CN100546646C - 含有lk8蛋白作为活性成分的抗癌剂 - Google Patents
含有lk8蛋白作为活性成分的抗癌剂 Download PDFInfo
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- CN100546646C CN100546646C CNB2004800048895A CN200480004889A CN100546646C CN 100546646 C CN100546646 C CN 100546646C CN B2004800048895 A CNB2004800048895 A CN B2004800048895A CN 200480004889 A CN200480004889 A CN 200480004889A CN 100546646 C CN100546646 C CN 100546646C
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Abstract
本发明涉及一种包含LK8蛋白作为活性成分的抗癌剂。本发明的抗癌剂有效抑制恶性肿瘤的生长和转移。因此，该药剂不仅可有效用于癌转移的抑制剂，也可有效用作原发性恶性肿瘤的治疗剂。
Description
发明领域
本发明涉及一种含某蛋白作为活性成分的抗癌剂，更明确地，涉及一种抗癌剂，该药剂含一种与载脂蛋白(a)的半胱氨酸卷曲区(kringle)KV38相对应的蛋白作为活性成分。
背景
肿瘤由失控的混乱的异常的细胞增殖发展而成。如果肿瘤显示出破坏性的生长、入侵和转移，则被认为是恶性肿瘤。入侵性是一种浸润或破坏周围组织的特性，尤其，形成组织边界的基底层被这种特性破坏，导致局部扩散及有时肿瘤通过循环系统流入。转移指肿瘤细胞从原发病灶通过淋巴管或血管向其它区域的扩散。广义上讲，转移性也指肿瘤细胞通过腹腔或其它空间直接扩散。
迄今为止，外科手术、放射性治疗和化学治疗已单独或联合应用于癌症治疗。外科手术是除去患病组织的一种方法。这样，特定区域内的肿瘤，如乳房、结肠和皮肤，可由外科手术有效去除。然而，脊椎内的肿瘤或分散性肿瘤如白血病则不能通过外科手术进行适当的治疗。
化学治疗阻断了细胞复制或代谢，并已被应用于乳腺癌、肺癌和睾丸癌的治疗。然而，接受化学治疗的癌症患者严重遭受了全身化疗的副作用的痛苦。其中普遍但严重的例子是晕动病和呕吐。化学治疗的副作用甚至会影响患者的生活，因为这些副作用可能使患者的适应性迅速降低。另外，剂量限制毒性(DLT)也是化学治疗的一个主要副作用，它引起了对药品服用的仔细的关注。粘膜炎就是针对抗癌药的DLT的一个例子，这些抗癌药如抗代谢细胞毒剂5-氟尿嘧啶、甲氨蝶呤、以及抗癌抗生素如多柔比星等。如果患者受化疗副作用影响严重，他或她应该住院并被施以镇痛剂以减轻痛苦。所以，化学治疗或放射性治疗的副作用是癌症患者治疗中的最大问题。
因此，急需开发一种源于生物体内的抗癌剂以减轻化学治疗的副作用。特别地，在一种生物体内产生的物质中，发现了一种有希望的物质，它不直接攻击癌症细胞，但通过对众多辅助癌细胞生长的内皮细胞发生作用而阻止癌细胞生长。所以，含此物质的抗癌剂不仅可以治疗癌症还可以阻止转移。
Kringle是一种包含80个氨基酸和三个分子内二硫键的蛋白结构。Kringle结构在许多蛋白中都有发现，如凝血酶原(Walz，D.A.等，Proc.Natl.Acad.Sci.，74：1069-1073，1977)、尿激酶(Pennica，D.等，Nature，301：579-582，1983)、肝细胞生长因子(Lukker，N.A.等，Protein Eng.，7：895-903，1994)和载脂蛋白(a)(下文中称为’apo(a)’)(McLean，J.W.等，Nature，330：132-137，1987)。Kringle包含一个单独的折叠单元。Kringle的功能尚未完被清楚阐明。
