CN101272694B - 新颖的乳蛋白组分及其用于预防或治疗慢性炎症疾病的应用 - Google Patents
新颖的乳蛋白组分及其用于预防或治疗慢性炎症疾病的应用 Download PDFInfo
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Abstract
本发明涉及富集TGF-β的乳蛋白组分、其制备方法、及其用于制备用来预防和/或治疗慢性炎症尤其是牛皮癣的药物和/或食品组合物的应用。
Description
技术领域
本发明的主题是一种新颖的富集TGF-β的乳蛋白组分、其制备方法、及其用于制备用来预防和/或治疗慢性炎症疾病尤其是牛皮癣的药物和/或食品组合物的应用。
背景技术
牛皮癣是一种慢性皮肤病症状,其特征为红斑-鳞屑状出疹,爆发式发展,主要出现在肘、膝和头皮部。
从生物学观点看,牛皮癣是一种慢性炎症过程,其特点在于角化细胞的非正常增殖和分化,与T淋巴细胞和多核中性粒细胞对真皮和表皮的渗透有关,这种渗透在角质层内形成微脓肿。
对牛皮癣在个体内出现的原因的了解还很少。这里列举一些促进牛皮癣出现的因素:
-遗传因素,
-心理因数(压力,激素变化,……),
-免疫紊乱。
最后,一些药物和细菌或病毒感染能够触发牛皮癣发作。
目前,还没有治愈牛皮癣的方法。目前已知的治疗方法仅仅能够延缓和/或削弱牛皮癣的症状。
对于仅限于少数斑点的牛皮癣,可开药维生素D,可选地局部联合施以类皮质激素。也可以局部施以类维生素A。
在极端严重的情况下,可采用光照疗法,或可选地用甲氨蝶呤或类维生素A以常规途径给药。后一治疗伴随着较大副作用。
因此目前还没有治疗牛皮癣的满意方法。
其他慢性炎症疾病,如变形性关节炎、骨关节炎、克罗恩氏(Crohn′s)病,多发性硬化、红斑狼疮,对从业者提出了同样的问题:还没有能够治愈这些病理症状的治疗方法,现存的治疗这些病理症状的治疗方法或者是不充分的,或者是具有非常严重的副作用。慢性炎症部分还存在于自身免疫疾病中。
众所周知,生长因子诸如趋化因子和细胞因子会对炎症过程产生影响。这些影响包括对炎症过程的增强和抑制。
尤其是,已知乳中特定的生长因子对炎症过程具有调节活性。
例如,文献WO 96/34614描述了将乳制品用于制备用以预防因化疗或放疗引起的消化道粘膜损害的药物。
最好采取使乳通过阳离子交换色谱柱来获得该提取物。它最好包含乳铁传递蛋白和/或乳过氧化物酶和生长因子,尤其是TGF-β。
TGF-β(转化生长因子)是肽生长因子,其能够调节细胞生长和分化。它是一个25kD的二聚分子,其可以以多种同种型存在:TGFβ1,TGFβ2和TGFβ3。TGF-β因其调节能力而为人所知,尤其是降低免疫和炎症反应的特定阶段方面(G.Prud’homme et al.,J.Autoimmun.(2000),14,23-42;M.Shull et al.,Nature(1992),359,693-699;J.Graycar et al.,Mol.Endocrinol.(1989),3,1977-1986;J.Letterio et al.,Annu.Rev.Immunol.(1998),16,137-161)。其以潜伏形式(非生物学活性)或活性形式存在。
它在乳中以微量(30μg/l乳)存在。然而,乳中存在众多其它活性不同于TGF-β的因子,它们的活性可能与TGF-β相反,其后果是,包含在乳中的TGF-β对炎症过程没有明显的作用。
此外,TGF-β从乳中的分离在工业规模上几乎不可行,首先因为成本考虑的原因,其次是因为分离过程经常导致靶蛋白质的变性,从而导致生产出的乳蛋白组分已丧失所含蛋白质的所有生物学性能。
文献EP 0 313 515描述了一种从乳中分离TGF-β的方法。然而,这一方法包含很多个阶段、复杂的特性,因此从经济观点看几乎是不可能的。
为获得在炎症过程中表现出生物活性(即对炎症过程的降低或抑制)的乳蛋白组分,需要能够从乳开始就增加TGF-β相对于其他蛋白质的含量,同时除去至少一些能够阻断TGF-β在乳中时的抗炎性能的拮抗因子。这一方法的难处在于耗时巨大,因为确切的TGF-β拮抗物是未知的。
几位作者集中于富集TGF-β的乳蛋白质组分的分离问题上。如专利申请和专利WO96/34614,EP 0 545 946,WO 01/25276,WO 03/008447,FR 2 827 240,EP 0 869 134。
