CN107349202B - Lgk974在制备治疗多囊肾病的产品中的应用 - Google Patents
Lgk974在制备治疗多囊肾病的产品中的应用 Download PDFInfo
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Abstract
本发明公开了LGK974在制备治疗多囊肾病的产品中的应用。本发明以Vil‑Cre；Pkd2f3/f3小鼠作为ADPKD动物模型，结果表明Wnt信号通路抑制剂—LGK974可减弱Vil‑Cre；Pkd2f3/f3小鼠的肾囊肿程度，可降低Vil‑Cre；Pkd2f3/f3小鼠的肾脏体重比，可降低Vil‑Cre；Pkd2f3/f3小鼠的肾脏囊肿指数，可降低Vil‑Cre；Pkd2f3/f3小鼠的血液中肌酐和尿素氮的含量。本发明对于ADPKD发病机制的分析，对于治疗ADPKD的药物的研发均具有重要意义。
Description
技术领域
本发明属于生物医药领域，具体涉及LGK974在制备治疗多囊肾病的产品中的应用，特别涉及LGK974在制备治疗常染色体显性遗传性多囊肾疾病的产品中的应用。
背景技术
常染色体显性遗传性多囊肾(Autosomal Dominant Polycystic KidneyDisease，ADPKD)疾病是最常见的单基因遗传性肾病之一。其主要临床症状为双侧肾脏形成大量大小不等的球形液性囊肿，并进行性增大，破坏肾脏的结构和功能，最终导致肾功能衰竭。ADPKD通常在成人中发病，其发病率为1:500-4000，是目前世界上终末肾衰竭的第四大主要原因。除肾病表型外，ADPKD患者的非肾脏部位病变表型也十分明显：83％的ADPKD患者出现肝囊肿，10％的ADPKD患者出现胰腺囊肿，16％的患者出现脑动脉瘤。其他病理表型包括主动脉根，胸部动脉异常，二尖瓣脱垂以及腹壁疝气。约50％的患者年满60岁后将出现终末肾衰竭。正常人的尿素氮(BUN)参考范围为2.14-7.14mmol/L，血肌酐(Cr)参考范围为44-133μmol/L，当血肌酐超过133μmol/L为炎症损伤期，超过186μmol/L为肾功能损伤期，超过451umol/L为肾功能衰竭期。最新研究结果表明，ADPKD致病基因PKD1和PKD2的缺失可以导致经典Wnt信号通路的异常激活，经典Wnt/β-catenin信号通路很可能密切的参与肾脏囊肿形成过程的调节。
LGK974是有效的特异性Porcn的抑制剂，IC50为4nM。LGK974通过特异性抑制Porcn介导的酰基化修饰从而抑制Wnt配体分子如Wnt-3a的分泌。LGKP74可抑制哺乳动物Porcn酰基转移酶活性，抑制Wnts配体的激活并减少Wnt受体LPR6的磷酸化从而促使Wnt信号通路靶基因表达降低，进而抑制经典Wnt通路的活性。LGK974作为有效的Porcn特异性抑制剂，现用于治疗依赖Wnt配体的恶性肿瘤。目前，该药已用于临床I期实验。Wnt-C59也是特异性Porcn的抑制剂，作用于Wnt3A介导的多聚TCF结合位点驱动的荧光素酶的激活。Wnt-C59可在纳摩尔水平抑制哺乳动物Porcn酰基转移酶活性。在HEK293细胞中IC50为74pM。
发明内容
本发明的一个目的是提供LGK974的新用途。
本发明提供了LGK974在制备治疗常染色体显性遗传性多囊肾的产品中的应用。
本发明还提供了LGK974在制备如下(1)-(4)中至少一种产品中的应用：
(1)减弱患病机体肾囊肿程度的产品；
(2)降低患病机体肾脏体重比的产品；
(3)降低患病机体的肾脏囊肿指数的产品；
(4)降低患病机体血液中肌酐和/或尿素氮含量的产品。
本发明的另一个目的是提供一种产品。