Apo(a)包含两种kringle域，KIV和KV，和一个无活性的蛋白酶样区域。Kringle域KIV根据氨基酸同源性分为10个亚型(KIV-1～KIV-10)，15～40个拷贝数的该域发现于许多人apo(a)基因的等位基因中。Apo(a)通过与apo B-100共价结合形成一种脂蛋白(a)(下文中称为’Lp(a)’)，apo B-100是低密度脂蛋白(LDL)的一种主要蛋白成分(Fless，G.M.，J.Biol.Chem.，261：8712-8717，1986)。细胞质中Lp(a)的增加本身就是动脉粥样硬化的主要风险因子(Armstrong，V.W.et al.，Artherosclerosis，62：249-257，1986；Assmann，G.，Am.J.Cardiol.，77：1179-1184，1996)。
基于动脉粥样硬化和癌细胞生长依赖于血管发生的事实，本发明人研究了KV38的抗癌活性，KV38是人apo(a)kringle结构的一种。结果显示，本发明人已完成了本发明，证实此蛋白可有效用作一种抗癌剂，这是由于其通过一种bFGF样的内源生长因子抑制了血管生成，血管生成在癌细胞生长中是必需的。
发明概述
本发明的一个目的是提供一种包含人apo(a)kringle KV38(下文中称为LK8蛋白)作为活性成分的抗癌剂。
优选实施方案详述
为了实现本发明的上述目的，本发明提供一种包含LK8蛋白作为活性成分的抗癌剂。
下文中，本发明被详述。
本发明的抗癌剂的特征在于含有具有SEQ.ID.No.1所示氨基酸序列的LK8蛋白作为活性成分。优选使用此药剂作为转移抑制剂，更优选地，其被应用于抑制结肠癌或直肠癌向肝的轩移。
另外，该抗癌剂优选用于原发性肿瘤的治疗。更优选地，此药剂用于治疗从由前列腺癌、肺癌、结肠癌和直肠癌的组中选择的癌症。
本发明中抗癌剂优选地含有0.1～100mg/kg LK8蛋白，更优选地含1～50mg/kg LK8蛋白，施药次数为每天1～4次。但组成不限于上述，并可能根据病人情况、疾病的种类和进展速度而改变。
本发明中，“KV38”指一种apo(a)kringle，“LK8”指KV38的重组蛋白。然而，除非特别声明，KV38和LK8蛋白都通称为LK8蛋白。
本发明中的LK8蛋白是一个与许多apo(a)kringle域的KV38kringle相对应的域，通过体外以及体内抑制内皮细胞的活性而具有对癌细胞增殖和分化以及转移的抑制效应。如本发明优选实施方案所述，LK8蛋白的全身用药导致了对原发性肿瘤及其转移的抑制(见图2～图6)。因此，由于其抑制肿瘤生长和转移的功能，本发明中的LK8蛋白可有效用作一种针对原发性肿瘤的抗癌剂及一种转移抑制剂。
如果本发明中LK8蛋白与常规化学治疗或放射治疗共同使用则其治疗效果将被增强。放射性治疗优选破坏原发性肿瘤，如果在放射性治疗中施用LK8蛋白，可更有效的阻止转移。对于化学治疗，大剂量化学抗癌剂产生的细胞毒性是最大的问题。如果在化学治疗中施用本发明的LK8蛋白，降低了剂量的化学抗癌剂将带来相同甚至改善了的抗癌效果并减轻了细胞毒性。
总之，如果施用LK8与外科手术、放射性治疗、化学治疗或免疫治疗同时进行，治疗效果将被最大化。更进一步，LK8蛋白的连续施用延长了微转移的休眠，抑制了原发性肿瘤的生长并稳定了病情。许多常规抗癌剂被设计用于长期服用，导致诸如连续生产蛋白和产品的高价等问题。它们的替代者是基因治疗。如果用于基因治疗，我们仍然预期LK8蛋白能使抗癌剂或转移抑制剂的效果最大化。
本发明含LK8蛋白的抗癌剂可口服或肠胃外给药并用于药物制剂的一般形式。
通过与通用的填充剂、膨胀剂、粘合剂、湿润剂、崩解剂、稀释剂如表面活性剂、或赋形剂相混合，本抗癌剂可制备用于口服或肠胃外给药。口服给药的固体制剂包括片剂、丸剂、散剂、粒剂或胶囊。这些固体制剂通过混合一种或多种合适的赋形剂制备，如淀粉、碳酸钙、蔗糖或乳糖、明胶等。除了简单的赋形剂，也可使用硬脂酸镁、滑石等润滑剂。口服给药的液体制剂为混悬剂、溶液、乳剂和糖浆，上述制剂在通用的稀释剂如水和液体石蜡外可含有多种赋形剂如湿润剂、甜味剂、芳香剂和防腐剂。肠胃外给药制剂为无菌的水溶液、水不溶性赋形剂、混悬剂、乳剂和栓剂。水不溶性赋形剂和混悬剂可含有，除一种或多种活性化合物外，丙二醇、聚乙二醇、植物油如橄榄油、可注射酯如油酸乙酯等。栓剂可含有，除一种或多种活性化合物外，witepsol、聚乙二醇、吐温61、可可脂、月桂脂、甘油明胶等。
LK8蛋白的LD50约为1,000mg/kg，说明本发明的抗癌剂非常安全(见表2)。
附图简述
本发明中的优选实施方案参考附图可被最好理解，其中：