文献EP 0 545 946描述了一种从乳清中提取生长因子的方法;该方法包括下述步骤:过滤乳清以去除不溶物,调整pH值在6.5-8之间,使用琼脂糖凝胶型阳离子交换树脂来吸收主要的碱性成分,洗脱主要的酸性蛋白质,用缓冲溶液平衡树脂,将滤液施加在树脂上,用高离子强度的缓冲溶液洗脱,过滤以除去盐,最后浓缩。
文献EP 0 869 134描述了一种从乳或乳衍生物中回收一种或多种生长因子的方法,通过吸附到阳离子交换剂上,分级洗脱,制得富集了生长因子的组分,然后在pH值3.5-4.5下处理。该方法最好在施加高表面速度和高液相负荷下进行。
文献WO 01/25276描述了一种提取TGF-β和IGF-1的方法,包括步骤:通过阳离子交换色谱法回收乳中碱性组分,使回收的组分通过羟磷灰石柱,用至少两种盐浓度增加的洗脱液洗脱以获得两个组分:
-富集IGF-1的组分,IGF-1/TGF-β>10
-富集TGF-β的组分,TGF-β/IGF-1>5,其相对于蛋白质总量含有30-50%的免疫球蛋白。
文献FR 2 827 240描述了一种从富含可溶性蛋白的乳水相溶液中制得活性形式的、富集TGF-β的蛋白质组分的方法,包括:a)将可溶性蛋白质浓度调节至5-30g/l,b)采用酸处理沉淀,c)微滤-渗滤,d)回收微滤渗余物,e)干燥。
文献WO 03/008447描述了一种分离生长因子TGF-β,IGF-1,乳过氧化物酶(lactoperoxidase)和免疫球蛋白。通过阳离子交换色谱法回收乳中的碱性蛋白组分。使获得的组分通过疏水性相互作用色谱分离。
然而,这些方法都不能在工业可行的条件下获得对慢性炎症病理学条件及其表现或症状、尤其是对牛皮癣具有真正效力的乳蛋白组分。
因此,就本申请人发现了对慢性炎症过程、尤其是对牛皮癣具有功效的新颖的乳蛋白组分而言,是出人意外的。
发明内容
本发明的乳蛋白组分可以采用包含下述步骤的方法获得:
乳或乳清用作起始材料。
(a)该乳或乳清可选地经过微滤或热处理;
(b)使来自步骤(a)的乳或乳清沉积在阳离子交换树脂上;
(c)用软化水冲洗该阳离子交换树脂;
(d)用浓度升高的盐水溶液洗脱该树脂;
(e)回收对应于电导率在21.0-22.0mS/cm之间的盐水溶液的洗脱液。
本发明的方法还可包含下述一个或多个步骤:
(f)通过超滤来浓缩(e)中获得的洗脱液,回收渗余物;
(g)通过微滤对(f)中获得的渗余物进行灭菌,回收渗透物;
(h)对(g)中获得的渗透物进行喷雾干燥。
作为根据本发明的方法中的起始材料,可以使用乳或者乳清,优选取自奶牛。乳清是从乳或酪乳中提取出蛋白质和脂肪之后得到的残液。通常能够区分出三种乳清。前两种是根据乳清的酸度划分,其可以是小于或大于1.8g乳酸/l;甜乳清,在制造蒸煮或不蒸煮压制乳酪中产生(Emmenthal,Saint-Paulin等),以及酸乳清,来源于酪蛋白或来源于混合得到的其他乳酪或乳胶凝(软干酪,清爽干酪(fromages frais))。甜乳清的平均组成(供参考)是,每kg乳清中61g干物质,42-48g乳糖,8g蛋白质,2g脂肪,5-7g矿物质,1-5g乳酸,其他为矿物质和维生素。
乳经由平均孔隙度为0.1μm的载体而微滤得到的理想乳清也是已知的。
根据本发明的第一变型,使用乳、尤其是牛乳作为起始材料,因其组成按照本发明的方法有可能获得具有更好生物学性能的蛋白分离物。这一变型还可以获得比乳清作为起始材料时更大的蛋白质产率。
根据本发明的第二种变型,酪蛋白制造中产生的酸乳清被用作起始材料。这一变型具有经济效益,因为该起始材料是工业开发的副产品,因此具有低成本。
作为起始材料的乳可以是牛乳、山羊乳、绵羊乳、水牛乳。并可以采用离心作用以已知的方式脱脂。
该乳或乳清经过处理,从而有可能去除细菌源的污染。为达到这一目的,优选使乳经过不超过68℃的热处理,或接受第一微滤步骤的处理,例如在孔隙率为约1.4μm的过滤器上进行。
然后,微滤的渗透物、或直接是乳或乳清,被沉积在离子交换树脂柱上。
上述方法中使用的树脂由聚阴离子纳米孔合成聚合物。聚合物优选为立体的球形或类球的形状。它以强酸阴离子的共轭碱的形式来载有该强酸型官能团。其优选被SO3 -官能团化。此外,制造该树脂的聚合物必须具备机械抗性,使其经得起因优选的洗脱参数导致的液压应力。
优选在步骤(b)中,柱的供给流速在10-15m3/h之间,线速度在2.8-4.5m/h之间。
在步骤(b)中,乳对树脂的体积比优选在100-200之间。