本发明提供的产品的活性成分为LGK974；所述产品的用途为如下(a)-(e)中的至少一种：
(a)治疗常染色体显性遗传性多囊肾；
(b)减弱患病机体肾囊肿程度；
(c)降低患病机体肾脏体重比；
(d)降低患病机体的肾脏囊肿指数；
(e)降低患病机体血液中肌酐和/或尿素氮含量。
上述应用或上述产品中，所述患病机体为具有常染色体显性遗传性多囊肾临床症状的机体。
上述应用或上述产品中，所述产品为药物。
本发明以Vil-Cre；Pkd2f3/f3小鼠作为ADPKD动物模型，进行了Wnt信号通路抑制剂—LGK974和Wnt-C59对常染色体显性遗传性多囊肾(ADPKD)治疗的效果研究，结果表明，Wnt信号通路抑制剂—LGK974对于治疗常染色体显性遗传性多囊肾具有一定的效果，具体体现在：(1)可减弱Vil-Cre；Pkd2f3/f3小鼠的肾囊肿程度；(2)可降低Vil-Cre；Pkd2f3/f3小鼠的肾脏体重比；(3)可降低Vil-Cre；Pkd2f3/f3小鼠的血液中肌酐和/或尿素氮的含量。本发明对于常染色体显性遗传性多囊肾(ADPKD)发病机制的分析，对于治疗常染色体显性遗传性多囊肾(ADPKD)的药物的研发均具有重要意义。
附图说明
图1为回交后代小鼠基因型的PCR检测结果。其中，图1A为针对Pkd2基因PCR检测结果，Pkd2f3/f3为Pkd2基因纯合，Pkd2+/f3为Pkd2基因杂合；图1B为针对Cre基因的PCR检测结果，Vil-Cre为表达Vil-Cre；WT为不表达Vil-Cre。
图2为Vil-Cre；Pkd2f3/f3小鼠模拟人类ADPKD的疾病表型。其中，图2A为Vil-Cre；Pkd2f3/f3小鼠肾脏形态；图2B为Vil-Cre；Pkd2f3/f3小鼠肝脏形态；图2C为Vil-Cre；Pkd2f3/f3小鼠胰腺形态；图2D为Vil-Cre；Pkd2f3/f3小鼠的肾脏横切面；图2E为Pkd2f3/f3小鼠的肾脏横切面。
图3为两组小鼠的肾脏肉眼观察和HE染色结果。其中，图3a为对照组肉眼观察结果；图3b为治疗组肉眼观察结果；图3c为对照组HE染色结果；图3d为治疗组HE染色结果。
图4为两组小鼠的肾脏体重比测定结果。
图5为两组小鼠的肾脏囊肿指数测定结果。
图6为两组小鼠的血液中尿素氮(BUN)与肌酐(Cr)的含量测定结果。其中，图6A为肌酐含量；图6B为尿素氮含量。
具体实施方式
下述实施例中所使用的实验方法如无特殊说明，均为常规方法。
下述实施例中所用的材料、试剂等，如无特殊说明，均可从商业途径得到。
下述实施例中的Pkd2f3/f3小鼠记载于文献“lngyu Kim,Tianbing Ding,YulongFu,et al.Conditional Mutation of Pkd2Causes Cystogenesis and Upregulatesβ-Catenin.J Am Soc Nephrol,2009(20):2556-2569”中。其构建及鉴定步骤如下：首先，在Pkd2基因的3号外显子两侧插入两个loxP位点，在阳性选择性Neo的两侧有FRT位点，由于该结构没有完全阻断正常Pkd2基因的表达，所以用这种载体通过基因打靶技术产生的Pkd2nf3小鼠可避免死于胚胎期；再将Pkd2nf3小鼠与ACTB-Flep小鼠杂交，即产生没有Neo元件的Pkd2f3/f3小鼠。鉴于在Pkd2f3/f3小鼠中没有观察到多囊表型，PC2的表达也没有发生改变，认为这种Pkd2f3等位基因不会导致Pkd2的表达失活。
下述实施例中的带有Vil-Cre重组酶的转基因小鼠购自The JacksonLaboratory，Strain Name：B6.SJL-Tg(Vil-cre)997Gum/J，Stock Number：004586，详见网址：http://jaxmice.jax.org/strain/004586.html。
