷酸。The set of modified nucleotides and/or the set of flanking nucleotides may comprise a number of nucleotides, wherein the number is selected from the group comprising 1 nucleotide to 10 nucleotides. In conjunction with any ranges described herein, it is to be understood that each range discloses any individual integer between the individual numbers defining the range including the two numbers defining the range. Thus, in the context of the present invention, the group comprises one nucleotide, two nucleotides, three nucleotides, four nucleotides, five nucleotides, six nucleotides, seven nucleotides acid, eight nucleotides, nine nucleotides and ten nucleotides.
该第一链的经修饰核苷酸类型可与该第二链的经修饰核苷酸类型相同，且可与该第二链的类型对准。此外，该第一链的类型可藉由相对于第二链类型的一种或多种核苷酸而改变。The modified nucleotide type of the first strand can be the same as the modified nucleotide type of the second strand and can be aligned with the type of the second strand. Additionally, the type of the first strand can be altered by one or more nucleotides relative to the type of the second strand.
上文所讨论的修饰可选自包含胺基、氟基、甲氧基、烷氧基及烷基的组。The modifications discussed above may be selected from the group comprising amine, fluoro, methoxy, alkoxy and alkyl groups.
可使一侧或两侧的siRNA双链结构末端平整。更明确言之，可使由第一链的5′末端及第二链的3′末端界定的双链结构侧的双链结构末端平整，或可使由第一链的3′末端及第二链的5′末端界定的双链结构侧的双链结构末端平整。The ends of the siRNA duplex can be blunt on one or both sides. More specifically, the end of the double-stranded structure on the side of the double-stranded structure defined by the 5' end of the first strand and the 3' end of the second strand may be blunt, or the end defined by the 3' end of the first strand and the second The ends of the double-stranded structure on the side of the double-stranded structure defined by the 5' ends of the strands are blunt.
此外，两条链中的至少之一可在5′末端具有至少一个核苷酸的悬垂物；该悬垂物可由至少一个脱氧核糖核苷酸组成。该链中的至少之一亦可任选在3′末端具有至少一个核苷酸的悬垂物。Additionally, at least one of the two strands may have an overhang of at least one nucleotide at the 5' end; the overhang may consist of at least one deoxyribonucleotide. At least one of the strands may also optionally have an overhang of at least one nucleotide at the 3' end.
siRNA双链结构的长度通常为约17至21个碱基且更优选为18或19个碱基。此外，该第一链的长度和/或该第二链的长度可彼此独立地选自包含约15至约23个碱基、17至21个碱基及18或19个碱基的范围的组。The length of the siRNA duplex structure is typically about 17 to 21 bases and more preferably 18 or 19 bases. In addition, the length of the first strand and/or the length of the second strand may be independently selected from a group comprising ranges of about 15 to about 23 bases, 17 to 21 bases, and 18 or 19 bases .
此外，极佳地可在该第一链与靶向核酸之间形成互补，或形成于第一链与靶向核酸之间的双链体可包含至少15个核苷酸，其中形成该双链结构的该第一链与靶向核酸之间存在一个错配或两个错配。Furthermore, complementarity can preferably be formed between the first strand and the target nucleic acid, or a duplex formed between the first strand and the target nucleic acid can comprise at least 15 nucleotides, wherein the duplex formed There is one mismatch or two mismatches between the first strand of the structure and the target nucleic acid.
在某些情形下，第一链及第二链各包含至少一组经修饰核苷酸及至少一组侧接核苷酸，其中各组经修饰核苷酸包含至少一个核苷酸且其中各组侧接核苷酸包含至少一个核苷酸，其中第一链的各组经修饰核苷酸与第二链的侧接核苷酸的组对准，其中第一链的最末端5′核苷酸为经修饰核苷酸的组的核苷酸，且第二链的最末端3′核苷酸为侧接核苷酸的组的核苷酸。各组经修饰核苷酸可由单个核苷酸组成和/或各侧接核苷酸的组可由单个核苷酸组成。In certain instances, the first strand and the second strand each comprise at least one set of modified nucleotides and at least one set of flanking nucleotides, wherein each set of modified nucleotides comprises at least one nucleotide and wherein each The set of flanking nucleotides comprises at least one nucleotide, wherein each set of modified nucleotides of the first strand is aligned with a set of flanking nucleotides of the second strand, wherein the most terminal 5' core of the first strand The nucleotides are nucleotides of the group of modified nucleotides, and the most terminal 3' nucleotides of the second strand are nucleotides of the group of flanking nucleotides. Each set of modified nucleotides may consist of a single nucleotide and/or each set of flanking nucleotides may consist of a single nucleotide.
此外，在第一链上形成侧接核苷酸的组的核苷酸可能为相对于形成经修饰核苷酸的组的核苷酸排列在3′方向的未经修饰核苷酸，且在第二链上形成经修饰核苷酸的组的核苷酸可能为相对于形成侧接核苷酸的组的核苷酸排列在5′方向的经修饰核苷酸。In addition, the nucleotides forming the group of flanking nucleotides on the first strand may be unmodified nucleotides arranged in the 3' direction relative to the nucleotides forming the group of modified nucleotides, and in The nucleotides forming the group of modified nucleotides on the second strand may be modified nucleotides arranged in the 5' direction relative to the nucleotides forming the group of flanking nucleotides.
此外，siRNA的第一链可包含8至12个、优选9至11个组的经修饰核苷酸，且第二链可包含7至11个、优选8至10个经修饰核苷酸的组。Furthermore, the first strand of the siRNA may comprise a set of 8 to 12, preferably 9 to 11 modified nucleotides, and the second strand may comprise a set of 7 to 11, preferably 8 to 10 modified nucleotides .
第一链与第二链可藉由可包含非核酸聚合物(诸如聚乙二醇)的环结构键联。或者，环结构可包含核酸。The first strand and the second strand may be linked by a loop structure which may comprise a non-nucleic acid polymer such as polyethylene glycol. Alternatively, the loop structure may comprise nucleic acid.
此外，siRNA第一链的5′末端可键联至第二链的3′末端，或第一链的3′末端可键联至第二链的5′末端，该键联是经由长度在10-2000个核碱基(nucleobase)之间的核酸连接体。In addition, the 5' end of the first strand of the siRNA can be linked to the 3' end of the second strand, or the 3' end of the first strand can be linked to the 5' end of the second strand via - Nucleic acid linkers between 2000 nucleobases.
本发明siRNA的特定说明Specific Description of siRNAs of the Invention
本发明提供具有以下结构(结构A)的化合物：The present invention provides compounds having the following structure (Structure A):
5′(N)x-Z3′(反义链)5′(N)x -Z3′ (antisense strand)
3′Z′-(N′)y5′(有义链)3'Z'-(N')y 5' (sense strand)
其中各N及N′为糖残基中可经修饰或未经修饰的核糖核苷酸，且(N)x及(N′)y为各连续N或N′藉由共价键接合另一N或N′的寡聚物；wherein each N and N' is a ribonucleotide that may be modified or not modified in the sugar residue, and (N)x and (N')y are each consecutive N or N' bonded to another by a covalent bond N or N'oligomers;
其中x及y的每一个均为19与40之间的整数；wherein each of x and y is an integer between 19 and 40;
其中Z及Z′的每一个均可存在或不存在，但若存在，则可为dTdT且共价连接在其所存在的链的3′末端；wherein each of Z and Z' may or may not be present, but if present, may be dTdT and is covalently attached to the 3' end of the chain in which it is present;
且其中(N)x序列包含RTP801基因的cDNA的互补序列。And wherein (N)x sequence comprises the complementary sequence of cDNA of RTP801 gene.
具体说，本发明提供上文化合物，其中(N)x序列包含一或多种存在于表A、B、C及D、尤其表D中的反义序列。In particular, the present invention provides the above compounds, wherein the (N)x sequence comprises one or more of the antisense sequences present in Tables A, B, C and D, especially Table D.
具体说，本发明提供上文化合物，其中共价键为磷酸二酯键，其中x＝y，优选地其中x＝y＝19，其中Z及Z′均缺乏，其中至少一种核糖核苷酸在其糖残基中的2′位上经修饰，其中2′位上的部分为甲氧基(2′-O-甲基)，其中交替核糖核苷酸在反义链及有义链中均经修饰，且其中反义链的5′及3′末端上的核糖核苷酸在其糖残基中经修饰，且有义链的5′及3′末端上的核糖核苷酸在其糖残基中未经修饰。In particular, the present invention provides the above compounds, wherein the covalent bond is a phosphodiester bond, wherein x=y, preferably wherein x=y=19, wherein both Z and Z' are absent, wherein at least one ribonucleotide Modified at the 2' position in its sugar residue, where the part at the 2' position is methoxy (2'-O-methyl), where alternating ribonucleotides are in the antisense and sense strands Both are modified, and wherein the ribonucleotides on the 5' and 3' ends of the antisense strand are modified in their sugar residues, and the ribonucleotides on the 5' and 3' ends of the sense strand are modified in their Unmodified in sugar residues.
具体说，用于本发明的siRNA为寡核糖核苷酸，其中一条链包含具有SEQ ID NO：3-52或SEQ ID NO：103-174或SEQ ID NO：247-295或SEQ ID NO：345-440(有义链)中所陈述的5′至3′序列的连续核苷酸(其中复数个碱基可优选藉由2-O-甲基修饰来修饰)或其同系物，其中在多达2个核苷酸中各末端区域中的碱基均改变。Specifically, the siRNA used in the present invention is an oligoribonucleotide, one of which has a chain comprising SEQ ID NO: 3-52 or SEQ ID NO: 103-174 or SEQ ID NO: 247-295 or SEQ ID NO: 345 - Contiguous nucleotides of the 5' to 3' sequence stated in -440 (sense strand) (wherein a plurality of bases may be modified preferably by 2-O-methyl modification) or homologues thereof, wherein in multiple The bases in each terminal region were changed up to 2 nucleotides.
此外，本发明提供一种用于治疗罹患呼吸病症、眼病、微血管病症、听力障碍、褥疮或其它卧床不起相关的皮肉伤或脊髓损伤或疾病的患者的方法，该方法包括将治疗有效量的包含上文结构(A)的化合物(具有上文提及的任一特性)的药物组合物给药于患者以藉此治疗患者。此外，本发明亦提供治疗有效量的上文结构(A)(具有上文提及的任一特性)用于制备用于促进罹患呼吸病症、眼病、微血管病症、听力障碍、褥疮或其它卧床不起相关的皮肉伤或脊髓损伤或疾病的患者恢复的药剂的用途。In addition, the present invention provides a method for treating a patient suffering from a respiratory disorder, eye disease, microvascular disorder, hearing impairment, decubitus ulcer or other bedridden related muscular or spinal cord injury or disease, the method comprising administering a therapeutically effective amount of A pharmaceutical composition comprising a compound of structure (A) above (having any of the properties mentioned above) is administered to a patient to thereby treat the patient. In addition, the present invention also provides a therapeutically effective amount of the above structure (A) (having any of the above-mentioned characteristics) for the preparation of a drug for promoting respiratory disorders, eye diseases, microvascular disorders, hearing impairment, bedsores or other ambulatory conditions. Use of a medicament for the recovery of patients with related muscular or spinal cord injuries or diseases.
本发明的另一方面提供包含上文结构(A)的化合物的药物组合物，其用于治疗本文所提及的任何疾病及病状。Another aspect of the present invention provides a pharmaceutical composition comprising a compound of structure (A) above for use in the treatment of any of the diseases and conditions mentioned herein.
此外，此方面亦提供包含两种或两种以上上文结构(A)的化合物的药物组合物以治疗本文所提及的任何疾病及病状，其中在药物组合物中该两种化合物可以产生相同或有益活性的量以物理方式混合于一起，或可藉由长度介于2-100、优选2-50或2-30个核苷酸之间的核酸连接体共价或非共价结合或接合于一起。因此，该siRNA分子包含如本文所述的双链核酸结构，其中选自表A-D且优选选自表A ID No：14、22、23、25、27、39、41、42、49及50或表D的siRNA No：257、260-262及264-268的两个siRNA序列是藉由连接体共价或非共价结合或接合以形成串联siRNA分子。该包含两个siRNA序列的串联siRNA分子通常具有38-150个核苷酸的长度，更优选具有38或40-60个核苷酸的长度，且若串联分子中包括两个以上siRNA序列，则因此长度更长。包含两个或两个以上编码经由内部细胞加工而产生的siRNA的较长序列的较长串联分子(例如，长dsRNA)亦被视作编码两个或两个以上shRNA的串联分子。该串联分子亦被视为本发明的部分，且关于其的其它信息在下文中给出。In addition, this aspect also provides a pharmaceutical composition comprising two or more compounds of structure (A) above to treat any of the diseases and conditions mentioned herein, wherein the two compounds in the pharmaceutical composition can produce the same or amounts of beneficial activity are physically mixed together, or can be covalently or non-covalently bound or joined by a nucleic acid linker with a length between 2-100, preferably 2-50 or 2-30 nucleotides together. Accordingly, the siRNA molecule comprises a double-stranded nucleic acid structure as described herein, wherein it is selected from Tables A-D and preferably selected from Table A ID Nos: 14, 22, 23, 25, 27, 39, 41, 42, 49 and 50 or The two siRNA sequences of siRNA Nos: 257, 260-262 and 264-268 in Table D are covalently or non-covalently bonded or joined by a linker to form a tandem siRNA molecule. The tandem siRNA molecule comprising two siRNA sequences typically has a length of 38-150 nucleotides, more preferably 38 or 40-60 nucleotides in length, and if more than two siRNA sequences are included in the tandem molecule, then Hence the longer length. A longer tandem molecule (eg, a long dsRNA) comprising two or more longer sequences encoding siRNAs produced through internal cellular processing is also considered a tandem molecule encoding two or more shRNAs. This tandem molecule is also considered part of the invention and further information about it is given below.
该经组合或串联的结构具有各siRNA的毒性和/或脱靶效应最小化而效率增加的优势。This combined or tandem structure has the advantage of minimizing toxicity and/or off-target effects of each siRNA while increasing efficiency.
具体地说，用于实例中的siRNA已经修饰，使得2′-O-Me基团存在于反义链的第一、第三、第五、第七、第九、第十一、第十三、第十五、第十七及第十九核苷酸上，其中存在极类似修饰，亦即2′-O-Me基团存在于有义链的第二、第四、第六、第八、第十、第十二、第十四、第十六及第十八核苷酸上。此外，应注意在本发明的这类特定核酸情形下，第一链段与第一链一致且第二链段与第二链一致，且这类核酸亦经末端平整化。虽然所用的siRNA已经磷酸化，但观察到未经磷酸化的形式可更易于大规模制备且发现CNV模型中该未经磷酸化的REDD14(称为REDD-14NP)与REDD-14具有相同生物活性(参见实施例6)。用于实施例6-8的实验中的此siRNA序列为REDD14的序列，亦即具有内部参考号14的序列(参见表A)。实施例14的实验中所用的siRNA称为801_1及801_4；801_1在表D中具有内部参考号257(SEQ ID NO：429[有义]及525[反义])且801_4在表D中具有内部参考号260(SEQ IDNO：432[有义]及528[反义])(参见表D)。Specifically, the siRNAs used in the examples have been modified such that 2'-O-Me groups are present on the first, third, fifth, seventh, ninth, eleventh, thirteenth antisense strands , on the fifteenth, seventeenth and nineteenth nucleotides, where there is a very similar modification, that is, the 2'-O-Me group exists on the second, fourth, sixth, eighth of the sense strand , Tenth, twelfth, fourteenth, sixteenth and eighteenth nucleotides. Furthermore, it should be noted that in the case of such specific nucleic acids of the invention, the first segment is identical to the first strand and the second segment is identical to the second strand, and such nucleic acids are also blunt-ended. Although the siRNA used was already phosphorylated, it was observed that the unphosphorylated form could be more easily produced on a large scale and this unphosphorylated REDD14 (referred to as REDD-14NP) was found to have the same biological activity as REDD-14 in the CNV model (See Example 6). The siRNA sequence used in the experiments of Examples 6-8 was the sequence of REDD14, ie the sequence with internal reference number 14 (see Table A). The siRNAs used in the experiments of Example 14 were designated 801_1 and 801_4; 801_1 has internal reference number 257 in Table D (SEQ ID NO: 429 [sense] and 525 [antisense]) and 801_4 has internal reference number 257 in Table D Reference number 260 (SEQ ID NO: 432 [sense] and 528 [antisense]) (see Table D).
该sRNA亦可经磷酸化或未经磷酸化。The sRNA can also be phosphorylated or unphosphorylated.
寡核苷酸的末端区域是指19-mer序列中的碱基1-4和/或16-19(下文表A、B及D)及21-mer序列中的碱基1-4和/或18-21(下文表C)。The terminal region of the oligonucleotide refers to bases 1-4 and/or 16-19 in the 19-mer sequence (Tables A, B and D below) and bases 1-4 and/or in the 21-mer sequence 18-21 (Table C below).
此外，本发明所使用的siRNA为寡核糖核苷酸，其中一条链包含具有SEQ ID NO：53-102或SEQ ID NO：175-246或SEQ ID NO：296-344或SEQ ID NO：441-536(反义链)中所陈述的5′至3′序列的连续核苷酸或其同系物，其中在多达2个核苷酸中各末端区域中的碱基均改变。In addition, the siRNA used in the present invention is an oligoribonucleotide, and one of the strands contains a sequence with SEQ ID NO: 53-102 or SEQ ID NO: 175-246 or SEQ ID NO: 296-344 or SEQ ID NO: 441- Contiguous nucleotides of the 5' to 3' sequence stated in 536 (antisense strand), or a homologue thereof, wherein the bases in each terminal region are changed in up to 2 nucleotides.
因此，在特定方面中，寡核苷酸包含双链结构，其中该双链结构包含第一链及第二链，其中第一链包含第一连续核苷酸链段且第二链包含第二连续核苷酸链段，其中第一链段与编码基因RTP801的核酸序列互补或一致且其中第二链段与编码RTP801的核酸序列一致或互补。该第一链段包含至少14个核苷酸、优选至少18个核苷酸且甚至更优选19个核苷酸或甚至至少21个核苷酸。在一实施方式中，第一链段包含约14至40个核苷酸，优选约18至30个核苷酸，更优选约19至27个核苷酸且最佳约19至23个核苷酸。在一实施方式中，第二链段包含约14至40个核苷酸，优选约18至30个核苷酸，更优选约19至27个核苷酸且最佳约19至23个核苷酸或甚至约19至21个核苷酸。在一实施方式中，第一链段的第一核苷酸对应于编码RTP801的核酸序列的核苷酸，其中第一链段的最终一个核苷酸对应于编码RTP801的核酸序列的核苷酸。在一实施方式中，第一链段包含寡核苷酸的至少14个连续核苷酸序列，其中该寡核苷酸是选自包含SEQ.ID.No：3-536的组，优选选自包含具有表A中序号14、22、23、25、27、39、41、42、49及50或表D中序号260-262及264-268中的任一个的序列的寡核苷酸的组。此外，本发明所用的siRNA分子的说明可提供寡核糖核苷酸，其中二核苷酸dTdT共价连接至3′末端，和/或在至少一个核苷酸中糖残基可能经包含2′-O-甲基修饰的修饰来修饰。此外，2′OH基团可经选自包含-H-OCH3、-OCH2CH3、-OCH2CH2CH3、-NH2及F的组的基团或部分置换。此外，如上文所揭示的本发明的优选化合物可经磷酸化或未经磷酸化。Thus, in particular aspects, an oligonucleotide comprises a double-stranded structure, wherein the double-stranded structure comprises a first strand and a second strand, wherein the first strand comprises a first stretch of contiguous nucleotides and the second strand comprises a second Contiguous nucleotide segments, wherein the first segment is complementary or identical to the nucleic acid sequence encoding gene RTP801 and wherein the second segment is identical or complementary to the nucleic acid sequence encoding RTP801. The first stretch comprises at least 14 nucleotides, preferably at least 18 nucleotides and even more preferably 19 nucleotides or even at least 21 nucleotides. In one embodiment, the first stretch comprises about 14 to 40 nucleotides, preferably about 18 to 30 nucleotides, more preferably about 19 to 27 nucleotides and optimally about 19 to 23 nucleosides acid. In one embodiment, the second stretch comprises about 14 to 40 nucleotides, preferably about 18 to 30 nucleotides, more preferably about 19 to 27 nucleotides and optimally about 19 to 23 nucleosides acid or even about 19 to 21 nucleotides. In one embodiment, the first nucleotide of the first segment corresponds to the nucleotide of the nucleic acid sequence encoding RTP801, wherein the last nucleotide of the first segment corresponds to the nucleotide of the nucleic acid sequence encoding RTP801 . In one embodiment, the first segment comprises at least 14 contiguous nucleotide sequences of oligonucleotides, wherein the oligonucleotides are selected from the group comprising SEQ.ID.No: 3-536, preferably selected from The set comprising oligonucleotides having the sequence of any of the
此外，本发明所用的siRNA可为寡核糖核苷酸，其中在交替核苷酸中经修饰的糖定位于两条链中。具体地说，寡核糖核苷酸可包含一条有义链，其中末端5′及3′核苷酸中糖未经修饰；或一条反义链，其中末端5′及3′核苷酸中糖经修饰。In addition, siRNAs used in the present invention may be oligoribonucleotides in which the modified sugars are located in both strands in alternating nucleotides. Specifically, oligoribonucleotides may comprise a sense strand in which the sugars in the terminal 5' and 3' nucleotides are unmodified; or an antisense strand in which the sugars in the terminal 5' and 3' nucleotides are unmodified; Modified.
此外，本发明欲用的其它核酸包含SEQ.ID.NO：3至536的任一个的至少14个连续核苷酸，且更优选包含在包含如上文所述的第一链段及第二链段的双链结构的任一末端处的14个连续核苷酸碱基对。本领域技术人员应了解，给定本发明核酸及尤其形成本发明的该核酸的个别链段的潜在长度，对各侧面而言有可能存在相对于如SEQ IDNO：1中详述的RTP801基因的编码序列的某些转变，其中在两个方向上该转变可多达1、2、3、4、5及6个核苷酸，且其中因此产生的双链核酸分子亦应在本发明内。In addition, other nucleic acids to be used in the present invention comprise at least 14 consecutive nucleotides of any one of SEQ.ID.NO: 3 to 536, and more preferably comprise the first strand and the second strand as described above. 14 contiguous nucleotide base pairs at either end of the double-stranded structure of the segment. It will be appreciated by those skilled in the art that, given the potential length of the nucleic acid of the present invention and in particular the individual segments forming the nucleic acid of the present invention, it is possible for each aspect to have an encoding relative to the RTP801 gene as detailed in SEQ ID NO: 1 Certain shifts in sequence, where the shifts can be up to 1, 2, 3, 4, 5 and 6 nucleotides in both directions, and where the resulting double-stranded nucleic acid molecules are also within the invention.
本发明的另一方面涉及包含双链结构的功能核酸，其中该双链结构包含第一链及第二链，其中第一链包含第一连续核苷酸链段且第二链包含第二连续核苷酸链段，其中第一链段与编码RTP801的核酸序列互补或一致且其中第二链段与编码RTP801的核酸序列一致或互补。Another aspect of the present invention relates to a functional nucleic acid comprising a double-stranded structure comprising a first strand and a second strand, wherein the first strand comprises a first contiguous stretch of nucleotides and the second strand comprises a second contiguous Nucleotide chain segments, wherein the first chain segment is complementary or identical to the nucleic acid sequence encoding RTP801 and wherein the second chain segment is identical or complementary to the nucleic acid sequence encoding RTP801.
在一实施方式中，核酸下调RTP801，其中RTP801的下调是选自包含RTP801功能下调、RTP801蛋白下调及RTP801 mRNA表达下调的组。In one embodiment, the nucleic acid down-regulates RTP801, wherein the down-regulation of RTP801 is selected from the group comprising down-regulation of RTP801 function, down-regulation of RTP801 protein, and down-regulation of RTP801 mRNA expression.
在一实施方式中，第一链段包含至少14个核苷酸、优选至少18个核苷酸且甚至更优选19个核苷酸。In one embodiment, the first stretch comprises at least 14 nucleotides, preferably at least 18 nucleotides and even more preferably 19 nucleotides.
在一实施方式中，第一链段包含约14至40个核苷酸，优选约18至30个核苷酸，更优选约19至27个核苷酸且最佳约19至23个核苷酸。In one embodiment, the first stretch comprises about 14 to 40 nucleotides, preferably about 18 to 30 nucleotides, more preferably about 19 to 27 nucleotides and optimally about 19 to 23 nucleosides acid.
在一实施方式中，第二链段包含约14至40个核苷酸，优选约18至30个核苷酸，更优选约19至27个核苷酸且最佳约19至23个核苷酸。In one embodiment, the second stretch comprises about 14 to 40 nucleotides, preferably about 18 to 30 nucleotides, more preferably about 19 to 27 nucleotides and optimally about 19 to 23 nucleosides acid.
在一实施方式中，第一链段的第一核苷酸对应于编码RTP801的核酸序列的核苷酸，其中第一链段的最终一个核苷酸对应于编码RTP801的核酸序列的核苷酸。In one embodiment, the first nucleotide of the first segment corresponds to the nucleotide of the nucleic acid sequence encoding RTP801, wherein the last nucleotide of the first segment corresponds to the nucleotide of the nucleic acid sequence encoding RTP801 .
在一实施方式中，一条链段包含核酸序列的至少14个连续核苷酸的序列，其中该核酸序列是选自表A-D中所揭示的序列，优选选自包含SEQ.ID.NO：53、66、67、72、73、74、75、76、77、91、92、93、94、96、101、102、525、528、529、530、532、533、534、535及536的组，更优选选自包含SEQ.ID.No：66、75、79、91、94、101、102、525及528的组，且最佳选自包含SEQ.ID.No：66、74、75、79、525及528的组。In one embodiment, a segment comprises a sequence of at least 14 consecutive nucleotides of a nucleic acid sequence, wherein the nucleic acid sequence is selected from the sequences disclosed in Tables A-D, preferably selected from sequences comprising SEQ.ID.NO:53,
在一实施方式中，另一链段包含核酸序列的至少14个连续核苷酸的序列，其中该核酸序列是选自表A-D中所揭示的序列，优选选自包含SEQ.ID.NO：3、16、22、23、24、25、26、27、29、41、42、43、44、45、46、51、52、429、432、433、434、436、437、438、439及440的组，更优选选自包含SEQ.ID.No：16、24、25、29、41、44、51及52的组，且最佳选自包含SEQ.ID.No：16、24、25及29的组。In one embodiment, another segment comprises a sequence of at least 14 consecutive nucleotides of a nucleic acid sequence, wherein the nucleic acid sequence is selected from the sequences disclosed in Tables A-D, preferably selected from the sequences comprising SEQ.ID.NO:3 ,16,22,23,24,25,26,27,29,41,42,43,44,45,46,51,52,429,432,433,434,436,437,438,439 and 440 The group, more preferably selected from the group comprising SEQ.ID.No: 16, 24, 25, 29, 41, 44, 51 and 52, and most preferably selected from the group comprising SEQ.ID.No: 16, 24, 25 and 29 groups.
在一实施方式中In one embodiment
第一链段具有SEQ.ID.NO.53的序列且第二链段具有SEQ.ID.NO.3的序列；The first segment has the sequence of SEQ.ID.NO.53 and the second segment has the sequence of SEQ.ID.NO.3;
第一链段具有SEQ.ID.NO.66的序列且第二链段具有SEQ.ID.NO.16的序列；The first segment has the sequence of SEQ.ID.NO.66 and the second segment has the sequence of SEQ.ID.NO.16;
第一链段具有SEQ.ID.NO.67的序列且第二链段具有SEQ.ID.NO.17的序列；The first segment has the sequence of SEQ.ID.NO.67 and the second segment has the sequence of SEQ.ID.NO.17;
第一链段具有SEQ.ID.NO.72的序列且第二链段具有SEQ.ID.NO.22的序列；The first segment has the sequence of SEQ.ID.NO.72 and the second segment has the sequence of SEQ.ID.NO.22;
第一链段具有SEQ.ID.NO.73的序列且第二链段具有SEQ.ID.NO.23的序列；The first segment has the sequence of SEQ.ID.NO.73 and the second segment has the sequence of SEQ.ID.NO.23;
第一链段具有SEQ.ID.NO.74的序列且第二链段具有SEQ.ID.NO.24的序列；The first segment has the sequence of SEQ.ID.NO.74 and the second segment has the sequence of SEQ.ID.NO.24;
第一链段具有SEQ.ID.NO.75的序列且第二链段具有SEQ.ID.NO.25的序列；The first segment has the sequence of SEQ.ID.NO.75 and the second segment has the sequence of SEQ.ID.NO.25;
第一链段具有SEQ.ID.NO.76的序列且第二链段具有SEQ.ID.NO.26的序列；The first segment has the sequence of SEQ.ID.NO.76 and the second segment has the sequence of SEQ.ID.NO.26;
第一链段具有SEQ.ID.NO.77的序列且第二链段具有SEQ.ID.NO.27的序列；The first segment has the sequence of SEQ.ID.NO.77 and the second segment has the sequence of SEQ.ID.NO.27;
第一链段具有SEQ.ID.NO.79的序列且第二链段具有SEQ.ID.NO.29的序列；The first segment has the sequence of SEQ.ID.NO.79 and the second segment has the sequence of SEQ.ID.NO.29;
第一链段具有SEQ.ID.NO.91的序列且第二链段具有SEQ.ID.NO.41的序列；The first segment has the sequence of SEQ.ID.NO.91 and the second segment has the sequence of SEQ.ID.NO.41;
第一链段具有SEQ.ID.NO.92的序列且第二链段具有SEQ.ID.NO.42的序列；The first segment has the sequence of SEQ.ID.NO.92 and the second segment has the sequence of SEQ.ID.NO.42;
第一链段具有SEQ.ID.NO.93的序列且第二链段具有SEQ.ID.NO.43的序列；The first segment has the sequence of SEQ.ID.NO.93 and the second segment has the sequence of SEQ.ID.NO.43;
第一链段具有SEQ.ID.NO.94的序列且第二链段具有SEQ.ID.NO.44的序列；The first segment has the sequence of SEQ.ID.NO.94 and the second segment has the sequence of SEQ.ID.NO.44;
第一链段具有SEQ.ID.NO.95的序列且第二链段具有SEQ.ID.NO.45的序列；The first segment has the sequence of SEQ.ID.NO.95 and the second segment has the sequence of SEQ.ID.NO.45;
第一链段具有SEQ.ID.NO.96的序列且第二链段具有SEQ.ID.NO.46的序列；The first segment has the sequence of SEQ.ID.NO.96 and the second segment has the sequence of SEQ.ID.NO.46;
第一链段具有SEQ.ID.NO.101的序列且第二链段具有SEQ.ID.NO.51的序列；且The first segment has the sequence of SEQ.ID.NO.101 and the second segment has the sequence of SEQ.ID.NO.51; and
第一链段具有SEQ.ID.NO.102的序列且第二链段具有SEQ.ID.NO.52的序列。The first segment has the sequence of SEQ.ID.NO.102 and the second segment has the sequence of SEQ.ID.NO.52.
在一实施方式中，第一链段具有选自包含SEQ.ID.NO.53、66、72、73、74、75、76、77、79、91、92、93、94、95、96、101、102、525、528、529、530、532、533、534、535及536的组的核酸序列。In one embodiment, the first segment has a sequence selected from the group consisting of SEQ.ID.NO. Nucleic acid sequences of groups 101, 102, 525, 528, 529, 530, 532, 533, 534, 535, and 536.
应了解虽然术语＂第一＂及＂第二＂链段结合本发明的核酸使用，但其仅为获得便利性而使用，且描述为具有序列X的第一链段及序列Y的第二链段的本发明的任何核酸分子亦可同等描述为具有序列Y的第一链段及序列X的第二链段，只要了解一条链段包含于必须反义于RTP801基因之一部分编码序列的反义链中，且另一链包含于必须与反义链互补(虽然非100％互补)的有义链中，所有均根据本文所陈述的定义及说明。It should be understood that although the terms "first" and "second" strands are used in connection with the nucleic acids of the invention, they are used for convenience only and are described as having a first strand of sequence X and a second strand of sequence Y Any nucleic acid molecule of the present invention of the segment can also be equally described as having a first segment of sequence Y and a second segment of sequence X, as long as it is understood that one segment is included in the antisense that must be antisense to a part of the coding sequence of the RTP801 gene strand, and the other strand is contained in the sense strand which must be complementary (though not 100% complementary) to the antisense strand, all according to the definitions and descriptions set forth herein.
在一实施方式中，第一和/或第二链包含在3′末端处与编码RTP801的核酸序列的相应核苷酸互补或一致的至少一个悬垂物核苷酸。In one embodiment, the first and/or second strand comprises at least one overhang nucleotide at the 3' end that is complementary or identical to the corresponding nucleotide of the nucleic acid sequence encoding RTP801.
在一实施方式中，第一和/或第二链包含3′末端处的1至15个悬垂物核苷酸，优选地第一和/或第二链包含3′末端处的1至10个悬垂物核苷酸，更优选地第一和/或第二链包含3′末端处的1至5个悬垂物核苷酸，且最佳地第一和/或第二链包含3′末端处的1至2个悬垂物核苷酸。In one embodiment, the first and/or second strand comprises 1 to 15 overhang nucleotides at the 3' end, preferably the first and/or second strand comprises 1 to 10 nucleotides at the 3' end Overhang nucleotides, more preferably the first and/or second strand comprises 1 to 5 overhang nucleotides at the 3' end, and optimally the first and/or second strand comprises 1 to 5 overhang nucleotides at the 3'
在一实施方式中，第一链和/或第二链包含不同于编码RTP801的核酸序列的相应核苷酸的至少一个悬垂物核苷酸。In one embodiment, the first strand and/or the second strand comprises at least one overhang nucleotide that is different from the corresponding nucleotide of the nucleic acid sequence encoding RTP801.
在一实施方式中，第一链包含不同于编码RTP801的核酸序列的相应核苷酸的两个悬垂物核苷酸。In one embodiment, the first strand comprises two overhang nucleotides that are different from the corresponding nucleotides of the nucleic acid sequence encoding RTP801.
在一实施方式中，第一链仅由第一链段组成。In one embodiment, the first chain consists of only the first segment.
在一实施方式中，第二链仅由第二链段组成。In one embodiment, the second chain consists only of the second segment.
在一实施方式中，第一链段和/或第一链包含核糖核苷酸。In one embodiment, the first strand segment and/or the first strand comprises ribonucleotides.
在一实施方式中，第二链段和/或第二链包含核糖核苷酸。In one embodiment, the second strand segment and/or the second strand comprises ribonucleotides.
在一实施方式中，第一链段和/或第二链由核糖核苷酸组成。In one embodiment, the first strand and/or the second strand consist of ribonucleotides.
在一实施方式中，某些或所有核苷酸经修饰。In one embodiment, some or all nucleotides are modified.
在一优选实施例中，该修饰涉及核苷酸的核碱基部分、核苷酸的糖部分和/或核苷酸的磷酸酯部分。In a preferred embodiment, the modification involves the nucleobase moiety of the nucleotide, the sugar moiety of the nucleotide and/or the phosphate moiety of the nucleotide.
在一更优选实施例中，修饰为糖部分的修饰且修饰为2′位上的修饰，其中2′OH基团藉由选自包含-H-OCH3、-OCH2CH3、-OCH2CH2CH3、-NH2及-F的组的基团或部分置换。In a more preferred embodiment, the modification is a modification of the sugar moiety and the modification is a modification at the 2' position, wherein the 2'OH group is selected from the group consisting of -H-OCH3 , -OCH2 CH3 , -OCH2 Group or partial substitution of the group of CH2 CH3 , —NH2 , and —F.
在一实施方式中，修饰为核碱基部分的修饰且修饰或经修饰的核碱基是选自包含下列各物的组：肌苷、黄嘌呤、次黄嘌呤、2-胺基腺嘌呤、6-甲基、2-丙基及其它烷基腺嘌呤、5-卤基尿嘧啶、5-卤基胞嘧啶、5-卤基胞嘧啶、6-氮杂胞嘧啶、6-氮杂胸腺嘧啶、假尿嘧啶、4-硫尿嘧啶、8-卤基腺嘌呤、8-胺基腺嘌呤、8-硫醇腺嘌呤、8-硫代烷基腺嘌呤、8-羟基腺嘌呤及其它8-取代腺嘌呤、8-卤基鸟嘌呤、8-胺基鸟嘌呤、8-硫醇鸟嘌呤、8-硫代烷基鸟嘌呤、8-羟基鸟嘌呤及其它经取代的鸟嘌呤、其它氮杂及脱氮腺嘌呤、其它氮杂及脱氮鸟嘌呤、5-三氟甲基尿嘧啶及5-三氟胞嘧啶。In one embodiment, the modification is a modification of a nucleobase moiety and the modified or modified nucleobase is selected from the group comprising inosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl, 2-propyl and other alkyl adenine, 5-halouracil, 5-halocytosine, 5-halocytosine, 6-azacytosine, 6-azathymine , pseudouracil, 4-thiouracil, 8-haloadenine, 8-aminoadenine, 8-thiol adenine, 8-thioalkyladenine, 8-hydroxyadenine and other 8- Substituted adenine, 8-haloguanine, 8-aminoguanine, 8-thiolguanine, 8-thioalkylguanine, 8-hydroxyguanine and other substituted guanine, other aza And deazaadenine, other aza and deazaguanine, 5-trifluoromethyluracil and 5-trifluorocytosine.