图1是LK8基因的表达载体‘pMBRI-LK8(8.25kb)’的示意图，其中LK8cDNA(261bp)插入于AOX1启动子和AOX1终止子之间。
图2是一组照片和一幅图，显示了小鼠(C57BL/6)尾静脉中注射的鼠黑素瘤细胞系B16F10的肺部转移被LK8蛋白的治疗所抑制。
(a)只用PBS处理的小鼠肺部。
(b)用1mg/kg LK8蛋白处理的小鼠肺部。
(c)图显示B16F10细胞向小鼠肺部的转移被LK8蛋白处理所抑制。
图3是一组照片和一幅图，显示了植入小鼠脾脏的结直肠癌细胞系CT-26向肝的转移被LK8蛋白处理所抑制。
(a)经PBS(对照)处理和LK8蛋白(10mg/kg/天)处理的小鼠肝脏。
(b)图显示转移入用PBS(对照)处理和LK8蛋白(10mg/kg/天)处理的小鼠肝脏中的CT-26细胞的克隆数。
(c)照片显示转移入用PBS(对照)处理和LK8蛋白(10mg/kg/天)处理的小鼠肝脏中的CT-26癌细胞的分布，用苏木精&曙红染色。
图4是一组图显示随着LK8蛋白的施用，植入人前列腺癌PC-3细胞的小鼠内肿瘤尺寸的变化。
(a)图显示用100mg/kg/天LK8蛋白处理后肿瘤尺寸的变化。
(b)图显示用50mg/kg/天LK8蛋白处理后肿瘤尺寸的变化。
图5是一幅图显示随着LK8蛋白的施用，植入人肺癌A549细胞的小鼠内肿瘤尺寸的变化。
图6是一组图显示随着LK8蛋白的施用，植入人直肠结肠癌LS174T细胞的小鼠内肿瘤尺寸(a)和肿瘤重量(b)的变化。
实施例
本发明的实际的和目前优选的实施方案例示于下述实施例中。
然而，本领域内的技术人员应当理解，由于本公开，可在本发明的精神和范围内做出修改和改进。
实施例1：LK8蛋白制备
<1-1>LK8表达载体pMBRI-LK8的构建
为了有效制备LK8蛋白，本发明人首先构建了LK8表达载体。
为了表达LK8基因，pPIC9载体(8.0kb，Invitrogen，Netherland)被用作基础载体。如示意图所示，pPIC9表达载体顺序包含可提供甲醇诱导的高表达的AOX1启动子、可使被表达蛋白分泌的α-因子分泌信号、3’AOX1(TT)、可使转录有效终止并多腺苷酸化的AOX1多腺苷酸化信号、以及一个编码野生型Pichia pastoris组氨醇脱氢酶的DNA片断用作上述菌株转化子的选择标记。
首先，LK8基因以SEQ.ID.No 2中所示引物‘LK8N-Xhol’和SEQ.ID.No 3中所示引物‘LK8C-EcoRI’进行PCR扩增，以pET15b/LK8(见PCT/KR99/00554)为模板。PCR产物用限制性内切酶XhoI和EcoRI消化，然后将消化产物插入用相同限制性内切酶消化过的pPIC9载体中。最后，构建成为LK8基因表达载体‘pMBRI-LK8(8.25kb)(图1)。
<1-2>含pMBRI-LK8的转化子的制备
Pichia pastoris，一种甲基营养型酵母，用作制备重组转化子的宿主。
具体地，LK8基因表达载体‘pMBRI-LK8’用限制性内切酶SacI处理，形成线性。此载体通过同源重组插入上述宿主菌株染色体的AOXI基因中。此时，用电穿孔法进行转化。从组氨酸-缺乏培养基中通过检查菌落形成挑选重组酵母转化子。用PCR验证LK8cDNA是否已经插入所选重组转化子的AOX1区。然后，重组转化子经过培养并通过甲醇诱导LK8基因的表达。结果，LK8基因的表达被验证，说明该蛋白在培养基中被大量分泌。
本发明中的分泌LK8蛋白由SEQ.ID.No 1所示氨基酸序列组成。
<1-3>重组菌株的培养
<1-3-1>种子培养
本发明中，通过将LK8基因插入Pichia pastoris得到一个重组菌株。所构建的菌株进行24小时的种子培养以得到适量的生物量及活性(当稀释20倍时，OD600为0.8-1.2)。
种子培养在YDP培养基(1％酵母浸出物，2％蛋白胨，2％葡萄糖)中进行24小时振荡培养。使用75L发酵罐。初始培养基体积为20L，通过补料分批培养法将培养物终体积调节至40L。
<1-3-2>主培养
在YPD培养基中完成种子培养后，以种子培养基作为初始培养基的30％进行主培养。主培养在含有如下表1所示成分的发酵罐中进行。当发酵被甲醇供应所饱和时，回收10％以上的发酵物以生产LK8蛋白。同时，甲醇被连续加入以诱导蛋白的持续表达。重复此过程以生产LK8蛋白。碳源的消耗速率与细胞的数量成正比。所以，当发酵物的一部分被回收时，甲醇的供应速度自动在±20％内进行调节。通过上述培养过程的重复，其中发酵连续进行200小时以上，得到了分泌的250mg/L LK8的蛋白培养液。
