CN108434150B - Zstk474用于制备ean治疗药物的应用 - Google Patents
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Abstract
本发明提供了ZSTK474用于制备EAN治疗药物的应用。该技术方案以EAN动物模型为实验对象,考察了ZSTK474对EAN的治疗作用,并进一步研究了ZSTK474对EAN若干病理特征的具体作用靶点。实验结果表明,ZSTK474改善了该疾病的严重程度,表现为神经功能缺损症状减轻、炎性细胞浸润减少,坐骨神经髓鞘脱失情况改善。具体来看,ZSTK474减少了炎性细胞Th1/Th17的数量,降低了促炎因子白介素(IL)‑1α,IL‑1β,IL‑17,IL‑23,干扰素(IFN)‑γ及肿瘤坏死因子(TNF)‑α的水平。与此同时,本发明还发现ZSTK474的神经保护作用可能是通过PI3K/AKT/mTORC1通路实现的。基于以上有益的发现,本发明确定了利用其制备EAN或GBS治疗药物的用途,从而拓展了该疾病治疗药物的可选范围。
Description
技术领域
本发明涉及医药技术领域,进一步涉及物质的新的医药用途,具体涉及 ZSTK474用于制备EAN治疗药物的应用。
背景技术
实验性自身免疫性神经炎(EAN)是一种主要由T细胞介导的周围神经系统脱髓鞘性自身免疫疾病。由于EAN可以模拟吉兰-巴雷综合征(GBS)临床、免疫、病理及电生理各方面的特点,因此业已成为研究人类吉兰-巴雷综合征的理想动物模型,在EAN环境下的药理研究结果对该药物在GBS中的作用具有指导意义。
EAN的症状主要包括血神经屏障破坏、周围神经脱髓鞘、炎细胞比如T细胞和巨噬细胞浸润等。现有技术中,对EAN或GBS的治疗方法主要包括血浆置换、注射丙种球蛋白等,但不管是否采用上述疗法,仍有20%的患者留有严重后遗症,5%的患者死亡,也就是说,目前针对EAN或GBS的治疗药物效果并不理想,而且以糖皮质激素为代表的部分药物在EAN或GBS治疗中缺乏药理学依据,不同病例之间效果差异甚大。此外,现有技术的治疗药物缺乏对具体症状的针对性,专用于EAN、GBS患者炎性损伤、神经脱髓鞘保护等具体症状的药物目前尚处于空白阶段。
ZSTK474,I类PI3K抑制剂,是一种很有前景、无毒的抗肿瘤药。近年来, ZSTK474被用于佐剂诱导的关节炎、骨关节炎、胶原诱导的关节炎、实验性自身免疫性脑脊髓炎、脑缺血再灌注损伤的动物模型上,表现出特定的抗炎和免疫调节作用。目前,尚没有ZSTK474用于治疗EAN或GBS的相关报道,而且,由于病理环境的差异,ZSTK474在上述关节炎、脑缺血再灌注损伤等疾病中所表现出的性质,在其他疾病环境中并无直接的借鉴意义。
发明内容
本发明旨在针对现有技术的技术缺陷,提供ZSTK474用于制备EAN治疗药物的应用,以拓展自身免疫性神经炎、吉兰-巴雷综合征治疗药物的可选范围。
本发明要解决的另一技术问题是现有技术中ZSTK474的适应症范围较为局限。
本发明要解决的再一技术问题是如何实现对EAN或GBS患者的神经功能缺损、炎性细胞浸润、坐骨神经髓鞘脱失等具体病理症状的治疗。
本发明要解决的又一技术问题是揭示ZSTK474对EAN或GBS治疗的作用机理。
为实现以上技术目的,本发明采用以下技术方案:
本发明首先提供了ZSTK474在制备自身免疫性神经炎治疗药物中的应用。
与此同时,基于EAN模型与GBS病理特征的同一性,本发明进一步提供了 ZSTK474用于制备吉兰-巴雷综合征治疗药物的应用。
作为优选,所述药物是针对EAN患者或GBS患者神经功能缺损症状的治疗药物。
作为优选,上述对神经功能缺损症状的治疗是通过PI3K/AKT/mTORC1信号通路实现的。
作为优选,所述药物是针对EAN患者或GBS患者炎性细胞浸润症状的治疗药物。
作为优选,所述药物降低CD4+T细胞中炎性细胞Th1/Th17的数量。
作为优选,所述药物降低促炎性细胞因子,IFN-γ以及TNF-α的水平。