在一实施方式中，修饰为磷酸酯部分的修饰，其中经修饰的磷酸酯部分是选自包含硫代磷酸酯的组。In one embodiment, the modification is a modification of a phosphate moiety, wherein the modified phosphate moiety is selected from the group comprising phosphorothioate.
在一实施方式中，第一链段和/或第二链段包含复数个在2′位上具有修饰的经修饰核苷酸的组，其中在链段内各组经修饰核苷酸通过核苷酸的侧接基团侧接于一或两侧，其中形成核苷酸的侧接基团的侧接核苷酸为未经修饰的核苷酸或具有不同于经修饰核苷酸的修饰的核苷酸。In one embodiment, the first segment and/or the second segment comprises a plurality of groups of modified nucleotides having modifications at the 2' position, wherein within the segment each group of modified nucleotides passes through the core The side group of the nucleotide is flanked on one or both sides, wherein the side nucleotides forming the side group of the nucleotide are unmodified nucleotides or have modifications other than modified nucleotides of nucleotides.
在一优选实施例中，第一链段和/或第二链段由核糖核苷酸组成。In a preferred embodiment, the first segment and/or the second segment consist of ribonucleotides.
在一更优选实施例中，第一链段及第二链段包含复数个组的经修饰核苷酸。In a more preferred embodiment, the first segment and the second segment comprise plural sets of modified nucleotides.
在一实施方式中，第一链段包含该复数个组的经修饰核苷酸。In one embodiment, the first stretch comprises the plurality of sets of modified nucleotides.
在一实施方式中，第二链段包含该复数个组的经修饰核苷酸。In one embodiment, the second segment comprises the plurality of sets of modified nucleotides.
在一实施方式中，各组经修饰核苷酸和/或各组侧接核苷酸包含大量核苷酸，其中数目是选自包含1个核苷酸至10个核苷酸的组。In one embodiment, each set of modified nucleotides and/or each set of flanking nucleotides comprises a plurality of nucleotides, wherein the number is selected from the group comprising 1 nucleotide to 10 nucleotides.
在一实施方式中，第一链段包含第一类型的经修饰核苷酸，且第二链段包含第二类型的经修饰核苷酸。In one embodiment, the first stretch comprises a first type of modified nucleotide and the second stretch comprises a second type of modified nucleotide.
在一实施方式中，第一类型与第二类型为相同类型。In one embodiment, the first type and the second type are the same type.
在另一实施方式中，第一类型与第二类型对准。In another embodiment, the first type is aligned with the second type.
在一优选实施例中，第一类型相对于第二类型存在一或多个核苷酸改变。In a preferred embodiment, the first type has one or more nucleotide changes relative to the second type.
在一实施方式中，各组经修饰核苷酸由一经修饰核苷酸组成且各组侧接核苷酸由一未经修饰的核苷酸或具有不同于经修饰核苷酸的修饰的核苷酸组成。In one embodiment, each set of modified nucleotides consists of a modified nucleotide and each set of flanking nucleotides consists of an unmodified nucleotide or a core with a modification different from the modified nucleotide. nucleotide composition.
在一优选实施例中，经修饰的核苷酸在2′位上具有-OMe基团。In a preferred embodiment, the modified nucleotide has an -OMe group at the 2' position.
在一优选实施例中，侧接核苷酸为具有2′OH基团的核糖核苷酸。In a preferred embodiment, the flanking nucleotides are ribonucleotides with a 2'OH group.
在一实施方式中，第一链段起始于5′末端的经修饰核苷酸，且该链段的所有其它核苷酸亦为经修饰的核苷酸，而起始于5′末端的第二核苷酸及所有其它核苷酸为未经修饰的核苷酸或具有不同于经修饰核苷酸的修饰的核苷酸。In one embodiment, the first segment starts at the modified nucleotide at the 5' end, and all other nucleotides of the segment are also modified nucleotides, and the first segment at the 5' end The second nucleotide and all other nucleotides are either unmodified nucleotides or nucleotides with modifications other than modified nucleotides.
在一实施方式中，第一链段是在编码RTP801的核酸序列的反义方位。In one embodiment, the first segment is in the antisense position of the nucleic acid sequence encoding RTP801.
本发明的另一方面涉及包含本发明的第一方面的核酸和/或本发明的第二方面的载体及优选可药用载体的药物组合物；该组合物任选用于全身或局部给药。Another aspect of the present invention relates to a pharmaceutical composition comprising the nucleic acid of the first aspect of the present invention and/or the carrier of the second aspect of the present invention and preferably a pharmaceutically acceptable carrier; the composition is optionally for systemic or local administration .
在一实施方式中，该组合物用于治疗疾病，其中疾病是选自包含肿瘤疾病的组。In one embodiment, the composition is for the treatment of a disease, wherein the disease is selected from the group comprising neoplastic diseases.
在另一方面中，本发明的潜在问题是由一种用于预防和/或治疗需该预防和/或治疗的患者的方法来解决，该方法包括给药本发明核苷酸和/或本发明载体和/或本发明的药物组合物。In another aspect, the underlying problem of the present invention is solved by a method for prophylaxis and/or treatment of a patient in need of such prophylaxis and/or treatment, the method comprising administering a nucleotide of the invention and/or Inventive carrier and/or pharmaceutical composition of the present invention.
在另一实施方式中，本发明的核酸和/或本发明的载体用于制造药剂。药剂可用于预防和/或治疗疾病，其中该疾病是选自包含肿瘤疾病的组。肿瘤疾病可选自包含实体肿瘤、转移性肿瘤(包括PTEN阴性肿瘤)、具有药物抗性的肿瘤及其中RTP801抑制可用于敏化的肿瘤。此外，肿瘤疾病可为晚期肿瘤疾病或可涉及肿瘤抑制剂阴性细胞；该肿瘤抑制剂可为PTEN。In another embodiment, the nucleic acid of the invention and/or the vector of the invention are used in the manufacture of a medicament. The medicament can be used to prevent and/or treat a disease, wherein the disease is selected from the group comprising neoplastic diseases. The neoplastic disease can be selected from the group consisting of solid tumors, metastatic tumors (including PTEN negative tumors), tumors with drug resistance and tumors in which RTP801 inhibition can be used for sensitization. Furthermore, the neoplastic disease can be an advanced neoplastic disease or can involve tumor suppressor negative cells; the tumor suppressor can be PTEN.
本发明的另一方面是由用于设计或筛检适于下调RTP801的核酸的方法来解决，该方法包括下列步骤：Another aspect of the present invention is addressed by a method for designing or screening nucleic acids suitable for down-regulating RTP801, the method comprising the steps of:
a)设计或筛检适于下调RTP801的核酸；a) designing or screening nucleic acids suitable for down-regulating RTP801;
b)评估本发明的任一上述方面的核酸的缺陷；及b) assessing the nucleic acid of any of the above aspects of the invention for deficiencies; and
c)比较步骤a)中核酸的作用与步骤b)中核酸的作用。c) comparing the effect of the nucleic acid in step a) with the effect of the nucleic acid in step b).
在一实施方式中，作用为下调RTP801。In one embodiment, the effect is to downregulate RTP801.
本发明的另一方面为本发明的核酸用作敏化剂、尤其用作治疗疾病的敏化剂的用途，其中该疾病优选是选自包含肿瘤且更具体地说对使用化学疗法和/或放射疗法的治疗具有抗性的肿瘤的组。本发明的核酸可充当敏化剂的额外疾病揭示于本文中。Another aspect of the invention is the use of a nucleic acid according to the invention as a sensitizer, especially as a sensitizer for the treatment of a disease, preferably selected from the group consisting of tumors and more particularly for the use of chemotherapy and/or A group of tumors that are resistant to radiation therapy. Additional diseases for which nucleic acids of the invention may act as sensitizers are disclosed herein.
此申请案揭示，包含对RTP801具有特异性的双链结构的核酸为适于抑制血管生成/脉管结构生长及血管渗漏(均来自现有脉管结构及正在生长的脉管结构)的工具。此外，此申请案揭示(不受理论限制)应力诱发蛋白RTP801(藉由低氧、氧化应力、热应力、ER应力诱发)是在回应能量不平衡的细胞微调中起作用的因子。因此，藉由该双链核酸来抑制RTP801可适于治疗应自归因于应力条件的细胞凋亡救出细胞的任何疾病(例如伴随正常细胞死亡的疾病)或归因于RTP801表达的改变而适应应力条件的细胞(例如肿瘤细胞)应被杀死的任何疾病。因此，在后一情形下，在藉由该双链核酸抑制RTP801后，此具有低氧细胞、更具体地说低氧癌细胞中的抗细胞凋亡功能的存活因子不能使缺乏RTP801介导的保护的细胞发生细胞凋亡。此外，当其它细胞凋亡促进因子存在时，此亦可发生。该其它细胞凋亡促进因子包括化学疗法及放射疗法。换言之，本发明的双链核酸可单独地有效治疗癌症(单一疗法)且亦可有效用作补充疗法。This application discloses that nucleic acids comprising a double-stranded structure specific for RTP801 are suitable tools for the inhibition of angiogenesis/vascular growth and vascular leakage, both from existing and growing vasculature . Furthermore, this application discloses (without being bound by theory) that the stress-induced protein RTP801 (induced by hypoxia, oxidative stress, heat stress, ER stress) is a factor that plays a role in cellular fine-tuning in response to energy imbalances. Therefore, inhibition of RTP801 by this double-stranded nucleic acid may be suitable for the treatment of any disease in which cells should be rescued from apoptosis due to stress conditions (such as diseases accompanied by normal cell death) or due to changes in the expression of RTP801. Any disease in which cells under stress conditions (such as tumor cells) should be killed. Therefore, in the latter case, after inhibition of RTP801 by the double-stranded nucleic acid, this survival factor with anti-apoptotic function in hypoxic cells, more specifically hypoxic cancer cells, cannot make the lack of RTP801-mediated Protected cells undergo apoptosis. Furthermore, this can also occur when other apoptosis-promoting factors are present. Such other apoptosis promoting factors include chemotherapy and radiation therapy. In other words, the double-stranded nucleic acid of the present invention is effective alone in the treatment of cancer (monotherapy) and is also effective as a complementary therapy.
该双链结构包含第一链及第二链，其中第一链包含第一连续核苷酸链段且第二链包含第二连续核苷酸链段，其中第一链段与编码RTP801的核酸序列互补或一致且其中第二链段与编码RTP801的核酸序列一致或互补。尤其藉由将RTP801用作该种双链核酸的目标，因此亦可能立即分别处理涉及脉管结构生长及发育及血管生成的级联中及因此与由VEGF抑制剂(诸如VEGF抗体)使用的路径相比不同的途径中的目标。不愿受任何理论限制，本发明的发明者假定本发明的核酸可在提供涉及任何疾病(其中发生不良、尤其低氧诱发的血管生成和/或脉管结构生长或发育)或结合其所观测到的背景的那些细胞中发挥其功能。此理解藉由下列发现支持：在非低氧条件下，RTP801基因剔除小鼠不会显示不同于野生型小鼠的任一表型。仅在如患病病状(诸如肿瘤生长)中观测的低氧诱发后，RTP801相关基因剔除会导致类似于在经受此种疾病的人类中所观测到的病理的病理。The double-stranded structure comprises a first strand and a second strand, wherein the first strand comprises a first continuous nucleotide segment and the second strand comprises a second continuous nucleotide segment, wherein the first strand is compatible with the nucleic acid encoding RTP801 The sequences are complementary or identical, and the second segment is identical or complementary to the nucleic acid sequence encoding RTP801. In particular by using RTP801 as a target for this double-stranded nucleic acid, it is therefore also possible to address at once separately in the cascades involved in the growth and development of vascular structures and angiogenesis and thus with the pathways used by VEGF inhibitors such as VEGF antibodies compared targets in different pathways. Without wishing to be bound by any theory, the inventors of the present invention hypothesize that the nucleic acids of the present invention may be useful in providing or in connection with any disease in which adverse, especially hypoxia-induced angiogenesis and/or vascular structure growth or development occurs, or in combination with the observed function in those cells that are in the background. This understanding is supported by the finding that under non-hypoxic conditions, RTP801 knockout mice do not display any phenotype that differs from wild-type mice. Knockout of the RTP801 -associated gene results in pathology similar to that observed in humans afflicted with this disease only after induction of hypoxia as observed in diseased conditions such as tumor growth.
应了解本发明的核酸优选为功能核酸。如本文所用的术语功能核酸优选应意谓功能不同于在细胞中用作任一hnRNA、mRNA或任何其它转录产物的转录模板的核酸，其中该hnRNA、mRNA或任何其它转录产物或本发明的核酸分别经受转译过程，优选经受细胞转译过程，从而导致生物活性RTP801蛋白。应了解，如本文优选使用的功能核酸能够减少靶向核酸的表达。更优选地，该减少是基于靶向核酸的转录后基因沉默过程。甚至更优选地，该减少是基于RNA干扰。功能核酸的最佳形式为siRNA分子或具有与siRNA分子作用相同的作用的任何其它分子。该另一分子是选自包含siRNA、合成siRNA、shRNA及合成shRNA的组。如本文所用的siRNA可额外包含表达载体衍生的siRNA，其中在一优选实施例中表达载体为病毒，诸如腺病毒、腺病毒相关的病毒、疱疹病毒及豆状病毒。如本文所用的shRNA优选应意谓短的发夹型RNA。该shRNA可合成制成或可使用载体编码表达系统、优选使用RNA聚合酶III启动子而产生。因此，应了解本发明的功能核酸是针对RTP801，RTP801在本文中亦优选称为靶向且编码该靶向的核酸是称为靶向核酸。It will be appreciated that the nucleic acids of the invention are preferably functional nucleic acids. The term functional nucleic acid as used herein shall preferably mean a nucleic acid that functions differently in a cell as a template for transcription of any hnRNA, mRNA or any other transcription product or nucleic acid of the invention respectively subjected to a translation process, preferably a cellular translation process, resulting in a biologically active RTP801 protein. It will be appreciated that functional nucleic acids as preferably used herein are capable of reducing the expression of targeted nucleic acids. More preferably, the reduction is based on a nucleic acid-targeted post-transcriptional gene silencing process. Even more preferably, the reduction is based on RNA interference. The best form of functional nucleic acid is an siRNA molecule or any other molecule that has the same effect as an siRNA molecule. The other molecule is selected from the group comprising siRNA, synthetic siRNA, shRNA and synthetic shRNA. siRNA as used herein may additionally comprise siRNA derived from an expression vector, wherein in a preferred embodiment the expression vector is a virus, such as adenovirus, adeno-associated virus, herpes virus, and bean virus. shRNA as used herein shall preferably mean short hairpin RNA. The shRNA can be made synthetically or can be produced using a vector-encoded expression system, preferably using an RNA polymerase III promoter. Therefore, it should be understood that the functional nucleic acid of the present invention is directed against RTP801, RTP801 is also preferably referred to herein as targeting and the nucleic acid encoding this targeting is referred to as targeting nucleic acid.
如本文优选使用，本发明核酸的双链结构包含任一双链结构，其中该双链结构优选藉由具有基础设计的核酸提供的第一链段及第二链段产生。双链结构可包含一或多个错配。该双链结构藉由Watson-Crick碱基配对和/或Hoogsteen碱基配对和/或类似碱基配对机制形成。基于本发明核酸的基础设计，一条链段最好在相对于编码RTP801的核酸序列或其部分的反义方位上，而另一链段最好在相对于编码RTP801的核酸序列或其部分的有义方位上。因此，一条链段与编码RTP801的核酸序列或其部分互补，且另一链段与编码RTP801的核酸序列或其部分一致。因此，应了解术语一致当然亦意谓部分一致，其中同一性(表示为同源性)为至少80％、优选90％、更优选95％、96％、97％、98％、99％或100％。类似于同一性的定义，互补性可根据同源性来定义，其中若互补链根据Watson-Crick碱基配对原则转译成类似链，则该同源性与同一性范围相同。在一替代实施例中，一条链段与编码RTP801的核酸序列或其部分一致，且另一链段与编码RTP801的核酸序列或其部分互补。As preferably used herein, the double-stranded structure of the nucleic acid of the invention comprises any double-stranded structure, wherein the double-stranded structure is preferably produced by the first and second strands provided by the nucleic acid having the basic design. The double-stranded structure may contain one or more mismatches. The double-stranded structure is formed by Watson-Crick base pairing and/or Hoogsteen base pairing and/or similar base pairing mechanisms. Based on the basic design of the nucleic acid of the present invention, one segment is preferably in an antisense position relative to the nucleic acid sequence encoding RTP801 or a part thereof, and the other segment is preferably in an antisense position relative to the nucleic acid sequence encoding RTP801 or a part thereof. Righteous orientation. Thus, one segment is complementary to a nucleic acid sequence encoding RTP801 or a portion thereof and the other segment is identical to a nucleic acid sequence encoding RTP801 or a portion thereof. Accordingly, it will be understood that the term identity also means partial identity, wherein the identity (expressed as homology) is at least 80%, preferably 90%, more preferably 95%, 96%, 97%, 98%, 99% or 100%. %. Similar to the definition of identity, complementarity can be defined in terms of homology, which is the same extent as identity if the complementary strand translates into an analogous strand according to the Watson-Crick base pairing principles. In an alternative embodiment, one segment is identical to a nucleic acid sequence encoding RTP801, or a portion thereof, and the other segment is complementary to a nucleic acid sequence encoding RTP801, or a portion thereof.
在一优选实施例中，本发明的核酸下调RTP801功能。RTP801功能的下调优选藉由蛋白水平和/或mRNA水平上的表达水平减少而发生，其中与在本发明的核酸未给药或不具有功能活性的条件下的表达相比，优选在蛋白水平上减少的该表达水平可少至5％且可高达100％。该条件优选为表达系统、优选RTP801的表达系统的条件或存在于其中的条件。该表达系统优选为可为活体外转译系统的转译系统，更优选为细胞、器官和/或有机体。更优选地，有机体为多细胞有机体，更优选为哺乳动物，其中该哺乳动物优选是选自包含人、猴、小鼠、大鼠、豚鼠、兔、猫、狗、绵羊、母牛、马、牛及猪的组。结合下调，应了解该下调可随时间变化，亦即下调作用无需在给药或功能活化本发明的核酸后立即观测到，而可延长时间以及空间(亦即在多种细胞、组织和/或器官中)。该延迟可介于5％-100％、优选10％至50％之间。本领域技术人员应了解，较长时间的5％减少可与较短时间的100％减少同样有效。本领域技术人员亦应了解，该延长强烈地取决于实际上所用的特定功能核酸以及靶向细胞群体，且因此最终取决于待根据本申请案的技术教导而治疗和/或预防的疾病。在此范围内，较长时间的5％减少可与较短时间的100％减少同样有效。本领域技术人员亦应了解，该延长可以如上文所概述的任何程度而发生，亦即功能延长(其中该功能为RTP801显示的任一功能)、蛋白表达的延长或mRNA表达水平的延长。In a preferred embodiment, the nucleic acid of the present invention down-regulates the function of RTP801. The downregulation of RTP801 function preferably occurs by a reduction of the expression level at the protein level and/or at the mRNA level, preferably at the protein level compared to the expression under conditions in which the nucleic acid according to the invention is not administered or has no functional activity This reduced level of expression can be as little as 5% and can be as high as 100%. The conditions are preferably those of the expression system, preferably that of RTP801, or conditions present therein. The expression system is preferably a translation system which may be an in vitro translation system, more preferably a cell, organ and/or organism. More preferably, the organism is a multicellular organism, more preferably a mammal, wherein the mammal is preferably selected from the group consisting of human, monkey, mouse, rat, guinea pig, rabbit, cat, dog, sheep, cow, horse, Set of cows and pigs. In connection with down-regulation, it is understood that down-regulation may vary over time, i.e. down-regulation need not be observed immediately following administration or functional activation of a nucleic acid of the invention, but may be extended over time as well as over space (i.e. in a variety of cells, tissues and/or in organs). This delay may be between 5% and 100%, preferably between 10% and 50%. Those skilled in the art will appreciate that a 5% reduction over a longer period of time may be as effective as a 100% reduction over a shorter period of time. The person skilled in the art will also understand that this prolongation strongly depends on the specific functional nucleic acid actually used and the target cell population, and thus ultimately on the disease to be treated and/or prevented according to the technical teaching of the present application. Within this range, a 5% reduction for a longer period of time can be as effective as a 100% reduction for a shorter period of time. Those skilled in the art will also appreciate that this elongation may occur to any extent as outlined above, ie functional elongation (where the function is any function exhibited by RTP801), protein expression or mRNA expression level.
在一优选实施例中，第一链段包含至少14个核苷酸、优选14个连续核苷酸。本领域技术人员亦应了解第一链段应具有适于允许特定处理编码RTP801的核酸序列且更特定处理编码存在于RTP801表达应减少的转译系统中的RTP801的核酸的长度。亦不愿受理论或本发明核酸的任何作用模式限制，似乎本发明核酸与编码RTP801的核酸序列之间优选在转录程度上(亦即自编码RTP801的各自核酸序列产生mRNA后)存在相互作用。归因于本发明核酸的任一序列与转译系统的基因组或转录组所含的序列一致或互补的可能性，因此第一链段的长度应长至确保下列情形：假定编码RTP801的核酸与本发明核酸之一条链之间的某种碱基配对实际上存在，则仅基因组或转录组(transcriptome)的编码RTP801的序列而非其它编码序列、当然亦非其它基本编码序列针对或藉由该碱基配对来处理。根据此长度，脱靶效应的发生可减少且优选消除。为增加此种特定处理RTP801及编码其的核酸序列的严格性，第一链段优选具有至少18或19个核苷酸的长度。第一链段长度的上限优选少于50个核苷酸，然而，该长度可显著更大且可包含100、200或甚至500个核苷酸或其间任一长度。除此之外，尤其若本发明的核酸为化学合成的，则本领域技术人员将青睐相对较短的第一链段，序列愈短，其合成时间及消耗的物质愈少，且不当核苷酸插入各自序列中的比率愈低。确定第一链段长度所考虑的另一因素是在长度超过50个或50个以上核苷酸时，通常可观测到非特异性干扰素反应。对此种非特异性干扰素反应是否耐受将取决于待治疗的特定病状。举例而言，若干扰素反应和/或干扰素基因表达可限于病理细胞，则干扰素反应可耐受。In a preferred embodiment, the first stretch comprises at least 14 nucleotides, preferably 14 consecutive nucleotides. Those skilled in the art will also understand that the first segment should have a length suitable to allow specific treatment of the nucleic acid sequence encoding RTP801 and more specific treatment of the nucleic acid encoding RTP801 present in the translation system in which the expression of RTP801 should be reduced. Without wishing to be bound by theory or any mode of action of the nucleic acids of the invention, it appears that the interaction between the nucleic acids of the invention and the RTP801-encoding nucleic acid sequence is preferably at the transcriptional level (ie following production of mRNA from the respective RTP801-encoding nucleic acid sequence). Due to the possibility that any sequence of the nucleic acid of the present invention is identical or complementary to a sequence contained in the genome or transcriptome of the translation system, the length of the first strand should be long enough to ensure the following: Assuming that the nucleic acid encoding RTP801 is identical to the present A certain base pairing between one strand of the nucleic acid of the invention actually exists, then only the sequence encoding RTP801 of the genome or transcriptome (transcriptome) but not other coding sequences, and certainly not other basic coding sequences are aimed at or by this base base pairing. Depending on this length, the occurrence of off-target effects can be reduced and preferably eliminated. To increase the stringency of this specific treatment of RTP801 and the nucleic acid sequence encoding it, the first stretch preferably has a length of at least 18 or 19 nucleotides. The upper limit of the length of the first segment is preferably less than 50 nucleotides, however, the length may be significantly greater and may comprise 100, 200 or even 500 nucleotides or any length in between. In addition, especially if the nucleic acid of the present invention is chemically synthesized, those skilled in the art will favor relatively short first strands. The lower the rate of acid insertion into the respective sequence. Another factor considered in determining the length of the first segment is that non-specific interferon responses are often observed when the length exceeds 50 nucleotides or more. Whether such a nonspecific interferon response is tolerated will depend on the particular condition being treated. For example, an interferon response can be tolerated if the interferon response and/or interferon gene expression can be restricted to pathological cells.
鉴于此，第一链段的更优选长度为约14至40个核苷酸、18至30个核苷酸、19至27个核苷酸、21至25个核苷酸及19至23个核苷酸。In view of this, more preferred lengths for the first segment are about 14 to 40 nucleotides, 18 to 30 nucleotides, 19 to 27 nucleotides, 21 to 25 nucleotides, and 19 to 23 cores glycosides.
与上文关于第一链段所概述的因素相同的因素可应用于第二链段，因此第二链段可包含如本文所述与第一链段相关的任一长度。亦在本发明内，第一链段的长度不同于第二链段的长度，然而，两条链段优选具有相同长度。The same factors as outlined above with respect to the first segment can be applied to the second segment, so the second segment can comprise any length as described herein in relation to the first segment. Also within the present invention, the length of the first segment is different from the length of the second segment, however, the two segments are preferably of the same length.
根据核酸的基本设计，第一链段及第二链段分别为本发明核酸的第一链及第二链的部分。应了解在每一末端(亦即在5′末端以及3′末端)，第一链和/或第二链可包含任何组合之一或多个核苷酸，优选额外核苷酸。According to the basic design of the nucleic acid, the first segment and the second segment are respectively part of the first strand and the second strand of the nucleic acid of the present invention. It is understood that at each end (ie at the 5' end as well as at the 3' end), the first strand and/or the second strand may comprise one or more nucleotides, preferably additional nucleotides, in any combination.
因此，应了解超出对应于各自链的链段的末端的个别链的那些核苷酸可用以分别进一步促进链段的互补性及同一性，且因此有助于特定处理编码RTP801的核酸序列。It will thus be appreciated that those nucleotides beyond the individual strands corresponding to the ends of the segments of the respective chains may be used to further facilitate the complementarity and identity of the segments, respectively, and thus facilitate the specific manipulation of the RTP801-encoding nucleic acid sequence.
应了解基本上基于本文所提供的技术教导，本发明的核酸可处理编码RTP801、尤其编码RTP801表达应减少的转译系统中的RTP801的核酸序列的任一部分。在此范围内，本发明包含具有如本文所定义的特征的任何核酸，其中本发明核酸的互补及一致链及链段可基本上起始于编码RTP801的核酸序列的任何核苷酸。因此，在本发明核酸的第一链段互补于编码RTP801的核酸序列(亦即为其反义链或在其反义方位上)的限制条件下，该链段的第一核苷酸(亦即在最5′末端的核苷酸)对应于(亦即对准)3′末端的编码RTP801的序列的最终一个核苷酸。在另一实施方式中，该最5′末端的核苷酸对应于编码RTP801的核酸的倒数第二个核苷酸，及其类似情形，直至到达最终一个位置，此在给定反义链长度下仍允许本发明核酸的反义链互补于编码RTP801的核酸序列。在此范围内，本发明的任何核酸均在本发明内，此可藉由扫描起始于其最5′末端核苷酸且覆盖本发明核酸的基础设计的编码RTP801的核酸序列且了解本发明的该核酸的特征而产生。相同考虑可应用于本文所揭示的实施例，其中本发明核酸的互补性及同一性不仅分别由第一链段及第二链段提供，该互补性及同一性亦分别包含除第一链段及第二链段以外之一或多个核苷酸，接着分别成为第一链及第二链的部分。It should be understood that the nucleic acid of the present invention can process any part of the nucleic acid sequence encoding RTP801, especially encoding RTP801 in a translation system in which the expression of RTP801 should be reduced, basically based on the technical teaching provided herein. Insofar, the invention encompasses any nucleic acid having the characteristics as defined herein, wherein complementary and consensus strands and stretches of the nucleic acid of the invention may start substantially from any nucleotide of the RTP801-encoding nucleic acid sequence. Therefore, under the restriction that the first chain segment of the nucleic acid of the present invention is complementary to the nucleic acid sequence encoding RTP801 (that is, its antisense strand or in its antisense position), the first nucleotide of the chain segment (that is, its antisense strand) That is, the nucleotide at the most 5' end) corresponds to (ie aligns with) the last nucleotide of the sequence encoding RTP801 at the 3' end. In another embodiment, the 5'-most nucleotide corresponds to the penultimate nucleotide of the nucleic acid encoding RTP801, and the like, until the final position is reached, at a given antisense strand length The antisense strand of the nucleic acid of the present invention is still allowed to be complementary to the nucleic acid sequence encoding RTP801. To the extent that any nucleic acid of the invention is within the invention, this can be done by scanning the nucleic acid sequence encoding RTP801 starting at its most 5' terminal nucleotide and covering the basic design of the nucleic acid of the invention and knowing the invention The characteristics of the nucleic acid produced. The same considerations apply to the embodiments disclosed herein, in which the complementarity and identity of the nucleic acids of the invention are not only provided by the first segment and the second segment, respectively, the complementarity and identity also include segments other than the first segment, respectively. and one or more nucleotides other than the second strand, which then become part of the first strand and the second strand, respectively.
在如本文所揭示的本发明的各种核酸中，具有内部参考号14、22、23、25、27、39、41、42、49及50(参见表A)及257、260-262及264-268(参见表D)的核酸尤其优选。因此，应注意可用于人类及动物模型(诸如大鼠和/或小鼠)的本发明的那些核酸尤其适用。本发明的这类特定核酸的惊人优势在于其在人类及动物模型中均有效，此意谓在动物模型中获得的测试结果可立即自动物模型转移至人类且更具体地说无需对人类序列作任何改变，而在设计本发明核酸(诸如)以包含在物种、更具体地说用于动物模型测试的物种与作为最终优选有机体或患者的人之间存在不同的序列的情形下有必要作出改变。另外优选地，这类核酸具有亦如实施例所述的修饰类型。Among the various nucleic acids of the invention as disclosed herein are
然而，亦在本发明内，根据SEQ.ID.NO.3、16-17、22-27、29、41-46、51-53、66-67、72-77、79、91-96、101-102、525、528-530、532-536及导致具有内部参考号14、22、23、25、27、39、41、42、49及50(表A)及257、260-262及264-268(表D)的本发明核酸分子的各自组合的任一序列仅部分含于本发明的另一核酸中。本发明的其它核苷酸优选包含SEQ.ID.NO.3、16-17、22-27、29、41-46、51-53、66-67、72-77、79、91-96、101-102、525、528-530、532-536的至少14个连续核苷酸，且更优选包含在包含上表所概述的第一链段及第二链段的双链结构的任一末端的14个连续核苷酸碱基对。本领域技术人员应了解，给定本发明核酸及尤其形成本发明的该核酸的个别链段的潜在长度，对各侧面而言有可能存在相对于RTP801的编码序列的某些转变，其中在两个方向上该转变可多达1、2、3、4、5及6个核苷酸，且其中因此产生的双链核酸分子亦应在本发明内。However, also within the present invention, according to SEQ.ID.NO. -102, 525, 528-530, 532-536 and leads with
在本发明之一优选实施例中，第一链段及第一链具有相同长度。类似地，第二链与第二链段最好具有相同长度，而第一链段与第二链段亦最好具有相同长度。在一更优选实施例中，第一链仅包含第一链段且第二链仅包含第二链段。在一甚至更优选实施例中，第一链段及因此第一链不含有悬垂物，而第二链段及因此第二链亦不包含悬垂物。换言之，亦在本发明内，优选在本发明核酸的双链结构的各末端处，本发明的双链核酸末端平整。结合本发明核酸的任何其它实施例、尤其其中本发明核酸具有修饰类型、更优选如本文所述的修饰类型的那些实施例，可了解该末端平整结构。In a preferred embodiment of the present invention, the first segment and the first chain have the same length. Similarly, the second chain is preferably the same length as the second segment, and the first segment is preferably the same length as the second segment. In a more preferred embodiment, the first chain contains only first segments and the second chain contains only second segments. In an even more preferred embodiment, the first segment, and thus the first chain, contains no overhangs, and the second segment, and thus the second chain, also does not contain overhangs. In other words, also within the present invention, the double-stranded nucleic acid of the present invention is preferably blunt-ended at each end of the double-stranded structure of the nucleic acid of the present invention. This blunt-ended structure can be understood in conjunction with any other embodiment of the nucleic acid of the invention, especially those in which the nucleic acid of the invention has a type of modification, more preferably a type of modification as described herein.
因此，在另一方面中，本发明的核酸具有在本发明核酸的双链结构的两个末端处提供平整末端的基础设计。然而，在本发明内亦存在悬垂物，亦即自双链结构突出之一或多个核苷酸的链段。原则上，悬垂物可在反义链的5′末端、反义链的3′末端、有义链的5′末端和/或有义链的3′末端。应注意了解该选择中的任一单个选择以及其任何组合均在本发明内。更优选为其中悬垂物位于反义链的3′末端及有义链的3′末端的组合。亦在本发明内，悬垂物位于反义链的5′末端及有义链的5′末端。此外，在本发明内，悬垂物仅位于双链结构的反义链上，更优选位于双链结构的反义链的3′末端。Thus, in another aspect, the nucleic acids of the invention have a basic design that provides blunt ends at both ends of the double-stranded structure of the nucleic acids of the invention. However, overhangs, ie stretches of one or more nucleotides protruding from the double-stranded structure, also exist within the present invention. In principle, the overhang can be at the 5' end of the antisense strand, the 3' end of the antisense strand, the 5' end of the sense strand and/or the 3' end of the sense strand. It should be noted that any single of these options as well as any combination thereof is understood to be within the invention. More preferred is a combination in which the overhang is located at the 3' end of the antisense strand and at the 3' end of the sense strand. Also within the present invention, an overhang is located at the 5' end of the antisense strand and at the 5' end of the sense strand. Furthermore, within the present invention, the overhang is located only on the antisense strand of the double-stranded structure, more preferably at the 3' end of the antisense strand of the double-stranded structure.
结合悬垂物，应注意悬垂物加上链段优选形成链，且本文中所提供的链段长度亦应用于这类实施例。与位置无关，个别悬垂物可由至少一个核苷酸组成。然而，个别悬垂物亦可包含多达10个核苷酸且优选为2个核苷酸长。在本发明内，在第一链互补于该编码RTP801的核酸序列且悬垂物在反义链的3′末端或5′末端的情形下形成悬垂物的各自核苷酸亦互补于该编码RTP801的核酸序列，或在第一链与编码RTP801的核酸序列一致的情形下悬垂物与编码RTP801的核酸序列一致。同样应用于位于本发明核酸的基础设计的第二链段的任何悬垂物，其中应了解第二链段上的悬垂物设计可独立于第一链段的悬垂物设计。In connection with pendants, it should be noted that pendants plus segments preferably form chains, and the segment lengths provided herein also apply to such embodiments. Independent of position, individual overhangs may consist of at least one nucleotide. However, individual overhangs may also comprise up to 10 nucleotides and are preferably 2 nucleotides long. Within the present invention, in the case where the first strand is complementary to the nucleic acid sequence encoding RTP801 and the overhang is at the 3' end or 5' end of the antisense strand, the respective nucleotides forming the overhang are also complementary to the RTP801 encoding The nucleic acid sequence, or overhang in the case of the first strand is identical to the nucleic acid sequence encoding RTP801, is identical to the nucleic acid sequence encoding RTP801. The same applies to any overhangs located on the second stretch of the basic design of the nucleic acids of the invention, with the understanding that the design of the overhang on the second stretch can be independent of the design of the overhang on the first stretch.
亦在本发明内，形成悬垂物的核苷酸与编码RTP801的核酸序列的相应核苷酸既不互补，亦不一致。如本文所用且优选在此实施例中的＂相应＂意谓跟随在具有编码RTP801的核酸上的核苷酸对应物的链段的5′末端和/或3′末端上的各自核苷酸。Also within the present invention, the nucleotides forming the overhang are neither complementary nor identical to the corresponding nucleotides of the nucleic acid sequence encoding RTP801. "Corresponding" as used herein and preferably in this embodiment means the respective nucleotides following the 5' end and/or the 3' end of the segment having the nucleotide counterpart on the nucleic acid encoding RTP801.
第一链优选在其3′末端包含两个核苷酸，优选脱氧核苷酸，且更优选两个TT，和/或此种核苷酸亦在第二链的3′末端，其中第一链段及第二链段的长度更优选为19个核苷酸。因此，链包含链段及悬垂物。在此实施例中，双链结构由19个碱基对及在个别链段的3′末端的各末端的两个核苷酸悬垂物组成。The first strand preferably comprises two nucleotides, preferably deoxynucleotides, and more preferably two TT at its 3' end, and/or such nucleotides are also at the 3' end of the second strand, wherein the first The length of the segment and the second segment is more preferably 19 nucleotides. Thus, a chain consists of segments and pendants. In this example, the double-stranded structure consists of 19 base pairs with two nucleotide overhangs at each end of the 3' end of the individual segments.