<表1>
主培养的培养基组成
| 培养基种类 | 成分 | 浓度 |
| 主培养基 | 甲醇微量金属溶液 | 500g/L8ml/L |
| 微量金属溶液培养基组成 | CuSO<sub>4</sub>·5H<sub>2</sub>OKIMnSO<sub>4</sub>·H<sub>2</sub>ONaMo·H<sub>2</sub>OH<sub>3</sub>BO<sub>4</sub>CoCl<sub>2</sub>ZnCl<sub>2</sub>FeSO<sub>4</sub>·H<sub>2</sub>O生物素H<sub>2</sub>SO<sub>4</sub> | 4g/L0.3g/L4g/L0.1g/L0.01g/L0.1g/L3g/L10g/L0.1g/L5ml/L |
实施例2：通过静脉注射B16F10鼠黑素瘤细胞进行肺转移实验
B16F10细胞(1.8×105)，小鼠黑素瘤(以下称为’黑素瘤细胞’)(American Type Culture Collection)，尾静脉注射于C57BL/6小鼠(Charles River Japan，Inc.)。从第二天起，将LK8蛋白，由上述<实施例1>制备，连续14天每天两次皮下注射(1mg/kg/天，0.2mg/kg/天)。对照组注射PBS而不是LK8蛋白。在细胞植入的第13天，切开小鼠取肺进行转移了的癌细胞(黑素瘤细胞)克隆计数。
结果，注射了PBS的对照小鼠组的肺中形成了巨大的克隆，说明黑素瘤细胞的转移(图2)。相反，注射了LK8蛋白的实验小鼠组中因黑素瘤细胞转移而形成的克隆的数量和尺寸小得多和少得多(图2b)。总之，与对照组相比，1mg/kg的LK8蛋白处理显示了53％的转移抑制(图2c)。
实施例3：通过脾内施用小鼠结直肠癌细胞CT-26进行肝转移实验
CT-26细胞(American Type Culture Collection)，小鼠结直肠癌细胞，被注射入脾内以诱导向肝的转移。然后，研究了LK8蛋白的转移抑制效果。特别地，CT-26细胞，在平板上长至80％成熟，用PBS洗涤，然后用0.02％EDTA分散。单细胞再次用PBS洗涤，然后小心重悬于PBS。悬液用锥虫蓝染色以细胞计数。将细胞密度调至5×105/ml，每只小鼠注射100μl。所用为6～8周龄BALB/c小鼠(CharlesRiver Japan，Inc.)。手术切开腹部右侧后，用30号针头将癌细胞悬液小心注射入脾脏。LK8蛋白皮下注射入实验组，一天两次，10mg/kg/天，类似地，将盐水注入对照组。14天后，处死小鼠检测其肝脏(图3a)，计数肝表面所见的克隆数量，其已用10％的福尔马林溶液固定。各组的重量无大的差异。然而，施以10mg/kg/天LK8蛋白的实验组中转移到肝脏中的克隆数量比对照组要少得多。注射了10mg/kg/天LK8蛋白的实验组中克隆数量与对照组相比降低约60％(图3b)。另外，福尔马林固定的肝组织切片用H&E染色进行观察。结果，用LK8处理的实验组中肿瘤区域比对照组有限得多(图3c)。
实施例4：原发性肿瘤的生长抑制
为了研究LK8蛋白对体内血管生成抑制效应，采用了一种异种移植肿瘤模型。各相关实验采用4周龄随机交叉雌性Balb/c nu/nu裸鼠(Charles River Japan，Inc.)，喂养于无菌条件下。
<4-1>人前列腺癌细胞(PC-3)
人前列腺癌PC-3细胞(American Type Culture Collection)培养于补充了10％FBS的RPMI 1640培养基(GIBCOTM，InvitrogenCorporation)，然后约5×106PC-3细胞皮下注射入裸鼠背部中心肌肉区。植入后的整10天后，LK8蛋白以100mg/kg/天进行注射。其间，对照组只注射PBS而不是LK8蛋白。此处理持续30天，然后，每3或4天测量肿瘤的尺寸。结果显示，肿瘤的生长被LK8蛋白所抑制，与对照组相比有约60％的抑制(图4a)。当LK8蛋白以50mg/kg/天注射时，肿瘤生长以相似的方式被抑制，与对照相比有超过60％的抑制(图.4b)。
<4-2>人肺癌细胞(A549)