作为优选,所述促炎性细胞因子包括IL-1α,IL-1β,IL-17,IL-23。
作为优选,所述药物是针对EAN患者或GBS患者坐骨神经髓鞘脱失症状的治疗药物。
作为优选,所述药物的剂型为口服剂。
作为优选,ZSTK474的每日剂量为30mg/kg体重。
作为优选,所述药物是ZSTK474悬浮于5%羟丙基纤维素中所形成固体分散体。
作为优选,所述药物从首次出现临床症状之日开始用药,通常从第7天到免疫后第16天或30天期间用药。
本发明以EAN动物模型为实验对象,考察了ZSTK474对EAN的治疗作用,并进一步研究了ZSTK474对EAN若干病理特征的具体作用靶点。实验结果表明, ZSTK474改善了该疾病的严重程度,表现为神经功能缺损症状减轻、炎性细胞浸润减少,坐骨神经髓鞘脱失情况改善。除此之外,ZSTK474减少了炎性细胞Th1/Th17的数量,降低了促炎因子白介素(IL)-1α,IL-1β,IL-17,IL-23,干扰素(IFN)-γ,及肿瘤坏死因子(TNF)-α的水平。与此同时,通过实验手段发现ZSTK474的神经保护作用可能是通过PI3K/AKT/mTORC1通路实现的。 ZSTK474有效地减少了Th1/Th17的数量,降低了促炎因子的水平,从而实现了对EAN的治疗。基于以上有益的发现,本发明确定了利用其制备EAN或GBS 治疗药物的用途,从而拓展了该疾病治疗药物的可选范围,并获得确切的治疗效果。
附图说明
图1说明了本发明具体实施方式中,ZSTK474对EAN大鼠临床症状、组织学损伤、炎症细胞聚集的缓解作用。
图2说明了本发明具体实施方式中,ZSTK474对EAN大鼠周围神经损伤的缓解作用。
图3说明了本发明具体实施方式中,ZSTK474对EAN大鼠CD4+T细胞分化的影响。
图4说明了本发明具体实施方式中,ZSTK474通过下调p-STAT3的表达,减轻EAN大鼠坐骨神经Th17细胞浸润。
图5说明了本发明具体实施方式中,ZSTK474对EAN大鼠细胞因子谱的影响。
图6说明了本发明具体实施方式中,ZSTK474对EAN大鼠PI3K/Akt/mTOR 途径的抑制作用。
图7说明了ZSTK474在EAN治疗过程中的作用原理。
具体实施方式
以下将对本发明的具体实施方式进行详细描述。为了避免过多不必要的细节,在以下实施例中对属于公知的结构或功能将不进行详细描述。
以下实施例中所使用的近似性语言可用于定量表述,表明在不改变基本功能的情况下可允许数量有一定的变动。因此,用“大约”、“左右”等语言所修正的数值不限于该准确数值本身。在一些实施例中,“大约”表示允许其修正的数值在正负百分之十(10%)的范围内变化,比如,“大约100”表示的可以是90到110之间的任何数值。此外,在“大约第一数值到第二数值”的表述中,大约同时修正第一和第二数值两个数值。在某些情况下,近似性语言可能与测量仪器的精度有关。
除有定义外,以下实施例中所用的技术和科学术语具有与本发明所属领域技术人员普遍理解的相同含义。
1材料与方法
1.1实验动物
野生型雄性Lewis大鼠(6-8周龄),购自维通利华实验动物技术有限公司 (中国北京)。将大鼠在自然昼夜循环下饲养1周适应环境后,将动物随机分配到治疗组或对照组。实验经天津医科大学动物实验伦理委员会批准。
1.2诱导EAN
为了诱导EAN,每只大鼠的每个后足垫皮下注射150μl含有150μg溶解的 P0180-199的乳化剂(Bio-Synthesis Corporation,Lewisville,TX,USA)。P0180-199多肽首先被溶解在磷酸盐缓冲液(PBS)(2mg/ml)里,然后用等体积的含弗氏完全佐剂 (Difco,Lawrence,KS,USA)及结核分支杆菌素(strain H37RA)的混合物乳化,P0 肽乳剂的终浓度为1mg/ml。免疫后,每日观察大鼠症状。瘫痪的严重程度按如下分级:0分,正常;1分,尾部变软;2分,尾部无法抬起;3分,体位不能自行纠正;4分,共济失调;5分,后肢轻度麻痹;6分,后肢中度麻痹;7分,后肢重度麻痹;8分,四肢轻瘫;9分,四肢重瘫;10分,死亡。