在一优选实施例中，第一链段和/或第一链包含核糖核苷酸，其中尤其优选地，第一链段全部由核糖核苷酸组成。同样分别应用于第二链段及第二链。与此结合，然而，在一优选实施例中第一链段及第二链段的每一及任一核苷酸分别经修饰。同样分别应用于第一链及第二链。与末端核苷酸为核糖核苷酸或脱氧核糖核苷酸无关，末端核苷酸尤其可具有同样可经修饰的OH基。该OH基可源自核苷酸的糖部分，更优选在5′OH基的情形下源自5′位和/或在3′OH基的情形下源自3′位，或源自连接于各自末端核苷酸的糖部分的磷酸酯基。原则上，磷酸酯基可连接至核苷酸的糖部分的任何OH基。磷酸酯基优选在游离5′OH基的情形下连接至糖部分的5′OH基，和/或在游离3′OH基的情形下连接至糖部分的3′OH基，仍假定5′OH基或3′OH基在本文中称为游离5′或3′OH基。In a preferred embodiment, the first chain segment and/or the first chain comprise ribonucleotides, wherein it is especially preferred that the first chain segment consists entirely of ribonucleotides. The same applies to the second segment and the second strand, respectively. In conjunction with this, however, in a preferred embodiment each and any nucleotide of the first stretch and the second stretch, respectively, are modified. The same applies to the first strand and the second strand respectively. Regardless of whether the terminal nucleotides are ribonucleotides or deoxyribonucleotides, the terminal nucleotides may in particular have OH groups which may likewise be modified. The OH group may be derived from the sugar moiety of the nucleotide, more preferably from the 5' position in the case of a 5'OH group and/or from the 3' position in the case of a 3'OH group, or from the The phosphate group of the sugar moiety of the respective terminal nucleotide. In principle, the phosphate group can be attached to any OH group of the sugar moiety of the nucleotide. The phosphate group is preferably attached to the 5'OH group of the sugar moiety in the case of a free 5'OH group, and/or to the 3'OH group of the sugar moiety in the case of a free 3'OH group, again assuming a 5'OH or 3'OH groups are referred to herein as free 5' or 3'OH groups.
在设计RNAi的任何策略或本文所揭示的RNAi的任何实施例下，如本文所用的术语＂末端修饰＂意谓添加至第一和/或第二链的最5′或3′核苷酸的化学实体。该末端修饰的实施例包括(但不限于)3′或5′磷酸酯、经转化的(脱氧)非碱基、胺基、氟基、氯基、溴基、CN、CF、甲氧基、咪唑、羧酸酯、硫代酯、C1至C10低级烷基、经取代的低级烷基、烷芳基或芳烷基、OCF3、OCN、O-、S-或N-烷基、O-、S-或N-烯基、SOCH3、SO2CH3、ONO2、NO2、N3、杂环烷基、杂环烷芳基、胺基烷胺基、聚烷胺基或经取代的硅烷基，如欧洲专利EP 0 586 520 B1或EP 0 618 925 B1中所述。Under any strategy for designing RNAi or any embodiment of RNAi disclosed herein, the term "terminal modification" as used herein means the addition of the most 5' or 3' nucleotides to the first and/or second strand. chemical entity. Examples of such terminal modifications include, but are not limited to, 3' or 5' phosphate, converted (deoxy) non-base, amine, fluoro, chloro, bromo, CN, CF, methoxy, Imidazole, carboxylate, thioester, C1 to C10 lower alkyl, substituted lower alkyl, alkaryl or aralkyl, OCF3 , OCN, O-, S- or N-alkyl, O-, S- or N-alkenyl, SOCH3 , SO2 CH3 , ONO2 , NO2 , N3 , heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino or Substituted silyl groups, as described in
如本文所用，烷基或包含＂烷基＂的任何术语优选意谓包含1至12个、优选1至6个且更优选1至2个碳原子的任何碳原子链。As used herein, alkyl or any term comprising "alkyl" preferably means any chain of carbon atoms comprising 1 to 12, preferably 1 to 6 and more preferably 1 to 2 carbon atoms.
另一末端修饰为生物素基团。该生物素基团优选可连接至第一和/或第二链的最5′或最3′核苷酸或两个末端。在一更优选实施例中，生物素基团与多肽或蛋白偶联。亦在本发明的范畴内，多肽或蛋白经由任何其它上述末端修饰来连接。多肽或蛋白可给予本发明的核酸分子其它特征。其中，多肽或蛋白可充当另一分子的配体。若该另一分子为受体，则受体功能及活性可藉由结合配体来活化。受体可显示使结合本发明核酸分子的配体有效转染的内在化活性。本发明的核酸分子所偶联的配体的实施例为VEGF且相应受体为VEGF受体。The other end was modified with a biotin group. The biotin group is preferably attachable to the most 5' or most 3' nucleotide or both termini of the first and/or second strand. In a more preferred embodiment, the biotin group is coupled to the polypeptide or protein. It is also within the scope of the present invention that the polypeptides or proteins are linked via any of the other above-mentioned terminal modifications. Polypeptides or proteins may impart additional characteristics to the nucleic acid molecules of the invention. Among other things, a polypeptide or protein can serve as a ligand for another molecule. If the other molecule is a receptor, receptor function and activity can be activated by binding the ligand. Receptors may exhibit internalization activity that permits efficient transfection of ligands that bind nucleic acid molecules of the invention. An example of a ligand to which a nucleic acid molecule of the invention is coupled is VEGF and the corresponding receptor is a VEGF receptor.
具有不同种类末端修饰的本发明RNAi的各种可能实施例呈现于下表1中。Various possible embodiments of RNAi according to the invention with different kinds of terminal modifications are presented in Table 1 below.
表1：本发明的干扰核糖核酸的各种实施例Table 1: Various embodiments of interfering ribonucleic acid of the present invention
如本文所揭示的各种末端修饰均优选位于本发明核酸的核苷酸的核糖部分。更具体地说，末端修饰可连接或置换核糖部分的任何OH基，包括(但不限于)2′OH、3′OH及5′OH位，其限制条件在于因此经修饰的核苷酸为末端核苷酸。经转化的非碱基为核苷酸(脱氧核糖核苷酸或核糖核苷酸)，其不具有核碱基部分。此种化合物描述于Sternberger，M.，Schmiedeknecht，A.，Kretschmer，A.，Gebhardt，F.，Leenders，F.，Czauderna，F.，Von Carlowitz，I.，Engle，M.，Giese，K.，Beigelman，L.& Klippel，A.(2002).Antisense Nucleic Acid DrugDev，12，131-43中。The various terminal modifications as disclosed herein are preferably located at the ribose moiety of the nucleotides of the nucleic acids of the invention. More specifically, terminal modifications may attach or replace any OH group of the ribose moiety, including but not limited to the 2'OH, 3'OH, and 5'OH positions, with the proviso that the modified nucleotide is thus a terminal Nucleotides. Converted non-bases are nucleotides (deoxyribonucleotides or ribonucleotides), which do not have a nucleobase portion. Such compounds are described in Sternberger, M., Schmiedeknecht, A., Kretschmer, A., Gebhardt, F., Leenders, F., Czauderna, F., Von Carlowitz, I., Engle, M., Giese, K. , Beigelman, L. & Klippel, A. (2002). Antisense Nucleic Acid DrugDev, 12, 131-43.
任何上述末端修饰均可与表1中所述的各种RNAi实施例结合使用；应注意上文所提及的5′末端修饰通常仅存在于siRNA分子的有义链中。Any of the above-mentioned terminal modifications can be used in conjunction with the various RNAi embodiments described in Table 1; it should be noted that the 5' terminal modifications mentioned above are generally only present in the sense strand of the siRNA molecule.
其它修饰可关于个别核苷酸的核碱基部分、糖部分或磷酸酯部分。Other modifications may pertain to the nucleobase moiety, sugar moiety or phosphate moiety of individual nucleotides.
核碱基部分的该修饰可使腺嘌呤、鸟嘌呤、胞嘧啶及胸苷及尿嘧啶的衍生物分别经修饰。尤其优选经修饰的核碱基是选自包含下列各物的组：肌苷、黄嘌呤、次黄嘌呤、2-胺基腺嘌呤、6-甲基、2-丙基及其它烷基腺嘌呤、5-卤基尿嘧啶、5-卤胞嘧啶、5-卤基胞嘧啶、6-氮杂胞嘧啶、6-氮杂胸腺嘧啶、假尿嘧啶、4-硫尿嘧啶、8-卤基腺嘌呤、8-胺基腺嘌呤、8-硫醇腺嘌呤、8-硫代烷基腺嘌呤、8-羟基腺嘌呤及其它8-取代腺嘌呤、8-卤基鸟嘌呤、8-胺基鸟嘌呤、8-硫醇鸟嘌呤、8-硫代烷基鸟嘌呤、8-羟基鸟嘌呤及其它经取代的鸟嘌呤、其它氮杂及脱氮腺嘌呤、其它氮杂及脱氮鸟嘌呤、5-三氟甲基尿嘧啶及5-三氟胞嘧啶。This modification of the nucleobase moiety allows modification of derivatives of adenine, guanine, cytosine, and thymidine and uracil, respectively. Especially preferred modified nucleobases are selected from the group comprising inosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl, 2-propyl and other alkyladenines , 5-Halouracil, 5-Halocytosine, 5-Halocytosine, 6-Azacytosine, 6-Azathymine, Pseudouracil, 4-thiouracil, 8-Haloadeno Purine, 8-aminoadenine, 8-thiol adenine, 8-thioalkyladenine, 8-hydroxyadenine and other 8-substituted adenine, 8-haloguanine, 8-aminoguanine Purine, 8-thiolguanine, 8-thioalkylguanine, 8-hydroxyguanine and other substituted guanine, other aza and deazaadenine, other aza and deazaguanine, 5 - Trifluoromethyluracil and 5-trifluorocytosine.
在另一优选实施例中，核苷酸的糖部分经修饰，其中该修饰优选地分别在核苷酸的核糖及脱氧核糖部分的2′位。2′OH基更优选由选自包含胺基、氟基、烷氧基及烷基的组的基团或部分来置换。烷氧基优选为甲氧基或乙氧基。烷基亦优选意谓甲基、乙基、丙基、异丁基、丁基及异丁基。甚至更优选地，不考虑修饰类型，核苷酸优选为核糖核苷酸。In another preferred embodiment, the sugar moiety of the nucleotide is modified, wherein the modification is preferably at the 2' position of the ribose and deoxyribose moieties of the nucleotide, respectively. The 2'OH group is more preferably replaced by a group or moiety selected from the group consisting of amine group, fluorine group, alkoxy group and alkyl group. Alkoxy is preferably methoxy or ethoxy. Alkyl also preferably means methyl, ethyl, propyl, isobutyl, butyl and isobutyl. Even more preferably, regardless of the type of modification, the nucleotides are preferably ribonucleotides.
磷酸酯部分的修饰优选是选自包含硫代磷酸酯的组。The modification of the phosphate moiety is preferably selected from the group comprising phosphorothioate.
本领域技术人员应了解，由大量核苷酸组成的本发明核酸因此可由经由磷酸二酯键或经由硫代磷酸酯键或两者的组合分别沿个别链及链段的核苷酸序列长度键联的核苷酸形成。Those skilled in the art will appreciate that the nucleic acids of the invention, which consist of a large number of nucleotides, can thus be formed by linkages along the length of the nucleotide sequence of individual strands and segments, respectively, via phosphodiester linkages or via phosphorothioate linkages or a combination of both. linked nucleotide formation.
所用核苷酸的另一形式实际上可为描述于国际专利申请案WO03/070918中的siRNA。Another form of nucleotides used may actually be siRNA as described in International Patent Application WO03/070918.
分别形成本发明核酸的第一链段及第一链的核苷酸可包含一或多个经修饰的核苷酸，其中个别经修饰的核苷酸具有的修饰优选为如本文所揭示的修饰。除特定修饰外，修饰亦可为或可包含某种标记，其中标记是选自化学发光标记、荧光标记及放射标记的组。这类标记种类为本领域技术人员所知且(例如)描述于Ausubel等人，CurrentProtocols in Molecular Biology，John Wiley and Sons，Baltimore，Maryland，1998中。因此经标记的本发明核酸亦可用于获得诊断目的或用于监控作用位点以及用于进行优选关于本文所揭示的任何疾病的任何治疗。The nucleotides forming the first strand and the first strand, respectively, of the nucleic acid of the invention may comprise one or more modified nucleotides, wherein the individual modified nucleotides have modifications preferably as disclosed herein . In addition to specific modifications, the modification may also be or include a certain label, wherein the label is selected from the group of chemiluminescent labels, fluorescent labels and radioactive labels. Such marker species are known to those skilled in the art and are described, for example, in Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, Maryland, 1998. The labeled nucleic acids of the invention can therefore also be used for diagnostic purposes or for monitoring the site of action and for carrying out any treatment, preferably with respect to any of the diseases disclosed herein.
在一优选实施例中，本发明的核酸经修饰，使得有义链段或链中的嘧啶核苷酸为2′O-甲基嘧啶核苷酸；且另外或其它，有义链段或链中的嘌呤核苷酸为2′-脱氧嘌呤核苷酸。在另一实施方式中，存在于有义链段或有义链中的嘧啶核苷酸为2′-脱氧-2′-氟嘧啶核苷酸。In a preferred embodiment, the nucleic acid of the invention is modified such that the pyrimidine nucleotides in the sense segment or chain are 2'O-methyl pyrimidine nucleotides; and additionally or alternatively, the sense segment or chain The purine nucleotides in are 2'-deoxypurine nucleotides. In another embodiment, the pyrimidine nucleotides present in the sense segment or sense strand are 2'-deoxy-2'-fluoropyrimidine nucleotides.
在一替代实施例中，修饰并非基于核苷酸的化学性质(亦即修饰取决于待修饰的核苷酸为嘌呤核苷酸或嘧啶核苷酸)，而主要基于本发明核酸的基础设计的总双链结构中个别核苷酸的空间排列。In an alternative embodiment, the modification is not based on the chemical nature of the nucleotide (i.e. the modification depends on whether the nucleotide to be modified is a purine nucleotide or a pyrimidine nucleotide), but is primarily based on the basic design of the nucleic acid of the invention The spatial arrangement of individual nucleotides in the overall double-stranded structure.
更具体地说，第一链及第一链段或第二链及第二链分别显示分别形成该链段及链的核苷酸的空间修饰类型。More specifically, a first strand and a first strand or a second strand and a second strand, respectively, exhibit the type of steric modification of the nucleotides forming the segment and strand, respectively.
首先关注第一链段，存在一种类型的经修饰核苷酸的组及未经修饰核苷酸的组。这类未经修饰核苷酸的组在本文中亦称为核苷酸的侧接基团的组。更优选地，该类型由经修饰核苷酸的组及未经修饰核苷酸的组组成。该类型甚至更优选为规则类型且甚至更优选为沿本发明核酸的第一链段的长度的交替类型。经修饰核苷酸的组可由一或多个经修饰且优选在2′位上经修饰(亦即在糖部分上修饰)的核苷酸组成。此修饰更优选为2′-O-Me修饰。Focusing first on the first segment, there is one type of set of modified nucleotides and a set of unmodified nucleotides. Such groups of unmodified nucleotides are also referred to herein as groups of flanking groups of nucleotides. More preferably, the type consists of groups of modified nucleotides and groups of unmodified nucleotides. The pattern is even more preferably a regular pattern and even more preferably an alternating pattern along the length of the first stretch of the nucleic acid of the invention. The set of modified nucleotides may consist of one or more nucleotides that are modified, preferably at the 2' position, ie on the sugar moiety. This modification is more preferably 2'-O-Me modification.
未经修饰核苷酸的组可由一或多个未经修饰的核苷酸组成，其中未经修饰的核苷酸优选为核糖核苷酸或未修饰核苷酸为经修饰的核苷酸，其中该修饰不同于形成经修饰核苷酸的组的核苷酸所显示的修饰。未修饰核苷酸甚至更优选为核糖核苷酸。应注意若未相反说明，则术语未修饰核苷酸及未经修饰的核苷酸可交替使用。本发明核酸的第一链段可起始于经修饰核苷酸的组或起始于如本文所定义的未经修饰核苷酸的组。然而，第一链段优选起始于经修饰核苷酸的组。经修饰核苷酸的组最佳由单一核苷酸组成。结合此实施例，第一链段优选在编码RTP801的核酸的反义方位上。亦在本发明内，如形成经修饰核苷酸的组的核苷酸所显示的修饰与所有存在于第一链段中的经修饰核苷酸的组相同。然而，亦在本发明之外，某一组经修饰核苷酸的修饰不同于存在于第一链段上之一或多组经修饰核苷酸的修饰。The group of unmodified nucleotides may consist of one or more unmodified nucleotides, wherein the unmodified nucleotides are preferably ribonucleotides or the unmodified nucleotides are modified nucleotides, wherein the modification is different from the modification exhibited by the nucleotides forming the group of modified nucleotides. Unmodified nucleotides are even more preferably ribonucleotides. It should be noted that the terms unmodified nucleotide and unmodified nucleotide are used interchangeably unless stated to the contrary. The first stretch of a nucleic acid of the invention may start from a group of modified nucleotides or from a group of unmodified nucleotides as defined herein. However, the first stretch preferably starts from a group of modified nucleotides. The set of modified nucleotides optimally consists of a single nucleotide. In conjunction with this embodiment, the first segment is preferably in the antisense position of the nucleic acid encoding RTP801. Also within the present invention, the modification as shown by the nucleotides forming the group of modified nucleotides is identical to all the groups of modified nucleotides present in the first stretch. However, it is also outside the invention that the modification of a certain set of modified nucleotides is different from the modification of one or more sets of modified nucleotides present on the first stretch.
在本发明核酸的第二链上，亦可了解如关于第一链段所述的类型。在一实施方式中，亦可在第二链段上了解与结合第一链段所述特征相同的特征，其中在第二链在相对于编码RTP801的核酸序列的有义方位上的限制条件下，本发明核酸的第二链最好起始于未经修饰核苷酸的组。On the second strand of the nucleic acid according to the invention, the type as described for the first strand is also known. In one embodiment, the same features as those described in conjunction with the first strand can also be understood on the second strand, where under the constraint that the second strand is in a sense orientation relative to the nucleic acid sequence encoding RTP801 , the second strand of the nucleic acid of the invention preferably begins with a set of unmodified nucleotides.
包含双链结构的本发明核酸可包含具有如本文所述的修饰类型的第一链段。或者，本发明的双链核酸可包含具有如上文所概述的修饰类型的第二链段。然而，本发明的双链核酸最佳由第一链段及第二链段组成，其中第一链段及第二链段均具有如本文所述的空间修饰类型。A nucleic acid of the invention comprising a double-stranded structure may comprise a first strand having a modification type as described herein. Alternatively, the double-stranded nucleic acid of the invention may comprise a second stretch having the type of modification as outlined above. Preferably, however, the double-stranded nucleic acid of the invention consists of a first segment and a second segment, wherein both the first segment and the second segment have the type of steric modification as described herein.
在本发明内，就经修饰核苷酸的组及未经修饰核苷酸的组的大小及实际上所用的修饰种类而言，两条链段上的空间修饰类型的特征相同。优选地，改变第一链段上的空间修饰类型以使得第一链段上的经修饰核苷酸的组与第二链段上未经修饰核苷酸的组相对，反之亦然。然而，亦在本发明内使该类型精确对准，亦即第一链段上的经修饰核苷酸的组与第二链段上未经修饰核苷酸的组相对，且第一链段上的未经修饰核苷酸的组与第二链段上未经修饰核苷酸的组相对。亦在本发明内，第一链段及第二链段上的空间修饰类型相对于彼此改变以使得一条链段上之一组经修饰核苷酸的第一部分与另一链段上之一组未经修饰核苷酸的一部分相对，而该组经修饰核苷酸的第二部分与另一组经修饰核苷酸相对。在本发明内，本文所提供的关于本发明核酸的链段的空间修饰类型的揭示内容亦应用于本发明核酸的链。然而，核酸的链段优选包含该空间修饰类型且链包含该链段及一或多个如本文所揭示的悬垂物。悬垂物尤其优选为在反义链或有义链或两种链的3′末端上的磷酸酯基，其中磷酸酯基更优选在反义链及有义链的3′末端。在甚至更优选的实施例中，磷酸酯基为如本文所定义的磷酸酯基。Within the present invention, the type of steric modification on both segments is characterized identically with regard to the size of the groups of modified and unmodified nucleotides and the kind of modification actually used. Preferably, the type of steric modification on the first stretch is altered such that the set of modified nucleotides on the first stretch is opposed to the set of unmodified nucleotides on the second stretch, and vice versa. However, it is also within the present invention to make this type of precise alignment, that is, the set of modified nucleotides on the first stretch is opposed to the set of unmodified nucleotides on the second stretch, and the first stretch The set of unmodified nucleotides on is opposed to the set of unmodified nucleotides on the second stretch. Also within the present invention, the type of steric modification on the first segment and the second segment is changed relative to each other such that the first part of a set of modified nucleotides on one segment is the same as a set of modified nucleotides on the other segment. A portion of the unmodified nucleotides is opposed and a second portion of the set of modified nucleotides is opposed to another set of modified nucleotides. Within the present invention, the disclosures provided herein regarding the type of steric modification of segments of nucleic acids of the invention also apply to strands of nucleic acids of the invention. However, preferably the segment of nucleic acid comprises the type of steric modification and the strand comprises the segment and one or more overhangs as disclosed herein. The overhang is especially preferably a phosphate group on the 3' end of the antisense strand or the sense strand or both, with the phosphate group being more preferably on the 3' end of both the antisense and sense strands. In even more preferred embodiments, the phosphate group is a phosphate group as defined herein.
亦在本发明内，本发明的核酸可显示连接第一链及第二链的连接体。该连接体优选为聚合物。聚合物可为任何合成或天然聚合物。可能性合成连接体为PEG或聚核苷酸。该连接体优选经设计以(诸如)使第一链段部分或完全向后折迭至第二链段上，反之亦然。Also within the present invention, a nucleic acid of the present invention may exhibit a linker linking the first strand and the second strand. The linker is preferably a polymer. The polymer can be any synthetic or natural polymer. Possibilities synthetic linkers are PEG or polynucleotides. The linker is preferably designed such as to partially or fully fold back the first segment onto the second segment and vice versa.
最终，在本发明内，本发明的核酸为合成核酸、化学合成核酸、经分离的核酸或衍生自任何天然来源的核酸，诸如藉助于重组技术制备的核酸。如本领域技术人员所知，结合本发明的任何核酸的制备，如本文所揭示的任何修饰均可在制备本发明的各自核酸之前、期间或之后引入。Finally, within the present invention, nucleic acids of the invention are synthetic nucleic acids, chemically synthesized nucleic acids, isolated nucleic acids or nucleic acids derived from any natural source, such as nucleic acids prepared by means of recombinant techniques. As is known to those skilled in the art, in connection with the preparation of any nucleic acid of the invention, any modification as disclosed herein may be introduced before, during or after the preparation of the respective nucleic acid of the invention.
本发明的载体包含本发明的核酸。此外，载体可包括以本领域已知的细胞选择方式来控制该核酸的靶向、表达及转录的元素。质粒可包括用于控制异源物质(亦即本发明的核酸)转录的启动子，且可为允许选择性转录的构成或诱发性启动子。任选可包括获得必要转录程度可需的增强子。增强子一般为连续对编码序列起作用、继而改变藉由启动子指示的基础转录程度的任何未经转译的DNA序列。该构筑体的表达为本领域技术人员所知且可(例如)藉由提供各自串联构筑体或藉由具有分别转录本发明核酸的第一及第二链及第一及第二链段的不同启动子来进行。A vector of the invention comprises a nucleic acid of the invention. In addition, the vector may include elements to control the targeting, expression and transcription of the nucleic acid in a manner known in the art for cell selection. A plasmid may include a promoter for controlling transcription of heterologous material (ie, a nucleic acid of the invention), and may be a constitutive or inducible promoter allowing selective transcription. Optionally, enhancers may be included to obtain the necessary degree of transcription. An enhancer is generally any untranslated DNA sequence that acts continuously on a coding sequence, thereby altering the basal level of transcription directed by the promoter. Expression of such constructs is known to those skilled in the art and can be achieved, for example, by providing respective tandem constructs or by having differences in the first and second strands and the first and second strands of the nucleic acid of the invention respectively transcribed. promoter to proceed.
当本发明的核酸经制造或表达、优选在活体内表达、更优选在需要本发明核酸的患者中表达时，该制造或表达优选使用表达载体，优选为哺乳动物表达载体。表达载体在本领域为已知的且优选包含质粒、粘粒、病毒表达系统。优选病毒表达系统包括(但不限于)腺病毒、反转录病毒及豆状病毒。When the nucleic acid of the present invention is produced or expressed, preferably expressed in vivo, more preferably in a patient in need of the nucleic acid of the present invention, the production or expression preferably uses an expression vector, preferably a mammalian expression vector. Expression vectors are known in the art and preferably comprise plasmid, cosmid, viral expression systems. Preferred viral expression systems include, but are not limited to, adenoviruses, retroviruses, and beanuloviruses.
将载体引入细胞或组织中的方法在本领域为已知的。一般可发现该方法描述于下列文献中：Sambrook等人，Molecular cloning：ALaboratory Manual，Cold Springs Harbour Laboratory，New York(1983，1992)；或Ausubel等人，Current Protocols in MolecularBiology，John Wiley and Sons，Baltimore，Maryland，1998。Methods for introducing vectors into cells or tissues are known in the art. The method can generally be found described in: Sambrook et al., Molecular cloning: A Laboratory Manual, Cold Springs Harbor Laboratory, New York (1983, 1992); or Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore , Maryland, 1998.
适合方法包括转染、脂质体转染、电穿孔及用重组病毒载体感染。结合本发明，在一实施方式中，载体的另一特征为表达限制特征，诸如启动子及调节组件，两者分别对所需细胞类型具有特异性，因此仅在提供允许所需表达的背景后使得本发明的核酸序列表达。Suitable methods include transfection, lipofection, electroporation, and infection with recombinant viral vectors. In connection with the present invention, in one embodiment, another feature of the vector is expression restriction features, such as a promoter and a regulatory element, both of which are each specific to the desired cell type, thus only after providing a background that allows the desired expression This results in the expression of the nucleic acid sequences of the invention.
在另一方面中，本发明涉及包含本发明核酸和/或本发明载体及任选可药用载体、稀释剂或佐剂或其它媒剂的药物组合物。该载体、稀释剂、佐剂及媒剂优选为惰性且无毒的。在药物组合物的各种实施例中，使药物组合物适于各种给药方式。该给药包含全身及局部给药以及经口、皮下、胃肠外、静脉内、动脉内、肌肉内、腹膜内、鼻内及背内(intrategral)给药。In another aspect, the invention relates to a pharmaceutical composition comprising a nucleic acid of the invention and/or a carrier of the invention and optionally a pharmaceutically acceptable carrier, diluent or adjuvant or other vehicle. Such carriers, diluents, adjuvants and vehicles are preferably inert and nontoxic. In various embodiments of the pharmaceutical composition, the pharmaceutical composition is adapted for various modes of administration. The administration includes systemic and topical administration as well as oral, subcutaneous, parenteral, intravenous, intraarterial, intramuscular, intraperitoneal, intranasal and intragral administration.
本领域技术人员应了解，药物组合物及各自核酸及载体的量分别视个别患者的临床病状、给药的位点及方法、给药时程、患者年龄、性别、体重及医师已知的其它因素而定。因此，用于获得预防和/或治疗目的的医药学有效量应藉由医药技术中已知的该考虑来确定。该量优选地有效获得改善，包括(但不限于)改善患病病状或提供更快速恢复、改善或消除症状及由医药技术领域技术人员选为适合测量方法的其它指示。It will be appreciated by those skilled in the art that the amount of the pharmaceutical composition and the respective nucleic acid and carrier depends on the individual patient's clinical condition, site and method of administration, administration schedule, patient's age, sex, body weight and other factors known to the physician. factors. Accordingly, a pharmaceutically effective amount for achieving prophylactic and/or therapeutic purposes should be determined by such considerations as are known in the medical art. The amount is preferably effective to achieve an amelioration including, but not limited to, ameliorating a diseased condition or providing for more rapid recovery, ameliorating or eliminating symptoms and other indications as chosen by those skilled in the medical arts to be suitable for measurement.
在一优选实施例中，本发明的药物组合物可包含其它医药学活性化合物。该其它医药学活性化合物优选是选自包含下列化合物的组：允许吸收细胞内细胞传递的化合物、允许内体小泡释放的化合物、允许较长循环时间的化合物及允许靶向内皮细胞或致病细胞的化合物。用于内体小泡释放的优选化合物为氯喹及ATP依赖性H+泵的抑制剂。In a preferred embodiment, the pharmaceutical composition of the present invention may contain other pharmaceutically active compounds. The other pharmaceutically active compound is preferably selected from the group comprising compounds that allow uptake intracellular delivery, compounds that allow release from endosomal vesicles, compounds that allow longer circulation times, and compounds that allow targeting of endothelial cells or pathogenicity. Cellular compounds. Preferred compounds for endosomal vesicle release are chloroquine and inhibitors of the ATP-dependent H+ pump.
药物组合物的调配优选应得以提供单剂量给药或多剂量给药。The formulation of the pharmaceutical composition should preferably be such that single or multiple dosages are provided.
应了解本发明的药物组合物可用于涉及不良脉管结构发育或生长(包括血管生成)的任何疾病以及本文所述的任何疾病及病状。这类疾病优选为肿瘤疾病。在肿瘤疾病中，下列肿瘤最佳：子宫内膜癌、结肠直肠癌、神经胶质瘤、子宫内膜癌、腺癌、子宫内膜增生、考登综合征(Cowden′s syndrome)、遗传性非息肉病性结肠直肠癌、Li-Fraumene综合征、乳癌-卵巢癌、前列腺癌(Ali，I.U.，Journalof the National Cancer Institute，92卷，11期，2000年6月7日，第861-863页)、Bannayan-Zonana综合征、LDD(Lhermitte-Duklos综合征)(上文Macleod，K.)、错构瘤-巨头畸形病(包括考病(Cowdisease，CD)及Bannayan-Ruvalcaba-Rily综合征(BRR))、黏膜与皮肤损害(例如trichilemmonmas)、巨头畸形、智力迟钝、肠胃错构瘤、脂肪瘤、甲状腺瘤、乳房纤维囊性病、小脑发育不良性神经节细胞瘤及乳房及甲状腺恶性肿瘤(上文Vazquez，F.，Sellers，W.R.)。It is understood that the pharmaceutical compositions of the present invention are useful in any disease involving undesirable vascular development or growth, including angiogenesis, and any of the diseases and conditions described herein. Such diseases are preferably neoplastic diseases. Among neoplastic diseases, the following tumors are best: endometrial cancer, colorectal cancer, glioma, endometrial cancer, adenocarcinoma, endometrial hyperplasia, Cowden's syndrome, hereditary Nonpolyposis colorectal cancer, Li-Fraumene syndrome, breast-ovarian cancer, prostate cancer (Ali, I.U., Journal of the National Cancer Institute, Vol. 92, No. 11, June 7, 2000, pp. 861-863 ), Bannayan-Zonana syndrome, LDD (Lhermitte-Duklos syndrome) (Macleod, K. above), hamartoma-macrodystrophic disease (including Cowdisease, CD) and Bannayan-Ruvalcaba-Rily syndrome ( BRR)), mucosal and skin lesions (e.g. trichilemmonmas), macrocephaly, mental retardation, gastrointestinal hamartoma, lipoma, thyroid tumor, breast fibrocystic disease, cerebellar dysplastic ganglioneuroma, and breast and thyroid malignancies ( Vazquez, F., Sellers, W.R. supra).
应了解待用本发明的药物组合物治疗的任何肿瘤疾病均优选为晚期肿瘤疾病。在另一实施方式中，肿瘤疾病涉及肿瘤抑制剂阴性细胞，其中肿瘤抑制剂更优选为PTEN。It will be appreciated that any neoplastic disease to be treated with the pharmaceutical composition of the present invention is preferably an advanced neoplastic disease. In another embodiment, the neoplastic disease involves tumor suppressor negative cells, wherein the tumor suppressor is more preferably PTEN.
本发明的药物组合物亦可用于用以预防和/或治疗如本文所揭示的疾病的方法中，其中该方法包括关于本文所述的任何疾病给药本发明核酸、本发明载体或本发明的药物组合物或药剂。The pharmaceutical composition of the invention may also be used in a method for preventing and/or treating a disease as disclosed herein, wherein the method comprises administering a nucleic acid of the invention, a vector of the invention or a vector of the invention with respect to any of the diseases described herein. Pharmaceutical composition or medicament.
在另一方面中，本发明涉及一种用于设计或筛检适于下调RTP801、更具体地说适于下调RTP801功能的核酸的方法。此方法包括使用如本文所揭示的核酸序列且在适合检定中分析该核酸。该检定在本领域为已知的，且(例如)描述于本申请案的实施例部分。在另一步骤中，优选根据如本文指定的设计原则来设计适于下调RTP801的双链核酸，优选结合转录后基因沉默机制(诸如RNA干扰)。因此获得(亦即设计或筛检)的核酸亦在各自检定中经分析且比较结果，亦即本发明核酸及新设计或筛检的核酸在该检定中的作用。若经设计或筛检的核酸更稳定或更有效，优选两者兼有，则其更适合。应了解若将本发明的任何核酸用作起始点，则该方法尤其有效。因此，在本发明内，新颖核酸分子将基于本文所揭示的原则来设计，其中RTP801 mRNA上的靶向序列相对于本发明的相应核酸的RTP801 mRNA上的靶向序列略微改变。新核酸优选将在编码RTP801的mRNA的5′或3′方向上相对于靶向mRNA上的链段改变至少一或多个核苷酸。然而，在本发明内，改变同时发生在两个方向上，亦即新核酸并有用作起始点的本发明核酸。亦在本发明内，根据本发明且用作起始点的核酸的延长偏向3′末端或5′末端。在诸如偏向的情形下，新核酸的3′末端或5′末端更长，亦即比另一末端延伸更长。当新核酸分子藉由延伸反义链和/或有义链的3′末端或5′末端而产生时，通常应用下列步骤次序。若关于RTP801的mRNA的5′末端发生改变，则反义链的3′末端必须延伸RTP801的mRNA的5′末端所改变的核苷酸数量。因此，待添加至新颖核酸的反义链的3′末端的核苷酸与跟随在用作起始点的本发明核酸分子所用的RTP801 mRNA上的靶向序列的5′末端的那些核苷酸互补。有义链的情形亦必须相同。然而，待添加至有义链的核苷酸必须对应(亦即互补)于新添加至反义链的3′末端的核苷酸，亦即其必须添加至有义链的5′末端。然而，有义链上之后一步骤必须仅进行至除反义链外有义链亦应改变的程度，此为本发明的优选实施例中的情形。虽然此改变可进行至本领域技术人员所界定的程度，但优选应进行改变以使得新核酸亦仍含有如本文所揭示的任何核酸分子所显示的至少14个核苷酸、优选14个连续核苷酸的链段。In another aspect, the present invention relates to a method for designing or screening nucleic acids suitable for down-regulating RTP801, more specifically for down-regulating RTP801 function. This method involves using a nucleic acid sequence as disclosed herein and analyzing the nucleic acid in a suitable assay. Such assays are known in the art and are described, for example, in the Examples section of this application. In a further step, double-stranded nucleic acids suitable for down-regulation of RTP801 are preferably designed according to design principles as specified herein, preferably in combination with post-transcriptional gene silencing mechanisms such as RNA interference. The thus obtained (ie designed or screened) nucleic acids are also analyzed in the respective assay and the results compared, ie the effect of the inventive nucleic acid and the newly designed or screened nucleic acid in the assay. It is more suitable if the designed or screened nucleic acid is more stable or more efficient, preferably both. It will be appreciated that this method is especially effective if any nucleic acid of the invention is used as a starting point. Thus, within the present invention, novel nucleic acid molecules will be designed based on the principles disclosed herein, wherein the targeting sequence on the RTP801 mRNA is slightly altered relative to the targeting sequence on the RTP801 mRNA of the corresponding nucleic acid of the invention. The new nucleic acid will preferably be altered by at least one or more nucleotides in the 5' or 3' direction of the RTP801-encoding mRNA relative to the stretch on the targeted mRNA. However, within the present invention the alteration takes place in both directions simultaneously, ie the new nucleic acid and with the nucleic acid of the invention used as starting point. Also within the present invention, the elongation of the nucleic acid according to the invention and used as starting point is biased toward the 3' end or the 5' end. In cases such as bias, the 3' end or the 5' end of the new nucleic acid is longer, ie extends longer than the other end. When new nucleic acid molecules are generated by extending the 3' end or the 5' end of the antisense strand and/or the sense strand, the following sequence of steps generally applies. If the 5' end of the mRNA for RTP801 is altered, the 3' end of the antisense strand must be extended by the number of nucleotides that are altered at the 5' end of the RTP801 mRNA. Therefore, the nucleotides to be added to the 3' end of the antisense strand of the novel nucleic acid are complementary to those nucleotides at the 5' end of the targeting sequence following the RTP801 mRNA used in the nucleic acid molecule of the invention used as the starting point . The same must be true for the sense strand. However, the nucleotide to be added to the sense strand must correspond to (ie be complementary to) the nucleotide newly added to the 3' end of the antisense strand, ie it must be added to the 5' end of the sense strand. However, the latter step on the sense strand must only be performed to the extent that the sense strand should also be altered in addition to the antisense strand, which is the case in preferred embodiments of the invention. While this change can be made to an extent defined by one skilled in the art, preferably the change should be made such that the new nucleic acid also still contains at least 14 nucleotides, preferably 14 contiguous cores, as shown by any of the nucleic acid molecules disclosed herein. chains of nucleotides.