人肺癌A549细胞(American Type Culture Collection)培养于补充了10％FBS的DMEM培养基(GIBCOTM，Invitrogen Corporation)。然后1×107肿瘤细胞皮下注射入裸鼠背部的中心肌肉区。植入整5天后，以50mg/kg/天注射LK8蛋白。对照组只施用PBS而不是LK8蛋白。处理持续了46天，然后，每3或4天测量肿瘤尺寸。结果显示，肿瘤生长被LK8蛋白的治疗所抑制，与对照组相比抑制了61％(图5)。
<4-3>人结直肠癌细胞(LS 174T)
人结直肠癌LS 174T细胞(American Type Culture Collection)培养于补充了10％FBS的RPMI培养基(GIBCOTM，InvitrogenCorporation)。然后5×106肿瘤细胞皮下注射入裸鼠背部的中心肌肉区。植入整5天后，以50mg/kg/天注射LK8蛋白。对照组只施用PBS而不是LK8蛋白。处理持续了34天，然后，每3或4天测量肿瘤的尺寸。结果显示，肿瘤生长被LK8蛋白处理所抑制，与对照组相比有64％的抑制(图6a)。并且，实验最后一天测量显示，LK8蛋白处理使肿瘤的重量降低了68.7％(图.6b)
实施例5：LK8蛋白的急性毒性实验
5周龄SPF SD(Sprague Dawley)系大鼠被用于急性毒性的实验。将大鼠分为5组，这5组大鼠每组分别一次性通过静脉注射施以LK8蛋白260mg/kg，364mg/kg，510mg/kg，714mg/kg和1000mg/kg(表2)。14天后，观察大鼠中的实验材料、LK8蛋白、用药、死亡、临床症状和重量变化。进行血相检查和血液生化检查，尸检中用肉眼检查胃肠或胸腹部器官任何异常状况。结果显示，施以1000mg/kg LK8蛋白的组检测到微弱的毒性，但在其它组的大多数检测中既未发现毒性也未发生死亡，这些检测包括重量变化、血液检查、血相检查和血液生化检查、尸检等。因此，本实验中所用LK8蛋白被认为是安全的物质，因为其在大鼠内高至1,000mg/kg的水平时不会导致大鼠体内的任何毒性变化，而且其估计的LD50值在大鼠内远高于1,000mg/kg(表2)。
<表2>
LK8蛋白施用后随天数变化的死亡数量
工业应用
如上文所述，LK8蛋白具有对转移的抑制效应，尤其，当全身施用时，对人前列腺癌、肺癌、结肠癌和直肠癌的生长抑制。因此，包含本发明LK8蛋白的抗癌剂可有效用作原发性肿瘤的治疗剂或转移抑制剂。
本领域内的技术人员应当理解在前述的描述中公开的概念和特定的实施方案可容易地用作修改或设计其它实现与本发明相同目的的实施方案的基础。本领域内的技术人员还将理解这些等价的实施方案没有背离本发明在所附权利要求中所阐明的精神及范围。
序列表
<110>财团法人牧岩生命工学研究所
<120>含有LK8蛋白作为活性成分的抗癌剂
<130>PA054409
<140>200480004889.5
<141>2005-08-22
<150>KR2003-10797
<151>2003-02-20
<160>3
<170>Kopatentln 1.71
<210>1
<211>86
<212>PRT
<213>人
<400>1
Glu Gln Asp Cys Met Phe Gly Asn Gly Lys Gly Tyr Arg Gly Lys Lys
1 5 10 15
Ala Thr Thr Val Thr Gly Thr Pro Cys Gln Glu Trp Ala Ala Gln Glu
20 25 30
Pro His Arg His Ser Thr Phe Ile Pro Gly Thr Asn Lys Trp Ala Gly
35 40 45
Leu Glu Lys Asn Tyr Cys Arg Asn Pro Asp Gly Asp Ile Asn Gly Pro
50 55 60
Trp Cys Tyr Thr Met Asn Pro Arg Lys Leu Phe Asp Tyr Cys Asp Ile
65 70 75 80
Pro Leu Cys Ala Ser Ser
85
<210>2
<211>34
<212>DNA
<213>人工序列
<220>
<223>LK8N-Xhol引物
<400>2