下述实施例中的LGK974是Selleck公司的产品，产品目录号S7143。分子量：396.44；化学式：C23H20N6O；CAS号：1243244-14-5；溶解性(25℃)：DMSO 79mg/mL，水<1mg/mL，乙醇<1mg/mL；稳定性：3年-20℃粉状，6月-80℃溶于溶剂。
下述实施例中的Wnt-C59是Selleck公司的产品，产品目录号为S7037。分子量：379.45；化学式：C25H21N3O；CAS号：1243243-89-1；溶解性(25℃)：DMSO 76mg/mL，水<1mg/mL，乙醇<1mg/mL；稳定性：3年-20℃粉状，6月-80℃溶于溶剂。
实施例1、Vil-Cre；Pkd2f3/f3小鼠模型的构建及鉴定
Vil-Cre；Pkd2f3/f3小鼠是利用Cre-loxP系统建立的可以条件性敲除Pkd2的小鼠模型，可产生与人类常染色体显性遗传性多囊肾(Autosomal Dominant Polycystic KidneyDisease，ADPKD)类似临床表型，并早期死于肾衰竭，可作为ADPKD动物模型。其构建及鉴定过程如下：
一、Vil-Cre；Pkd2f3/f3小鼠模型的构建
将Pkd2f3/f3小鼠与带有Vil-Cre重组酶的转基因小鼠(B6.SJL-Tg(Vil-cre)997Gum/J)进行杂交，得到F1代(理论上有1/2后代的基因型为Vil-Cre:Pkd2+/f3；1/2后代的基因型为Pkd2+/f3)；将F1代中Vil-Cre:Pkd2+/f3小鼠(针对Cre基因进行PCR鉴定，具体方法参见下文，经鉴定得到大小为650bp目的条带的F1代小鼠)与Pkd2f3/f3小鼠进行回交，得到回交后代。
从以上所得回交后代中，筛选得到Pkd2基因纯合，同时可表达Vil-Cre的小鼠，具体操作如下：
提取回交后代鼠尾基因组，以其为模板，分别对Pkd2基因以及Cre基因进行PCR鉴定，其中针对Pkd2基因的PCR所用引物序列为如下三条：
5’-TCTGACTTGCAGACTGTGGG-3’，
5’-AGGTAGGGGAAGGTCAGGGTT GG-3’；
5'-TTTACGTCCAGCCAAGCT-3'；
针对Cre基因的PCR所用引物序列为如下两条：
5’-CCAGGTTACGGATATAGTTCATG-3’；
5’-TGCCACGACCAAGTGACAGC-3’。
当采用针对Pkd2基因的三条引物在同一反应体系中进行PCR扩增时，如果得到大小分别为650bp和350bp的两个条带，则对应的回交后代小鼠为Pkd2基因杂合(Pkd2+/f3)，如果得到大小为650bp的单一条带，则对应的回交后代小鼠为Pkd2基因纯合(Pkd2f3/f3)；当采用针对Cre基因的两条引物进行PCR扩增时，如果得到大小为650bp的目的条带，则对应的回交后代小鼠表达Vil-Cre，如果没有得到大小为650bp的目的条带，则对应的回交后代小鼠不表达Vil-Cre。
结果如图1所示，图1A中的Pkd2f3/f3为Pkd2基因纯合，图1B中的Vil-Cre为可表达Vil-Cre。经过以上鉴定，从回交后代中得到的Pkd2基因纯合，同时可表达Vil-Cre的小鼠，即为Vil-Cre:Pkd2f3/f3小鼠。Vil-Cre为受控于Villin1启动子的Cre酶，其中Villin1启动子表达于小鼠胚胎期第12.5天的肠上皮细胞，小鼠在Cre酶的作用下，可在细胞水平上产生Pkd2d3/d3，由于Pkd2d3/d3等位基因上的3号外显子已被剪切掉，进而产生移码突变，导致3号外显子下游不远处产生终止子，进而使Pkd2基因产物PC2成为截断蛋白。