本文所述的任何核酸的合成均在本领域技术人员的技能内。该合成描述于下列文献中：Beaucage S.L.及Iyer R.P.，Tetrahedron1992；48：2223-2311；Beaucage S.L.及Iyer R.P.，Tetrahedron1993；49：6123-6194；及Caruthers M.H.等人，Methods Enzymol.1987；154：287-313，硫代酯的合成描述于Eckstein F.，Annu.Rev.Biochem.1985；54：367-402中，RNA分子的合成描述于Sproat B，由Herdewijn P.编辑的Humana Press 2005；Kap.2：17-31，且各自下游过程描述于Pingoud A.等人，由Oliver R.W.A.编辑的IRL Press1989；Kap.7：183-208及Sproat B，由Herdewijn P.编辑的HumanaPress 2005；Kap.2：17-31(上文)中。Synthesis of any of the nucleic acids described herein is within the skill of those in the art. The synthesis is described in: Beaucage S.L. and Iyer R.P., Tetrahedron 1992;48:2223-2311; Beaucage S.L. and Iyer R.P., Tetrahedron 1993;49:6123-6194; and Caruthers M.H. et al., Methods Enzymol.1987;154:287 -313, the synthesis of thioesters is described in Eckstein F., Annu.Rev.Biochem.1985;54:367-402, the synthesis of RNA molecules is described in Sproat B, Humana Press 2005 edited by Herdewijn P.; Kap. 2:17-31, and the respective downstream processes are described in Pingoud A. et al., IRL Press 1989 edited by Oliver R.W.A.; Kap.7:183-208 and Sproat B, HumanaPress 2005, edited by Herdewijn P.; Kap.2: 17-31 (above).
RTP801的siRNA可使用如上所述的本领域已知的方法基于已知RTP801序列(SEQ ID NO：1)而制成且可藉由如上所述的各种修饰使的更稳定。其它信息参见实施例9。The siRNA of RTP801 can be prepared based on the known RTP801 sequence (SEQ ID NO: 1) using methods known in the art as described above and can be made more stable by various modifications as described above. See Example 9 for additional information.
此外，相对于如本文所述的本发明方法，额外RNA分子可用于该方法，例如本发明的抑制性RNA分子包括优选包含存在于表A-D中所详述的序列中的至少7-10个连续核苷酸链段的单链寡核糖核苷酸，该寡核糖核苷酸能够形成(和/或包含)呈藉由细胞内复合物识别的特定构型的双链区域，从而导致该寡核糖核苷酸降解成能够抑制相应内源性基因的较小RNA分子及编码该RNA分子的DNA分子。相应内源性基因优选为801基因且另外可为VEGF基因和/或VEGF-R1基因。本发明亦提供一种组合物，其包含载体、优选可药用载体中的以上单链寡核糖核苷酸。Furthermore, additional RNA molecules may be used in the methods relative to the methods of the invention as described herein, for example the inhibitory RNA molecules of the invention comprise preferably at least 7-10 contiguous sequences present in the sequences detailed in Tables A-D. A single-stranded oligoribonucleotide of a nucleotide chain segment capable of forming (and/or comprising) a double-stranded region in a specific configuration recognized by an intracellular complex, resulting in the oligoribonucleotide Nucleotides are degraded into smaller RNA molecules capable of inhibiting the corresponding endogenous gene and DNA molecules encoding the RNA molecules. The corresponding endogenous gene is preferably the 801 gene and may additionally be the VEGF gene and/or the VEGF-R1 gene. The present invention also provides a composition comprising the above single-stranded oligoribonucleotide in a carrier, preferably a pharmaceutically acceptable carrier.
此外，本发明提供用于本文所揭示的所有病状且尤其涉及脉络膜新血管生成的病状的组合疗法。在该组合疗法中，RTP801及VEGFR基因均受到抑制以改善所治疗疾病的症状。这类基因可用一或多种siRNA或抗体或适体的组合来抑制。因此，本发明亦提供包含RTP801抑制剂及VEGF或VEGF-1抑制剂的新颖药物组合物，RTP801抑制剂优选为siRNA，更优选为表A-D中详述的siRNA分子，且具体地说表A的siRNANo：14、22、23、25、27、39、41、42、49及50或表D的siRNA No：257、260-262及264-268，且VEGF/VEGFR-1抑制剂任选为抗体或适体。该化合物(亦即RTP801 siRNA及VEGF抗体或本文所揭示的任何其它组合实施例)在药剂制备中的组合使用亦为本发明的部分。Furthermore, the present invention provides combination therapy for all the conditions disclosed herein and especially those involving choroidal neovascularization. In this combination therapy, both the RTP801 and VEGFR genes are inhibited to improve the symptoms of the disease being treated. Such genes can be inhibited with one or more siRNA or a combination of antibodies or aptamers. Accordingly, the present invention also provides novel pharmaceutical compositions comprising an RTP801 inhibitor, preferably an siRNA, more preferably an siRNA molecule as detailed in Tables A-D, and specifically Table A, and a VEGF or VEGF-1 inhibitor. siRNANos: 14, 22, 23, 25, 27, 39, 41, 42, 49 and 50 or siRNA Nos of Table D: 257, 260-262 and 264-268, and the VEGF/VEGFR-1 inhibitor is optionally an antibody or fit. The combined use of this compound (ie RTP801 siRNA and VEGF antibody or any other combination embodiment disclosed herein) in the preparation of a medicament is also part of the invention.
因此，RTP801 siRNA(诸如表A-D中所详述的siRNA分子且具体地说表A的siRNA No：14、22、23、25、27、39、41、42、49及50或表D的siRNA No：257、260-262及264-268)可与靶向VEGF或VEGF受体1(VEGFR1)的药剂一起给药。该药剂目前存在于市场上或在不同机构的不同阶段批准及证实中。抗体及抗体片段(诸如兰尼单抗(ranibizumab，Lucentis，Genentech))连接至经释放的VEGF以抑制VEGF结合至活性受体。可充当配体/抗体的适体(麦可净(Macugen)，Eyetech/Pfizer，其针对湿式AMD，最近由FDA批准)亦为可能的。麦可净在细胞外结合VEGF以阻断其活性。这类药物藉由玻璃体内注射来局部给药。以抗VEGF siRNA为主的化合物(诸如Acuity的Cand5 VEGF抑制剂或VEGFR-1的SIRNA 027抑制剂)亦可得。此外，据报导全身性给药的小分子胺基甾醇鲨胺(Squalamine，Genaera)会干扰血管生成过程的多个方面，包括在内皮细胞中抑制VEGF及其它生长因子信号传输。Thus, RTP801 siRNAs (such as the siRNA molecules detailed in Tables A-D and specifically Table A's siRNA Nos: 14, 22, 23, 25, 27, 39, 41, 42, 49 and 50 or Table D's siRNA Nos. : 257, 260-262 and 264-268) can be administered with agents targeting VEGF or VEGF receptor 1 (VEGFR1). The agent currently exists on the market or is in various stages of approval and validation by different agencies. Antibodies and antibody fragments (such as ranibizumab (Lucentis, Genentech)) bind to released VEGF to inhibit VEGF binding to active receptors. Aptamers that can act as ligands/antibodies (Macugen, Eyetech/Pfizer for wet AMD, recently approved by FDA) are also possible. Michelin binds VEGF extracellularly to block its activity. These drugs are administered locally by intravitreal injection. Anti-VEGF siRNA-based compounds such as Acuity's Cand5 VEGF Inhibitor or VEGFR-1 SIRNA 027 Inhibitor are also available. In addition, the systemically administered small molecule sterol squalamine (Squalamine, Genaera) has been reported to interfere with various aspects of the angiogenic process, including inhibition of VEGF and other growth factor signaling in endothelial cells.
RTP801抑制剂、优选siRNA与任何以上VEGF/VEGFR-1抑制剂的组合给药均可产生协同效应，其中无论单一疗法选项的剂量，该组合治疗均比藉由这类个别组合物的任一个进行的治疗有效。此协同效应亦由如实施例6中详述的初步结果支持。Combination administration of an RTP801 inhibitor, preferably siRNA, and any of the above VEGF/VEGFR-1 inhibitors may result in a synergistic effect, wherein the combination treatment is performed more efficiently than by either of these individual compositions regardless of the dose of the monotherapy option treatment is effective. This synergistic effect is also supported by preliminary results as detailed in Example 6.
RTP801i具有不同作用机制且与VEGF-VEGFR抑制剂潜在协作。RTP801 KO小鼠中的研究表明，尽管眼中的VEGF mRNA表达与在WT小鼠中同样高，但KO小鼠中的保护性表型亦持续。我们的额外初步数据表明在视网膜病理的治疗中，RTP801的抑制可与VEGF-VEGFR调节轴的抑制协作。本发明的发明者已发现在适当实验中，在AMD模型中给药对抗RTP801的siRNA(参见下文实例6)不仅导致RTP801自身下调，亦因此导致抗血管生成及神经保护因子PEDF上调以及MCP1(巨噬细胞趋化蛋白)的表达下调。因此，RTP801的抑制同时给予抗血管生成、神经保护及消炎作用。RTP801i has a different mechanism of action and potentially cooperates with VEGF-VEGFR inhibitors. Studies in RTP801 KO mice showed that although VEGF mRNA expression in the eye was as high as in WT mice, the protective phenotype persisted in KO mice. Our additional preliminary data suggest that inhibition of RTP801 may cooperate with inhibition of the VEGF-VEGFR regulatory axis in the treatment of retinal pathologies. The inventors of the present invention have found that in appropriate experiments, administration of siRNA against RTP801 in an AMD model (see Example 6 below) not only results in down-regulation of RTP801 itself, but also consequently up-regulation of the anti-angiogenic and neuroprotective factor PEDF and MCP1 (major Phage chemoattractant protein) expression was down-regulated. Thus, inhibition of RTP801 confers anti-angiogenic, neuroprotective and anti-inflammatory effects simultaneously.
如本文所揭示的适体亦可结合本文所揭示的新颖siRNA用于本发明中。举例而言，适体可与本文所揭示的任何siRNA一起用于治疗本文所揭示的任何病状的组合疗法中。该组合疗法所用的新颖药物组合物亦为本发明的部分，其可包含共价或非共价连接至适体的本发明siRNA。适体为RNA或DNA单链或双链寡核酸，该寡核酸结合靶向蛋白且一般不显示非特异性作用。根据本文所揭示和/或本领域技术人员所知的任何核酸修饰，适体可经修饰以获得稳定性或其它所需品质。适体的修饰可引入分子中的任何位置(诸如5′或3′末端)或任何内部定义的修饰位点。举例而言，RNA适体可用经2′-氟基或2′-胺基修饰的嘧啶稳定。适体亦可连接至报导分子或连接体化学分子且若需要，则可连接至珠粒或其它固体载体(例如5′或3′胺基、硫醇酯或生物素基团)。硫代适体为在特定核苷酸间磷酰基位点含有硫修饰的适体，且可具有增强的稳定性、抗核酸酶性、靶亲和性和/或选择性。硫代适体的实例包括单硫代磷酸酯(S-ODN)及二硫代磷酸酯(S2-ODN)寡脱氧硫代适体。关于适体及硫代适体的其它信息参见美国专利第5,218,088及6,423,493号。Aptamers as disclosed herein may also be used in the present invention in conjunction with the novel siRNAs disclosed herein. For example, aptamers can be used with any of the siRNAs disclosed herein in combination therapy to treat any of the conditions disclosed herein. Novel pharmaceutical compositions for this combination therapy are also part of the invention, which may comprise the siRNA of the invention linked covalently or non-covalently to an aptamer. Aptamers are RNA or DNA single- or double-stranded oligonucleotides that bind a targeted protein and generally do not exhibit non-specific effects. Aptamers may be modified for stability or other desirable qualities according to any nucleic acid modification disclosed herein and/or known to those skilled in the art. Modifications of the aptamer can be introduced at any position in the molecule (such as the 5' or 3' end) or at any internally defined modification site. For example, RNA aptamers can be stabilized with 2'-fluoro or 2'-amine modified pyrimidines. Aptamers can also be linked to reporter molecules or linker chemical molecules and, if desired, to beads or other solid supports (such as 5' or 3' amine groups, thiol ester or biotin groups). Thioaptamers are aptamers that contain sulfur modifications at specific internucleotide phosphoryl positions and may have enhanced stability, nuclease resistance, target affinity and/or selectivity. Examples of thioaptamers include phosphoromonothioate (S-ODN) and phosphorodithioate (S2-ODN) oligodeoxythioaptamers. Additional information on aptamers and thioaptamers is found in US Patent Nos. 5,218,088 and 6,423,493.
应了解在本发明的内容中，藉由内源性细胞复合物(诸如DICER-参见上文)加工以形成本文所揭示的siRNA分子或包含本文所揭示的siRNA分子的分子的本文所揭示的任何siRNA分子或任何长度双链RNA分子(通常25-500个核苷酸长)均可用于治疗本文所揭示的疾病或病症。It is to be understood in the context of the present invention that any of the siRNA molecules disclosed herein are processed by endogenous cellular complexes (such as DICER - see above) to form the siRNA molecules disclosed herein or molecules comprising the siRNA molecules disclosed herein. siRNA molecules or double-stranded RNA molecules of any length (typically 25-500 nucleotides in length) can be used to treat the diseases or conditions disclosed herein.
可藉由本发明的分子及组合物治疗的额外病症包括所有类型的脉络膜新血管生成(CNV)，CNV不仅发生在湿式AMD中，亦发生在其它眼部病理中，诸如眼部组织浆菌病综合征、血管样条纹、布鲁赫氏膜破裂、近视性变性、眼部肿瘤及某些视网膜退化疾病。Additional disorders treatable by the molecules and compositions of the invention include all types of choroidal neovascularization (CNV), which occurs not only in wet AMD but also in other ocular pathologies such as ocular histoplasmosis syndrome vascular striae, rupture of Bruch's membrane, myopic degeneration, ocular tumors, and certain retinal degenerative diseases.
本发明的另一方面提供用于治疗细胞凋亡相关的疾病的方法。提供用于治疗与不受控病理细胞生长相关的疾病或病症(例如癌症、牛皮癣、自体免疫疾病)的方法及用于治疗与缺血及缺乏适当血流量相关的疾病(例如心肌梗塞(MI)及中风)的方法。＂癌症＂或＂肿瘤＂是指不受控制的异常细胞生长块。这类术语包括可为良性或恶性的原发性肿瘤以及继发性肿瘤或已扩散至身体内其它位点的转移。癌症型疾病的实例包括癌(例如乳房、结肠及肺)、白血病(诸如B细胞白血病)、淋巴瘤(诸如B细胞淋巴瘤)、胚细胞瘤(诸如神经母细胞瘤及黑素瘤)。Another aspect of the invention provides methods for treating apoptosis-related diseases. Methods are provided for treating diseases or conditions associated with uncontrolled pathological cell growth (e.g., cancer, psoriasis, autoimmune diseases) and for treating diseases associated with ischemia and lack of proper blood flow (e.g., myocardial infarction (MI) and stroke) method. "Cancer" or "tumor" refers to an uncontrolled mass of abnormal cell growth. Such terms include primary tumors, which may be benign or malignant, as well as secondary tumors or metastases that have spread to other sites in the body. Examples of cancer-type diseases include carcinomas (eg, breast, colon, and lung), leukemias (such as B-cell leukemia), lymphomas (such as B-cell lymphoma), blastomas (such as neuroblastoma and melanoma).
本发明亦提供一种组合物，其包含在载体、优选可药用载体中的一或多种本发明的化合物。此组合物可包含不同基因的两种或两种以上siRNA或相同基因的不同RNA的混合物。预计包含RTP801基因的siRNA及VEGF基因和/或VEGF-R1基因的siRNA的组合物。The invention also provides a composition comprising one or more compounds of the invention in a carrier, preferably a pharmaceutically acceptable carrier. The composition may comprise two or more siRNAs of different genes or a mixture of different RNAs of the same gene. Compositions comprising siRNA of the RTP801 gene and siRNA of the VEGF gene and/or VEGF-R1 gene are contemplated.
本发明的另一化合物包含共价或非共价结合本发明之一或多个化合物(结构A)的以上本发明化合物(结构A)。此化合物可在载体、优选可药用载体中传递，且可藉由内源性细胞复合物在细胞内加工以产生一或多个本发明的siRNA。本发明的另一化合物包含共价或非共价结合另一基因、尤其VEGF基因和/或VEGF-R1基因的siRNA的以上本发明化合物(结构A)。Another compound of the invention comprises a compound of the invention above (Structure A) covalently or non-covalently bound to one or more compounds of the invention (Structure A). The compound can be delivered in a carrier, preferably a pharmaceutically acceptable carrier, and can be processed intracellularly by endogenous cellular complexes to produce one or more siRNAs of the invention. Another compound of the invention comprises the above compound of the invention covalently or non-covalently bound to siRNA of another gene, especially the VEGF gene and/or VEGF-R1 gene (structure A).
本发明亦包含新颖化学实体RTP801抑制剂，优选为siRNA，其共价或非共价地化学结合至以上VEGF/VEGFR-1抑制剂的任一个。所预计的特定化学实体为共价结合VEGF或VEGF受体-1的抗体的siRNA RTP801抑制剂。所预计的另一化学实体为靶向共价结合本文所揭示的RTP801siRNA的VEGF受体-1的经修饰或未经修饰DNA适体。不受理论限制，该药物组合物的适体部分应结合VEGF受体-1且内在化至细胞中，因此分子的siRNA部分将抑制细胞中的RTP801表达。因此，该药物将具有选择性及靶向增加的益处以及如本文所揭示的组合疗法的额外益处。产生该新颖化学实体的方法为本领域技术人员所知。The present invention also encompasses novel chemical entities RTP801 inhibitors, preferably siRNAs, chemically bound to any of the above VEGF/VEGFR-1 inhibitors, either covalently or non-covalently. The specific chemical entities predicted are siRNA RTP801 inhibitors that covalently bind to antibodies to VEGF or VEGF receptor-1. Another chemical entity contemplated is a modified or unmodified DNA aptamer targeting VEGF receptor-1 covalently bound to the RTP801 siRNA disclosed herein. Without being bound by theory, the aptamer portion of the pharmaceutical composition should bind VEGF receptor-1 and be internalized into the cell, thus the siRNA portion of the molecule will inhibit RTP801 expression in the cell. Thus, the drug will have the added benefit of selectivity and targeting with the added benefit of combination therapy as disclosed herein. Methods for producing such novel chemical entities are known to those skilled in the art.
本发明亦包含一种串联双链结构，该串联双链结构包含两种或两种以上siRNA序列，其在细胞内经加工以形成两种或两种以上不同siRNA，在相关方面中一种抑制801且第二种抑制VEGF/VEGFR-1；本发明亦包含一种串联双链结构，该串联双链结构包含两种或两种以上siRNA序列，其在细胞内降解以形成两种或两种以上均抑制801的不同siRNA。The invention also encompasses a tandem double-stranded structure comprising two or more siRNA sequences that are processed in the cell to form two or more different siRNAs, in a related aspect one inhibiting 801 And the second inhibits VEGF/VEGFR-1; the present invention also includes a tandem double-stranded structure comprising two or more siRNA sequences, which are degraded in cells to form two or more All inhibited different siRNAs of 801.
具体说，预计包含一或多个茎及环结构(其中茎区域包含本发明的寡核苷酸的序列)的长寡核苷酸(通常长约80-500个核苷酸)可在载体、优选可药用载体中传递，且可藉由内源性细胞复合物(例如，如上文所述的DROSHA及DICER)在细胞内加工以产生作为本发明的寡核苷酸的一或多个较小双链寡核苷酸(siRNA)。此寡核苷酸可称为串联shRNA构筑体。预计此长寡核苷酸为包含一或多个茎及环结构的单链寡核苷酸，其中各茎区域包含801基因的有义及相应反义siRNA序列。具体说，预计此寡核苷酸包含如表A-D的任一个中所述的有义及反义siRNA序列。或者，串联shRNA构筑体可包含801基因的有义及相应反义siRNA序列及另外不同基因(诸如VEGF或VEGF-R1)的有义及相应反义siRNA序列。In particular, long oligonucleotides (typically about 80-500 nucleotides in length) comprising one or more stem and loop structures, wherein the stem region comprises the sequence of an oligonucleotide of the invention, are expected to be present in vectors, Preferably, it can be delivered in a pharmaceutically acceptable carrier, and can be processed intracellularly by endogenous cellular complexes (eg, DROSHA and DICER as described above) to produce one or more comparative oligonucleotides that are oligonucleotides of the invention. Small double-stranded oligonucleotides (siRNA). This oligonucleotide can be referred to as a tandem shRNA construct. This long oligonucleotide is expected to be a single-stranded oligonucleotide comprising one or more stem and loop structures, where each stem region comprises the sense and corresponding antisense siRNA sequences of the 801 gene. In particular, the oligonucleotides are expected to comprise sense and antisense siRNA sequences as described in any of Tables A-D. Alternatively, the tandem shRNA construct may comprise the sense and corresponding antisense siRNA sequences for the 801 gene and the sense and corresponding antisense siRNA sequences for an additional different gene such as VEGF or VEGF-R1.
如本文所提及，对抗RTP801的siRNA可为药物组合物中的主要活性组份，或可为含有两种或两种以上siRNA(或编码或内源性产生两种或两种以上siRNA的分子，编码两种或两种以上siRNA的分子混合物或一或多个串联分子)的药物组合物的一种活性组份，该药物组合物进一步包含一或多种靶向一或多种其它基因的其它siRNA分子。RTP801及该(等)其它基因的同时抑制可能具有添加或协同效应以治疗本文所揭示的疾病，根据下列情形：As mentioned herein, the siRNA against RTP801 may be the main active ingredient in a pharmaceutical composition, or may be a molecule containing two or more siRNAs (or encoding or endogenously producing two or more siRNAs) , an active ingredient of a pharmaceutical composition encoding two or more siRNA molecules or one or more tandem molecules), the pharmaceutical composition further comprising one or more targeting one or more other genes Other siRNA molecules. Simultaneous inhibition of RTP801 and the other gene(s) may have additive or synergistic effects to treat the diseases disclosed herein, depending on the following:
急性肾衰竭(ARF)及其它微血管病症以及听力障碍及褥疮：用于治疗ARF的药物组合物可包含下列化合物组合：1)RTP801 siRNA与p53siRNA的二聚体，2)RTP801与Fas siRNA的二聚体，3)RTP801与BaxsiRNA的二聚体，4)RTP801与Fas siRNA的二聚体，5)RTP801与BaxsiRNA的二聚体，6)RTP801与Noxa siRNA的二聚体，7)RTP801与PumasiRNA的二聚体，8)RTP801(REDD1)与RTP801L(REDD2)siRNA的二聚体，9)RTP801 siRNA、Fas siRNA与RTP801L siRNA p53 siRNA、BaxsiRNA、Noxa siRNA或Puma siRNA的任一个以形成三聚体或多聚体(亦即，编码三种或三种以上siRNA的串联分子)。Acute renal failure (ARF) and other microvascular disorders as well as hearing impairment and decubitus: The pharmaceutical composition for the treatment of ARF may comprise the following compound combinations: 1) dimer of RTP801 siRNA and p53 siRNA, 2) dimer of RTP801 and Fas siRNA 3) Dimer of RTP801 and BaxsiRNA, 4) Dimer of RTP801 and Fas siRNA, 5) Dimer of RTP801 and BaxsiRNA, 6) Dimer of RTP801 and Noxa siRNA, 7) Dimer of RTP801 and PumasiRNA Dimer, 8) dimer of RTP801(REDD1) and RTP801L(REDD2) siRNA, 9) RTP801 siRNA, Fas siRNA and any one of RTP801L siRNA p53 siRNA, BaxsiRNA, Noxa siRNA or Puma siRNA to form a trimer or Multimers (ie, molecules encoding three or more siRNAs in tandem).
此外，用于组合治疗ARF、听力障碍、褥疮及本文所揭示的任何其它疾病或病状的其它药物组合物亦可包含两种siRNA，其中第一种siRNA为优选选自表A-D的RTP801 siRNA，且第二种siRNA可共价或非共价地结合第一种siRNA或与第一种siRNA混合，该siRNA为靶向选自以下群的基因的siRNA：肿瘤蛋白p53结合蛋白2(TP53BP2)、富含亮氨酸的重复序列及死亡域(含LRDD)、细胞色素b-245、α多肽(CYBA)、活化转录因子3(ATF3)、卡斯蛋白酶2、细胞凋亡相关的半胱氨酸肽酶(经神经前体细胞表达、发育性下调2)(CASP2)、NADPH氧化酶3(NOX3)、harakiri、BCL2相互作用蛋白(HRK)、互补组份1、q亚组份结合蛋白(C1QBP)、BCL2/腺病毒E1B19kDa相互作用蛋白3(BNIP3)、分裂素活化的蛋白激酶8(MAPK8)、分裂素活化的蛋白激酶14(MAPK14)、ras相关的C3肉毒杆菌毒素基质1(rho家族，小GTP结合蛋白Rac1)、肝糖合成酶激酶3β(GSK3B)、嘌呤型受体P2X配体门控性离子通道7(P2RX7)、瞬时受体电位阳离子通道子族M成员2(TRPM2)、聚(ADP-核糖)糖苷水解酶(PARG)、CD38分子(CD38)、STEAP家族成员4(STEAP4)、骨形态发生蛋白2-BMP2、缝隙连接蛋白α1(43kDa)(connexin43)(GJA1)、TYRO蛋白酪氨酸激酶结合蛋白(TYROBP)、结缔组织生长因子(CTGF)、分泌磷蛋白1(骨质素(osteopontin)、骨唾液酸蛋白I、早期T淋巴细胞活化1)(SPP1)、网霉素4受体(RTN4R)、连接素A2(annexinA2，ANXA2)、ras同源基因家族成员A(RHOA)及双氧化酶1(DUOX1)。溶质载体家族5(钠/葡萄糖共转运蛋白)、膜1(SLC5A1)、溶质载体家族2(易化的葡萄糖转运蛋白)、膜2(SLC2A2)、aldo-keto还原酶家族1、膜B1(醛糖还原酶)(AKR1B1)、山梨醇脱氢酶(SORD)、溶质载体家族2(易化的葡萄糖转运蛋白)、膜1(SLC2A1)和膜金属-内肽酶(中性内肽酶、脑啡肽酶)(MME)。In addition, other pharmaceutical compositions for combination treatment of ARF, hearing impairment, decubitus and any other disease or condition disclosed herein may also comprise two siRNAs, wherein the first siRNA is an RTP801 siRNA preferably selected from Tables A-D, and The second siRNA can be covalently or non-covalently bound or mixed with the first siRNA, which is an siRNA targeting a gene selected from the group consisting of: tumor protein p53 binding protein 2 (TP53BP2), rich Leucine-containing repeats and death domains (including LRDD), cytochrome b-245, alpha polypeptide (CYBA), activated transcription factor 3 (ATF3), caspase 2, apoptosis-related cysteine peptides Enzymes (expressed by neural progenitor cells, developmentally downregulated 2) (CASP2), NADPH oxidase 3 (NOX3), harakiri, BCL2 interacting protein (HRK), complementarity component 1, q subcomponent binding protein (C1QBP) , BCL2/adenovirus E1B19kDa interacting protein 3 (BNIP3), mitogen-activated protein kinase 8 (MAPK8), mitogen-activated protein kinase 14 (MAPK14), ras-related C3 botulinum toxin substrate 1 (rho family, Small GTP-binding protein Rac1), glycogen synthase kinase 3β (GSK3B), purinergic receptor P2X ligand-gated ion channel 7 (P2RX7), transient receptor potential cation channel subfamily M member 2 (TRPM2), poly (ADP-ribose) glycoside hydrolase (PARG), CD38 molecule (CD38), STEAP family member 4 (STEAP4), bone morphogenetic protein 2-BMP2, gap junction protein α1 (43kDa) (connexin43) (GJA1), TYRO protein Tyrosine kinase binding protein (TYROBP), connective tissue growth factor (CTGF), secreted phosphoprotein 1 (osteopontin, bone sialoprotein I, early T lymphocyte activation 1) (SPP1), netomycin 4 receptor (RTN4R), annexin A2 (annexinA2, ANXA2), ras homologous gene family member A (RHOA) and double oxygenase 1 (DUOX1). Solute carrier family 5 (sodium/glucose cotransporter), membrane 1 (SLC5A1), solute carrier family 2 (facilitated glucose transporter), membrane 2 (SLC2A2), aldo-
黄斑变性(MD)、糖尿病性视网膜病(DR)、脊髓损伤：用于治疗MD、DR及脊髓损伤的药物组合物可包含下列化合物组合：1)与VEGFsiRNA、VEGF-R1 siRNA、VEGF R2 siRNA、PKCbeta siRNA、MCP1 siRNA、eNOSsiRNA、KLF2 siRNA、RTP801L siRNA中任一个组合的RTP801 siRNA(以物理方式混合或在串联分子中)；2)与以上清单的两种或两种以上siRNA组合的RTP801 siRNA(以物理方式混合或在编码三种siRNA的串联分子中或其组合)。Macular degeneration (MD), diabetic retinopathy (DR), spinal cord injury: the pharmaceutical composition for treating MD, DR and spinal cord injury may comprise the following compound combination: 1) with VEGFsiRNA, VEGF-R1 siRNA, VEGF R2 siRNA, RTP801 siRNA combined with any of PKCbeta siRNA, MCP1 siRNA, eNOS siRNA, KLF2 siRNA, RTP801L siRNA (mixed physically or in tandem molecules); 2) RTP801 siRNA combined with two or more siRNAs listed above ( physically mixed or in tandem molecules encoding the three siRNAs or combinations thereof).
COPD及呼吸病症：用于治疗呼吸病症的药物组合物可包含下列化合物组合：与对抗一或多种以下基因的siRNA组合的RTP801：弹性蛋白酶、基质金属蛋白酶、磷脂酶、卡斯蛋白酶、神经鞘磷脂酶及神经酰胺合酶。COPD and Respiratory Disorders: Pharmaceutical compositions for the treatment of respiratory disorders may comprise the following combinations of compounds: RTP801 in combination with siRNA against one or more of the following genes: Elastase, Matrix Metalloproteinase, Phospholipase, Caspase, Neurosheath Phospholipase and ceramide synthase.
此外，RTP801 siRNA或包含或编码RTP801 siRNA的任何核酸分子可连接(共价或非共价)抗体或适体以获得增强的靶向作用，从而治疗本文所揭示的疾病，根据下列情形：In addition, RTP801 siRNA or any nucleic acid molecule comprising or encoding RTP801 siRNA can be linked (covalently or non-covalently) to an antibody or aptamer for enhanced targeting to treat the diseases disclosed herein, according to the following:
ARF：抗Fas抗体(优选为中和抗体)。ARF: anti-Fas antibody (preferably neutralizing antibody).
黄斑变性、糖尿病性视网膜病、脊髓损伤：抗Fas抗体或适体、抗MCP1抗体或适体、抗VEGFR1及抗VEGFR2抗体或适体。抗体优选应为中和抗体。Macular degeneration, diabetic retinopathy, spinal cord injury: anti-Fas antibody or aptamer, anti-MCP1 antibody or aptamer, anti-VEGFR1 and anti-VEGFR2 antibody or aptamer. Antibodies should preferably be neutralizing antibodies.
包含本文所揭示的siRNA序列(具有适当核酸修饰)的任何分子(诸如反义DNA分子)尤其理想，且可以与其相应siRNA相同的能力用于本文所揭示的所有用途及方法。Any molecule comprising an siRNA sequence disclosed herein (with appropriate nucleic acid modifications), such as an antisense DNA molecule, is especially desirable and can be used in the same capacity as its corresponding siRNA for all uses and methods disclosed herein.
本发明亦包含治疗罹患诸如本文所述病症的患者的方法，该方法包括将治疗有效量的以上组合物或化合物给药于患者以藉此治疗患者。The present invention also encompasses methods of treating a patient suffering from a condition such as described herein, the method comprising administering to the patient a therapeutically effective amount of the above composition or compound, thereby treating the patient.
术语＂反义＂(AS)或＂反义片段＂意谓具有抑制反义活性的聚核苷酸片度(包含脱氧核糖核苷酸、核糖核苷酸或两者的混合物)，该活性使相应基因(在此情形下为RTP801)的内源性基因复本的表达减少。RTP801AS聚核苷酸是包含具有足够长度且与存在于SEQ ID NO：1所陈述的RTP801基因序列中的序列同源的序列的连续核苷酸的聚核苷酸以使AS与基因杂交。设计AS的序列以互补所关注的靶向mRNA且形成RNA：AS双链体。此双链体形成可预防相关mRNA的加工、剪接、转运或转译。此外，某些AS核苷酸序列在与其靶向mRNA杂交时可引发细胞核糖核酸酶H活性，导致mRNA降解(Calabretta等人，1996：Antisensestrategies in the treatment of leukemias.Semin Oncol.23(1)：78-87)。在该情形下，核糖核酸酶H将裂解双链体的RNA组份且可潜在释放AS以进一步与靶向RNA的其它分子杂交。另一作用模式源自AS与基因组DNA的相互作用以形成可经转录失活的三螺旋结构。特定AS片段为编码本文所述RTP801的特定片段的DNA的AS。The term "antisense" (AS) or "antisense fragment" means a fragment of a polynucleotide (comprising deoxyribonucleotides, ribonucleotides, or a mixture of both) that inhibits antisense activity that renders The expression of the endogenous gene copy of the corresponding gene (in this case RTP801) is reduced. The RTP801 AS polynucleotide is a polynucleotide comprising contiguous nucleotides of sufficient length and sequence homology to the sequence present in the RTP801 gene sequence set forth in SEQ ID NO: 1 to allow AS to hybridize to the gene. The sequence of AS is designed to complement the targeted mRNA of interest and form an RNA:AS duplex. This duplex formation prevents the processing, splicing, transport or translation of the associated mRNA. In addition, certain AS nucleotide sequences can trigger cellular RNase H activity when hybridized to their targeted mRNA, resulting in mRNA degradation (Calabretta et al., 1996: Antisense strategies in the treatment of leukemias. Semin Oncol. 23(1): 78-87). In this case, RNase H will cleave the RNA component of the duplex and could potentially release AS for further hybridization with other RNA-targeting molecules. Another mode of action arises from the interaction of AS with genomic DNA to form a transcriptionally inactive triple helix structure. The specific AS segment is the AS of the DNA encoding the specific segment of RTP801 described herein.
多篇评论已报导了反义(AS)技术及其治疗潜力的主要方面(Wright & Anazodo，1995.Antisense Molecules and TheirPotential For The Treatment Of Cancer and AIDS.Cancer J.8：185-189)。存在对此技术的化学(Crooke，1995.Progress inantisense therapeutics，Hematol.Pathol.2：59；Uhlmann及Peyman，1990.Antisense Oligonucleotides：A New TherapeuticPrinciple.Chem Rev 90(4)：543-584)、细胞(Wagner，1994.Geneinhibition using antisense oligodeoxynucleotides.Nature372：333)及治疗(Hanania等人1995.Recent advances in theapplication of gene therapy to human disease.Am.J.Med.99：537；Scanlon等人，1995.Oligonucleotides-mediatedmodulation of mammalian gene expression.FASEB J.9：1288；Gewirtz，1993.Oligodeoxynucleotide-based therapeutics forhuman leukemias，Stem Cells Dayt.11：96)方面的评论。Several reviews have reported major aspects of antisense (AS) technology and its therapeutic potential (Wright & Anazodo, 1995. Antisense Molecules and Their Potential For The Treatment Of Cancer and AIDS. Cancer J. 8:185-189). There is chemistry for this technique (Crooke, 1995. Progress inantisense therapeutics, Hematol. Pathol. 2: 59; Uhlmann and Peyman, 1990. Antisense Oligonucleotides: A New Therapeutic Principle. Chem Rev 90 (4): 543-584), cells ( Wagner, 1994.Gene inhibition using antisense oligodeoxynucleotides.Nature372:333) and treatment (Hanania et al. 1995.Recent advances in the application of gene therapy to human disease.Am.J.Med.99:537; Scanlon et al., 1995.Oligonucleotides- mediated modulation of mammalian gene expression. FASEB J.9: 1288; Gewirtz, 1993. Oligodeoxynucleotide-based therapeutics for human leukemias, Stem Cells Dayt. 11: 96).
反义干扰特异性基因的表达可藉由使用合成AS寡核苷酸序列来获得(Lefebvre-d′Hellencourt等人，1995.Immunomodulation bycytokine antisense oligonucleotides.Eur.Cytokine Netw.6：7；Agrawal，1996.Antisense oligonucleotides：towards clinicaltrials，TIBTECH，14：376；Lev-Lehman等人，1997.AntisenseOligomers in vitro and in vivo.In Antisense Therapeutics，A.Cohen及S.Smicek编(Plenum Press，New York))。设计AS寡核苷酸序列以互补所关注的靶向mRNA且形成RNA：AS双链体。此双链体形成可预防相关mRNA的加工、剪接、转运或转译。此外，某些AS核苷酸序列在与其靶向mRNA杂交时可引发细胞核糖核酸酶H活性，导致mRNA降解(Calabretta等人，1996：Antisense strategies in the treatmentofleukemias.Semin.Oncol.23：78)。在该情形下，核糖核酸酶H将裂解双链体的RNA组份且可潜在释放AS以进一步与靶向RNA的其它分子杂交。另一作用模式源自AS与基因组DNA的相互作用以形成可经转录失活的三螺旋结构。Antisense interference specific gene expression can be obtained by using synthetic AS oligonucleotide sequences (Lefebvre-d'Hellencourt et al., 1995. Immunomodulation by cytokine antisense oligonucleotides. Eur. Cytokine Netw. 6:7; Agrawal, 1996. Antisense oligonucleotides: towards clinicaltrials, TIBTECH, 14:376; Lev-Lehman et al., 1997. Antisense Oligomers in vitro and in vivo. In Antisense Therapeutics, edited by A. Cohen and S. Smicek (Plenum Press, New York)). AS oligonucleotide sequences were designed to complement the target mRNA of interest and form RNA:AS duplexes. This duplex formation prevents the processing, splicing, transport or translation of the associated mRNA. In addition, certain AS nucleotide sequences can trigger cellular RNase H activity when hybridized to their targeted mRNA, resulting in mRNA degradation (Calabretta et al., 1996: Antisense strategies in the treatment of leukemias. Semin. Oncol. 23:78). In this case, RNase H will cleave the RNA component of the duplex and could potentially release AS for further hybridization with other RNA-targeting molecules. Another mode of action arises from the interaction of AS with genomic DNA to form a transcriptionally inactive triple helix structure.