在乳或乳清沉积之后,使用软化水冲洗离子交换柱,优选采用10-15l水/l树脂。
然后使用盐浓度为10-14g/l、优选为11-13g/l的盐水溶液。
盐水的流速为1.5-2m3/h,盐水溶液的线速度优选为0.4-0.6m/h。盐水对树脂的体积比在2.5-3.5之间。
然后通过一个或更多超滤步骤、以及可选的一个或更多渗滤步骤浓缩,从而回收洗脱液。这些步骤优选在低温(2到6℃)下进行。
由此获得的渗余物经过微滤和喷雾干燥。
然而,还可以想到所属技术领域的专业人员熟知的其他脱矿质和浓缩的方法。
由此获得的乳蛋白组分具有如下特性:
-相对于蛋白质的总重量,TGF-β的含量在0.010-0.025wt%之间;
-相对于蛋白质的总重量,IgG(免疫免疫球蛋白)的含量<25wt%;
-TGF-β/IGF1比率≥5w/w。
最好是,它们还具有下述一个或多个特性:
-相对于蛋白质的总重量,乳过氧化物酶的含量在35-45wt%之间。
这些乳蛋白组分构成了本发明的另一个主题。
令人意外的是,在对试验小鼠的脾脏细胞进行评价时发现,本发明的乳蛋白组分导致了淋巴细胞增殖的下降:使用氯化铵对这些细胞进行处理并经过冲洗,在作为本发明主题的蛋白质组分和促有丝分裂剂如伴刀豆球蛋白A的存在下,培养48小时,并在培养的末期往细胞培养基中添加5-溴脱氧尿苷。通过进入到细胞中的5-溴脱氧尿核苷的量来评价淋巴细胞增殖。
了解到患牛皮癣的表皮是活化T淋巴细胞流入的处所,作为本发明主题的蛋白质组分可以用来预防或治疗牛皮癣,以及其他状况如慢性炎症病理学状况和/或它们的症状。这些病理学状况中,有牛皮癣,还可以提及变形性关节炎、骨关节炎、克罗恩氏病、多发性硬化、红斑狼疮。此外,它们对自身免疫疾病的炎症部分有功效。因此它们能够用于自身免疫性疾病的预防和/或治疗,尤其是自身免疫性疾病的炎症部分的预防和/或治疗。
此外,本发明的方法易于大规模实施,操作方便且仅包含有限个步骤。
本发明的方法的另一个优点是有可能回收必然具有产业利益的其它乳蛋白组分。通过使用浓度升高的盐水溶液洗脱该柱,可选地在回收步骤e)的组分之后,可以回收多种乳蛋白组分。在步骤e′)中,用电导率为50.5~51.5mS/cm的盐水溶液洗脱的组分是富集乳铁传递蛋白的组分。这有可能在单个色谱步骤中回收富含乳铁传递蛋白的乳蛋白组合物。这一方法优选在下述一个或多个条件下施行:
-盐水对树脂的体积比在2.5-3.5之间,
-盐水的流速在1.5-2m3/h之间,
-洗脱的线速度在0.4-0.6m/h之间。
此外还可包含下面的依序步骤:超滤、渗滤、微滤和/或干燥,尤其是喷雾干燥。
这种分离富集乳铁传递蛋白的乳蛋白组分的方法构成了本发明的另一个主题。
本发明的另一个主题是包含了本发明的乳蛋白组分和药学可接受载体的药物组合物。
根据所涉及的病理状况和病理状况的严重性,制剂和载体的模式将由所属技术领域的专业人员制定:局部施用(乳膏,洗液,贴片),口服给药(胶囊,糖浆,药片,水溶液或分散体),注射(注射溶液)。
还可以看到,本发明的乳蛋白组分还可用于食品组合物的制备,尤其是食疗组合物,尤为特别的是针对遭受慢性炎症病理学状况、尤其是牛皮癣或自身免疫性疾病的人。这种食品组合物包含本发明的乳蛋白组分,用以代替常规使用的乳蛋白。它们可以是蛋白质饮料、乳制品等。它们构成了本发明的另一个主题。
根据病理状况、严重性、病人的年龄和体重,由所属技术领域的专业人员来制定所要给药的乳蛋白组分的数量和给药频率。
本发明的药物或食疗组合物可被用来治疗慢性炎症病理状况,尤其是在发病期间的牛皮癣或自身免疫性疾病。它们也可以在症状缓解期间使用,用以预防和/或避免和/或延迟急性传染的新周期的出现。
具体实施方式
实施例1:乳蛋白组分的制备
预先将250m3的脱脂乳在68℃下热处理15秒,然后使其通过具有2000l的阳离子交换树脂(BIOSEPRA的SPEC 70)的色谱柱(下行方向),该阳离子交换树脂具备如下特性:
用SO3 -基团官能团化的全合成大孔聚阴离子聚合物;该聚合物为三维球形或类球形;其粒度大于261μm;载体是由AMPS:2-丙烯酰胺-2-甲基丙烷磺酸聚合得到的。
乳对柱的供给流速为14m3/h,线速度为3.2m/h。
在乳通过之后,用23,000升的软化水冲洗该柱(下行方向)。
然后用12g/l的盐水溶液从树脂上提取结合的蛋白质。其流速为1900l/h,线速度为0.6m/h;得到的洗脱液体积接近8000升。