二、Vil-Cre；Pkd2f3/f3小鼠模型的鉴定
常规饲养步骤一得到的Vil-Cre；Pkd2f3/f3小鼠，观察其存活状态。待小鼠四月龄，取其肾脏、肝脏、胰腺，观察病变情况；另外对肾脏进行HE染色分析(具体操作参见实施例2相关步骤)。同时设置Pkd2f3/f3小鼠作为对照。
结果如图2所示，其中，图2A为Vil-Cre；Pkd2f3/f3小鼠肾脏形态；图2B为Vil-Cre；Pkd2f3/f3小鼠肝脏形态；图2C为Vil-Cre；Pkd2f3/f3小鼠胰腺形态；图2D为Vil-Cre；Pkd2f3/f3小鼠的肾脏横切面；图2E为Pkd2f3/f3小鼠的肾脏横切面。从图中可以看出，步骤一得到的Vil-Cre；Pkd2f3/f3小鼠在第4～5月左右死亡，其肾脏、肝脏、胰腺均出现严重的囊肿，而且肾脏的HE染色结果进一步表明肾脏无肾实质，肾功能严重受损，判定Vil-Cre；Pkd2f3/f3小鼠死于肾囊肿导致的肾衰竭，而同窝同龄的Pkd2f3/f3小鼠则表型正常。
以上结果表明，步骤一得到的Vil-Cre；Pkd2f3/f3小鼠表型与人类ADPKD患者相似，可作为ADPKD动物模型。
实施例2、LGK974和Wnt-C59在治疗常染色体显性遗传性多囊肾中的应用
本实施例以实施例1获得的Vil-Cre；Pkd2f3/f3小鼠作为ADPKD动物模型，研究分析LGK974和Wnt-C59在治疗常染色体显性遗传性多囊肾中的应用。具体如下：
一、实验方法
1、实验分组及处理
(1)LGK974溶液
将LGK974用溶液A溶解，至其浓度为3mg/mL，后用生理盐水稀释至0.3mg/ml。溶液A的溶剂为水，溶剂为甲基纤维素和吐温80，其中，甲基纤维素在溶液A中的质量分数为0.5％，吐温80在溶液A中的体积分数为0.5％。
(2)Wnt-C59溶液
将Wnt-C59用溶液A溶解，至其浓度为3mg/mL，后用生理盐水稀释至1mg/ml。
(3)分组及处理
按照是否用药物处理分为LGK974治疗组，Wnt-C59治疗组和对照组(安慰剂)，每组随机选取至少20只Vil-Cre:Pkd2f3/f3小鼠。从小鼠出生后第三十天开始，进行连续两个月的灌胃给药，每天给药直至小鼠三月龄停药，治疗组每只小鼠按3mg/kg(每kg体重给3mgLGK974)和10mg/kg(每kg体重给10mg Wnt-C59)的剂量给药；对照组每只小鼠每天注射等体积的含有等量0.5％甲基纤维素/0.5％吐温80的生理盐水。
2、各组小鼠治疗情况分析
(1)HE染色分析两组小鼠的肾囊肿表型
在小鼠两月龄停药后，每组随机选取不少于10只小鼠，处死后，摘除双侧肾脏。在电子天平上精确称重，计算肾脏体重比(g/g)，即双侧肾脏重量(g)/体质量(g)。后将双侧肾脏沿着矢状面切开，用10％中性福尔马林溶液固定24小时，后梯度乙醇脱水，然后用石蜡包埋机常规包埋，蜡块待后行切片及HE染色。
A.脱水包埋
具体步骤：①脱水：70％乙醇1h→75％乙醇1h→80％乙醇1h→85％乙醇1h→90％乙醇1h→95％乙醇1h→100％乙醇1h→二甲苯15min×2；②包埋：石蜡包埋机进行包埋。
B.切片
将肾脏蜡块在轮转式切片机上做成3μm厚度的连续肾组织切片，用毛笔轻轻扶起，在温水中将其充分展开，再用多聚赖氨酸处理过的玻片捞起肾组织切片，电热干燥机烤干，然后放于电热恒温鼓风干燥箱里中，70℃恒温烘烤1小时，以使组织切片与玻片紧密粘连，置于盒中备用。
C.HE病理染色
原理：苏木精(hematoxylin)染液为碱性，主要使细胞核内的染色质与胞质内的核糖体着紫蓝色；伊红(eosin)为酸性染料，主要使细胞质和细胞外基质中的成分着红色。
步骤：
①二甲苯脱蜡，后用100％乙醇洗去；
②不同浓度的乙醇依次水化；