选择反义寡核苷酸的序列靶向片段以使得序列显示关于寡核苷酸与其互补模板形成双链体的重要的适合能量相关特征，且显示自体二聚或自体互补的低潜能(Anazodo等人，1996)。举例而言，计算机程序OLIGO(Primer Analysis Software，3.4版本)可用以确定反义序列熔融温度、自由能性质且评估潜在自体二聚体形成及自体互补性质。该程序确定此两个参数(潜在自体二聚体形成及自体互补)的定性评估且提供＂无潜能＂或＂某些潜能＂或＂基本上完全潜能＂的指示。使用此程序，一般选择评估在这类参数上无潜能的靶向片段。然而，可使用具有该类别之一的＂某些潜能＂的片段。Sequence-targeted fragments of antisense oligonucleotides are selected such that the sequence exhibits suitable energy-related features important for duplex formation of the oligonucleotide with its complementary template, and exhibits a low potential for self-dimerization or self-complementation (Anazodo et al. People, 1996). For example, the computer program OLIGO (Primer Analysis Software, version 3.4) can be used to determine antisense sequence melting temperature, free energy properties and assess potential self-dimer formation and self-complementation properties. The program determines a qualitative assessment of these two parameters (potential self-dimerization and self-complementation) and provides an indication of "no potential" or "some potential" or "essentially full potential". Using this procedure, target fragments are generally selected for evaluation with no potential on such parameters. However, fragments with "some potential" of one of the classes may be used.
如本领域所知，在选择中使用该参数的平衡。此外，亦在有需要时选择寡核苷酸以使类似取代大体上不影响功能。A balance of this parameter is used in the selection, as is known in the art. In addition, oligonucleotides are also selected, where necessary, such that similar substitutions do not substantially affect function.
在有效且在动物中显示足够药物动力学半衰期的浓度下，硫代磷酸酯反义寡核苷酸通常不显示显著毒性(Agrawal，1996.Antisenseoligonucleotides：towards clinical trials，TIBTECH，14：376)及耐核酸酶性。细胞发育相关的反义诱发的功能损失表型已关于胶质纤维酸性蛋白(GFAP)显示以建立鸡中的顶盖板形成(Galileo等人，1991.J.Cell.Biol.，112：1285)，且关于N-myc蛋白(负责维持神经外胚培养物中的细胞异质)(上皮细胞与成神经细胞相比，两者不同在于其菌落形成能力、致瘤性及黏附特性)显示(Rosolen等人，1990.Cancer Res.50：6316；Whitesell等人，1991.Episome-generatedN-myc antisense RNA restricts the differentiation potential ofprimitive neuroectodermal cell lines.Mol.Cell.Biol.11：1360)。基本成纤维细胞生长因子(bFgF)(具有致有丝分裂及血管生成性质)的反义寡核苷酸抑制以可饱和且特定的方式抑制神经胶质瘤细胞的80％生长(Morrison，1991.Suppression of basic fibroblastgrowth factor expression by antisense oligonucleotidesinhibits the growth of transformed human astrocytes.J.Biol.Chem.266：728)。疏水性反义寡核苷酸与磷脂膜充分地相互作用(Akhter等人，1991.Interactions of antisense DNAoligonucleotide analogs with phospholipid membranes(liposomes)Nuc.Res.19：5551-5559)。在反义寡核苷酸与细胞质膜相互作用后，其以经预测涉及特异性受体的可饱和机制(Yakubov等人，1989.PNAS USA 86：6454)主动(或被动)转运至活细胞中(Loke等人，1989.Characterization of oligonucleotide transport intoliving cells.PNAS USA 86：3474)。Phosphorothioate antisense oligonucleotides generally show no significant toxicity (Agrawal, 1996.Antisense oligonucleotides: towards clinical trials, TIBTECH, 14:376) and resistance to oligonucleotides at concentrations that are effective and show sufficient pharmacokinetic half-life in animals. nuclease. Cell development-associated antisense-induced loss-of-function phenotypes have been shown for glial fibrillary acidic protein (GFAP) to establish tectum formation in chickens (Galileo et al., 1991. J. Cell. Biol., 112:1285) , and with respect to the N-myc protein (responsible for maintaining cellular heterogeneity in neuroectodermal cultures) (epithelial cells differ from neuroblasts in their colony-forming ability, tumorigenicity, and adhesive properties) (Rosolen et al., 1990. Cancer Res. 50: 6316; Whitesell et al., 1991. Episome-generated N-myc antisense RNA restricts the differentiation potential of primitive neuroectodermal cell lines. Mol. Cell. Biol. 11: 1360). Antisense oligonucleotide inhibition of basic fibroblast growth factor (bFgF), which has mitogenic and angiogenic properties, suppressed 80% of the growth of glioma cells in a saturable and specific manner (Morrison, 1991. Suppression of basic fibroblast growth factor expression by antisense oligonucleotides inhibits the growth of transformed human astrocytes. J. Biol. Chem. 266: 728). Hydrophobic antisense oligonucleotides interact substantially with phospholipid membranes (Akhter et al., 1991. Interactions of antisense DNA oligonucleotide analogs with phospholipid membranes (liposomes) Nuc. Res. 19:5551-5559). After antisense oligonucleotides interact with the plasma membrane, they are actively (or passively) transported into living cells by a saturable mechanism predicted to involve specific receptors (Yakubov et al., 1989. PNAS USA 86:6454) (Loke et al., 1989. Characterization of oligonucleotide transport into living cells. PNAS USA 86:3474).
＂核糖核酸酶＂为具有RNA催化能力(参见Cech评论)且裂解靶向RNA中的特定位点的RNA分子。A "ribonuclease" is an RNA molecule that has RNA catalytic capabilities (see review by Cech) and that targets specific sites in the RNA for cleavage.
根据本发明，裂解RTP801 mRNA的核糖核酸酶可用作RTP801抑制剂。在反向疗法受到化学计量考虑限制的情形下，此可为必需的(Sarver等人，1990，Gene Regulation and Aids，第305-325页)。According to the present invention, ribonucleases that cleave RTP801 mRNA can be used as RTP801 inhibitors. This may be necessary where reverse therapy is limited by stoichiometric considerations (Sarver et al., 1990, Gene Regulation and Aids, pp. 305-325).
接着可使用将靶向RTP801序列的核糖核酸酶。藉由核糖核酸酶裂解的RNA分子的数目大于藉由化学计量学预测的数目(Hampel及Tritz，1989；Uhlenbeck，1987)。A ribonuclease that will target the RTP801 sequence can then be used. The number of RNA molecules cleaved by ribonucleases is greater than that predicted by stoichiometry (Hampel and Tritz, 1989; Uhlenbeck, 1987).
核糖核酸酶催化RNA的磷酸二酯键裂解。已鉴别出若干核糖核酸酶结构家族，包括最初衍生自烟草环斑病毒卫星RNA(sTRSV)负链的I族内含子、核糖核酸酶P、肝炎δ病毒核糖核苷酸、锤头型核糖核酸酶及发夹型核糖核酸酶(Sullivan，1994；美国专利第5,225,347号，第4-5行)。后两个家族衍生自类病毒及拟病毒，其中据信核糖核酸酶自在滚环复制期间产生的寡聚体分离出单体(Symons，1989及1992)。对基因疗法而言，锤头型及发夹型核糖核酸酶主结构最常适于mRNA的逆裂解(Sullivan，1994)。如本领域已知，选择用于本发明的核糖核酸酶类型。发夹型核糖核酸酶现在临床试验中且为优选类型。核糖核酸酶一般长为30-100个核苷酸。核糖核酸酶的传递类似于AS片段和/或siRNA分子的传递。Ribonucleases catalyze the cleavage of phosphodiester bonds in RNA. Several structural families of ribonucleases have been identified, including the group I intron originally derived from the negative strand of tobacco ringspot virus satellite RNA (sTRSV), RNase P, hepatitis delta virus ribonucleotides, hammerhead ribonucleases Enzymes and hairpin ribonucleases (Sullivan, 1994; US Patent No. 5,225,347, lines 4-5). The latter two families are derived from viroids and viroids, in which ribonucleases are believed to separate monomers from oligomers produced during rolling circle replication (Symons, 1989 and 1992). For gene therapy, the hammerhead and hairpin ribonuclease main structures are most commonly adapted for reverse cleavage of mRNA (Sullivan, 1994). The type of ribonuclease used in the present invention is selected as known in the art. Hairpin ribonucleases are now in clinical trials and are the preferred type. Ribonucleases are generally 30-100 nucleotides long. Delivery of ribonucleases is similar to delivery of AS fragments and/or siRNA molecules.
应注意用于本发明的所有聚核苷酸均可经历修饰以具有改善的治疗性质。可引入核苷酸的修饰或类似物以改善聚核苷酸的治疗性质。改善的性质包括增加的抗核酸酶性和/或增加的渗透细胞膜能力。若需要，则抗核酸酶性应藉由本领域已知的任何方法来提供，该方法不干扰使用且传递该方法所需的AS聚核苷酸siRNA、cDNA和/或核糖核酸酶的生物活性(Iyer等人，1990；Eckstein，1985；Spitzer及Eckstein，1988；Woolf等人，1990；Shaw等人，1991)。可对寡核苷酸进行修饰以增强抗核酸酶性，该修饰包括修饰磷酸酯主链中的磷或氧杂原子。这类修饰包括制备膦酸甲酯、硫代磷酸酯、二硫代磷酸酯及吗啉寡聚物。在一实施方式中，提供键联在4至6个3′末端核苷酸碱基之间的硫代磷酸酯键。或者，硫代磷酸酯键会连接所有核苷酸碱基。It should be noted that all polynucleotides used in the present invention may undergo modification to have improved therapeutic properties. Nucleotide modifications or analogs can be introduced to improve the therapeutic properties of polynucleotides. Improved properties include increased resistance to nucleases and/or increased ability to penetrate cell membranes. If desired, nuclease resistance should be provided by any method known in the art that does not interfere with the biological activity of the AS polynucleotide siRNA, cDNA and/or ribonuclease required for use and delivery of the method ( Iyer et al., 1990; Eckstein, 1985; Spitzer and Eckstein, 1988; Woolf et al., 1990; Shaw et al., 1991). Oligonucleotides can be modified to enhance nuclease resistance, including modification of phosphorus or oxygen heteroatoms in the phosphate backbone. Such modifications include the preparation of methyl phosphonates, phosphorothioates, phosphorodithioates, and morpholine oligomers. In one embodiment, phosphorothioate linkages between 4 and 6 3' terminal nucleotide bases are provided. Alternatively, phosphorothioate linkages link all nucleotide bases.
本领域已知的其它修饰可用于保持生物活性但大体上增加核酸酶稳定性的情况。Other modifications known in the art may be used where biological activity is retained but substantially increased nuclease stability.
聚核苷酸的所有类似物或修饰均可用于本发明，其限制条件为该类似物或修饰大体上不影响聚核苷酸的功能。核苷酸可选自天然产生或合成的经修饰碱基。天然产生的碱基包括腺嘌呤、鸟嘌呤、胞嘧啶、胸腺嘧啶及尿嘧啶。经修饰的核苷酸碱基包括下列各物：肌苷、黄嘌呤、次黄嘌呤、2-胺基腺嘌呤、6-甲基、2-丙基及其它烷基腺嘌呤、5-卤基尿嘧啶、5-卤基胞嘧啶、6-氮杂胞嘧啶及6-氮杂胸腺嘧啶、假尿嘧啶、4-硫尿嘧啶、8-卤基腺嘌呤、8-胺基腺嘌呤、8-硫醇腺嘌呤、8-硫代烷基腺嘌呤、8-羟基腺嘌呤及其它8-取代腺嘌呤、8-卤基鸟嘌呤、8-胺基鸟嘌呤、8-硫醇鸟嘌呤、8-硫代烷基鸟嘌呤、8-羟基鸟嘌呤及其它经取代的鸟嘌呤、其它氮杂及脱氮腺嘌呤、其它氮杂及脱氮鸟嘌呤、5-三氟甲基尿嘧啶及5-三氟胞嘧啶。All analogs or modifications of polynucleotides can be used in the present invention, provided that the analogs or modifications do not substantially affect the function of the polynucleotides. Nucleotides may be selected from naturally occurring or synthetic modified bases. Naturally occurring bases include adenine, guanine, cytosine, thymine, and uracil. Modified nucleotide bases include the following: inosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl, 2-propyl and other alkyladenines, 5-halo Uracil, 5-halocytosine, 6-azacytosine and 6-azathymine, pseudouracil, 4-thiouracil, 8-haloadenine, 8-aminoadenine, 8- Thiol adenine, 8-thiol adenine, 8-hydroxyadenine and other 8-substituted adenine, 8-haloguanine, 8-aminoguanine, 8-thiolguanine, 8- Thioalkylguanine, 8-hydroxyguanine and other substituted guanine, other aza and deazaadenine, other aza and deazaguanine, 5-trifluoromethyluracil and 5-trifluoromethyluracil Flucytosine.
此外，可制备聚核苷酸的类似物，其中核苷酸的结构基本上改变且该聚核苷酸类似物更适用作治疗或实验试剂。核苷酸类似物的实例为肽核酸(PNA)，其中DNA(或RNA)中脱氧核糖(或核糖)磷酸酯主链已用类似于肽中所发现的主链的聚酰胺主链置换。PNA类似物已显示出对抗酶降解性且具有活体内及活体外的延长寿命。此外，PNA已显示出比DNA分子更强地结合互补DNA序列。此观测归因于PNA链与DNA链之间电荷排斥的缺乏。可对寡核苷酸进行的其他修饰包括聚合物主链、环状主链或非环状主链。In addition, analogs of polynucleotides can be prepared in which the structure of the nucleotides is substantially altered and which are more suitable for use as therapeutic or experimental reagents. An example of a nucleotide analog is peptide nucleic acid (PNA) in which the deoxyribose (or ribose) phosphate backbone in the DNA (or RNA) has been replaced with a polyamide backbone similar to that found in peptides. PNA analogs have been shown to be resistant to enzymatic degradation and have extended lifetimes in vivo and in vitro. Furthermore, PNAs have been shown to bind complementary DNA sequences more strongly than DNA molecules. This observation is attributed to the lack of charge repulsion between the PNA strands and the DNA strands. Other modifications that can be made to oligonucleotides include polymeric backbones, cyclic backbones, or acyclic backbones.
用于本发明的多肽亦可经修饰，任选经化学修饰以改善其治疗活性。当涉及多肽时，＂经化学修饰＂意谓多肽的至少一个氨基酸残基藉由天然方法(诸如加工或其它转译后修饰)或藉由本领域熟知的化学修饰技术来修饰。在大量已知的修饰中，典型但非唯一的实例包括乙酰化、酰化、酰胺化、ADP核糖基化、糖基化、GPI锚形成、脂质或脂质衍生物的共价连接、甲基化、肉豆蔻酰化、聚乙二醇化、异戊二烯基化、磷酸化、泛素化或任何类似方法。Polypeptides for use in the invention may also be modified, optionally chemically, to improve their therapeutic activity. "Chemically modified" in reference to a polypeptide means that at least one amino acid residue of the polypeptide has been modified by natural means, such as processing or other post-translational modifications, or by chemical modification techniques well known in the art. Among the large number of known modifications, typical but not exclusive examples include acetylation, acylation, amidation, ADP ribosylation, glycosylation, GPI anchor formation, covalent attachment of lipids or lipid derivatives, formazan Myristoylation, pegylation, prenylation, phosphorylation, ubiquitination, or any similar method.
其它可能性多肽修饰(诸如源自核酸序列改变的修饰)包括下列修饰：Other possible polypeptide modifications, such as those resulting from nucleic acid sequence changes, include the following modifications:
＂保守性取代＂是指一种氨基酸藉由相同种类氨基酸取代，其中一种类别藉由通用物理化学氨基酸侧链性质及天然发现的同源多肽中的高取代频率(例如藉由标准Dayhoff频率交换矩阵或BLOSUM矩阵测定)来定义。已分成6种一般氨基酸侧链且其包括：I类(Cys)、II类(Ser、Thr、Pro、Ala、Gly)、III类(Asn、Asp、Gin、Glu)、IV类(His、Arg、Lys、)、V类(Ile、Leu、Val、Met)及VI类(Phe、Tyr、Trp)。举例而言，用另一III类残基(诸如Asn、Gln或Glu)取代Asp为保守性取代。＂非保守性取代＂是指用一类氨基酸取代另一类氨基酸；举例而言，用III类残基(诸如Asp、Asn、Glu或Gln)取代II类残基Ala。"Conservative substitution" refers to the substitution of an amino acid by an amino acid of the same class, one of which is characterized by common physicochemical amino acid side chain properties and high substitution frequencies in homologous polypeptides found in nature (e.g., by standard Dayhoff frequency exchange). matrix or BLOSUM matrix measurement) to define. It has been divided into 6 general amino acid side chains and they include: Class I (Cys), Class II (Ser, Thr, Pro, Ala, Gly), Class III (Asn, Asp, Gin, Glu), Class IV (His, Arg , Lys,), V class (Ile, Leu, Val, Met) and VI class (Phe, Tyr, Trp). For example, substitution of Asp with another class III residue such as Asn, Gln or Glu is a conservative substitution. "Non-conservative substitution" refers to the substitution of one class of amino acids for another class of amino acids; for example, the substitution of a class III residue, such as Asp, Asn, Glu or Gln, for a class II residue, Ala.
＂缺失＂-核苷酸或氨基酸序列的改变，其中一或多个核苷酸或氨基酸残基分别缺乏。"Deletion" - an alteration in a nucleotide or amino acid sequence in which one or more nucleotide or amino acid residues, respectively, are absent.
＂插入＂或＂添加＂-核苷酸或氨基酸序列的变化，其导致分别与天然产生的序列相比，添加一或多个核苷酸或氨基酸残基。"Insertion" or "Addition" - A change in a nucleotide or amino acid sequence resulting in the addition of one or more nucleotide or amino acid residues, respectively, compared to the naturally occurring sequence.
＂取代＂-藉由不同核苷酸或氨基酸分别置换一或多个核苷酸或氨基酸。关于氨基酸序列，取代可为保守性或非保守性的。"Substitution" - replacement of one or more nucleotides or amino acids by different nucleotides or amino acids, respectively. With respect to amino acid sequences, substitutions may be conservative or non-conservative.
在本发明的另一实施方式中，RTP801多肽或聚核苷酸可用以诊断或检测受检者的黄斑变性。检测方法通常应包含在得自受检者的样品中检定RTP801mRNA或RTP801多肽。In another embodiment of the present invention, the RTP801 polypeptide or polynucleotide can be used to diagnose or detect macular degeneration in a subject. The detection method will generally comprise the detection of RTP801 mRNA or RTP801 polypeptide in a sample obtained from a subject.
＂检测＂-是指检测疾病的方法。此术语可指疾病诱因的检测或疾病严重程度的检测。"Detection"-refers to a method of detecting a disease. The term can refer to detection of disease predisposition or detection of disease severity.
如本发明所用的＂同源/同源性＂意谓至少约70％、优选至少约75％同源、有利地至少约80％同源、更有利地至少约90％同源、甚至更有利地至少约95％(例如至少约97％、约98％、约99％或甚至约100％)同源。本发明亦包含这类聚核苷酸及多肽可以与本文或上述聚核苷酸及多肽相同的方式使用。"Homology/homology" as used in the present invention means at least about 70%, preferably at least about 75% homology, advantageously at least about 80% homology, more advantageously at least about 90% homology, even more advantageously are at least about 95% (eg, at least about 97%, about 98%, about 99%, or even about 100%) homologous. The invention also encompasses that such polynucleotides and polypeptides may be used in the same manner as herein or described above.
其它或另外，序列的＂同源性＂可指一致核苷酸或氨基酸残基的位置数除以两个序列中的较短序列的核苷酸或氨基酸残基数，其中两个序列的对准可根据Wilbur and Lipman算法((1983)Proc.Natl.Acad.Sci.USA 80：726)来测定；例如使用20个核苷酸的窗口尺寸、4个核苷酸的字长及4个空隙处罚，使用市售程序可便利地实施序列数据的计算机辅助分析及解释(包括对准)(例如Intelligenetics Suite，Intelligenetics Inc.，CA)。当RNA序列据说类似或与DNA序列在一定程度上具有序列同一性或同源性时，认为DNA序列中的胸腺嘧啶(T)与RNA序列中的尿嘧啶(U)相等。本发明范畴内的RNA序列可藉由用尿嘧啶(U)取代DNA序列中的胸腺嘧啶(T)而衍生自DNA序列或其互补序列。Alternatively or additionally, "homology" of sequences may refer to the number of positions of identical nucleotides or amino acid residues divided by the number of nucleotides or amino acid residues of the shorter of the two sequences, where a pair of Accuracy can be determined according to the Wilbur and Lipman algorithm ((1983) Proc. Natl. Acad. Sci. USA 80:726); for example using a window size of 20 nucleotides, a word length of 4 nucleotides, and 4 gaps For punishment, computer-aided analysis and interpretation (including alignment) of sequence data can be conveniently performed using commercially available programs (eg, Intelligenetics Suite, Intelligenetics Inc., CA). When the RNA sequence is said to be similar to or have some degree of sequence identity or homology to the DNA sequence, thymine (T) in the DNA sequence is considered equivalent to uracil (U) in the RNA sequence. An RNA sequence within the scope of the present invention may be derived from a DNA sequence or its complement by substituting uracil (U) for thymine (T) in the DNA sequence.
另外或其它，可(例如)使用BlastP程序(Altschul等人，Nucl.Acids Res.25：3389-3402，可在NCBI获得)来测定氨基酸序列的类似性或同源性。下列文献提供用于比较两种多肽的氨基酸残基的相对同一性或同源性的算法，且另外或其它，关于前述内容，这类文献中的教导可用于测定同源性百分比：Smith等人，(1981)Adv.Appl.Math.2：482-489；Smith等人，(1983)Nucl.Acids Res.11：2205-2220；Devereux等人，(1984)Nucl.Acids Res.12：387-395；Feng等人，(1987)J.Molec.Evol.25：351-360；Higgins等人，(1989)CABIOS5：151-153；及Thompson等人，(1994)Nucl.Acids Res.22：4673-4680。Alternatively or alternatively, amino acid sequence similarity or homology can be determined, for example, using the BlastP program (Altschul et al., Nucl. Acids Res. 25:3389-3402, available at NCBI). The following documents provide algorithms for comparing the relative identity or homology of amino acid residues of two polypeptides, and additionally or alternatively, with respect to the foregoing, the teachings in such documents can be used to determine percent homology: Smith et al. , (1981) Adv.Appl.Math.2:482-489; Smith et al., (1983) Nucl.Acids Res.11:2205-2220; Devereux et al., (1984) Nucl.Acids Res.12:387- 395; Feng et al., (1987) J. Molec. Evol. 25:351-360; Higgins et al., (1989) CABIOS 5:151-153; and Thompson et al., (1994) Nucl. Acids Res. 22:4673 -4680.
＂具有至少X％同源性＂-关于两个氨基酸或核苷酸序列，是指当两个序列最适对准时，两个序列中相同残基的百分比。因此，90％氨基酸序列一致意谓二或多个最适对准的多肽序列中90％氨基酸相同。"Having at least X% homology"-with reference to two amino acid or nucleotide sequences, refers to the percentage of residues in the two sequences that are identical when the two sequences are optimally aligned. Thus, 90% amino acid sequence identity means that two or more optimally aligned polypeptide sequences are 90% identical in amino acids.
本发明的另一实施例涉及一种包含治疗有效量的RTP801抑制剂作为活性成份及可药用载体的药物组合物。抑制剂可为生物抑制剂、有机分子、化学分子等。该药物组合物可包含一种RTP801抑制剂为一个聚核苷酸，其包含的连续核苷酸具有一个序列为图1所陈述序列(SEQID NO：1)的反义序列。此外，RTP801抑制剂可为一种包含这类聚核苷酸的载体。此外，RTP801抑制剂可为一种单克隆抗体，其特异性结合于一个包含图2所陈述的4-25个氨基酸(SEQ ID No：2)的抗原决定簇，或为一种RNA分子，其靶向RTP801基因mRNA，诸如siRNA分子(任选描述于表A-D中，特别是表A的siRNA Nos：22、23、25、27、39、41、42、49及50或表D的siRNA Nos：257、260-262及264-268)或一种核糖核酸酶。Another embodiment of the present invention relates to a pharmaceutical composition comprising a therapeutically effective amount of an RTP801 inhibitor as an active ingredient and a pharmaceutically acceptable carrier. Inhibitors can be biological inhibitors, organic molecules, chemical molecules, and the like. The pharmaceutical composition may comprise an RTP801 inhibitor as a polynucleotide, and the contiguous nucleotides contained therein have an antisense sequence of the sequence set forth in Figure 1 (SEQ ID NO: 1). Additionally, the RTP801 inhibitor can be a vector comprising such polynucleotides. In addition, the RTP801 inhibitor can be a monoclonal antibody that specifically binds to an antigenic determinant comprising 4-25 amino acids (SEQ ID No: 2) set forth in Figure 2, or an RNA molecule that Target RTP801 gene mRNA, such as siRNA molecules (optionally described in Tables A-D, particularly siRNA Nos of Table A: 22, 23, 25, 27, 39, 41, 42, 49 and 50 or siRNA Nos of Table D: 257, 260-262 and 264-268) or a ribonuclease.
药物组合物的活性成份可包括具有实施本发明所需的核酸酶抗性的寡核苷酸或其显示具有相同靶向适当序列和/或核糖核酸酶作用的片段。可使用如本发明所揭示的活性成份的组合物，包括反义序列的组合物。The active ingredient of the pharmaceutical composition may comprise oligonucleotides or fragments thereof exhibiting the same targeting appropriate sequence and/or ribonuclease action with the nuclease resistance required to practice the invention. Combinations of active ingredients as disclosed herein, including combinations of antisense sequences, may be used.
本发明的另一实施例提供治疗有效量的RTP801抑制剂用于制备一种用于促进罹患脊髓疾病或损伤的患者恢复的药剂的用途。在一实施方式中，抑制剂优选为siRNA。在另一实施方式中，抑制剂优选为本文所述的结构A。Another embodiment of the present invention provides the use of a therapeutically effective amount of an RTP801 inhibitor for the preparation of a medicament for promoting recovery of a patient suffering from a spinal cord disease or injury. In one embodiment, the inhibitor is preferably siRNA. In another embodiment, the inhibitor is preferably structure A as described herein.
本发明已以例示性方式来描述，且应了解意欲所使用的术语具有描述词语的性质而非限制性。The present invention has been described in an exemplary manner, and it is understood that the terminology which has been used is intended to be words of description rather than limitation.
根据上文教导，显然可能存在本发明的多种修改及变化。因此，应了解在随附申请专利范围的范畴内，本发明的实施可与特定描述不同。Obviously many modifications and variations of the present invention are possible in light of the above teachings. It is therefore to be understood that within the scope of the appended claims the invention may be practiced otherwise than as specifically described.
在整个本申请案中，各种公开案(包括美国专利)均由作者引用且年份及专利藉由数字来引用。这类公开案及专利及专利申请案的揭示内容均以全文引用的方式并入本申请案中，以更全面描述本发明所属的当前技术。Throughout this application, various publications, including US patents, are cited by the authors and the year and patent are cited by number. The disclosures of such publications and patents and patent applications in their entireties are incorporated by reference into this application to more fully describe the state of the art to which this invention pertains.
附图简述Brief description of the drawings
图1详述RTP801基因的编码序列(SEQ ID NO：1)；Fig. 1 details the coding sequence (SEQ ID NO: 1) of RTP801 gene;
图2详述RTP801多肽的氨基酸序列(SEQ ID NO：2)；Figure 2 details the amino acid sequence (SEQ ID NO: 2) of the RTP801 polypeptide;
图3为描述具有人类特异性或人类、小鼠及大鼠平行特异性的各种核酸分子的外显子、CDS、人类SNP及位置的图；Figure 3 is a diagram depicting exons, CDS, human SNPs and positions of various nucleic acid molecules with human specificity or parallel specificity for human, mouse and rat;
图4A-H描述对第一人类细胞株应用本发明的多种双链核酸所获得的西方墨点分析结果的面板，其中实验实施两次，称为实验1及实验2，且其中p110a及p85的表达水平表示负载对照组且RTP801带的强度(密度)是测量所用特定双链核酸的抑制活性的方法；4A-H depict panels of Western blot analysis results obtained using various double-stranded nucleic acids of the invention on a first human cell line, where the experiment was performed twice, referred to as
图5A-F描述对第二人类细胞株应用本发明的多种双链核酸所获得的西方墨点分析结果的面板，其中实验实施两次，称为实验1及实验2，且其中p110a及p85的表达水平表示负载对照组且RTP801带的密度是测量所用特定双链核酸的抑制活性的方法；5A-F depict panels of Western blot analysis results obtained using various double-stranded nucleic acids of the invention on a second human cell line, where the experiment was performed twice, referred to as
图6A-C描述对不同浓度(亦即10nM(5A)、5nM(5B)及1nM(5C))的第一人类细胞株应用本发明的多种双链核酸所获得的西方墨点分析结果的面板，其中实验实施两次，称为实验1及实验2，且其中p110a及p85的表达水平表示负载对照组且RTP801带的密度是测量所用特定双链核酸的抑制活性的方法；Figure 6A-C depicts the Western blot analysis results obtained by applying various double-stranded nucleic acids of the present invention to different concentrations (i.e., 10 nM (5A), 5 nM (5B) and 1 nM (5C)) of the first human cell line A panel in which the experiments were performed twice, called
图7描述对小鼠细胞株应用本发明的多种双链核酸所获得的西方墨点分析结果的面板，其中实验实施两次，称为实验1及实验2，且其中p110a及p85的表达水平表示负载对照组且RTP801带的密度是测量所用特定双链核酸的抑制活性的方法；Figure 7 depicts a panel of Western blot analysis results obtained using various double-stranded nucleic acids of the present invention on mouse cell lines, wherein the experiment was performed twice, referred to as
图8显示在小鼠AMD模型系统中的实验结果；Figure 8 shows the experimental results in the mouse AMD model system;
图9显示在小鼠AMD模型系统中的其它实验结果；Figure 9 shows other experimental results in the mouse AMD model system;
图10显示在非人类灵长类动物AMD模型系统中的实验结果；Figure 10 shows the experimental results in the non-human primate AMD model system;
图11A-B显示在非人类灵长类动物AMD模型系统中的其它实验结果；Figures 11A-B show other experimental results in a non-human primate AMD model system;
图12A-B显示在非人类灵长类动物AMD模型系统中的其它实验结果；Figures 12A-B show other experimental results in a non-human primate AMD model system;
图13A-B表示在非人类灵长类动物AMD模型中所获得的实验结果的分析；13A-B represent an analysis of experimental results obtained in a non-human primate AMD model;
图14表示在非人类灵长类动物AMD模型中所获得的实验结果的另一分析；Figure 14 represents another analysis of the experimental results obtained in the non-human primate AMD model;
图15A-C显示包括气管内滴注表达质粒的RTP801至小鼠中的实验的结果；Figures 15A-C show the results of experiments involving intratracheal instillation of RTP801 expressing plasmids into mice;
图16A-C显示在RTP801 KO及WT小鼠中的短期(7天)吸烟模型的结果；Figures 16A-C show the results of a short-term (7 day) smoking model in RTP801 KO and WT mice;
图17A-C显示在用活性抗RTP801(REDD14)及对照(REDD8)siRNA滴注的WT小鼠中的短期吸烟模型的结果；Figures 17A-C show the results of a short-term smoking model in WT mice instilled with active anti-RTP801 (REDD14) and control (REDD8) siRNA;
图18显示长期CS模型中的RTP801 KO小鼠的实验结果；Figure 18 shows the experimental results of RTP801 KO mice in the long-term CS model;
图19显示在小鼠ARF模型系统中的实验结果；Figure 19 shows the experimental results in the mouse ARF model system;
图20显示在小鼠糖尿病性视网膜病模型系统中的实验结果；Figure 20 shows the experimental results in the mouse diabetic retinopathy model system;
图21显示在小鼠糖尿病性视网膜病模型系统中的其它实验结果；Figure 21 shows other experimental results in the mouse diabetic retinopathy model system;
图22显示在小鼠糖尿病性视网膜病模型系统中的其它实验结果；Figure 22 shows other experimental results in the mouse diabetic retinopathy model system;
图23显示在小鼠CNV模型系统中的组合RTP801/VEGF抑制实验的结果；Figure 23 shows the results of combined RTP801/VEGF inhibition experiments in the mouse CNV model system;
图24显示在小鼠CNV模型系统中的其它组合RTP801/VEGF抑制实验的结果；Figure 24 shows the results of other combined RTP801/VEGF inhibition experiments in the mouse CNV model system;
图25显示研究RTP801 siRNA对RPE及神经视网膜中的基因表达的影响的实验的结果；Figure 25 shows the result of the experiment of researching the effect of RTP801 siRNA on the gene expression in RPE and neural retina;
图26A-B显示研究RTP801 siRNA对RPE及神经视网膜中的基因表达的影响的实验的其它结果；及Figures 26A-B show other results of experiments investigating the effect of RTP801 siRNA on gene expression in the RPE and neural retina; and
图27显示证实RT801NP与RTP801具有同样活性的实验结果；Figure 27 shows the experimental results confirming that RT801NP has the same activity as RTP801;
图28显示与801_1(SEQ ID No 430及527)及801_4(SEQ ID No 433及530)相关的功效结果；Figure 28 shows the efficacy results associated with 801_1 (SEQ ID No 430 and 527) and 801_4 (SEQ ID No 433 and 530);
图29显示与801_1(SEQ ID No 430及527)及801_4(SEQ ID No 433及530)相关的功效结果；Figure 29 shows the efficacy results associated with 801_1 (SEQ ID No 430 and 527) and 801_4 (SEQ ID No 433 and 530);
图30显示与801_1(SEQ ID No 430及527)及801_4(SEQ ID No 433及530)相关的其它功效结果。Figure 30 shows other efficacy results associated with 801_1 (SEQ ID No 430 and 527) and 801_4 (SEQ ID No 433 and 530).
具体实施方式Detailed ways
无进一步详细说明，相信本领域技术人员使用前述描述可最大程度利用本发明。因此，下列优选的特定实施例可解释为仅具例示性且不以任何方式限制本发明。Without further elaboration, it is believed that one skilled in the art can, using the preceding description, utilize the present invention to its fullest extent. Accordingly, the following preferred specific examples are to be construed as merely illustrative and not restrictive of the invention in any way.
本文未特定描述的本领域已知的标准分子生物学方案一般基本上根据下列文献：Sambrook等人，Molecular cloning：A laboratorymanual，Cold Springs Harbor Laboratory，New-York(1989，1992)；及Ausubel等人，Current Protocols in Molecular Biology，JohnWiley and Sons，Baltimore，Maryland(1988)。Standard molecular biology protocols known in the art not specifically described herein are generally based essentially on the following documents: Sambrook et al., Molecular cloning: A laboratory manual, Cold Springs Harbor Laboratory, New-York (1989, 1992); and Ausubel et al. , Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, Maryland (1988).
本文未特定描述的本领域已知的标准有机合成方案一般基本上根据下列文献：Organic syntheses：1-79卷，编辑不同，J.Wiley，NewYork，(1941-2003)；Gewert等人，Organic synthesis workbook，Wiley-VCH，Weinheim(2000)；Smith及March，Advanced OrganicChemistry，Wiley-Interscience；第5版(2001)。Standard organic synthesis schemes known in the art not specifically described herein are generally based essentially on the following documents: Organic syntheses: Volumes 1-79, edited differently, J. Wiley, New York, (1941-2003); Gewert et al., Organic synthesis workbook, Wiley-VCH, Weinheim (2000); Smith and March, Advanced Organic Chemistry, Wiley-Interscience; 5th edition (2001).
本文未特定描述的本领域已知的标准医药化学方法一般基本上根据多名作者及编辑的由Pergamon Press公开的＂ComprehensiveMedicinal Chemistry＂系列。Standard medicinal chemistry methods known in the art not specifically described herein are generally based essentially on the "Comprehensive Medicinal Chemistry" series published by Pergamon Press by various authors and editors.
揭示于说明书、申请专利范围和/或图式中的本发明特征可单独及以其任何组合用于了解本发明的各种形式。The features of the invention disclosed in the specification, claims and/or drawings can be used alone and in any combination to understand the various forms of the invention.