然后使用UF(DSS,400m GR10D膜)对洗脱液在4℃下进行浓缩,使用的单位体积浓度因子使得渗余物的电导率大于15mS/cm。得到的渗余物再次采用UF和渗滤(配备有相同的膜单表面积仅为34m2)进行浓缩,直到获得的渗余物的电导率为5mS/cm。
将由此获得的渗余物在孔隙率为1.4μm的17m2的微滤模件上升温至30℃;得到的渗透物的干提出物接近8%,并在进口温度为180℃、塔出口温度为80℃的单效喷嘴塔中进行喷雾干燥。
回收得到了1650g粉末状的乳蛋白组分。这一组分具有下面的特性:
水分 5.4%
蛋白质 94.2%
灰分 0.9%
TGF-β含量 110μg/g蛋白质
乳过氧化物酶含量 407mg/g蛋白质
IgG含量 <190mg/g蛋白质
实施例2:乳蛋白组分对细胞生长的影响
实施例1中获得的乳蛋白组分的影响在来源于乳腺癌胸膜积液MCF7的细胞培养物上进行测试。进行了两个实验。以7000000个细胞/皿(实验1)和200000个细胞/皿(实验2)的浓度将MCF7细胞接种于25cm2的培养皿中。放置24小时后,吸出培养基,取而代之的是7ml含有从实施例1中获得的蛋白质组分的培养基,蛋白质组分浓度为218μg/ml,109μg/ml或0μg/ml(对照培养基)。然后,以每24小时更换一次培养基的方式培养细胞72小时。结果对应于在每个试验中4个培养皿上得到的计数的平均值。
实验确实表明,实施例1中得到的乳蛋白组分对MCF7细胞生长的剂量依赖型抑制效果。
培养基中蛋白质组分的浓度(μg/ml) | 218 | 109 | 0(对照) | |
实验1 | 细胞数(106)相对于对照样的减少% | 3.64±0.4646.0% | 4.64±0.4031.2% | 6.74±0.70 |
实验2 | 细胞数(106)相对于对照样的减少% | 0.87±0.0547.9% | 1.15±0.0831.1% | 1.67±0.08 |
实施例3:含有该乳蛋白组分的营养增补剂的制备
通过结合实施例1中获得的乳蛋白组分,制备出胶囊形式的膳食补充剂。
每胶囊(350mg) | |
实施例1中获得的乳蛋白组分Polaris公司的Omegacaps(含有20%ω-3脂肪酸的鱼油粉末)维生素PP(烟酸)硬脂酸镁Emcocel | 125mg125mg(其中含25mg的ω-3)1.25mg5mg93.75mg |
实施例4:含有该乳蛋白组分的营养增补剂对患有牛皮癣的人的功效
测试了实施例3中制备的营养增补剂的功效。20位牛皮癣患者(女性8名,男性12名,年龄23-70岁)每日摄取8粒胶囊(早晨4粒胶囊晚上4粒胶囊)实施例3制备的营养增补剂,持续60天。换句话说,实施例1中获得的乳蛋白组分的日摄取量为1g,相当于110μg的TGF-β。在营养增补剂摄取的60日前后进行症状对照和血液分析。
结果:
-牛皮癣症状:在摄取营养增补剂60日后,观察到有80%的人症状明显改善(在牛皮癣斑点、发炎、脱落和瘙痒的减轻)。
牛皮癣症状的综合评定 | 对20人 | |
很明显的改善明显的改善没有改善恶化 | 61040 | 30%50%20%0% |
-安全性:该营养增补剂耐药力良好。在摄取的整个60日期间,没有观察到严重意外情况。对肝功参数(ASAT,ALAT,GGT)、肾功参数(尿素,肌酸酐)和血液学参数(白血球,多核,淋巴细胞,单核细胞,血小板)没有观察到异常,证实了该营养增补剂的安全性。
Claims (15)
1.一种制备乳蛋白组分的方法,其特征在于,包括如下步骤:
乳或乳清用作起始材料;
(a)所述乳或乳清可选地经过微滤或热处理;
(b)使来自步骤(a)的乳或乳清沉积在阳离子交换树脂上,所述阳离子交换树脂的组成为用强酸阴离子的共轭碱型官能团进行官能团化的纳米孔合成聚合物;线速度在2.8-4.5m/h之间和乳的体积对树脂的体积之比为100-200;
(c)用软化水冲洗所述阳离子交换树脂;
(d)用浓度升高的盐水溶液洗脱所述树脂;
(e)回收对应于电导率在21.0-22.0mS/cm之间的盐水溶液的洗脱液。
2.如权利要求1所述的方法,其特征在于,还包含下述一个或多个步骤:
(f)通过超滤以浓缩(e)中获得的洗脱液,并回收渗余物;
(g)通过微滤对(f)中获得的渗余物进行灭菌,回收渗透物;
(h)对(g)中获得的渗透物进行喷雾干燥。
3.如权利要求1或2所述的方法,其特征在于,所述起始材料是牛乳。
4.如权利要求1-3中任一项所述方法,其特征在于,所述聚合物用SO3 -基团官能团化。