③磷酸缓冲液摇床缓洗5分钟；
④苏木精浸提8次；
⑤流水冲洗5分钟；
⑥盐酸酒精浸提8次；
⑦流水冲洗2分钟；
⑧伊红染色10分钟；
⑨不同浓度的乙醇依次脱水；
⑩二甲苯透明，后用中性树胶封片；显微镜观察。
⑾结果显示：细胞浆红色，细胞核蓝紫色。
(2)小鼠的肾脏囊肿指数的测定
LGK974治疗组，Wnt-C59治疗组和对照组小鼠HE染色的肾脏切片拍照后，图片运行于GNU Image Manipulation Program。首先将图片转换为灰度图，然后调整图片像素至800×598。将调整后的图片运行添加网格程序，设定每个网格为大小为22×22像素。计算囊肿区域网格数量和肾脏区域总网格数量比例即可得到囊肿指数。
(3)LGK974治疗组，Wnt-C59治疗组和对照组小鼠ADPKD相关生化指标的测定
本发明的发明人为了准确反应出治疗组小鼠肾肝功能的改善程度，进一步对LGK974治疗组，Wnt-C59治疗组和对照组小鼠血液中尿素氮(BUN)与肌酐(Cr)的含量进行了测定，以此确切反应肾功能的变化情况，反映出囊肿的严重程度。具体操作如下：
在小鼠三月龄时，将LGK974治疗组，Wnt-C59治疗组和对照组小鼠乙醚麻醉，从下腔静脉抽取小鼠血液，离心后获取上清即为血清。对血清进行化验即可测得肌酐及尿素氮含量。
二、实验结果
1、LGK974治疗组，Wnt-C59治疗组和对照组小鼠的肾囊肿表型
肉眼观察和HE染色结果如图3所示，从图中可以看出，LGK-974治疗组较对照组(安慰剂)具有更多的肾实质，且肾囊肿表型明显少于对照组，但Wnt-C59治疗后与对照组没有显著性差异。另外，LGK974治疗组，Wnt-C59治疗组和对照组小鼠的肾脏体重比(g/g)测定结果如图4所示，结果显示LGK974治疗组的肾脏体重比(g/g)显著小于对照组(p＝0.000015)，具有统计学意义，但Wnt-C59治疗组的肾脏体重比(g/g)较对照组相比虽有下降，但没有统计学意义(p＝0.11)。
2、LGK974治疗组，Wnt-C59治疗组和对照组小鼠的肾脏囊肿指数
LGK974治疗组，Wnt-C59治疗组和对照组小鼠的肾脏囊肿指数测定结果如图5所示，LGK974治疗组较对照组相比肾脏囊肿指数显著降低(p＝0.000019)，具有统计学意义,但Wnt-C59治疗组的肾脏囊肿指数较对照组相比虽有下降，但没有统计学意义(p＝0.09)。
3、LGK974治疗组，Wnt-C59治疗组和对照组小鼠ADPKD相关生化指标的测定
LGK974治疗组，Wnt-C59治疗组和对照组小鼠的血液中尿素氮(BUN)与肌酐(Cr)的含量测定结果如图6所示，结果显示，LGK974治疗组小鼠的血液中尿素氮(BUN)与肌酐(Cr)的含量较对照组相比，均有下降趋势，LGK974治疗组的血液中尿素氮和血肌酐显著小于对照组(p＝0.00005，p＝0.00174)，具有统计学意义，但Wnt-C59治疗组的尿素氮和血肌酐较对照组相比虽有下降，但没有统计学意义(p＝0.08，p＝0.012)。
综合本实施例的实验结果，可见LGK974在治疗常染色体显性遗传性多囊肾(ADPKD)中具有一定作用。
Claims (3)
1. LGK974在制备治疗常染色体显性遗传性多囊肾的产品中的应用。
2.根据权利要求1所述的应用，其特征在于：LGK974具备如下（1)-（4）至少一种作用：
（1）减弱患病机体肾囊肿程度；
（2）降低患病机体肾脏体重比；
（3）降低患病机体的肾脏囊肿指数；
（4）降低患病机体血液中肌酐和/或尿素氮含量。
3.根据权利要求1或2所述的应用，其特征在于：所述产品为药物。
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