实施例1Example 1
通用物质及方法General Materials and Methods
若未相反说明，则下列物质及方法用于实施例1-5中：If not stated otherwise, the following materials and methods were used in Examples 1-5:
细胞培养：Cell culture:
如下培养第一人类细胞株，亦即海拉细胞(HeLa cell)(AmericanType Culture Collection)：海拉细胞(American Type CultureCollection)如Czauderna F等人(Czauderna，F.，Fechtner，M，Aygun，H.，Arnold，W.，Klippel，A.，Giese，K.& Kaufmann，J.(2003).Nucleic Acids Res，31，670-82)中所述来培养。The first human cell line, namely HeLa cells (American Type Culture Collection), was cultured as follows: HeLa cells (American Type Culture Collection) as described by Czauderna F. et al. (Czauderna, F., Fechtner, M, Aygun, H. , Arnold, W., Klippel, A., Giese, K. & Kaufmann, J. (2003). Nucleic Acids Res, 31, 670-82).
第二人类细胞株为如下培育的人类角质细胞细胞株：The second human cell line is a human keratinocyte cell line grown as follows:
在含有10％FCS的杜尔贝科改性伊格尔培养基(Dulbecco′smodified Eagle medium，DMEM)中，于37℃下培养人类角质细胞。Human keratinocytes were cultured at 37°C in Dulbecco's modified Eagle medium (DMEM) containing 10% FCS.
小鼠细胞株为在含有10％FCS的杜尔贝科改性伊格尔培养基(DMEM)中于37℃下培养的B16V(American Type Culture Collection)。培养条件描述于Methods Find Exp Clin Pharmacol.1997年5月；19(4)：231-9中。The mouse cell line was B16V (American Type Culture Collection) cultured at 37°C in Dulbecco's Modified Eagle's Medium (DMEM) containing 10% FCS. Culture conditions are described in Methods Find Exp Clin Pharmacol. 1997 May;19(4):231-9.
在各情形下，在每孔约50,000个细胞的密度下使细胞经受如本文所述的实验，且本发明的双链核酸以20nM来添加，其中使用1μg/ml的专有脂质来复合双链核酸。In each case, the cells were subjected to the experiment as described herein at a density of about 50,000 cells per well, and the double-stranded nucleic acid of the invention was added at 20 nM, wherein 1 μg/ml of a proprietary lipid was used to complex the double-stranded nucleic acid. strand nucleic acid.
类缺氧条件的诱发induction of hypoxia-like conditions
如下将细胞用CoCl2处理以诱发类缺氧条件：如(Czauderna等人，2003；Kretschmer等人，2003)所述，在10cm培养盘(30-50％融合)中实施siRNA转染。简言之，藉由将GB及脂质于无血清培养基中的预形成10x浓复合物添加至完全培养基中的细胞中来转染siRNA。总转染体积为10ml。最终脂质浓度为1.0μg/ml；除非另外说明，否则最终siRNA浓度为20nM。低氧反应的诱发藉由在溶解前24h直接添加CoCl2(100μM)至组织培养基中来实施。Cells were treated withCoCl2 to induce hypoxia-like conditions as follows: siRNA transfections were performed in 10 cm culture dishes (30-50% confluency) as described (Czauderna et al., 2003; Kretschmer et al., 2003). Briefly, siRNA was transfected by adding preformed 10x concentrated complexes of GB and lipids in serum-free medium to cells in complete medium. The total transfection volume was 10 ml. Final lipid concentration was 1.0 μg/ml; final siRNA concentration was 2OnM unless otherwise stated. Induction of the hypoxic response was performed by addingCoCl2 (100 μM) directly to the tissue culture medium 24 h before lysis.
细胞萃取物及免疫印迹的制备Preparation of cell extracts and immunoblotting
细胞萃取物的制备及免疫墨点分析基本上均如Klippel等人(Klippel，A.，Escobedo，M.A.，Wachowicz，M.S.，Apell，G.，Brown，T.W.，Giedlin，M.A.，Kavanaugh，W.M.& Williams，L.T.(1998).Mol Cell Biol，18，5699-711；Klippel，A.，Reinhard，C，Kavanaugh，W.M.，Apell，G.，Escobedo，M.A.& Williams，L.T.(1996).Mol Cell Biol，16，4117-27)所述来实施。对抗全长RTP801的多克隆抗体藉由用自pET19-b表达载体产生细菌的重组RTP801蛋白免疫兔而产生(Merck Biosciences GmbH，Schwalbach，Germany)。鼠科动物单株抗p110a及抗p85抗体已由Klippel等人(前述)描述。The preparation of cell extracts and immunoblot analysis were basically as described by Klippel et al. L.T.(1998).Mol Cell Biol, 18, 5699-711; Klippel, A., Reinhard, C, Kavanaugh, W.M., Apell, G., Escobedo, M.A. & Williams, L.T.(1996).Mol Cell Biol, 16, 4117-27) to implement. Polyclonal antibodies against full-length RTP801 were generated by immunizing rabbits with recombinant RTP801 protein from pET19-b expression vector-producing bacteria (Merck Biosciences GmbH, Schwalbach, Germany). Murine monoclonal anti-p110a and anti-p85 antibodies have been described by Klippel et al. (supra).
实施例2Example 2
第一人类细胞株中RTP801表达的减少Reduction of RTP801 expression in a first human cell line
制备多种双链核酸。其相对于mRNA及CDS以及人类SNP在编码人类RTP801的核酸中的位置(数据库寄存编号NM_019058)描述于图3中。将第一人类细胞株与如实施例1中所述的该双链核酸接触。在类低氧条件诱发且用该双链核酸处理后，细胞溶解且细胞溶解产物经受免疫印迹。将PI3激酶的催化单元p110a以及p85用作负载对照组。如使用RTP801多克隆抗体可视化的RTP801带强度是根据RTP801表达水平的减少来测量个别双链核酸活性的方法。A variety of double-stranded nucleic acids are prepared. Its position relative to mRNA and CDS and human SNPs in the nucleic acid encoding human RTP801 (database accession number NM_019058) is depicted in FIG. 3 . A first human cell line is contacted with the double-stranded nucleic acid as described in Example 1. After induction of hypoxic-like conditions and treatment with the double-stranded nucleic acid, cells are lysed and cell lysates are subjected to immunoblotting. The catalytic unit p110a of PI3 kinase as well as p85 were used as loading controls. The intensity of the RTP801 band as visualized using the RTP801 polyclonal antibody is a measure of the activity of individual double-stranded nucleic acids in terms of the reduction in RTP801 expression levels.
每一及任一双链核酸均已经修饰，使得2′O-Me基团存在于反义链的第一、第三、第五、第七、第九、第十一、第十三、第十五、第十七及第十九核苷酸上，其中存在极类似修饰，亦即2′O-Me基团存在于有义链的第二、第四、第六、第八、第十、第十二、第十四、第十六及第十八核苷酸上。此外，应注意在本发明的这类特定核酸情形下，第一链段与第一链一致且第二链段与第二链一致且这类核酸亦经末端平整化。Each and any double-stranded nucleic acid has been modified such that a 2'O-Me group is present on the first, third, fifth, seventh, ninth, eleventh, thirteenth,
将实验实施两次且个别结果显示于图4A至H中，其中该实验分别命名为实验1及实验2。The experiment was performed twice and the individual results are shown in Figures 4A to H, where the experiments are named
图4A至H中的表示h、hr及hmr表明设计各自双链核酸以(例如)处理具有人类RTP801 mRNA(h)特异性的RTP801 mRNA区，处理具有人类及大鼠RTP801 mRNA(hr)特异性的RTP801 mRNA区，且处理具有人类、小鼠及大鼠RTP801 mRNA(hmr)特异性的RTP801 mRNA区。将称为no.40.1的双链核酸用作正性对照且将未经处理的细胞(UT+)用作负性对照。The representations h, hr and hmr in Figure 4A to H indicate that the respective double stranded nucleic acids were designed to, for example, process the RTP801 mRNA region specific for human RTP801 mRNA (h), and the processing has human and rat RTP801 mRNA (hr) specificity RTP801 mRNA region, and processing RTP801 mRNA region specific for human, mouse and rat RTP801 mRNA (hmr). A double-stranded nucleic acid called no.40.1 was used as a positive control and untreated cells (UT+) were used as a negative control.
根据结果，证实下列双链核酸尤其适用于下调RTP801的表达：no.14、no.15、no.20、no.21、no.22、no.23、no.24、no.25、no.27、no.39、no.40、no.41、no.42、no.43、no.44、no.49及no.50(参见表A)。According to the results, it is confirmed that the following double-stranded nucleic acids are especially suitable for down-regulating the expression of RTP801: no.14, no.15, no.20, no.21, no.22, no.23, no.24, no.25, no. 27, no.39, no.40, no.41, no.42, no.43, no.44, no.49 and no.50 (see Table A).
实施例3Example 3
第二人类细胞株中RTP801表达的减少Reduction of RTP801 expression in a second human cell line
使用如实施例1中所指定的第二人类细胞株重复如实施例2所述的实验，且结果描述于图5A至F中。The experiment as described in Example 2 was repeated using a second human cell line as specified in Example 1 and the results are depicted in Figures 5A-F.
如可自这类图中推断，使用此第二人类细胞株证实如实施例2中所述的实验所得的结果。As can be inferred from such figures, this second human cell line was used to confirm the results obtained from the experiments described in Example 2.
实施例4Example 4
RTP801特异性双链核酸的剂量作用Dose effect of RTP801-specific double-stranded nucleic acids
在此实验中，研究RTP801特异性双链核酸的剂量作用。In this experiment, the dose effect of RTP801-specific double-stranded nucleic acids was investigated.
为获得此目的，使海拉细胞如实施例2及3经处理，其中培养肉汤中双链核酸的浓度为10nM、5nM及1nM。将no.40.1的双链核酸用作正性对照，将未经处理的细胞(UT+)用作负性对照。读出与实施例2及3中所述相同。所用特定双链核酸为具有内部参考号14、22、23及27的双链核酸(针对人类、小鼠及大鼠共享的RTP801 mRNA上的链段)及具有内部参考号39及42的双链核酸(针对具有人类RTP801特异性的RTP801mRNA的链段)。For this purpose, HeLa cells were treated as in Examples 2 and 3, wherein the concentration of double-stranded nucleic acid in the culture broth was 10 nM, 5 nM and 1 nM. The double-stranded nucleic acid of no.40.1 was used as a positive control, and untreated cells (UT+) were used as a negative control. Readout was the same as described in Examples 2 and 3. The specific double-stranded nucleic acids used were double-stranded nucleic acids with
结果显示于图6A至C中。根据该图，可得出对RTP801具有特异性的双链核苷酸的作用明显视浓度而定，其中具有内部参考号1、15、20、21、24、40、41、43、44、22、23、27、39、42、40.1、44.1及14、优选22、23、27、39、42、40.1及44.1且更优选14、23及27的核酸分子且优选具有如在本文实验部分所述的特定修饰类型的该核酸分子的每一个尤其有效。The results are shown in Figures 6A-C. From this figure, it can be concluded that the effect of double-stranded nucleotides specific for RTP801 is clearly dependent on concentration, with
实施例5Example 5
RTP801特异性双链核酸的物种特异性Species specificity of RTP801-specific double-stranded nucleic acids
已设计对抗各种物种中的相同或不同RTP801 mRNA链段的本发明的双链核酸。为测试RTP801特异性双链核酸的物种特异性是否存在，根据使用如实施例1及2中所指定的相同方法及读出下调RTP801来比较具有内部参考号14、22、23及27的处理人类、小鼠及大鼠RTP801 mRNA中的保守RTP801 mRNA链段的双链核酸与具有内部参考号39及42的处理人类RTP801 mRNA的特异性RTP801 mRNA链段(亦即处理不存在于小鼠或大鼠中的链段)的双链核酸。Double-stranded nucleic acids of the invention have been engineered against the same or different RTP801 mRNA segments in various species. In order to test whether the species specificity of RTP801-specific double-stranded nucleic acids exists, according to using the same method as specified in Examples 1 and 2 and reading down-regulated RTP801 to compare the treatment humans with
虽然所用的所有双链核酸原则上活性对抗人类mRNA，且如前述实施例中所示，其亦适于下调RTP801的表达，但在使用小鼠细胞株后，仅亦特异性针对小鼠RTP801 mRNA的那些双链核酸(亦即双链核酸no.14、22、23及27)有效减少RTP801表达。Although all double-stranded nucleic acids used are in principle active against human mRNA and, as shown in the previous examples, are also suitable for downregulating the expression of RTP801, only mouse RTP801 mRNA is also specific after using mouse cell lines Those double-stranded nucleic acids (ie, double-stranded nucleic acid no. 14, 22, 23 and 27) effectively reduced RTP801 expression.
根据此结果，可得出可能设计处理特异性针对一或多种物种的双链核酸的RTP801。此允许使用在动物模型及人中极类似的分子。From this result, it was possible to design RTP801 to process double-stranded nucleic acids specific for one or more species. This allows the use of molecules that are very similar in animal models as well as in humans.
实施例6Example 6
与黄斑变性相关的实验模型、方法及结果Experimental models, methods and results related to macular degeneration
在以下脉络膜新血管生成(CNV)的动物模型中测试本发明的化合物。湿式AMD的此特征藉由激光处理在模型动物中诱发。The compounds of the invention were tested in the following animal model of choroidal neovascularization (CNV). This feature of wet AMD was induced in model animals by laser treatment.
A)小鼠模型A) mouse model
脉络膜新血管生成(CNV)诱发Choroidal neovascularization (CNV) induced
脉络膜新血管生成(CNV)为湿式AMD的特征，其藉由在第0天藉由并不知晓药物组分配的单独个体在各小鼠的双眼上实施激光凝固(532nm，200mW，100ms，75μm)(OcuLight GL，Iridex，Mountain View，CA)来引发。在视神经周围以标准化方式施加激光光斑，其中使用裂缝灯传递系统及盖玻片作为隐形眼镜。Choroidal neovascularization (CNV), a characteristic of wet AMD, was determined by laser photocoagulation (532nm, 200mW, 100ms, 75μm) on both eyes of each mouse on
治疗组therapy group
在下列各组小鼠(6-8周大的雄性小鼠)中诱发CNV：CNV was induced in the following groups of mice (6-8 week old male mice):
(1)12只WT小鼠；(1) 12 WT mice;
(2)12只RTP801基因剔除小鼠；(2) 12 RTP801 knockout mice;
(3)在0天及7天，在一只眼睛中用0.25μg合成稳定活性抗RTP801 siRNA(REDD14)注射及在另一只眼睛中用失活抗RTP801siRNA(REDD8-负性对照)注射的12只WT小鼠；(3) On
(4)在0天及7天，在一只眼睛中用0.25μg合成稳定活性抗RTP801 siRNA(REDD14)注射及在另一只眼睛中用失活抗GFP siRNA(负性对照)注射的12只WT小鼠；(4) On
(5)在0天及7天，在一只眼睛中用0.1μg合成稳定活性抗RTP801 siRNA(REDD14)注射及在另一只眼睛中用PBS(负性对照)注射的12只WT小鼠；(5) 12 WT mice injected with 0.1 μg of synthetic stable active anti-RTP801 siRNA (REDD14) in one eye and PBS (negative control) in the other eye at
(6)在0天及7天，在一只眼睛中用0.05μg合成稳定活性抗RTP801 siRNA(REDD14)注射及在另一只眼睛中用PBS(负性对照)注射的12只WT小鼠。(6) Twelve WT mice injected with 0.05 μg of synthetic stable active anti-RTP801 siRNA (REDD14) in one eye and PBS (negative control) in the other eye on
各小鼠的双睛均经激光处理。所注射的体积为2μl。Both eyes of each mouse were laser treated. The volume injected was 2 μl.
评估Evaluate
1.在第14天终止实验。为进行评估，将眼睛摘出且用4％聚甲醛在4℃下固定30min。将感觉神经视网膜分离且自视神经切断。将残余RPE-脉络膜-巩膜复合物平坦封固于Immu-Mount(Vectashield封固培养基、载体)中且盖片。将平坦封固件用扫描激光共聚焦显微镜(TCSSP，Leica，Germany)下检查。藉由用蓝色氩激光激发来可视化脉管。自RPE-脉络膜-巩膜复合物的表面获得水平眼区(1μm级距)。判断出其中连接损害的周围脉络膜血管网可经鉴别的最深焦平面为损害层。在经激光处理的区域中且在此参考平面表面的任何血管经判断为CNV。各区的影像经数字储存。CNV相关的荧光区藉由计算机化影像分析使用Leica TCS SP软件来测量。整个荧光区在各水平区中的总数用作CNV体积的指数。1. Terminate the experiment on
2.对自RPE/脉络膜或自神经视网膜萃取的RNA使用实时PCR，将单独WT小鼠(每组5只眼睛)用于评估CNV中的RTP801 mRNA表达(以及相关于AMD的其它基因的表达)(未经治疗及经siRNA治疗)。2. Individual WT mice (5 eyes per group) were used to assess RTP801 mRNA expression (and expression of other genes associated with AMD) in CNV using real-time PCR on RNA extracted from RPE/choroid or from neural retina (untreated and siRNA-treated).
结果result
1.在CNV诱发后，RTP801 KO小鼠显示与WT小鼠相比30％减少的血管渗漏。1. After CNV induction, RTP801 KO mice showed 30% reduced vascular leakage compared with WT mice.
2.对抗RTP801的合成稳定siRNA(REDD14)引起CNV体积的剂量依赖性减少。在每只眼睛0.25μg剂量的REDD14(表A中序列号14，SEQ IDNo.16(有义)及66(反义))下，获得与注射PBS的眼睛相比约70％抑制的最大值。在相同剂量下，两个负性对照siRNA(REDD8及抗GFP siRNA)分别显示仅27％及33％的CNV体积减少，此支持REDD14的较高功效以及其作用的特异性。2. A synthetic stable siRNA against RTP801 (REDD14) caused a dose-dependent reduction in CNV volume. At a dose of 0.25 μg per eye of REDD14 (SEQ ID No. 14, SEQ ID No. 16 (sense) and 66 (antisense) in Table A), a maximum of about 70% inhibition was obtained compared to PBS-injected eyes. At the same dose, two negative control siRNAs (REDD8 and anti-GFP siRNA) showed only 27% and 33% reduction in CNV volume, respectively, which supports the higher potency of REDD14 and the specificity of its action.
B)非人类灵长类动物模型B) Non-human primate models
CNV诱发CNV induced
将八只2-6岁大的雄性石蟹猕猴(食蟹猴)用于研究。脉络膜新血管生成(CNV)藉由在剂量给药前双眼的黄斑周围激光处理来诱发。用激光(具有IRIS 
治疗treat
在激光治疗后，使所有动物的双眼立即经受单一玻璃体内注射。给予左眼最终体积为50μl的350μg抗RTP801的合成稳定siRNA(与用于小鼠研究中者相同)，而对侧眼接收50μl PBS(媒剂)。Immediately after laser treatment, both eyes of all animals underwent a single intravitreal injection. The left eye was given 350 μg of synthetic stable siRNA against RTP801 (the same as used in the mouse study) in a final volume of 50 μl, while the fellow eye received 50 μl of PBS (vehicle).
评估Evaluate
1.使所有动物经受食物消耗及体重测量的每日检查。1. Subject all animals to daily checks for food consumption and body weight measurements.
2.CNV诱发后，在第6天对2只猴实施安乐死。摘出其眼且使后极变平。接着切割凹区且将其分成脉络膜及神经视网膜，将脉络膜及神经视网膜(每只动物)单独冷冻于液氮中以随后用于RNA萃取及RTP801表达的实时PCR评估。2. After CNV induction, 2 monkeys were euthanized on
3.在研究前及在CNV诱发后1、2及3周结束时实施荧光素血管造影。使用眼底摄影机(TRC-50EX视网膜摄影机)拍摄照片。使用TOPCONIMAGEnetTM系统捕获影像。经由血管进口注射荧光素染料(10％荧光素钠，约0.1mL/kg)。在染料注射后若干时间点拍摄照片以包括动脉阶段、早期动静脉阶段及若干晚期动静脉阶段，以评估新血管生成且监控与CNV损害相关的荧光素渗漏。荧光素血管造影的解释及分析独立由两名眼科医师进行。3. Fluorescein angiography was performed before the study and at the end of 1, 2 and 3 weeks after CNV induction. Photographs were taken with a fundus camera (TRC-50EX retinal camera). Images were captured using the TOPCONIMAGEnet™ system. Fluorescein dye (10% sodium fluorescein, approximately 0.1 mL/kg) was injected via the vascular inlet. Pictures were taken at several time points after dye injection to include the arterial phase, early arteriovenous phase, and several late arteriovenous phases to assess neovascularization and monitor fluorescein leakage associated with CNV lesions. Interpretation and analysis of fluorescein angiography were performed independently by two ophthalmologists.
在早期血管造影中分析新血管生成(NV)且根据下列方案对每一光斑分级：Neovascularization (NV) was analyzed on early angiography and each spot was graded according to the following scheme:
0 -无NV标记0 - no NV flag
0.5 -可疑光斑0.5 - Suspicious facula
1 -＂热＂光斑1 - "Hot" spot
2 -激光灼伤中的NV2 - NV in laser burns
3 -明显NV3 - Obvious NV
根据下列方案分析渗漏：Leakage is analyzed according to the following scheme:
0 -无渗漏0 - no leakage
0.5 -可疑光斑0.5 - Suspicious facula
1 -明显小光斑渗漏1 - Obvious small spot leakage
2 -随时间生长的渗漏2 - Leakage over time
3 -比先前边界大(明显)的渗漏3 - Larger (significant) leak than previous boundary
此外，使用形态测量法在早期血管造影与晚期血管造影之间比较每一光斑尺寸，且计算源自渗漏的光斑尺寸增加。In addition, each spot size was compared between early and late angiograms using morphometry, and the increase in spot size resulting from leakage was calculated.
4.在研究前及在3周结束时，根据Sierra′s SOP及研究特异性SOP，使用Epic 2000视网膜电流描记器来记录视网膜电流图(ERG)，包括使用Ganzfield设备。藉由兽医眼科医师来评估制成表的ERG数据。4. Electroretinograms (ERGs) were recorded using an Epic 2000 electroretinograph, including using a Ganzfield device, according to Sierra's SOPs and study-specific SOPs prior to the study and at the end of 3 weeks. Tabulated ERG data were evaluated by a veterinary ophthalmologist.
在CNV诱发后21天终止研究。在包括眼睛的器官及组织上实施总体尸体解剖及组织检查。The study was terminated 21 days after CNV induction. A gross autopsy and histological examination are performed on organs and tissues including the eyes.
结果result
1.对抗RTP801的siRNA减少经激光治疗的动物的RPE/脉络膜中RTP801表达，如藉由实时PCR在CNV诱发后第6天所测量(参见图10)。1. siRNA against RTP801 reduces RTP801 expression in the RPE/choroid of laser-treated animals as measured by real-time PCR at
2.在各猴的同侧眼之间比较渗漏及新血管生成的光斑等级，揭示与对照组相比此两个病理特征在用RTP801 siRNA注射的眼中减少(渗漏结果，参见图11；新血管生成结果，参见图12)。2. Compare leakage and the spot level of neovascularization between the ipsilateral eyes of each monkey, and reveal that these two pathological features are reduced in eyes injected with RTP801 siRNA compared with the control group (leakage results, see Figure 11; Neovascularization results, see Figure 12).
3.与所有注射PBS的眼睛相比，在所有注射siRNA的眼睛中对具有渗漏或新血管生成的更高临床相关等级(2及3)的光斑的总数进行计算，再次揭示注射siRNA的眼睛更少受影响(参见图13，a+b)。3. The total number of spots with higher clinically relevant grades (2 and 3) of leakage or neovascularization were calculated in all siRNA-injected eyes compared to all PBS-injected eyes, again revealing siRNA-injected eyes Less affected (see Figure 13, a+b).
4.对光斑渗漏及新血管生成的总体等级数据进行统计学评估。siRNA与对照治疗之间差异的存在是藉由计算对照右(R)眼与注射siRNA的左(L)眼的平均光斑等级之间的δ(δ＝R-L)来分析。使用无参数统计学方法威尔卡逊秩和测试(Wilcoxon signed ranks test，单尾测试)来计算差异的显著性。每周(1、2及3)分别分析不同阶段的血管造影(早期动脉、动脉静脉及晚期静脉)。4. Statistically evaluate the overall grade data of spot leakage and neovascularization. The presence of differences between siRNA and control treatments was analyzed by calculating the delta (delta = R-L) between the mean spot level of the control right (R) eye and the siRNA-injected left (L) eye. The significance of differences was calculated using the nonparametric statistical method Wilcoxon signed ranks test (one-tailed test). Angiograms at different stages (early arterial, arteriovenous, and late venous) were analyzed weekly (1, 2, and 3).
表2显示各组不同于0的渗漏等级的显著性(单尾测试)(小于0.05的p值加下划线)。在晚期血管造影中2周及3周，相对于右眼(经安慰剂治疗)，在左眼(经siRNA治疗)中发现显著渗漏等级减少。Table 2 shows the significance (one-tailed test) of the levels of leakage different from 0 for each group (p-values less than 0.05 are underlined). Significant reductions in leak grade were found in the left eye (siRNA-treated) relative to the right eye (placebo-treated) at late angiography at 2 and 3 weeks.
表2Table 2
注意晚期血管造影常用于评估渗漏参数。Note that late angiography is often used to assess leakage parameters.
表3显示各组中不同于0的新血管生成(NV)等级的显著性(单尾测试)(小于0.05的p值加下划线)。Table 3 shows the significance (one-tailed test) of neovascularization (NV) grades different from 0 in each group (p values less than 0.05 are underlined).
表3table 3
相对于2周及3周的右眼，在2周的早期及动静脉时期于左眼中发现显著NV等级减少。Significant reductions in NV grade were found in the left eye at the early and arteriovenous phases at 2 weeks relative to the right eye at 2 weeks and 3 weeks.
注意早期血管造影常用于评估新血管生成参数。Note that early angiography is often used to assess parameters of neovascularization.
5.对归因于渗漏而出现在早期(动脉阶段)及晚期(静脉阶段)血管造影之间的光斑的面积增加进行量化形态测量评估，揭示与对照组(右眼，OD)相比在注射siRNA的眼(左眼，OS)内激光光斑中此参数显著减少。两个实施例显示于图14中。该图证实动物3315及3300号的左眼及右眼中每一光斑面积的相对增加(以％计)。5. Quantitative morphometric assessment of the area increase of the facula between early (arterial phase) and late (venous phase) angiograms due to leakage reveals that compared with control (right eye, OD) in This parameter was significantly reduced in the laser spot in the siRNA-injected eye (left eye, OS). Two examples are shown in FIG. 14 . The graph demonstrates the relative increase (in %) of each spot area in the left and right eyes of
此外，注意在以上所有研究中，抗RTP801 siRNA对视网膜电流图(ERG)、眼睛组织学或其它器官及系统的结构及功能并无不利影响。In addition, note that in all of the above studies, anti-RTP801 siRNA did not adversely affect the electroretinogram (ERG), ocular histology, or the structure and function of other organs and systems.
概述以上实验及结果：Summarize the above experiments and results:
1.遗传(RTP801-/-)及治疗siRNA抑制RTP801在患有湿式年龄相关的黄斑变性(湿式AMD)的激光诱发的CNV模型中的表达，导致CNV体积显著减少。1. Genetic (RTP801-/-) and therapeutic siRNA inhibit RTP801 expression in a laser-induced CNV model with wet age-related macular degeneration (wet AMD), resulting in a significant reduction in CNV volume.
2.在小鼠及非人类灵长类动物模型中获得正性结果。2. Obtain positive results in mouse and non-human primate models.
3.猴中病理及ERG检查并未揭示眼睛或任何其它器官或系统中任何siRNA介导的毒性。3. Pathological and ERG examinations in monkeys did not reveal any siRNA-mediated toxicity in the eye or any other organ or system.
C)RTP801 siRNA(REDD14)及抗VEGF抗体的组合疗法的功效C) Efficacy of combination therapy of RTP801 siRNA(REDD14) and anti-VEGF antibody
在以上小鼠CNV模型中测试RTP801 siRNA(REDD14)及抗VEGF抗体的组合疗法在治疗CNV发生的疾病中的功效。The combination therapy of RTP801 siRNA(REDD14) and anti-VEGF antibody was tested in the above mouse CNV model for the efficacy in the treatment of CNV-occurring diseases.
A)CNV体积研究A) CNV volume study
藉由如先前所述的共焦荧光显微镜法(Sakurai等人，IOVS 2003；44：3578-85；及Sakurai等人，IOVS 2003；44：2743-2749)来计算激光损伤后3周脉络膜新血管生成(CNV)的体积。Choroidal neovascularization was counted 3 weeks after laser injury by confocal fluorescence microscopy as previously described (Sakurai et al.,
在先前研究中，发现抗VEGF-A抗体(Ab)以剂量依赖方式使CNV体积减少。针对REDD14+VEGF-A Ab组合研究，选择1ng VEGF-A Ab的剂量，因为此剂量具有中间抑制作用：VEGF-A Ab(1ng)会使CNV尺寸减少26±6％。In a previous study, anti-VEGF-A antibody (Ab) was found to reduce CNV volume in a dose-dependent manner. For the REDD14+VEGF-A Ab combination study, a dose of 1 ng VEGF-A Ab was chosen because of its intermediate inhibitory effect: VEGF-A Ab (1 ng) reduced CNV size by 26±6%.
REDD14+VEGF-A抗体(Ab)研究的主要发现为：The main findings of the REDD14+VEGF-A antibody (Ab) study are:
与单独VEGF-A Ab相比，添加低于0.05μg剂量的REDD14会使CNV尺寸减少27±4％。Addition of REDD14 at doses lower than 0.05 μg reduced CNV size by 27±4% compared to VEGF-A Ab alone.
与单独VEGF-A Ab相比，添加高于0.25μg剂量的REDD14会使CNV尺寸减少55±3％。Addition of REDD14 at doses higher than 0.25 μg reduced CNV size by 55±3% compared to VEGF-A Ab alone.
B)CNV渗漏研究B) CNV Leakage Study
实验1
设计此实验以鉴别在患有激光诱发的脉络膜新血管生成的小鼠模型中抑制VEGF及RTP801的潜在添加或协同治疗效应。This experiment was designed to identify potential additive or synergistic therapeutic effects of inhibiting VEGF and RTP801 in a mouse model of laser-induced choroidal neovascularization.
物质：substance:
·REDD14(RTP801 siRNA)· REDD14 (RTP801 siRNA)
·REDD8(负性对照)· REDD8 (negative control)
·抗VEGF抗体Anti-VEGF antibody
·非特异性IgG(负性对照)·Non-specific IgG (negative control)
如上所述在第0天诱发CNV；在第0天及第7天，将测试物质注射至受检者中。CNV was induced on
藉由1、2、3周荧光血管造影及藉由第3周CNV体积测量来评估结果。每一测试组均由10只眼睛构成。Results were assessed by fluorescein angiography at 1, 2, and 3 weeks and by CNV volume measurement at
实验组：test group:
·每只眼睛VEGF Ab 0.5ngVEGF Ab 0.5ng per eye
·每只眼睛VEGF Ab 1ngVEGF Ab 1ng per eye
·每只眼睛VEGF Ab 2ngVEGF Ab 2ng per eye
·每只眼睛VEGF Ab 4ngVEGF Ab 4ng per eye
·每只眼睛REDD14 0.05μg· REDD14 0.05 μg per eye
·每只眼睛REDD14 0.1μg· REDD14 0.1 μg per eye
·每只眼睛REDD14 0.25μg· REDD14 0.25 μg per eye
·每只眼睛REDD14 0.05μg+每只眼睛VEGF Ab 1ng· REDD14 0.05μg per eye + VEGF Ab 1ng per eye
·每只眼睛REDD14 0.1μg+每只眼睛VEGF Ab 1ng· REDD14 0.1μg per eye + VEGF Ab 1ng per eye
·每只眼睛REDD14 0.25μg+每只眼睛VEGF Ab 1ng· REDD14 0.25μg per eye + VEGF Ab 1ng per eye
对照组control group
·PBS·PBS
·每只眼睛非特异性IgG 2ng·Non-specific IgG 2ng per eye
·每只眼睛REDD8 0.1μg· REDD8 0.1 μg per eye
·每只眼睛REDD8 0.1μg+每只眼睛VEGF Ab 1ng· REDD8 0.1μg per eye + VEGF Ab 1ng per eye
结果result
以上实验结果呈现于图23-24中。这类结果显示VEGF Ab及REDD14的同时玻璃体内给药会导致增加性及剂量依赖性脉络膜新血管生成及脉络膜血管渗漏抑制，如等级4光斑的减少发生率及等级1光斑的增加发生率所示。使用对先前公开的半量化等级(1-4)方案的修改(Sakurai等人，IOVS 2003；44：2743-2749)将血管造影分级。将等级1损害视为未形成，亦即等于完全预防。将等级4损害视为病理上显著的，亦即等于在患者中需要治疗的损害。VEGF-A Ab(1ng)会使每只眼睛等级4损害的发生率减少38±8％且使每只眼睛等级1损害的发生率增加66±43％。REDD14+VEGF-A Ab组合渗漏研究的主要发现为：The above experimental results are presented in Figures 23-24. These results show that simultaneous intravitreal administration of VEGF Ab and REDD14 leads to increased and dose-dependent inhibition of choroidal neovascularization and choroidal vascular leakage, as indicated by reduced incidence of
·与单独VEGF-A Ab相比，添加低于0.05μg剂量的REDD14会使等级4损害的发生率减少66±12％。The addition of REDD14 at doses below 0.05 μg reduced the incidence of
·与单独VEGF-A Ab相比，添加高于0.25μg剂量的REDD14会使等级4损害的发生率减少60±12％。The addition of REDD14 at doses above 0.25 μg reduced the incidence of
·与单独VEGF-A Ab相比，添加高于0.25μg剂量的REDD14使等级1损害的发生率加倍(100±34％)。• Addition of REDD14 at doses higher than 0.25 μg doubled the incidence of
实验2
设计此实验以研究REDD14对RPE及神经视网膜中的基因表达的影响。This experiment was designed to investigate the effect of REDD14 on gene expression in the RPE and neural retina.
实验设计experimental design
组：Group:
·PBS·PBS
·REDD14 0.25mg·REDD14 0.25mg
组大小为5只眼睛。在第0天，藉由如上所述的激光处理来诱发CNV；在第0天亦注射测试物质，且在第0天及第5天藉由qPCR分析RPE及神经视网膜中的基因表达来评估影响。The group size was 5 eyes. On
结果result
以上实验的结果呈现于图25中。这类结果显示给药REDD14会引起：The results of the above experiments are presented in FIG. 25 . These results show that administration of REDD14 causes:
·RPE及神经视网膜中低于基线的RTP801表达的约40％下调(亦参见图26)；Approximately 40% downregulation of RTP801 expression below baseline in the RPE and neural retina (see also Figure 26);
·神经视网膜中超过基线的PEDF表达的约70％上调(注意：在注射PBS的眼睛中，低于基线的PEDF表达下调40％)；Approximately 70% upregulation of PEDF expression over baseline in the neural retina (note: 40% downregulation of PEDF expression below baseline in PBS-injected eyes);
·RPE中低于基线的VEGF164表达的约40％下调(注意：在注射PBS眼睛中，VEGF164的表达下调20％)；Approximately 40% downregulation of VEGF164 expression below baseline in RPE (Note: 20% downregulation of VEGF164 expression in PBS injected eyes);
·在RPE/脉络膜中MCP1表达减少约50％(图26)。- About 50% reduction in MCP1 expression in RPE/choroid (Figure 26).
根据两个实验获得的总体结论：General conclusions obtained from two experiments:
·RTP801及VEGF的同时抑制会增强对脉络膜新血管生成及新血管渗漏的抑制作用。Simultaneous inhibition of RTP801 and VEGF enhanced inhibition of choroidal neovascularization and neovascular leakage.
·藉由REDD14抑制RTP801表达不仅预防CNV模型中的PEDF下调，与基线相比亦增强其表达。• Inhibition of RTP801 expression by REDD14 not only prevents PEDF downregulation in CNV models but also enhances its expression compared to baseline.
·RTP801表达的抑制会导致伴随的应具有消炎作用的MCP1下调。• Inhibition of RTP801 expression leads to concomitant downregulation of MCP1 which should have an anti-inflammatory effect.
·不受理论限制，藉由REDD14增加PEDF表达可构成所观测到的VEGF及RTP801同时抑制的合作效应的基础。(PEDF为熟知的抗血管生成及神经保护因子)。• Without being bound by theory, the increase in PEDF expression by REDD14 may underlie the observed cooperative effect of simultaneous inhibition of VEGF and RTP801. (PEDF is a well-known anti-angiogenic and neuroprotective factor).
·不受理论限制，藉由REDD14减少MCP1表达亦可构成所观测到的VEGF及RTP801同时抑制的合作效应的基础。(MCP1为涉及AMD发病机理的已知促炎症趋化细胞素)。• Without being bound by theory, reduction of MCP1 expression by REDD14 may also underlie the observed cooperative effect of simultaneous inhibition of VEGF and RTP801. (MCP1 is a known pro-inflammatory chemokine involved in AMD pathogenesis).
可用以测试本发明方法的额外AMD模型：Additional AMD models that can be used to test the methods of the invention:
·Ccl-2或Ccr-2缺乏的动物-这类蛋白的每一个的缺乏均会引起AMD的某些主要特征发展。这类蛋白缺乏的动物可用以测试本发明的方法。• Ccl-2 or Ccr-2 deficient animals - Deficiency in each of these proteins causes some of the cardinal features of AMD to develop. Animals deficient in such proteins can be used to test the methods of the invention.