tccgctcgag aaaagagaac aagactgtat gttt 34
<210>3
<211>31
<212>DNA
<213>人工序列
<220>
<223>LK8C-EcoRI引物
<400>3
cgaattctta agaggatgca cagagaggga t 31
Claims (8)
1.一种用于治疗癌症的药物组合物，所述癌症选自由前列腺癌、结肠癌、直肠癌和肺癌组成的组，其包含SEQ.ID.No 1所示的LK8蛋白作为有效成分。
2.如权利要求1中所述的药物组合物，其中LK8蛋白的有效剂量为0.1～100mg/kg。
3.如权利要求2中所述的药物组合物，其中LK8蛋白的有效剂量为1～50mg/kg。
4.用于抑制癌症转移的药物组合物，其包含SEQ.ID.No 1所示的LK8蛋白作为有效成分。
5.如权利要求4中所述的药物组合物，其中所述癌症选自结肠癌和直肠癌组成的组。
6.权利要求4的药物组合物，其中所述转移是黑素瘤转移到肺中的转移或结肠或直肠癌转移到肝脏中的转移。
7.SEQ ID NO：1所示的LK8蛋白在制备用于治疗癌症的药物中的应用，所述癌症选自由前列腺癌、结肠癌、直肠癌和肺癌组成的组。
8.SEQ ID NO：1所示的LK8蛋白在制备用于抑制癌症转移的药物中的应用，其中所述转移是黑素瘤转移到肺中的转移或结肠或直肠癌转移到肝脏中的转移。
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| KR1020030010797A KR100595364B1 (ko) | 2003-02-20 | 2003-02-20 | Ｌｋ8 단백질을 유효성분으로 포함하는 항암제 |
| KR1020030010797 | 2003-02-20 |
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| US7285277B2 (en) | 2002-03-15 | 2007-10-23 | Mogam Biotechnology Research Institute | Anticancer agent |
| KR20050090420A (ko) | 2002-12-26 | 2005-09-13 | 다케다 야쿠힌 고교 가부시키가이샤 | 메타스틴 유도체 및 이의 용도 |
| JP4660486B2 (ja) * | 2004-01-09 | 2011-03-30 | モガム バイオテクノロジー リサーチ インスティチュート | ヒトアポリポ蛋白質（ａ）クリングルＬＫ６８またはＬＫ８遺伝子を有効成分として含有する抗癌治療剤及びそれを使用した癌治療方法 |
| AR049938A1 (es) | 2004-06-25 | 2006-09-13 | Takeda Pharmaceutical | Derivados de metastina y utilizacion de los mismos |
| US8404643B2 (en) | 2005-12-22 | 2013-03-26 | Takeda Pharmaceutical Company Limited | Metastin derivatives and use thereof |
| AR058584A1 (es) | 2005-12-22 | 2008-02-13 | Takeda Pharmaceutical | Derivados de metastina y uso de los mismos |
| JO3048B1 (ar) | 2006-10-25 | 2016-09-05 | Takeda Pharmaceuticals Co | مشتقات متاستين واستخدامها |
| KR100888022B1 (ko) * | 2006-12-21 | 2009-03-09 | 재단법인 목암생명공학연구소 | 면역글로불린 Ｆｃ와 인간 아포리포단백질（ａ）크링글절편의 융합단백질 ＬＫ8Ｆｃ |
| ES2565512T3 (es) | 2007-07-19 | 2016-04-05 | bioMérieux | Procedimiento de ensayo de la proteína de unión a ácidos grasos del hígado, de ACE y de CA19-9 para el diagnóstico in vitro del cáncer colorrectal |