5.如前述任一项权利要求所述的方法,其特征在于,
步骤(b)中向所述柱供给的流速在10-15m3/h之间。
6.如前述任一项权利要求所述的方法,其特征在于,满足下述一种或多种条件:
-所述盐水溶液的盐浓度为10-14g/l;
-盐水流速为1.5-2m3/h;
-所述盐水溶液的线速度为0.4-0.6m/h;
-盐水的体积对树脂的体积之比在2.5-3.5之间。
7.一种能够使用如前述任一项权利要求所述的方法获得的乳蛋白组分,其特征在于,满足以下特征:
-相对于蛋白质的总重量,TGF-β的含量在0.010-0.025wt%之间;
-相对于蛋白质的总重量,IgG(免疫免疫球蛋白)的含量<25wt%;
-TGF-β/IGF 1之比率≥5w/w。
-相对于蛋白质的总重量,乳过氧化物酶的含量在35-45wt%之间。
8.如权利要求7所述的乳蛋白组分的应用,用以制备用于预防或治疗慢性炎症病理学状况和/或它们的症状的药物。
9.如权利要求8所述的应用,用以制备用于预防或治疗牛皮癣和/或其症状的药物。
10.如权利要求7所述的乳蛋白组分的应用,用以制备用于预防或治疗自身免疫性疾病的药物。
11.如权利要求7所述的乳蛋白组分的应用,用以制备用于预防或治疗自身免疫性疾病的炎症表现的药物。
12.一种含有如权利要求7所述的乳蛋白组分和药学可接受载体的药物组合物。
13.一种含有如权利要求7所述的乳蛋白组分的食品组合物,尤其是食疗组合物。
14.如权利要求1~6中任一项所述的方法,其特征在于,还包含用以分离富集乳铁传递蛋白的乳蛋白组分的步骤(e′),其中回收与电导率在50.5-51.5mS/cm之间的盐水溶液相对应的洗脱液。
15.如权利要求14所述的方法,其特征在于,所述方法的步骤(e′)在下述一个或多个条件下进行:
-盐水的体积对树脂的体积之比在2.5-3.5之间,
-盐水的流速在1.5-2m3/h之间,
-洗脱线速度在0.4-0.6m/h之间。
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FR0508177A FR2889068B1 (fr) | 2005-07-29 | 2005-07-29 | Nouvelles fractions proteiques laitieres et leur utilisation pour la prevention ou le traitement des maladies inflammatoires chroniques |
PCT/FR2006/001810 WO2007012748A2 (fr) | 2005-07-29 | 2006-07-25 | Nouvelles fractions proteiques laitieres et leur utilisation pour la prevention ou le traitement des maladies inflammatoires chroniques |
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JP2008189637A (ja) * | 2007-02-08 | 2008-08-21 | Snow Brand Milk Prod Co Ltd | 自己免疫疾患予防剤 |
EP2902409B1 (en) * | 2007-07-10 | 2018-01-17 | Glanbia Nutritionals (Ireland) Limited | Method for removing endotoxin from proteins |
US9247766B2 (en) * | 2008-05-14 | 2016-02-02 | Murray Goulburn Co-Operative Co., Limited | Angiogenin-enriched milk fractions |
US11109604B2 (en) | 2019-05-09 | 2021-09-07 | Memtec LLC | Dairy processing systems and methods |
FR3115037A1 (fr) | 2020-10-12 | 2022-04-15 | Compagnie Laitiere Europeenne | Procédé de purification de fraction de protéines cationiques et fraction ainsi obtenue |