1.3 ZSTK474处理
ZSTK474由Nippon Zenyaku Kogyo Co.Ltd(Koriyama,Japan)友情提供。将ZSTK474悬浮于5%羟丙基纤维素(HPC)中以形成固体分散体。对于治疗组 EAN大鼠,从首次出现临床症状的那一天开始用药,通常从第7天到免疫后第 16天或30天,通过灌胃(30mg/kg/天)给予ZSTK474,对照组给予等体积的 HPC。ZSTK474的剂量选择及给药方式是基于先前的研究确定的,先前研究表明灌胃给予这个剂量的ZSTK474,在体内可以完全抑制PI3K的活化。
1.4电生理研究
在免疫后的第16天,本发明采用全数字肌电图用来记录每只大鼠左侧坐骨神经的电生理指数。类似于以前的报道,本发明使用单盲试验方法记录坐骨神经的诱发复合肌肉动作电位(CMAP)波幅和潜伏期。将大鼠麻醉及局部消毒后,切开臀肌暴露左侧坐骨神经,然后将成对的电极针插入踝(远端)及坐骨切迹(近端)处。引出CMAP的1Hz脉冲属性为振幅5mA并且持续时间为0.3ms的脉冲。放在腓肠肌腹侧的记录电极记录了刺激坐骨神经的诱发电位。测量刺激电极与记录电极间的距离以用于计算运动神经传导速度(motor nerveconduction velocity,
MNCV)。在产生的CMAP曲线下,最大峰与基线间的距离为波幅。测量结束后将切口无菌缝合。
1.5组织病理评估
在免疫后第16天,将对照组和治疗组的大鼠处死并像之前一样取右侧坐骨神经。简而言之,使用水合氯醛将大鼠深度麻醉,然后使用PBS灌注心脏,仔细解剖取下右侧坐骨神经部分,经过多聚甲醛固定、脱水后将其包埋为石蜡包块并切片6μm于防脱载玻片上。室温储存切片,直到用于快蓝(LFB)和H&E染色,分别用来评估髓鞘脱失程度及炎细胞浸润的情况。使用光学显微镜(Nikon,Sendai, Japan)来观察染色结果,并用半定量方法来评估严重程度。采用单盲法观察切片血管周围炎性细胞浸润情况并进行病理评分。快蓝染色评分标准:0分,血管周围区域无炎性细胞浸润和脱髓鞘;1分,血管周围区域轻度的炎性细胞浸润;2 分,血管周围区域存在炎性细胞浸润和脱髓鞘;3分,整个切片视野均有炎性细胞浸润和脱髓鞘存在。每张切片随机取5个视野(200×magnification),进行细胞计数。
1.6免疫荧光检测
应用如上所述的方法收获右侧坐骨神经,并进行梯度脱水。然后将神经包埋在OCT中(Sakura,Torrance,CA,USA),并用液氮中迅速冷冻制成冰冻包块。应用冰冻切片机(Leica Microsystems LM3050S),将神经切成厚度8μm横向切片,并将组织切片贴附于防脱载玻片上。冷冻切片的荧光免疫荧光操作遵循抗体制造商提供的标准方案。简而言之,取出存放于-80℃冰箱中的冰冻切片,复温后浸于冰丙酮进行固定破膜,PBS洗片后,用3%牛血清白蛋白(BSA)37℃下封闭切片30分钟。然后孵育一抗过夜:小鼠抗大鼠CD4抗体(1:200)(Santa Cruz Biotechnology,Inc.Dallas,Texas,USA),兔抗大鼠IL-17抗体(1:200)(Abcam, Cambridge,United Kingdom)。第二天,载玻片用冰PBS洗涤至少5遍。然后滴加合适的二抗:Alexa594驴抗兔IgG(H+L)(1:1000)和Alexa488 驴抗小鼠IgG(H+L)(1:1000)。避光室温孵育1小时。PBS洗涤至少5遍。最后,用含DAPI(Abcam)的封片剂覆盖组织复染细胞核并封片。通过尼康显微镜(Nikon,Sendai,Japan)观察荧光成像。从四个随机微视野(200倍放大率)随机获取5 个视野并以每平方毫米计数阳性细胞数。计算其平均值。所有计数以单盲方式进行,观察并计数EAN大鼠坐骨神经的横断面中的阳性细胞。
1.7流式细胞学检测