| FR2919064B1 (fr) * | 2007-07-19 | 2009-10-02 | Biomerieux Sa | Procede de dosage de l'apolipoproteine all pour le diagnostic in vitro du cancer colorectal |
| FR2919061B1 (fr) | 2007-07-19 | 2009-10-02 | Biomerieux Sa | Procede de dosage de la plastine-i pour le diagnostic in vitro du cancer colorectal. |
| FR2919062B1 (fr) | 2007-07-19 | 2009-10-02 | Biomerieux Sa | Procede de dosage de l'aminoacylase 1 pour le diagnostic in vitro du cancer colorectal. |
| FR2919063B1 (fr) | 2007-07-19 | 2009-10-02 | Biomerieux Sa | Procede de dosage du leucocyte elastase inhibitor pour le diagnostic in vitro du cancer colorectal. |
| FR2919060B1 (fr) | 2007-07-19 | 2012-11-30 | Biomerieux Sa | Procede de dosage de l'ezrine pour le diagnostic in vitro du cancer colorectal. |
| FR2919065B1 (fr) | 2007-07-19 | 2009-10-02 | Biomerieux Sa | Procede de dosage de l'apolipoproteine ai pour le diagnostic in vitro du cancer colorectal |
| FR2933773B1 (fr) | 2008-07-10 | 2013-02-15 | Biomerieux Sa | Procede de dosage de la proteine disulfide isomerase pour le diagnostic in vitro du cancer colorectal |
| WO2012067427A2 (ko) * | 2010-11-16 | 2012-05-24 | 재단법인 목암생명공학연구소 | Lk8 단백질을 유효성분으로 포함하는 당뇨망막병증 또는 노인성 황반변성의 예방 또는 치료용 약학 조성물 |
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| EP1608395A4 (en) | 2007-10-31 |
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| EP1608395A1 (en) | 2005-12-28 |
| AU2004212856A1 (en) | 2004-09-02 |
| KR100595364B1 (ko) | 2006-07-03 |
| JP2006518342A (ja) | 2006-08-10 |
| BRPI0407611A (pt) | 2006-02-14 |
| KR20040075270A (ko) | 2004-08-27 |
| AU2004212856B2 (en) | 2007-07-19 |
| RU2005129273A (ru) | 2006-03-10 |
| CN1753685A (zh) | 2006-03-29 |
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