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WO2001025276A1 (en) * | 1999-10-06 | 2001-04-12 | Campina B.V. | Process for obtaining growth factor preparations (tgf-beta and igf-1) from milk products having low mutual cross-contamination |
CN1557482A (zh) * | 2004-02-04 | 2004-12-29 | 高春平 | 调节免疫功能、抗肿瘤的天然营养剂 |
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US5866418A (en) * | 1990-07-13 | 1999-02-02 | Gropep Pty. Ltd. | Milk protein mixture for promoting growth of animal cells or treating wounds and method of making and methods employing the mixture |
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FR2841747B1 (fr) * | 2002-07-02 | 2004-08-20 | Cie Laitiere Europeenne | Isolat de proteines de lait et procede pour sa preparation |
US20050208638A1 (en) * | 2004-03-22 | 2005-09-22 | Chao Wu | Process for preparing bioactive protein-enriched whey products |
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KR101404035B1 (ko) | 2014-06-19 |
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NZ565287A (en) | 2010-12-24 |
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PL1912513T3 (pl) | 2014-03-31 |
AU2006273912B2 (en) | 2013-01-24 |
ES2438591T3 (es) | 2014-01-17 |
EP1912513B1 (fr) | 2013-09-11 |
SI1912513T1 (sl) | 2014-01-31 |
CY1114811T1 (el) | 2016-12-14 |
US7982001B2 (en) | 2011-07-19 |
CA2616345A1 (fr) | 2007-02-01 |
KR20080048456A (ko) | 2008-06-02 |
FR2889068A1 (fr) | 2007-02-02 |
PT1912513E (pt) | 2013-12-17 |
AU2006273912A1 (en) | 2007-02-01 |
DK1912513T3 (da) | 2014-01-06 |
WO2007012748A2 (fr) | 2007-02-01 |
CN101272694A (zh) | 2008-09-24 |
CA2616345C (fr) | 2014-05-06 |
JP5709353B2 (ja) | 2015-04-30 |
FR2889068B1 (fr) | 2012-03-02 |
JP2009502892A (ja) | 2009-01-29 |
WO2007012748A3 (fr) | 2007-06-07 |
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