关于AMD动物模型的其它信息参见：Chader，Vision research 42(2002)393-399；Ambati等人，Nature Medicine 9(11)(2003)1390-1397；Tolentino等人，Retina 24(2004)132-138。For additional information on AMD animal models see: Chader, Vision research 42 (2002) 393-399; Ambati et al., Nature Medicine 9(11) (2003) 1390-1397; Tolentino et al., Retina 24 (2004) 132-138 .
D)在激光诱发的CNV模型中比较各链上具有3′磷酸酯基的REDD14抗RTP801 siRNA与缺乏3′磷酸酯基的相同分子(REDD14NP)的活性D) Comparison of the activity of REDD14 anti-RTP801 siRNA with a 3′ phosphate group on each strand and the same molecule lacking a 3′ phosphate group (REDD14NP) in a laser-induced CNV model
一般，如上所述实施且评估实验。将各小鼠(每组12只)之一只眼睛用0.25μg REDD14 siRNA注射，而将另一只眼睛用REDD14NP siRNA注射。In general, experiments were performed and evaluated as described above. One eye of each mouse (12 per group) was injected with 0.25 μg REDD14 siRNA, while the other eye was injected with REDD14NP siRNA.
结果result
两种siRNA同等有效地减小CNV体积(图27)。Both siRNAs were equally effective in reducing CNV size (Figure 27).
实施例7Example 7
关于COPD及气肿的模型及结果Models and results on COPD and emphysema
在以下动物模型中测试本发明的化合物：Compounds of the invention were tested in the following animal models:
*吸烟诱发的气肿模型：在若干动物(诸如小鼠、豚鼠)中，长期暴露于烟会引起气肿。*Smoking-induced emphysema model: Chronic exposure to smoke causes emphysema in several animals (eg mice, guinea pigs).
*作为气肿引发物的肺蛋白酶活性。*Pulmonary protease activity as an emphysema trigger.
*气肿的VEGFR抑制模型。*VEGFR inhibition model of emphysema.
*在啮齿动物中用人类嗜中性粒细胞/胰腺弹性酶滴注支气管。*Bronchoinstillation with human neutrophil/pancreatic elastase in rodents.
*MMP(基质金属蛋白酶)诱发的气肿。*MMP (matrix metalloproteinase)-induced emphysema.
*炎症诱发的气肿。*Inflammation-induced emphysema.
此外，气肿模型可经由遗传方式(例如携带TSK突变的小鼠)产生，且气肿性动物可藉由已知对气肿敏感的改性剂而产生，诸如肺损伤、肺泡发育不全、氧过多、糖皮质激素治疗及营养。In addition, emphysema models can be generated genetically (e.g., mice carrying TSK mutations), and emphysematous animals can be generated by modifiers known to be emphysema-sensitive, such as lung injury, alveolar hypoplasia, oxygen Overdose, glucocorticoid therapy, and nutrition.
A.缺乏RTP801对患有气肿的小鼠模型中疾病发展的影响的评估(使用RTP801基因剔除小鼠)A. Evaluation of the effect of lack of RTP801 on disease development in a mouse model with emphysema (using RTP801 knockout mice)
(1)吸烟(CS)诱发的炎症及细胞凋亡起始于5只RTP KO及5只对照野生型雄性小鼠(4个月大)。使小鼠经受强烈CS(如Rangasamy等人中所述，参见上文)历经7天。来自以上VEGFR抑制实验的未经KO及WT处理小鼠亦可用作此实验的未经治疗对照组。随后使肺经琼脂糖膨胀，固定且将其埋入石蜡中，且藉由以下步骤来分析KO小鼠中的发育氧化应力：(1) Cigarette smoking (CS)-induced inflammation and apoptosis were initiated in 5 RTP KO and 5 control wild-type male mice (4 months old). Mice were subjected to vigorous CS (as described in Rangasamy et al., supra) for 7 days. Untreated KO and WT treated mice from the above VEGFR inhibition experiments can also be used as untreated control groups for this experiment. Lungs were then expanded through agarose, fixed and embedded in paraffin, and the developmental oxidative stress in KO mice was analyzed by the following steps:
a)对肺切片中的8-氧代-dG进行免疫组织化学定位及量化；a) Immunohistochemical localization and quantification of 8-oxo-dG in lung sections;
b)使用特异性抗体对肺切片中的活性卡斯蛋白酶3进行免疫组织化学定位及量化，或对TUNEL阳性细胞数目进行量化评估；b) Immunohistochemical localization and quantification of
c)测量肺萃取物中的神经酰胺浓度；c) measuring the ceramide concentration in the lung extract;
d)测量肺萃取物中的卡斯蛋白酶活性。d) Measurement of caspase activity in lung extracts.
(2)长期吸烟的KO小鼠(2) Long-term smoking KO mice
在6个月时期内，使6只KO及6只年龄相符的WT雌性小鼠经受强烈吸烟(一天5小时)。接着杀死小鼠，且使用形态测量方法评估平均隔间直径(气肿发育的参数)。Six KO and six age-matched WT female mice were subjected to intense smoking (5 hours a day) over a 6-month period. Mice were then sacrificed and mean compartment diameter (a parameter of emphysema development) was assessed using morphometric methods.
B.藉由使用肺内传递RTP801失活siRNA抑制内源性RTP801来评估缺乏RTP801对患有气肿的小鼠模型中疾病进程的影响B. Assessment of the effect of RTP801 deficiency on disease progression in a mouse model of emphysema by intrapulmonary delivery of RTP801-inactivating siRNA to inhibit endogenous RTP801
在2组C57BL6小鼠(每组10只小鼠)中，藉由7天吸烟来诱发CS诱发的炎症。组1：CS+对照siRNA(REDD8)siRNA的传递；组2：CS+RTP801siRNA(REDD14)。将小鼠的对照组用每一类型的siRNA滴注但保持于室内空气条件下。如同以上用基因剔除小鼠进行的实验，评估动物。In 2 groups of C57BL6 mice (10 mice per group), CS-induced inflammation was induced by smoking for 7 days. Group 1: Delivery of CS+control siRNA (REDD8) siRNA; Group 2: CS+RTP801 siRNA (REDD14). A control group of mice was instilled with each type of siRNA but kept under room air conditions. Animals were evaluated as above for experiments with knockout mice.
方法method
暴露于吸烟(CS)exposure to smoking (CS)
藉由燃烧2R4F参考烟(每只烟2.45mg烟碱；购自Tobacco ResearchInstitute，University of Kentucky，Lexington，KY，USA)，使用吸烟机(模型TE-10，Teague Enterprises，Davis，CA，USA)来实施暴露(每天7h，每周7天)。以1.05L/min的流动速率吹开各闷烧的烟历经2s，每分钟总共八团烟，以提供35cm3的标准烟团。调节吸烟机以藉由同时燃烧5只烟来产生侧流烟(89％)及主流烟(11％)的混合物。监控腔室气氛以使总悬浮颗粒及一氧化碳浓度分别为90mg/m3及350ppm。By burning 2R4F reference cigarettes (2.45 mg nicotine per cigarette; purchased from Tobacco Research Institute, University of Kentucky, Lexington, KY, USA), using a smoking machine (model TE-10, Teague Enterprises, Davis, CA, USA) to Exposure was carried out (7h per day, 7 days per week). Each smoldering puff was blown away at a flow rate of 1.05 L/min for 2 s for a total of eight puffs per minute to provide a standard puff of 35 cm3 . The smoking machine was adjusted to produce a mixture of sidestream smoke (89%) and mainstream smoke (11%) by burning 5 cigarettes simultaneously. The chamber atmosphere was monitored so that the total suspended particulate and carbon monoxide concentrations were 90 mg/m3 and 350 ppm, respectively.
形态学及形态测量分析Morphology and Morphometric Analysis
在使小鼠暴露于CS或滴注表达质粒的RTP801后，用氟烷麻醉小鼠且如先前所述6在25cm的恒定压力下用0.5％低熔点琼脂糖使肺膨胀。将膨胀的肺固定于10％经缓冲的福尔马林中且埋入石蜡中。将切片(5μm)用苏木精及曙红染色。平均肺泡直径、肺泡长度及平均直线截距藉由具有Image Pro Plus软件(Media Cybernetics，Silver Spring，MD，USA)的计算机辅助形态测量来测定。编码各组的肺切片且由并不知晓载玻片身份的研究者用Nikon E800显微镜(20X透镜)获得代表性影像(每个肺切片15个影像)。After exposing mice to CS or instilling RTP801 expressing the plasmid, mice were anesthetized with halothane and the lungs were inflated with 0.5% low-melting point agarose under a constant pressure of 25 cm as previouslydescribed6 . Inflated lungs were fixed in 10% buffered formalin and embedded in paraffin. Sections (5 μm) were stained with hematoxylin and eosin. Mean alveolar diameter, alveolar length, and mean linear intercept were determined by computer-aided morphometry with Image Pro Plus software (Media Cybernetics, Silver Spring, MD, USA). Lung sections from each group were coded and representative images (15 images per lung section) were acquired with a Nikon E800 microscope (20X lens) by an investigator blinded to the slide's identity.
支气管肺泡灌洗(BAL)及表型Bronchoalveolar lavage (BAL) and phenotype
在暴露于CS或滴注表达质粒的RTP801后，将小鼠用戊巴比妥钠(sodium pentobarbital)麻醉。将自小鼠的肺收集的BAL液离心(4℃下，500′g)且将细胞小球再悬浮于磷酸盐缓冲的生理食盐水中。测定灌洗液中的细胞总数，且2×104个细胞经细胞离心(Shandon SouthernProducts，Pittsburgh，PA，USA)至玻璃载片上且用Wright-Giemsa染料染色。根据标准细胞学技术，在300个细胞上执行分化细胞计数。After exposure to CS or instillation of RTP801 expressing the plasmid, mice were anesthetized with sodium pentobarbital. BAL fluid collected from lungs of mice was centrifuged (500'g at 4°C) and the cell pellet was resuspended in phosphate buffered saline. The total number of cells in the lavage fluid was determined and 2 x104 cells were cytospun (Shandon Southern Products, Pittsburgh, PA, USA) onto glass slides and stained with Wright-Giemsa dye. Differentiation cell counts were performed on 300 cells according to standard cytology techniques.
鉴别肺中的肺泡细胞凋亡细胞群体Identifying alveolar cell apoptotic cell populations in the lung
为鉴别肺中经历细胞凋亡的不同肺泡细胞类型，在来自暴露于室内空气(RA)以及CS的小鼠的肺切片中实施活性卡斯蛋白酶3的免疫组织化学染色。为鉴别肺中细胞凋亡II型上皮细胞，在活性卡斯蛋白酶3标记后，将肺切片首先用抗小鼠表面蛋白C(SpC)抗体培育，且接着用抗兔德州红(Texas red)抗体培育。藉由首先用抗小鼠CD 31抗体且接着用生物素结合的兔抗小鼠二次抗体培育来鉴别细胞凋亡内皮细胞。在PBS中冲洗肺切片且接着用抗生蛋白链菌素-德州红结合的复合物来培育。藉由首先用大鼠抗小鼠Mac-3抗体培育切片且接着用抗大鼠德州红抗体培育来鉴别肺中的细胞凋亡巨噬细胞。最终，将DAPI应用于所有肺切片，培育5分钟，洗涤且用Vectashield HardSet封固培养基封固。分别在330-380nm及465-495nm下可视化DAPI及荧光素。用NikonE800显微镜(40X透镜)获得肺切片的影像。To identify different alveolar cell types undergoing apoptosis in the lung, immunohistochemical staining for
活性卡斯蛋白酶3的免疫组织化学定位Immunohistochemical localization of
使用抗活性卡斯蛋白酶3抗体来实施活性卡斯蛋白酶3检定的免疫组织化学染色，且用宏指令使用Image Pro Plus程序来计数活性卡斯蛋白酶3阳性细胞。藉由肺泡分布总数(本文称为肺泡长度且以μm表示)来标准化该计数。肺泡长度与平均直线截距反向相关，亦即当肺泡隔受到破坏时，平均直线截距随总肺泡长度(亦即总肺泡隔长度)减少而增加。Immunohistochemical staining for
卡斯蛋白酶3活性检定Caspase 3 activity assay
在肺组织萃取物中，使用荧光测定检定根据制造商说明书来测量卡斯蛋白酶3/7活性。将快速冷冻的肺组织(每组n＝3)用检定缓冲液均质化，接着超音波处理且在800×g下离心。移除细胞核及细胞碎片后，接着用前荧光基质在室温下培育上清液(300μg蛋白)1h且利用Typhoon影像分析仪(Amersham Biosciences，Inc.，Piscataway，NJ，USA)测量荧光强度。结果表示为特异性卡斯蛋白酶3基质分解的速度，以卡斯蛋白酶3酶活性的单位表示，藉由总蛋白浓度标准化。将活性重组卡斯蛋白酶3用作检定标准(0-4U)。将无基质的组织溶解产物、单独检定缓冲液及具有卡斯蛋白酶3抑制剂的溶解产物用作负性对照。In lung tissue extracts,
8-氧代-dG的免疫组织化学定位Immunohistochemical localization of 8-oxo-dG
为获得8-氧代-dG的免疫组织化学定位及量化，将来自暴露于CS或用表达质粒的RTP801滴注的小鼠肺切片用抗8-氧代-dG抗体培育，且使用小鼠抗体使用InnoGenexTM Iso-IHC DAB试剂盒来染色。用宏指令(使用Image Pro Plus)计数8-氧代-dG阳性细胞，且藉由所述的肺泡长度来标准化该计数。To obtain immunohistochemical localization and quantification of 8-oxo-dG, lung sections from mice exposed to CS or instilled with RTP801 expressing the plasmid were incubated with anti-8-oxo-dG antibody, and mouse antibody Use the InnoGenexTM Iso-IHC DAB Kit for staining. 8-Oxo-dG positive cells were counted with a macro (using Image Pro Plus) and the counts were normalized by the stated alveolar length.
将质粒DNA滴注至小鼠肺中Instillation of plasmid DNA into mouse lungs
使用无内毒素的DNA分离试剂盒来制备RTP801表达的质粒DNA及对照载体。针对气管内滴注，在80μl无菌全氟化碳中传递50μg质粒DNA。全氟化碳的携氧性使得其在这类体积下良好耐受，而当气管内滴注时其物理化学性质允许极有效的末端肺传递。藉由短暂吸入性氟烷暴露使小鼠麻醉，用镊子轻轻地将舌头向前拉，且经由钝的血管导管在舌头底部施加用全氟化碳溶液的基础上滴注气管。An endotoxin-free DNA isolation kit was used to prepare plasmid DNA for RTP801 expression and a control vector. For intratracheal instillation, 50 μg of plasmid DNA was delivered in 80 μl of sterile perfluorocarbon. The oxygen-carrying properties of perfluorocarbons make them well tolerated at such volumes, while their physicochemical properties allow for extremely efficient terminal lung delivery when instilled intratracheally. Mice were anesthetized by brief inhalational halothane exposure, the tongue was gently pulled forward with forceps, and an instillation based on perfluorocarbon solution was applied to the base of the tongue via a blunt vascular catheter.
将siRNA滴注至小鼠肺中Instillation of siRNA into mouse lungs
藉由腹膜内注射氯胺酮/甲苯噻嗪(Xylazine)(115/22mg/kg)使小鼠麻醉。藉由连续五次10μl传递，经鼻内滴注50μl体积的0.9％NaCl中的50μg siRNA。在鼻内滴注的末期，使小鼠头部直立向上1分钟以确保所有经滴注的溶液流入内部。Mice were anesthetized by intraperitoneal injection of Ketamine/Xylazine (115/22 mg/kg). 50 μg of siRNA in 0.9% NaCl in a volume of 50 μl was instilled intranasally by five consecutive 10 μl delivery. At the end of the intranasal instillation, the mice were held head upright for 1 minute to ensure that all instilled solution flowed inside.
其它信息参见Rangasamy T，Cho CY，Thimmulappa，RK，ZhenL，Srisuma SS，Kensler TW，Yamamoto M，Petrache I，Tuder RM，BiswalS.Geneticabl ation of Nrf2 enhances susceptibility tocigarette smoke-iduced emphysema in mice.Submitted to Journalof Clinincal Investigation；Yasunori Kasahara，Rubin M.Tuder，Carlyne D.Cool，David A.Lynch，Sonia C.Flores及Norbert F.Voelkel.Endothelial Cell Death and Decreased Expression ofVascular Endothelial Growth Factor and Vascular EndothelialGrowth Factor Receptor 2 in Emphysema.Am J Respir Crit CareMed，163卷，第737-744页，2001；Yasunori Kasahara，Rubin M.Tuder，Laimute Taraseviciene-Stewart，Timothy D.Le Cras，Steven Abman，Peter K.Hirth，Johannes Waltenberger及NorbertF.Voelkel.Inhibition of VEGF receptors causes lung cellapoptosis and emphysema.J.Clin.Invest.106：1311-1319(2000)；及关于主题的评论：Robin M.Tuder，Sharon McGrath及EnidNeptune，The pathological mechanisms of emphysema models：whatdo they have in common？，Pulmonary Pharmacology & Therpaeutics2002。For additional information see Rangasamy T, Cho CY, Thimmulappa, RK, ZhenL, Srisuma SS, Kensler TW, Yamamoto M, Petrache I, Tuder RM, Biswal S. Geneticablation of Nrf2 enhances susceptibility tocigarette smoke-iduced emphysema lin in our calin of mice. Submitted to Investigation; Yasunori Kasahara, Rubin M. Tuder, Carlyne D. Cool, David A. Lynch, Sonia C. Flores, and Norbert F. Voelkel. Endothelial Cell Death and Decreased Expression of Vascular Endothelial Growth Factor and Vascular Endothelial Growth. J Respir Crit CareMed, Vol. 163, pp. 737-744, 2001; Yasunori Kasahara, Rubin M. Tuder, Laimute Taraseviciene-Stewart, Timothy D. Le Cras, Steven Abman, Peter K. Hirth, Johannes Waltenberger, and Norbert F. Voelkel. Inhibition of VEGF receptors causes lung cellapoptosis and emphysema. J. Clin. Invest. 106: 1311-1319 (2000); and comments on the subject: Robin M. Tuder, Sharon McGrath and Enid Neptune, The pathological mechanisms of emphysema models: what do they have in common? , Pulmonary Pharmacology & Therpaeutics 2002.
结果result
1.滴注表达质粒的RTP801会导致小鼠肺中类气肿表型，类气肿表型藉由(1)支气管肺泡灌洗细胞数的增加(图15a)、(2)肺隔细胞的细胞凋亡(图15b)及肺泡直径的增加(图15c)来证实。1. Instillation of RTP801 expressing the plasmid will lead to emphysema-like phenotype in the lungs of mice. Apoptosis (Figure 15b) and an increase in alveolar diameter (Figure 15c) were confirmed.
2.滴注RTP801 siRNA(REDD14)会导致肺中RTP801表达减少(图17b)。2. Instillation of RTP801 siRNA (REDD14) would lead to a decrease in the expression of RTP801 in the lung (Fig. 17b).
3.吸烟6个月后RTP801 KO小鼠免于气肿发展，如肺泡直径扩大的缺乏所证实(图18)。3. RTP801 KO mice were free from emphysema development after 6 months of smoking, as evidenced by the lack of expansion in alveolar diameter (Fig. 18).
4.RTP801 KO小鼠免于吸烟诱发的炎症，如藉由炎症支气管肺泡细胞在1周吸烟后的数目减少(图16a-b)所证实。4. RTP801 KO mice were protected from smoking-induced inflammation, as evidenced by a reduction in the number of inflammatory bronchoalveolar cells after 1 week of smoking (Fig. 16a-b).
5.RTP801 KO小鼠免于吸烟诱发的肺隔细胞细胞凋亡，如藉由关于活化卡斯蛋白酶染色的肺切片所证实(图16c)。5. RTP801 KO mice were protected from smoking-induced septal cell apoptosis, as demonstrated by lung sections stained for activated caspases (Fig. 16c).
6.滴注REDD14的小鼠部分地免于吸烟诱发的炎症，如藉由炎症支气管肺泡细胞在1周吸烟后的数目减少(图17a)所证实。6. Mice instilled with REDD14 were partially protected from smoking-induced inflammation, as evidenced by a reduction in the number of inflammatory bronchoalveolar cells after 1 week of smoking ( FIG. 17 a ).
7.滴注REDD14的小鼠部分地免于吸烟诱发的肺隔细胞细胞凋亡，如藉由关于活化卡斯蛋白酶染色的肺切片及用抗活化卡斯蛋白酶3抗体获得的肺萃取物的免疫印迹所证实(图17c)。7. Mice instilled with REDD14 were partially protected from smoking-induced septal cell apoptosis, as demonstrated by immunization of lung sections stained for activated caspase and lung extracts obtained with
实施例8Example 8
微血管病症相关的模型及结果Models and results related to microvascular disorders
在如下所述的各种微血管病症的动物模型中测试本发明的化合物。Compounds of the invention are tested in animal models of various microvascular disorders as described below.
1.糖尿病性视网膜病1. Diabetic retinopathy
RTP801在活体外促进神经元细胞凋亡及活性氧产生。本发明的发明者亦发现在经受早产儿视网膜病(ROP)模型的RTP801基因剔除(KO)小鼠中，在缺氧条件下尽管VEGF升高但病理新血管生成NV仍减少，而此基因的缺乏并不影响生理上新生儿视网膜NV。此外，在此模型中，缺乏RTP801亦具保护性以对抗缺氧神经元细胞凋亡及高氧血管阻塞。RTP801 promotes neuronal apoptosis and reactive oxygen species production in vitro. The inventors of the present invention also found that in RTP801 knockout (KO) mice subjected to the retinopathy of prematurity (ROP) model, pathological neovascularization NV was reduced under hypoxic conditions despite elevated VEGF, whereas expression of this gene Deficiency did not affect physiologically neonatal retinal NV. Furthermore, in this model, lack of RTP801 was also protective against hypoxic neuronal apoptosis and hyperoxic vascular occlusion.
实验1
藉由腹膜内注射STZ在8周大的RTP801 KO及C57/129sv野生型(WT)同窝出生的小鼠中诱发糖尿病。4周后，在1小时黑暗适应期后自左眼获得ERG(单一白色闪光，1.4 x 10^4ftc，5ms)。使用伊文思蓝(Evans-blue)白蛋白渗透技术，根据双眼分析RVP。Diabetes was induced in 8-week-old RTP801 KO and C57/129sv wild-type (WT) littermates by intraperitoneal injection of STZ. 4 weeks later, ERGs (single white flash, 1.4 x 10^4ftc, 5ms) were obtained from the left eye after a 1-hour dark adaptation period. RVP was analyzed binocularly using the Evans-blue albumin permeation technique.
结果result
血糖在糖尿病性(DM)WT与DM KO之间并非不同(495±109与513±76mg/dl)，在非糖尿病性(NDM)WT与KO之间亦并非不同(分别130±10与135±31mg/dl)。与NDM WT(21.5±18.8μL/g/hr，n＝9，p＝0.055)相比，DM WT组中RVP增加138％(51.2±37.9μL/g/hr，n＝8)。相比之下，与DM WT小鼠相比，DM KO中RVP减少80％(9.5±8.5μL/g/hr，n＝6，p＝0.023)，从而导致糖尿病诱发的RVP减少140％。在DM WT小鼠中，与NDM WT相比，OP2(11％)、OP3(12％) & OP4(14％)及B波(23％)的振荡潜在内隐时间延长(p<0.05)。A波并无显著变化。与NDM KO相比，DM KO小鼠中OP3及OP4的这类变化标准化为约100％且B波为65％。Blood glucose was not different between diabetic (DM) WT and DM KO (495±109 versus 513±76 mg/dl), nor was it different between non-diabetic (NDM) WT and KO (130±10 versus 135± 31mg/dl). RVP increased by 138% in the DM WT group (51.2±37.9 μL/g/hr, n=8) compared to NDM WT (21.5±18.8 μL/g/hr, n=9, p=0.055). In contrast, RVP was reduced by 80% in DM KO compared to DM WT mice (9.5±8.5 μL/g/hr, n=6, p=0.023), resulting in a 140% reduction in diabetes-induced RVP. In DM WT mice, the oscillatory latent implicit time of OP2 (11%), OP3 (12%) & OP4 (14%) and B wave (23%) was prolonged compared with NDM WT (p<0.05). A wave did not change significantly. Such changes in OP3 and OP4 were normalized to approximately 100% and 65% for the B wave in DM KO mice compared to NDM KO mice.
结论：RTP801的基因剔除会改善小鼠中糖尿病诱发的RVP及ERG异常，此表明此缺氧诱发基因可在早期糖尿病性视网膜病的发病机理中起重要作用。Conclusion: Gene knockout of RTP801 can improve diabetes-induced RVP and ERG abnormalities in mice, suggesting that this hypoxia-induced gene may play an important role in the pathogenesis of early diabetic retinopathy.
实验2
在RTP801基因剔除小鼠及具有匹配基因背景的对照野生型小鼠中诱发糖尿病。此外，在C57B16小鼠中诱发糖尿病，随后将该C57B16小鼠用于玻璃体内注射抗RTP801及对照siRNA。关于糖尿病诱发，将小鼠用链菌素注射(隔夜禁食后，STZ 90mg/kg/d2天)。在整个研究期间，关于血糖、体重及血球比容的变化来监控动物生理学。将经媒剂注射的小鼠用作对照物。藉由玻璃体内注射1μg REDD14抗RTP801 siRNA或1μg抗GFP对照siRNA来处理适当动物。在研究过程中注射siRNA两次-在第0天(当实施第一次注射STZ时)及注射STZ后第14天。Diabetes was induced in RTP801 knockout mice and control wild-type mice with a matching genetic background. In addition, diabetes was induced in C57B16 mice, which were then used for intravitreal injection of anti-RTP801 and control siRNA. For diabetes induction, mice were injected with streptavidin (STZ 90 mg/kg/d for 2 days after an overnight fast). Animal physiology was monitored for changes in blood glucose, body weight and hematocrit throughout the study period. Vehicle-injected mice were used as controls. Appropriate animals were treated by intravitreal injection of 1 μg REDD14 anti-RTP801 siRNA or 1 μg anti-GFP control siRNA. siRNA was injected twice during the course of the study - on day 0 (when the first STZ injection was administered) and on
在糖尿病的4周持续时间后，使用伊文思蓝(EB)染料技术来测量动物的视网膜血管渗漏。在伊文思蓝(EB)测量前24小时，将导管植入小鼠右颈静脉中。在各动物的双眼中，视网膜渗透性测量是根据标准伊文思蓝方案来进行的。Following a 4-week duration of diabetes, the animals' retinal vascular leakage was measured using the Evans Blue (EB) dye technique. Twenty-four hours before Evans blue (EB) measurements, mice were catheterized in the right jugular vein. Retinal permeability measurements were performed according to the standard Evans blue protocol in both eyes of each animal.
结果result
1.与野生型糖尿病小鼠相比，RTP801 KO糖尿病小鼠中视网膜血管渗漏减少70％(参见图20)。1. Retinal vascular leakage was reduced by 70% in RTP801 KO diabetic mice compared to wild-type diabetic mice (see Figure 20).
2.RTP801的基因剔除使小鼠中的ERG异常正常化：在DM WT小鼠中，与NDM WT相比，OP2(11％)、OP3(12％)及OP4(14％)及B波(23％)的振荡潜在内隐时间延长(p<0.05)。A波无显著变化。与NDM RTP801 KO相比，DM RTP801 KO小鼠的OP3及OP4的这类变化标准化为约100％且B波为65％(参见图21)。2. Gene knockout of RTP801 normalized ERG abnormalities in mice: in DM WT mice, compared with NDM WT, OP2 (11%), OP3 (12%) and OP4 (14%) and B wave ( 23%) of the oscillatory latent implicit time was prolonged (p<0.05). There was no significant change in wave A. Such changes in OP3 and OP4 were normalized to approximately 100% and 65% for the B wave in DM RTP801 KO mice compared to NDM RTP801 KO (see FIG. 21 ).
3.类似于KO小鼠中的结果，与经玻璃体内注射抗GFP的对照siRNA的糖尿病小鼠相比，经玻璃体内注射抗RTP801的REDD14 siRNA糖尿病小鼠中视网膜血管渗漏减少50％(参见图22)。3. Similar to the results in KO mice, retinal vascular leakage was reduced by 50% in diabetic mice injected intravitreally with REDD14 siRNA against RTP801 compared to diabetic mice injected with control siRNA against GFP intravitreally (see Figure 22).
2.早产儿视网膜病2. Retinopathy of prematurity
藉由将测试动物暴露于缺氧及高氧条件来诱发早产儿视网膜病，且继而测试对视网膜的影响。结果表明RTP801 KO小鼠免于早产儿视网膜病，藉此证实RTP801抑制的保护作用。Retinopathy of prematurity was induced by exposing test animals to hypoxic and hyperoxic conditions, and the effect on the retina was then tested. The results demonstrate that RTP801 KO mice are protected from retinopathy of prematurity, thereby confirming the protective effect of RTP801 inhibition.
3.心肌梗塞3. Myocardial infarction
藉由小鼠中短期及长期左前降支动脉结扎(Left AnteriorDescending artery ligation)来诱发心肌梗塞。结果：在RTP801KO小鼠中，梗塞后24小时TnT及CPK-MB分数含量减少，且梗塞后28天超声心动图(射血分数体积)更优选。Myocardial infarction was induced by short-term and long-term left anterior descending artery ligation in mice. RESULTS: In RTP801KO mice, TnT and CPK-MB fraction levels were reduced at 24 hours post-infarction, and echocardiography (ejection fraction volume) at 28 days post-infarction was more preferred.
4.微血管缺血病状4. Microvascular ischemic conditions
用于分析缺血病状的动物模型包括：Animal models for analysis of ischemic conditions include:
1.闭锁性头部损伤(CHI)-实验性TBI产生一系列促进神经及神经代谢级联的事件，该事件与行为不足的程度及范围相关。在麻醉下诱发CHI，而允许重量自预先指定的高度自由下落(Chen等人，J.Neurotrauma 13，557，1996)至覆盖头颅中部平面中的左半球的暴露头骨上。1. Locked Head Injury (CHI)—Experimental TBI produces a series of events that promote a neural and neurometabolic cascade that correlates with the degree and extent of behavioral deficits. CHI was induced under anesthesia while weights were allowed to fall freely from a prespecified height (Chen et al.,
2.暂时性大脑中央动脉阻塞(MCAO)-在成年、雄性Sprague Dawley大鼠(300-370gr)中实施90至120分钟的暂时性病灶性缺血。所用的方法为腔内缝合MCAO(Longa等人，Stroke，30，84，1989；及Dogan等人，J.Neurochem.72，765，1999)。简言之，在氟烷麻醉下，将涂布聚L-赖氨酸的3-0-尼龙缝合物质经由颈外动脉中的洞插入右侧颈内动脉(ICA)中。将尼龙线推至ICA中至右侧MCA源点(20-23mm)。90-120分钟后，将线断开，缝合动物且使其恢复。2. Transient Central Cerebral Artery Occlusion (MCAO) - 90 to 120 minutes of transient focal ischemia in adult, male Sprague Dawley rats (300-370gr). The method used was endoluminal suture MCAO (Longa et al., Stroke, 30, 84, 1989; and Dogan et al., J. Neurochem. 72, 765, 1999). Briefly, under halothane anesthesia, a poly-L-lysine-coated 3-O-nylon suture material was inserted into the right internal carotid artery (ICA) through a hole in the external carotid artery. Push the nylon thread into the ICA to the right MCA source (20-23mm). After 90-120 minutes, the threads were broken and the animals were sutured and allowed to recover.
3.永久性大脑中央动脉阻塞(MCAO)-阻塞为永久性的，藉由MCA的电凝法单方面诱发。两种方法均导致大脑皮质同侧的病灶性脑缺血，而对侧完整(对照)。经由暂时部分颅骨切除术暴露左侧MCA，如TamuraA.等人，J Cereb Blood Flow Metab.1981；1：53-60关于大鼠所述。MCA及其豆纹状枝在接近嗅束的中间边界处闭合，伴随微双极凝结。缝合伤口，且使动物返回在26℃至28℃下加温的室中的笼中。在自动调温器下一直保持动物温度。3. Permanent central cerebral artery occlusion (MCAO) - The occlusion is permanent and induced unilaterally by electrocoagulation of the MCA. Both approaches resulted in focal cerebral ischemia on the ipsilateral side of the cerebral cortex, while the contralateral side was intact (control). The left MCA was exposed via temporary partial craniectomy as described for rats by Tamura A. et al., J Cereb Blood Flow Metab. 1981; 1:53-60. The MCA and its lentilized branches close close to the medial border of the olfactory tract with microbipolar condensation. The wound is closed and the animal is returned to its cage in a room warmed at 26°C to 28°C. Animal temperature was maintained under a thermostat at all times.
5.急性肾衰竭(ARF)5. Acute renal failure (ARF)
测试用于治疗ARF的活性siRNA可使用脓毒症诱发的ARF或缺血-再灌注诱发的ARF来进行。Testing active siRNAs for the treatment of ARF can be performed using sepsis-induced ARF or ischemia-reperfusion-induced ARF.
1.脓毒症诱发的ARF1. Sepsis-induced ARF
脓毒症诱发的ARF的两种预测性动物模型藉由Miyaji T，Hu X，Yuen PS，Muramatsu Y，Iyer S，Hewitt SM，Star RA，2003，Ethylpyruvate decreases sepsis-induced acute renal failure andmultiple organ damagein aged mice，KidneyInt.Nov；64(5)：1620-31描述。Two predictive animal models of sepsis-induced ARF by Miyaji T, Hu X, Yuen PS, Muramatsu Y, Iyer S, Hewitt SM, Star RA, 2003, Ethylpyruvate decreases sepsis-induced acute renal failure and multiple organ damagein aged mice, described in Kidney Int. Nov;64(5):1620-31.
此两种模型为小鼠、优选年老小鼠中的脂多醣给药及盲肠结扎穿孔术。These two models are lipopolysaccharide administration and cecal ligation and puncture in mice, preferably aged mice.
2.缺血-再灌注诱发的ARF2. Ischemia-reperfusion-induced ARF
此预测性动物模型藉由Kelly KJ，Plotkin Z，Vulgamott SL，Dagher PC，2003年1月.P53 mediates the apoptotic response toGTP depletion after renal ischemia-reperfusion：protectiverole of a p53 inhibitor，J Am Soc Nephrol；14(1)：128-38描述。This predictive animal model was improved by Kelly KJ, Plotkin Z, Vulgamott SL, Dagher PC, January 2003. P53 mediates the apoptotic response to GTP depletion after renal ischemia-reperfusion: protective role of a p53 inhibitor, J Am Soc Nephrol; 14( 1): 128-38 description.
在45分钟两侧肾动脉钳夹及随后释放夹以进行24小时再灌注后，在大鼠中诱发缺血-再灌注损伤。在钳夹前2小时及钳夹后30分钟，将250μg REDD14或GFP siRNA(负性对照)注射至颈静脉中。另外250μgsiRNA在钳夹后4及8小时经由尾静脉给予。将对抗GFP的siRNA用作负性对照。藉由在外科手术之前及之后24小时测量血清肌酸酐含量来监控ARF进程。在实验结束时，经由留置股节线用温PBS、接着用4％聚甲醛灌注大鼠。将左肾移除且储存于4％聚甲醛中以进行随后组织分析。急性肾衰竭常定义为血清肌酸酐含量自基线急性增加。至少0.5mg/dL或44.2μmol/L的血清肌酸酐增加被视为急性肾衰竭的指示。在外科手术前零时及在ARF外科手术后24小时，测量血清肌酸酐。Ischemia-reperfusion injury was induced in rats after 45 minutes of bilateral renal artery clamping followed by release of the clamps for 24 hours of reperfusion. 250 μg of REDD14 or GFP siRNA (negative control) was injected into the
为研究siRNA在大鼠肾中的分布，经静脉内给药糖残基中具有改变的O-甲基修饰的Cy3-标记的19-mer末端平整siRNA分子(2mg/kg)，历时3-5min，此后使用双质子共焦显微镜法进行活体内成像。共焦显微镜法分析揭示肾中大部分siRNA集中在近端肾小管细胞之内体小泡区室中。内体小泡及细胞质siRNA荧光在传递后起始2小时期间相对稳定，且在24小时之时消失。To study the distribution of siRNA in rat kidney, Cy3-labeled 19-mer blunt-ended siRNA molecules (2 mg/kg) with altered O-methyl modifications in sugar residues were administered intravenously for 3-5 min , after which in vivo imaging was performed using two-proton confocal microscopy. Confocal microscopy analysis revealed that most of the siRNA in the kidney was concentrated in the endosomal vesicular compartment of proximal tubular cells. Endosomal vesicle and cytoplasmic siRNA fluorescence was relatively stable during the initial 2 hours post-delivery and disappeared by 24 hours.
如图19所证实，在45min肾双侧动脉钳夹治疗(PBS治疗)后血清肌酸酐的含量增加10倍。4次注射801 siRNA(REDD14，SEQ In No.16及66)(钳夹前2小时及钳夹后30min、4h及8h)会使血清中的肌酸酐含量显著减少40％(P<0.02)。这类结果表明801 siRNA可保护肾组织免于缺血-再灌注损伤的影响且因此减少ARF严重程度。As demonstrated in Figure 19, serum creatinine levels increased 10-fold after 45 min bilateral renal artery clamping (PBS treatment). Four injections of 801 siRNA (REDD14, SEQ In No.16 and 66) (2 hours before clamp and 30min, 4h and 8h after clamp) will significantly reduce the creatinine content in serum by 40% (P<0.02). These results suggest that 801 siRNA can protect kidney tissue from ischemia-reperfusion injury and thus reduce ARF severity.