将两组EAN大鼠的脾脏无菌取出、研磨、制成单个核细胞(MNCs)悬液。将两组MNCs分为两份分别检测需要额外刺激的Th1、2和17以及不需要额外刺激的Treg。制备刺激混合物:2.5mg/ml Brefeldin A(BFA),40.5μM phorbol-12-myristate 13-acetate(PMA),669.3μM离子霉素(ionomycin)。然后每孔加入2μl刺激混合物于37℃,5%CO2的孵箱内刺激6小时,以抑制含有细胞因子的囊泡的分泌。接下来,收集每孔细胞置于流式管中,PBS清洗后,加入抗大鼠CD4(APC标记)抗体(eBioscience,San Diego,CA,USA),室温条件下孵育45min。然后固定破细胞膜,加入抗大鼠IFN-γ抗体(FITC标记)(BioLegend)、抗大鼠IL-4抗体(PE标记)(BioLegend)、抗大鼠IL-17A抗体(PerCP标记) (eBioscience),室温避光条件下孵育30min。两组的另一份细胞,加入抗大鼠CD4 抗体(APC标记)(eBioscience)、抗大鼠CD25抗体(PE标记)(eBioscience),室温避光条件下孵育45min。然后固定液(BioLegend)固定细胞后,加入破细胞核膜液(BioLegend)破膜。最后,加入PerCP连接的抗大鼠Foxp3抗体(eBioscience)。所有样品洗涤,重悬,滤网过滤后上机。在同一天收集和分析每组6只大鼠的细胞。数据用BD Accuri C6采集,用BD Accuri CFlow软件进行分析(BD Biosciences,San Jose,CA,USA)。
1.8 Western印迹分析
将大鼠的坐骨神经于液氮中取出,低温下用研钵研磨后,加入包含磷酸酶抑制剂PMSF的(Roche Diagnostics,Indianapolis,IN,USA)的裂解液RIPA,提取总蛋白,用蛋白质测定试剂盒测定浓度(Solarbio Life Sciences,Beijing,China)。样本加入12%聚丙烯酰胺凝胶,蛋白经过电泳,然后转到PVDF膜上(EMD Millipore,Billerica,MA,USA),转膜时间为1.5小时,80V。将PVDF膜用含 5%牛血清白蛋白封闭1小时后,加入一抗4℃过夜。使用了下列一抗:p-4EBP1 (4E-binding protein),p-P70S6K(ribosomal protein,S6kinase),p-Akt,p-mTOR, p-STAT3(Cell Signaling Technology,Danvers,MA,USA)(1:1000)和GAPDH(1: 1000,ZSGB-Bio,Beijing,China)。第二天,PVDF膜经TBST漂洗(3次,10min)。然后加入经辣根过氧化物酶(HRP)标记的二抗(1:2000)(TransGen Biotech, Beijing,China),室温1小时。使用图象分析仪(Bio-Rad公司)分析蛋白条带。
1.9酶联免疫吸附试验(ELISA)
如上所述,无菌制备EAN大鼠的脾脏单个核细胞,并调节浓度为2×106个/ml,加入P0180-199(终浓度10μg/mL)在37℃和5%CO2条件下的孵箱中孵育72h;离心后留取上清液存于-80℃冰箱中备用。按照多因子ELISArray试剂盒(Qiagen, Düsseldorf,Germany)说明书检测多种细胞因子和化学因子,包括IL-1α,IL-1β, IL-2,IL-4、IL-6、IL-10、IL-12、IL-13、IFN-γ,TNF-α,粒细胞巨噬细胞集落刺激因子(GM-CSF)和调节激活正常T细胞表达和分泌(RANTES)。用酶标仪(Thermo Scientific,Waltham,MA,USA)在450nm处测定其光密度值(OD值)。
1.10聚合酶链式反应(Real-time PCR,RT-PCR)