实施例9Example 9
siRNA的制备Preparation of siRNA
使用专有算法及已知的基因RTP801序列(SEQ ID NO：1)，产生多种潜在siRNA的序列。以上说明书的siRNA分子基本上如本文所述来制备。Using a proprietary algorithm and the known sequence of the gene RTP801 (SEQ ID NO: 1), the sequences of multiple potential siRNAs were generated. The siRNA molecules of the above specification are prepared essentially as described herein.
本发明的siRNA可藉由本领域熟知的任何用于合成核糖核酸(或脱氧核糖核酸)寡核苷酸的方法来合成。举例而言，可使用市售机器(购自Applied Biosystems)；根据本文所揭示的序列制备寡核苷酸。可使用本领域熟知的方法来结扎重迭对的化学合成片段(例如，参见美国专利第6,121,426号)。单独合成各链且接着使其在管中彼此退火。接着，藉由HPLC将双链siRNA自未退火的单链寡核苷酸(例如，因为其中之一者过量)中分离。关于本发明的siRNA或siRNA片段，两个或两个以上该序列可合成且连接在一起以用于本发明中。The siRNA of the present invention can be synthesized by any method well known in the art for synthesizing ribonucleic acid (or deoxyribonucleic acid) oligonucleotides. For example, commercially available machines (from Applied Biosystems) can be used; oligonucleotides can be prepared based on the sequences disclosed herein. The overlapping pairs of chemically synthesized fragments can be ligated using methods well known in the art (see, eg, US Patent No. 6,121,426). Each strand is synthesized separately and then annealed to each other in the tube. Next, double-stranded siRNA is separated from single-stranded oligonucleotides that are not annealed (eg, because one of them is in excess) by HPLC. Regarding the siRNA or siRNA fragment of the present invention, two or more such sequences can be synthesized and linked together for use in the present invention.
本发明的siRNA分子可藉由本领域已知的程序(例如描述于Usman等人，1987，J.Am.Chem.Soc，109，7845；Scaringe等人，1990，Nucleic Acids Res.，18，5433；Wincott等人，1995，Nucleic AcidsRes.23，2677-2684及Wincott等人，1997，Methods Mol.Bio.，74，59中的程序)合成且可利用常见核酸保护及偶联基团，诸如5′末端的二甲氧基三苯甲基及3′末端的胺基磷酸酯。必要时并入经修饰(例如2′-O-甲基化)的核苷酸及未经修饰的核苷酸。The siRNA molecules of the present invention can be prepared by procedures known in the art (for example, described in Usman et al., 1987, J. Am. Chem. Soc, 109, 7845; Scaringe et al., 1990, Nucleic Acids Res., 18, 5433; Wincott et al., 1995, Nucleic Acids Res.23, 2677-2684 and Wincott et al., 1997, Methods Mol.Bio., 74, 59 procedures) synthesis and can utilize common nucleic acid protection and coupling groups, such as 5 ' Dimethoxytrityl at the end and phosphoramidate at the 3' end. Modified (eg, 2'-O-methylated) nucleotides as well as unmodified nucleotides are incorporated as necessary.
或者，本发明的核酸分子可单独合成且在合成后(例如)藉由结扎(Moore等人，1992，Science 256，9923；Draper等人，国际PCT公开案第W093/23569号；Shabarova等人，1991，Nucleic Acids Research19，4247；Bellon等人，1997，Nucleosides & Nucleotides，16，951；Bellon等人，1997，Bioconjugate Chem.8，204)或藉由合成和/或去保护后杂交而接合在一起。Alternatively, the nucleic acid molecules of the invention can be synthesized separately and after synthesis, for example, by ligation (Moore et al., 1992, Science 256, 9923; Draper et al., International PCT Publication No. WO93/23569; Shabarova et al., 1991,
本发明的siRNA分子亦可经由串联合成方法来合成，如美国专利申请公开案第US2004/0019001号(McSwiggen)所述，其中两条siRNA链合成由可分解的连接体(其随后分解以提供杂交且使siRNA双链体纯化的单独siRNA片段或链)分隔的单个连续寡核苷酸片段或链。连接体可为聚核苷酸连接体或非核苷酸连接体。其它信息参见PCT公开案第WO2004/015107号(ATUGEN)。siRNA molecules of the present invention can also be synthesized via tandem synthesis methods, as described in U.S. Patent Application Publication No. US2004/0019001 (McSwiggen), in which two siRNA strands are synthesized by a cleavable linker (which is subsequently broken down to provide hybridization and separate siRNA fragments or strands to purify siRNA duplexes) separate a single contiguous oligonucleotide fragment or strand. A linker can be a polynucleotide linker or a non-nucleotide linker. For additional information see PCT Publication No. WO2004/015107 (ATUGEN).
如上所述，构筑表A(下文)的si RNA以使得替代的糖具有2′-O-甲基修饰，亦即因此替代核苷酸经修饰。在这类优选实施例中，在siRNA之一条链中，经修饰的核苷酸为第1、3、5、7、9、11、13、15、17及19号且在另一相对链中经修饰的核苷酸为第2、4、6、8、10、12、14、16及18号。因此，这类siRNA为具有如上所述的替代2′-O-甲基修饰的末端平整的19-mer RNA分子。表2及3(下文)的siRNA亦以此方式构筑；表B及D的siRNA为具有替代2′-O-甲基修饰的末端平整的19-merRNA分子；表C的siRNA为具有替代2′-O-甲基修饰的末端平整的21-merRNA分子。As described above, the siRNAs of Table A (below) were constructed such that the substituted sugar had a 2'-O-methyl modification, i.e. the substituted nucleotide was modified accordingly. In such preferred embodiments, the modified nucleotides are
表A详述所产生且随后合成基因RTP801的各种新颖siRNA分子。最终两行表明为检查新颖分子活性而实施的两个实验的结果。简言之，用待测试的特异性新颖siRNA转染海拉细胞或HaCat细胞。接着，使用对抗RTP801多肽的抗体藉由西方墨点法来测定RTP801多肽的表达。涉及表A的右手两行，＂-＂表示失活或低活性分子(其大体上不会抑制RTP801基因的表达)；＂+＂表示具有某些抑制活性的siRNA分子(抑制RTP801基因表达)；＂++＂表示具有较高抑制活性的分子等。本文所揭示的任一siRNA分子及具体地说表A中详述的活性分子均为新颖的且亦被视为本发明的部分。Table A details various novel siRNA molecules generated and subsequently synthesized for the gene RTP801. The final two rows show the results of two experiments performed to examine the activity of the novel molecule. Briefly, HeLa cells or HaCat cells were transfected with the specific novel siRNA to be tested. Next, the expression of the RTP801 polypeptide was determined by Western blotting using an antibody against the RTP801 polypeptide. Referring to the two right-hand rows of Table A, "-" indicates an inactive or low activity molecule (which generally does not inhibit the expression of the RTP801 gene); "+" indicates an siRNA molecule with some inhibitory activity (inhibits the expression of the RTP801 gene); "++" indicates molecules with higher inhibitory activity, etc. Any of the siRNA molecules disclosed herein, and in particular the active molecules detailed in Table A, are novel and are also considered part of the present invention.
表ATable A
注意在上表A中，siRNA 1-50的有义链分别具有SEQ ID NO：3-52且siRNA 1-50的反义链分别具有SEQ ID NO：53-102。称为REDD14的分子具有SEQ ID No 16(有义链)及66(反义链)。Note that in Table A above, the sense strands of siRNAs 1-50 have SEQ ID NOs: 3-52 and the antisense strands of siRNAs 1-50 have SEQ ID NOs: 53-102, respectively. The molecule called REDD14 has SEQ ID No 16 (sense strand) and 66 (antisense strand).
注意在上表B中，siRNA 51-122的有义链分别具有SEQ IDNO：103-174且siRNA 51-122的反义链分别具有SEQ ID NO：175-246。Note that in Table B above, the sense strands of siRNAs 51-122 have SEQ ID NOs: 103-174 and the antisense strands of siRNAs 51-122 have SEQ ID NOs: 175-246, respectively.
实施例10Example 10
药理学及药物传递Pharmacology and Drug Delivery
本发明的核苷酸序列可直接或用病毒载体或非病毒载体传递。当直接传递时，该序列一般提供抗核酸酶性。或者，该序列可并入表达卡闸或构筑体中，使得序列表达于如下文所讨论的细胞中。一般，构筑体含有适合调节序列或启动子以使序列表达于靶向细胞中。The nucleotide sequences of the invention can be delivered directly or with viral or non-viral vectors. This sequence generally provides nuclease resistance when delivered directly. Alternatively, the sequence can be incorporated into an expression lock or construct such that the sequence is expressed in the cell as discussed below. Typically, the construct contains suitable regulatory sequences or a promoter to allow expression of the sequence in the targeted cell.
根据良好医药规范来给药及给予本发明的化合物或药物组合物，其中应考虑个别患者的临床病状、待治疗的疾病、给药的位点及方法、给药时程、患者年龄、性别、体重及医师所知的其它因素。Dosing and administration of the compounds or pharmaceutical compositions of the present invention is in accordance with good pharmaceutical practice, taking into account the clinical condition of the individual patient, the disease to be treated, the site and method of administration, the schedule of administration, the patient's age, sex, Body weight and other factors known to the physician.
因此，获得本文目的的医药学＂有效量＂应根据本领域已知的该考虑来确定。此量必须有效地获得改善，包括(但不限于)改善存活率或更快恢复，或改善或消除症状及本领域技术人员选为适合测量标准的其它指示。Accordingly, a pharmaceutically "effective amount" to achieve the purposes herein should be determined in light of such considerations as are known in the art. This amount must be effective to achieve improvement including, but not limited to, improved survival or faster recovery, or improved or eliminated symptoms and other indications as those skilled in the art choose to measure as appropriate.
治疗一般具有与疾病过程长度及药物有效性及所治疗的患者物种相称的长度。应注意，人类一般比小鼠或本文所例示的其它实验动物具有更长治疗。Treatment is generally of a length commensurate with the length of the disease process and the effectiveness of the drug and the patient species being treated. It should be noted that humans generally have longer treatments than mice or other experimental animals exemplified herein.
本发明的化合物可藉由任何熟知给药途径来给药。应注意，该化合物可作为化合物或医药学上可接受的盐给药且可单独给药或可作为活性成份与可药用载体、溶剂、稀释剂、赋形剂、佐剂及媒剂组合给药。化合物可经口、经皮下或胃肠外(包括静脉内、动脉内、肌肉内、腹膜内及鼻内给药以及胸内及灌注技术)给药。化合物的植入物亦适用。可制备液体形式来进行注射，该术语包括皮下、经皮、静脉内、肌肉内、胸内及其它胃肠外给药途径。液体组合物包括具有及不具有有机共溶剂的水溶液、水性或油性悬浮液、具有可食用油的乳液以及类似医药媒剂。此外，在某些环境下，用于本发明的新颖治疗的组合物可以雾剂形成以进行鼻内及类似给药。所治疗的患者为温血动物且尤其为包括人的哺乳动物。可药用载体、溶剂、稀释剂、赋形剂、佐剂及媒剂以及植入物载体一般是指不与本发明的活性成份反应的惰性、无毒固体或液体填充剂、稀释剂或封囊物质。The compounds of the present invention may be administered by any of the well-known routes of administration. It should be noted that the compound can be administered as a compound or a pharmaceutically acceptable salt and can be administered alone or as an active ingredient in combination with pharmaceutically acceptable carriers, solvents, diluents, excipients, adjuvants and vehicles medicine. The compounds can be administered orally, subcutaneously or parenterally (including intravenous, intraarterial, intramuscular, intraperitoneal and intranasal administration as well as intrathoracic and infusion techniques). Compound implants are also suitable. Liquid forms may be prepared for injection, which term includes subcutaneous, transdermal, intravenous, intramuscular, intrathoracic and other parenteral routes of administration. Liquid compositions include aqueous solutions with and without organic co-solvents, aqueous or oily suspensions, emulsions with edible oils, and similar pharmaceutical vehicles. Additionally, under certain circumstances, compositions useful in the novel treatments of the present invention may be formulated as aerosols for intranasal and the like administration. The patients to be treated are warm-blooded animals and especially mammals including humans. Pharmaceutically acceptable carriers, solvents, diluents, excipients, adjuvants and vehicles, and implant carriers generally refer to inert, non-toxic solid or liquid fillers, diluents or sealants that do not react with the active ingredients of the present invention. capsule material.
当胃肠外给药本发明的化合物时，其一般以可注射单位剂型(溶液、悬浮液、乳液)经调配。适于注射的医药配方包括无菌水溶液或分散液及用于重建至无菌可注射溶液或分散液中的无菌粉末。载体可为含有(例如)水、乙醇、多元醇(例如甘油、丙二醇、液体聚乙二醇及其类似物)、其适合混合物及植物油的溶剂或分散介质。When the compounds of the invention are administered parenterally, they are generally formulated in injectable unit dosage forms (solutions, suspensions, emulsions). Pharmaceutical formulations suitable for injection include sterile aqueous solutions or dispersions and sterile powders for reconstitution into sterile injectable solutions or dispersion. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (such as glycerol, propylene glycol, liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils.
举例而言，可藉由使用涂层(诸如卵磷脂)、维持分散情形下所需粒径及使用界面活性剂来维持适当流动性。非水性媒剂亦可用作化合物组合物的溶剂系统，诸如棉花籽油、芝麻油、橄榄油、大豆油、玉米油、向日葵油或花生油及酯(诸如十四烷酸异丙酯)。此外，亦可添加增强组合物稳定性、无菌性及等渗性的各种添加剂，包括抗菌防腐剂、抗氧化剂、螯合剂及缓冲剂。预防微生物作用可藉由各种抗菌剂及抗真菌剂来确保，例如对氧苯甲酸酯、氯丁醇、苯酚、山梨酸及其类似物。在多种情形下，亦可需要包括等张剂，例如糖、氯化钠及其类似物。可注射医药形式的延长吸收可藉由使用延迟吸收的药剂(例如单硬脂酸铝及明胶)而引起。然而，根据本发明，所用的任何媒剂、稀释剂或添加剂均必须与化合物相容。Proper fluidity can be maintained, for example, through the use of coatings such as lecithin, maintaining the desired particle size in the case of dispersion, and the use of surfactants. Non-aqueous vehicles, such as cottonseed oil, sesame oil, olive oil, soybean oil, corn oil, sunflower oil, or peanut oil and esters such as isopropyl myristate, can also be used as solvent systems for compound compositions. In addition, various additives which enhance the stability, sterility and isotonicity of the compositions, including antimicrobial preservatives, antioxidants, chelating agents and buffering agents, can also be added. Prevention of the action of microorganisms can be ensured by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, and the like. In many instances it may also be desirable to include isotonic agents, for example sugars, sodium chloride, and the like. Prolonged absorption of the injectable pharmaceutical form can be brought about by the use of agents which delay absorption, for example aluminum monostearate and gelatin. However, any vehicle, diluent or additive used must be compatible with the compound in accordance with the present invention.
无菌可注射溶液可藉由将实施本发明所用的化合物并入具有各种所需的各种其它成份的所需量的适合溶剂中来制备。Sterile injectable solutions can be prepared by incorporating the compounds used in the practice of this invention in the required amount of a suitable solvent with various other ingredients as required.
本发明的药物配方可以含有任何可相容载体(诸如各种媒剂、佐剂、添加剂及稀释剂)的可注射配方形式给药于患者；或本发明所用的化合物可以缓慢释放皮下植入物或靶向传递系统(诸如单克隆抗体、载体传递、离子电渗、聚合物基质、脂质体及微球体)形式胃肠外给药于患者。适用于本发明的传递系统实例包括美国专利第5,225,182、5,169,383、5,167,616、4,959,217、4,925,678、4,487,603、4,486,194、4,447,233、4,447,224、4,439,196及4,475,196号。其它多种这类植入物、传递系统及组件亦为本领域技术人员熟知。The pharmaceutical formulations of the present invention may be administered to patients in the form of injectable formulations containing any compatible carriers such as various vehicles, adjuvants, additives, and diluents; or the compounds used in the present invention may be slow-release subcutaneous implants Or parenteral administration to patients in the form of targeted delivery systems such as monoclonal antibodies, vector delivery, iontophoresis, polymer matrices, liposomes, and microspheres. Examples of delivery systems suitable for use in the present invention include US Pat. Various other such implants, delivery systems and components are also known to those skilled in the art.
本发明所用的化合物的药物配方可经口给药于患者。可使用熟知方法，诸如以锭剂、悬浮液、溶液、乳液、胶囊、粉末、糖浆及其类似物的形式来给药化合物。经口或经静脉内传递且保留生物活性的已知技术为优选的。在一实施方式中，本发明的化合物最初可藉由静脉内注射来给药以使血液含量达到适合含量。接着，虽然亦可使用其它给药形式(视患者病状而定且如上文所示)，但患者含量可藉由口服剂型来维持。Pharmaceutical formulations of the compounds used in the present invention can be administered orally to patients. The compounds can be administered using well known methods such as in the form of tablets, suspensions, solutions, emulsions, capsules, powders, syrups and the like. Known techniques for oral or intravenous delivery with retention of biological activity are preferred. In one embodiment, compounds of the invention may be administered initially by intravenous injection to bring blood levels to appropriate levels. Patient levels may then be maintained by oral dosage forms, although other forms of administration may also be used (depending on the patient's condition and as indicated above).
用于人类的活性化合物剂量一般在每日1ng/kg至约20-100mg/kg体重、优选每日约0.01mg至约2-10mg/kg体重的范围内，给药方式为每日一次或每日两次或三次或三次以上给药，历经1-2周或更长时间，优选历经24至48小时，或给药方式为在1-2周或更长时期内连续灌注。The dosage of the active compound for humans is generally in the range of 1 ng/kg to about 20-100 mg/kg body weight per day, preferably about 0.01 mg to about 2-10 mg/kg body weight per day, administered once a day or once a day. Two or three or more times a day, over a period of 1-2 weeks or longer, preferably over 24 to 48 hours, or as a continuous infusion over a period of 1-2 weeks or longer.
将本发明化合物给药于眼睛Administration of Compounds of the Invention to the Eye
本发明的化合物可局部或以注射(诸如玻璃体内注射、视网膜下注射或双侧注射)形式给药于眼睛。关于本发明化合物的给药的其它信息可见于下列文献：Tolentino等人，Retina 24(2004)132-138；Reich等人，Molecular vision 9(2003)210-216。The compounds of the invention may be administered to the eye topically or by injection, such as intravitreal, subretinal or bilateral injections. Additional information on the administration of the compounds of the invention can be found in the following documents: Tolentino et al., Retina 24 (2004) 132-138; Reich et al., Molecular vision 9 (2003) 210-216.
本发明化合物的肺部给药Pulmonary Administration of Compounds of the Invention
本发明的治疗组合物优选应藉由吸入含有这类组合物/化合物的雾剂或藉由鼻内或气管内滴注该组合物而给药于肺中。于脂质体中调配该组合物可利于吸收。此外，组合物亦可包括PFC液体(诸如全氟碳)，且组合物可调配为本发明化合物与聚乙烯亚胺(PEI)的复合物。The therapeutic compositions of the present invention should preferably be administered to the lungs by inhalation of a mist containing such compositions/compounds or by intranasal or intratracheal instillation of the compositions. Absorption is facilitated by formulating the composition in liposomes. In addition, compositions may also include PFC fluids such as perfluorocarbons, and compositions may be formulated as complexes of compounds of the invention with polyethyleneimine (PEI).
关于药物组合物的肺部传递的其它信息参见下列文献：Weiss等人，Human gene therapy 10：2287-2293(1999)；Densmore等人，Molecular therapy 1：180-188(1999)；Gautam等人，Moleculartherapy 3：551-556(2001)；及Shahiwala及Misra，AAPSPharmSciTech 5(2004)。此外，siRNA的呼吸配方描述于Davis等人的美国专利申请案第2004/0063654号中。For additional information on pulmonary delivery of pharmaceutical compositions see the following: Weiss et al., Human gene therapy 10:2287-2293 (1999); Densmore et al., Molecular therapy 1:180-188 (1999); Gautam et al., Molecular therapy 3:551-556 (2001); and Shahiwala and Misra, AAPSPharmSciTech 5 (2004). In addition, respiratory formulations of siRNA are described in US Patent Application No. 2004/0063654 by Davis et al.
此外，在适当时(例如，在糖尿病性足部溃疡的情形下)亦可任选以脂质/脂质体配方形式局部给药本发明的化合物。Furthermore, the compounds of the invention may also optionally be administered topically in lipid/liposomal formulations, where appropriate (for example, in the case of diabetic foot ulcers).
给药本发明化合物于耳Administration of compounds of the present invention to the ear
优选的给药模式为局部传递RTP801抑制剂至耳蜗的圆窗膜上，举例而言，如Tanaka等人(Hear Res.2003 Mar；177(1-2)：21-31)所揭示。给药于耳的另一模式是经鼓膜注射。A preferred mode of administration is local delivery of an RTP801 inhibitor to the round window membrane of the cochlea, for example as disclosed by Tanaka et al. (Hear Res. 2003 Mar; 177(1-2):21-31). Another mode of administration to the ear is transtympanic injection.
虽然在褥疮或其它创伤治疗中，药物组合物优选藉由局部施加至损害区域来给药，但组合物亦可全身给药。Although in the treatment of bedsores or other wounds, the pharmaceutical compositions are preferably administered by topical application to the lesion area, the compositions may also be administered systemically.
用于改善本发明化合物的传递的其它配方可包括未经调配的化合物、共价结合胆固醇的化合物及结合靶向抗体的化合物(Song等人，Antibody mediated in vivo delivery of small interfering RNAsvia cell-surface receptors，Nat Biotechnol.2005年6月；23(6)：709-17)。Other formulations for improved delivery of compounds of the invention may include unformulated compounds, compounds covalently bound to cholesterol, and compounds bound to targeting antibodies (Song et al., Antibody mediated in vivo delivery of small interfering RNAs via cell-surface receptors , Nat Biotechnol. 2005 Jun;23(6):709-17).
本文公开的所有siRNA可以以非配方形式作为裸siRNA给药，用于治疗本文所提及的任何疾病或病状。术语“裸siRNA”指的是siRNA分子不带有任何递送载体，所述载体用于辅助、促进或有利于进入到细胞，其包括病毒序列、病毒质粒、脂质体配方、脂质体转然试剂、或沉淀剂等。例如，siRNA在PBS中为“裸siRNA”。All siRNAs disclosed herein can be administered over-the-counter as naked siRNA for the treatment of any of the diseases or conditions mentioned herein. The term "naked siRNA" refers to siRNA molecules without any delivery vehicle that aids, facilitates, or facilitates entry into cells, including viral sequences, viral plasmids, liposome formulations, liposome-transferred Reagents, or precipitants, etc. For example, siRNA is "naked siRNA" in PBS.
另外，给药裸siRNA、寡核苷酸、裸siRNA的组合或裸siRNA与另外的分子的组合的任一种来治疗本文提及的任何疾病或病状也在本发明的范围内。In addition, it is also within the scope of the invention to administer any of naked siRNA, oligonucleotides, a combination of naked siRNA, or a combination of naked siRNA with another molecule to treat any of the diseases or conditions mentioned herein.
然而，如本文所述，本发明的siRNA分子也可以在脂质体配方和脂质体转染剂配方等等中递送，和通过本领域技术人员已知的方法制备。这类方法例如公开于US专利号5,593,972；5,589,466和5,580,859，将这些文件以全文引入的方式加入本文。However, as described herein, siRNA molecules of the invention can also be delivered in liposomal formulations, lipofectamine formulations, and the like, and prepared by methods known to those skilled in the art. Such methods are disclosed, for example, in US Patent Nos. 5,593,972; 5,589,466 and 5,580,859, which are incorporated herein by reference in their entirety.
实施例11Example 11
顺铂诱发的耳毒性的活体外模型及RTP801 siRNA的保护作用An in vitro model of cisplatin-induced ototoxicity and the protective effect of RTP801 siRNA
顺铂诱发的耳毒性诱发于耳蜗及前庭器官型培养基中。将出生后3-4天的大鼠幼崽用于制备如Zhang等人，Neuroscience 120(2003)191-205中所详述的耳蜗及前庭器官型培养基。小心切开耳蜗且移除含有螺旋神经节、考蒂器官及中转的耳蜗组织以制备如Zhang等人中所述的器官型培养基。在治疗前将培养基置于CO2培育器中24h以进行调节。在调节后，单独用1、5或10μg/ml的顺铂将培养基处理48h。为研究RTP801 siRNA的保护作用，用10μg/ml的顺铂及具有或不具有Lipofectamine的不同浓度的RTP801 siRNA处理耳蜗外植体(每组5个耳蜗)48h。未经处理的对照培养基及用RTP801 siRNA处理48h的培养基平行进行实验。实验结束时，将培养基用10％福尔马林固定且用FITC结合的鬼笔环肽(phalloidin)染色。计数每0.25mm耳蜗长度之内毛细胞及外毛细胞的数目且测定每次治疗的毛细胞平均数目。Cisplatin-induced ototoxicity was induced in cochlear and vestibular organotypic media. Postnatal day 3-4 rat pups were used to prepare cochlear and vestibular organotypic media as detailed in Zhang et al., Neuroscience 120 (2003) 191-205. The cochlea was carefully dissected and cochlear tissue containing the spiral ganglion, organ of Coatie, and transit was removed to prepare organotypic medium as described in Zhang et al. The medium was conditioned by placing it in aCO incubator for 24 h before treatment. After conditioning, the medium was treated with cisplatin at 1, 5 or 10 μg/ml alone for 48 h. To study the protective effect of RTP801 siRNA, cochlear explants (5 cochleae per group) were treated with 10 μg/ml cisplatin and different concentrations of RTP801 siRNA with or without Lipofectamine for 48 h. The untreated control medium and the medium treated with RTP801 siRNA for 48 hours were tested in parallel. At the end of the experiment, the medium was fixed with 10% formalin and stained with FITC-conjugated phalloidin. The number of inner and outer hair cells per 0.25 mm of cochlear length was counted and the average number of hair cells per treatment was determined.
实施例12Example 12
庆大霉素诱发的耳毒性的活体内模型及RTP801 siRNA的保护作用In vivo model of gentamicin-induced ototoxicity and the protective effect of RTP801 siRNA
将南美栗鼠(chinchilla)用单独硫酸庆大霉素或与RTP801 siRNA组合的硫酸庆大霉素处理5天(125-300mg/kg，肌肉内)。在暴露于庆大霉素之前两天及暴露于庆大霉素期间，将siRNA分子局部给药于耳蜗的圆窗膜上。在治疗结束时，藉由使动物暴露于二氧化碳及断颈来杀死动物。移除耳蜗且预备用FITC结合的鬼笔环肽染色，以测定耳蜗组织中内毛细胞及外毛细胞的数目(如Ding等人，Hearing Research 164(2002)115-126；及Wang等人，The Journal of Neuroscience23(24)：8596-8607中所述)。Chinchillas were treated with gentamicin sulfate alone or in combination with RTP801 siRNA for 5 days (125-300 mg/kg, intramuscular). The siRNA molecules were administered topically on the round window membrane of the cochlea two days before and during the exposure to gentamicin. At the end of treatment, animals were sacrificed by exposing them to carbon dioxide and neck dislocation. The cochlea was removed and prepared for staining with FITC-conjugated phalloidin to determine the number of inner and outer hair cells in cochlear tissue (e.g. Ding et al., Hearing Research 164 (2002) 115-126; and Wang et al., described in The Journal of Neuroscience 23(24):8596-8607).
实施例13Example 13
声创伤的活体内模型及RTP801siRNA的保护作用An in vivo model of acoustic trauma and the protective effect of RTP801 siRNA
将经染色的豚鼠用于声创伤研究中(Wang等人，The Journal ofNeuroscience 23(24)：8596-8607中所述)。经7天的时期将RTP801siRNA分子局部给药于耳蜗的圆窗膜上。未经治疗的左耳蜗充当声创伤引起耳毛细胞损失及听力功能损失的效力的对照。将动物暴露于声创伤(6kHz，120dB声压级(SPL)，30min)，且双耳的听力图均得自经由慢性圆窗膜电极自耳神经记录的复合动作电位(CAP)。每日在苏醒动物中测量双耳的CAP听力图。声暴露后30d，将动物杀死，且准备其耳蜗以量化评估毛细胞损失，其中使用扫描电子显微镜以计数全长耳蜗管的所有毛细胞。Stained guinea pigs were used in acoustic trauma studies (described in Wang et al., The Journal of Neuroscience 23(24):8596-8607). RTP801 siRNA molecules were administered topically to the round window membrane of the cochlea over a period of 7 days. The untreated left cochlea served as a control for the efficacy of acoustic trauma to induce otic hair cell loss and loss of hearing function. Animals were exposed to acoustic trauma (6 kHz, 120 dB sound pressure level (SPL) for 30 min) and audiograms of both ears were obtained from compound action potentials (CAP) recorded from the ear nerve via chronic round window membrane electrodes. CAP audiograms were measured daily in both ears in awake animals. 30d after acoustic exposure, animals were sacrificed and their cochleae were prepared for quantitative assessment of hair cell loss using scanning electron microscopy to count all hair cells in the full length cochlear duct.
实施例14Example 14
与8011(表D中ID No.257，SEQ ID NO：527(反义链))及8014(表D中ID No.260，SEQ ID NO：530(反义链))相关的实验结果Experimental results related to 8011 (ID No.257 in Table D, SEQ ID NO:527 (antisense strand)) and 8014 (ID No.260 in Table D, SEQ ID NO:530 (antisense strand))
HEK293细胞中的活体外功效In vitro efficacy in HEK293 cells
将5、10及20nm的三种不同浓度siRNA转染至HEK293细胞中。将相同浓度的不相关siRNA(siTG)用作负性对照，而将相同浓度的REDD14用作正性对照。转染后72小时收集细胞；使RNA经纯化且将其用作qPCR反应的模板以量化测定内源性RTP801转录含量。将人类亲环素A(Cyclophilin A)用作qPCR的内部参考。使参考标准化的数据进一步经受生物标准化：对各siRNA浓度而言，所检测的RTP801含量表示为RTP801在相应负性对照样品中的百分比。结果显示于图28中。Three different concentrations of siRNA at 5, 10 and 20 nm were transfected into HEK293 cells. The same concentration of irrelevant siRNA (siTG) was used as a negative control, while the same concentration of REDD14 was used as a positive control. Cells were harvested 72 hours after transfection; RNA was purified and used as template for qPCR reactions to quantify endogenous RTP801 transcript levels. Human Cyclophilin A was used as an internal reference for qPCR. Reference normalized data were further subjected to biological normalization: for each siRNA concentration the detected RTP801 content was expressed as a percentage of RTP801 in the corresponding negative control sample. The results are shown in Figure 28.
BE2C细胞中的活体外功效In vitro efficacy in BE2C cells
虽然在HEK293细胞中分析基本RTP801表达水平，但为分析siRNA减少诱发的RTP801表达水平的能力，使用经由作为RTP801诱发剂的CoCl2处理的BE2C细胞。设计及数据评估基本上如上所述。siRNA转染后24小时，将BE2C细胞用10μM CoCl2另外处理24小时，且接着进行RNA萃取。结果显示于图29中。While basal RTP801 expression levels were analyzed in HEK293 cells, to analyze the ability of siRNAs to reduce induced RTP801 expression levels, BE2C cells treated with CoCl2 as an RTP801 inducer were used. Design and data evaluation were essentially as described above. 24 hours after siRNA transfection, BE2C cells were treated with 10 μM CoCl2 for an additional 24 hours, and then subjected to RNA extraction. The results are shown in Figure 29.
结果表明在活体外检定中，801_1及801_4至少如同REDD14一样有效对抗RTP801。The results indicated that 801_1 and 801_4 were at least as effective against RTP801 as REDD14 in an in vitro assay.
小鼠CNV模型中的活体内功效In vivo efficacy in a mouse CNV model
伴随下列实验组，如实施例6中所述进行实验：The experiment was carried out as described in Example 6 with the following experimental groups:
表4Table 4
结果呈现于图30中，且显示在小鼠CNV的活体内模型中801_4至少如同REDD14一样有效。The results are presented in Figure 30 and show that 801_4 is at least as effective as REDD14 in an in vivo model of mouse CNV.
实施例15Example 15
与耳聋相关的模型及结果Models and results related to deafness
例示性(p53)siRNA治疗对南美栗鼠耳蜗中声诱发的毛细胞死亡的影响Effect of Exemplary (p53) siRNA Treatment on Acoustic-Induced Hair Cell Death in the Chinchilla Cochlea
在南美栗鼠中研究例示性siRNA(QM5-抗p53)在声创伤模型中的活性。使一组7只动物经受声创伤。将动物暴露于集中在4kHz下的105dB噪声的倍频带中历经3h。将暴露于噪声的南美栗鼠的左耳用约20μL中的30μg siRNA处理；用媒剂处理右耳。在声创伤后2.5周测定自圆窗记录的复合动作电位的平均阈值，以测定经siRNA治疗的耳中的阈值是否低于(优于)未经治疗(媒剂)的耳中的阈值。与未经治疗的耳相比，经siRNA治疗的耳中平均阈值较低。4kHz下的差异在统计学上显著(p<0.033)。这类结果表明给药于耳蜗的圆窗的例示性p53siRNA能够减少藉由声创伤引起的损害。The activity of an exemplary siRNA (QM5-anti-p53) in an acoustic trauma model was studied in chinchillas. A group of 7 animals were subjected to acoustic trauma. Animals were exposed for 3 h to an octave band of 105 dB noise centered at 4 kHz. The left ear of the noise-exposed chinchilla was treated with 30 μg siRNA in approximately 20 μL; the right ear was treated with vehicle. Mean thresholds of compound action potentials recorded from the round window were determined 2.5 weeks after acoustic trauma to determine whether thresholds in siRNA-treated ears were lower (better) than those in untreated (vehicle) ears. Mean thresholds were lower in siRNA-treated ears compared to untreated ears. The difference at 4 kHz was statistically significant (p<0.033). These results indicate that exemplary p53 siRNA administered to the round window of the cochlea can reduce damage caused by acoustic trauma.
p53或801siRNA治疗对大鼠耳蜗中顺铂诱发的毛细胞死亡的影响Effect of p53 or 801 siRNA treatment on cisplatin-induced hair cell death in rat cochlea
在顺铂治疗前，关于敲击信号8、16及32kHz来测试雄性Wistar大鼠的基本听觉脑干反应阈值。在基本听觉脑干反应测试后，经30分钟以腹膜内灌注13mg/kg来给药顺铂。经治疗的耳接收直接施加至圆窗膜的每4毫升15μg例示性p53 siRNA(如上文)或RTP801 siRNAREDD14(表A中序列No.14，SEQ ID No.16(有义)及66(反义))。对照的耳经非相关GFP siRNA或PBS治疗。在顺铂给药之前3-5天给药siRNA分子以检测对耳蜗的保护作用。The basal auditory brainstem response thresholds of male Wistar rats were tested with respect to tap
在顺铂给药之后3天重复听觉脑干反应测试。比较治疗前与治疗后的听觉脑干反应阈值且阈值的改变在表5中加以说明。顺铂治疗后阈值的更高改变表明耳蜗中的毛细胞损失更严重。在重复听觉脑干反应测试后，将动物杀死且将耳蜗移除且处理以用于扫描电子显微镜(SEM)，从而量化钩状区(高频区)中的外毛细胞(OHC)损失。藉由用照片区域中损失或严重损害的细胞数目除以外毛细胞的总数目来计算外毛细胞损失的百分比。The auditory brainstem response test was repeated 3 days after cisplatin administration. The auditory brainstem response thresholds before and after treatment were compared and the changes in thresholds are illustrated in Table 5. Higher changes in threshold after cisplatin treatment indicated greater hair cell loss in the cochlea. After repeating the auditory brainstem response test, the animals were sacrificed and the cochlea was removed and processed for scanning electron microscopy (SEM) to quantify outer hair cell (OHC) loss in the uncinate region (high frequency region). The percentage of outer hair cell loss was calculated by dividing the total number of outer hair cells by the number of lost or severely damaged cells in the photographed area.
表5证实自经受顺铂诱发的损害的4只动物获得且分析钩状区中外毛细胞损失的结果。如结果所揭示，接收对抗p53或801的siRNA的动物显示较低外毛细胞损失且关于32kHz信号显示阈值的较小改变。两个参数均表明对抗p53或801的siRNA可具保护性以对抗耳蜗中顺铂诱发的损害。Table 5 demonstrates the results obtained from 4 animals subjected to cisplatin-induced lesions and analyzed for loss of outer hair cells in the uncinate zone. As revealed by the results, animals receiving siRNA against p53 or 801 showed lower outer hair cell loss and a smaller change in threshold with respect to the 32 kHz signal. Both parameters suggest that siRNA against p53 or 801 may be protective against cisplatin-induced damage in the cochlea.
表5：经顺铂治疗的大鼠耳蜗中毛细胞损失与阈值改变Table 5: Hair cell loss and threshold changes in the cisplatin-treated rat cochlea
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