将大鼠脾脏单个核细胞浸于Trizol试剂存于-80℃冰箱,并按Trizol说明书提取总RNA。用紫外分光光度法测定RNA的浓度。用TransScript First-Strand cDNA SynthesisSuperMix(北京转基因生物技术有限公司,北京,中国)将总RNA逆转录成互补的cDNA。使用Opticon 2 RT-PCR检测系统(Bio-Rad)和2-ΔΔCt方法分析mRNA表达。表1中列出了用于RT-PCR的引物序列。
表1 PCR引物序列
1.11统计分析
使用GraphPad Prism 5.0(GraphPad Software Inc.,La Jolla,CA,USA)进行统计分析。使用Mann-Whitney U检验分析神经学评分和组织学评分之间的差异;数据用中位数和四分位间距表示。未配对的t检验用于其他2组比较,数据用平均值±SEM表示。对于所有的统计分析来说,p<0.05被认为是差异显著的。
2实验结果
2.1 ZSTK474改善EAN大鼠临床症状,减轻组织学损伤和减少炎症细胞聚集
代表瘫痪严重程度的临床评分方面,治疗组明显低于对照组(图1A)。此外,高峰期神经功能障碍的严重程度也明显下降(图1B;p<0.01)。通过评估临床评分,本发明还记录到了治疗组中运动缺陷曲线下面积(AUC)的减少(图1C;p<0.01)。治疗组在疾病高峰期坐骨神经H&E染色中显示炎性细胞浸润减少(图1D,E)(p< 0.01),LFB染色显示脱髓鞘程度得到明显改善(图1F,G)(p<0.05)。
2.2 ZSTK474减轻EAN诱导的周围神经损伤
在疾病高峰期,EAN大鼠坐骨神经的电生理检测表现为CMAP波幅降低, MNCV减少和CMAP潜伏期延长(图2A,B)。与对照组相比,治疗组的MNCV 显着升高(图2C;p<0.01)。而且,治疗组的CMAP幅度也上升(图2D;p<0.05),并且,治疗组CMAP潜伏期缩短(图2E;p<0.01)。
2.3 ZSTK474抑制CD4+T细胞的分化
为了评估ZSTK474处理对CD4+T细胞分化的影响,如上所述从疾病高峰期的大鼠脾脏中分离出单个核细胞(MNC)。然后通过使用流式细胞术分析脾MNCs 中的CD4+T细胞亚群。与对照组相比,ZSTK474治疗组脾MNC中Th1CD4+ IFN-γ+细胞的百分比低于对照组(图3A,B)(p<0.01)。此外,ZSTK474处理降低了CD4+IL-17+Th17细胞的百分比(图3E,F,p<0.05)。与对照组相比,治疗组中Th2细胞CD4+IL-4+(图3C,D)和Treg细胞CD4+CD25+Foxp3+(图3G,H)的百分比没有显示显著差异。这些结果表明,ZSTK474的保护作用可能是通过抑制Th细胞分化成Th1/Th17细胞而产生的。
2.4 ZSTK474通过下调p-STAT3的表达,减轻EAN大鼠坐骨神经Th17细胞浸润
同样,在EAN大鼠的疾病高峰期,分离坐骨神经并进行荧光免疫组织化学染色以鉴定Th17细胞浸润(图4A)。用每平方毫米CD4+/IL-17+细胞的计数反映Th17细胞浸润的程度。结果显示,与对照组相比,治疗组中Th17细胞数目减少(图4B;p<0.01)。Western印迹检测分析坐骨神经中p-STAT3蛋白的表达,并使用GAPDH作为标准(图4C)。与对照相比,治疗组p-STAT3表达水平降低(图4D;p<0.01)。
2.5 ZSTK474改变细胞因子谱
多细胞因子ELISA试剂盒用于研究ZSTK474处理对脾脏MNCs产生的细胞因子的影响。与对照组相比,ZSTK474治疗组中促炎细胞因子例如IL-1α,IL-1β, IFN-γ和TNF-α的产生显著减少(图5A,p<0.05)。然而,在两组之间的IL-2,IL-4, IL-6,IL-10,IL-12,IL-13,GM-CSF或RANTES的水平中没有发现显著差异。当进一步使用RT-PCR在转录水平检测细胞因子,结果是相同的,即ZSTK474治疗组中IFN-γ和TNF-α的水平在显著下降。同时还发现ZSTK474治疗组中IL-17和 IL-23含量下降(图5B,p<0.01)。
2.6 ZSTK474介导的神经保护与PI3K/Akt/mTORC1途径有关
在EAN大鼠疾病高峰期,留取EAN大鼠的坐骨神经用于Western印迹分析。 ZSTK474减少了PI3K的主要靶标AKT的磷酸化。在这项研究中,ZSTK474不仅降低了ZSTK474治疗组中AKT磷酸化水平(图6A,B,p<0.05),而且降低了p-mTOR水平(图6A,C,p<0.01)。此外,本发明证实了mTOR的下游组分p-P70S6K(图6A,D p<0.01)和p-4EBP1(图6A,E,p<0.01)的水平也降低。根据Western印迹结果,ZSTK474通过抑制PI3K/Akt/mTOR途径来调节Th细胞的分化,ZSTK474在EAN治疗过程中的作用原理如图7所示。
3、结论
本发明旨在探索ZSTK474对GBS动物模型(即EAN)的治疗效果。与对照组相比,ZSTK474治疗改善了临床评分,减轻了疾病高峰期的瘫痪严重程度。此外, H&E和LFB染色的结果表明ZSTK474抑制坐骨神经的炎症细胞浸润并抑制其脱髓鞘作用。此外,ZSTK474显著缩短远端运动潜伏期,增加CMAP波幅,加速 MNCV,从而减轻EAN诱发的神经损伤。
具体来看,本发明发现在ZSTK474治疗作用下,EAN大鼠中促炎Th细胞(Th1 和Th17)的数目明显减少。尽管抗炎CD4+T细胞(如Th2和Treg)的数量没有变化,但与未治疗组相比,治疗组仍然表现出改善的结果。此外,实验结果显示治疗组中浸润到坐骨神经中的Th17细胞数量低于对照组,而且促炎细胞因子例如 IFN-γ,TNF-α,IL-23和IL-17的mRNA表达降低是显著的。该结果与脾MNCs培养后收集的上清液的ELISA结果一致。因此,ZSTK474降低了CD4+T细胞中Th1/ Th17细胞的数量,抑制了促炎细胞因子的释放,从而起到了保护EAN的作用。
此外,本发明发现,ZSTK474下调AKT和mTORC1的磷酸化,因此也降低了 p-P70S6K,p-4EBP1和p-STAT3的含量。PI3K/AKT/mTORC1途径可能是 ZSTK474调节Th1/Th17细胞功能的途径。本发明研究表明,ZSTK474在p-AKT, p-mTORC1和p-P70S6K减少的环境中抑制Th1分化,ZSTK474还减少了p-STAT3,从而抑制了Th17分化。
综上所述,本发明已经证明ZSTK474通过减少Th1/Th17细胞和促炎细胞因子的产生对EAN起保护作用,同时,还证实了ZSK474的保护作用是通过PI3K /AKT/mTORC1途径实现的。基于以上有益的发现,可以依据本领域常规的药物制备方法将ZSK474应用于其中,从而形成GBS或EAN治疗药物。
以上对本发明的实施例进行了详细说明,但所述内容仅为本发明的较佳实施例,并不用以限制本发明。凡在本发明的申请范围内所做的任何修改、等同替换和改进等,均应包含在本发明的保护范围之内。
Claims (2)
1.ZSTK474用于制备吉兰-巴雷综合征治疗药物的应用;所述药物缓解神经功能缺损症状;所述药物对神经功能缺损症状的缓解作用是通过PI3K/AKT/mT ORC1信号通路实现的;所述药物缓解炎性细胞浸润症状;所述药物降低CD4+T细胞中炎性细胞Th1/Th17的数量;所述药物降低促炎性细胞因子,IFN-γ以及TNF-α的水平;所述促炎性细胞因子包括IL-1α,IL-1β,IL-17,IL-23;所述药物缓解坐骨神经髓鞘脱失症状。
2.根据权利要求1所述的应用,其特征在于所述药物的剂型为口服剂;该应用中,ZSTK474的每日剂量为30mg/kg体重。
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WO2016142508A1 (fr) * | 2015-03-11 | 2016-09-15 | Centre Léon-Bérard | Composition pour le traitement des tumeurs neuroendocrines pancréatiques |
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