CN114080238A - 抑制肌肉生长抑制素基因的mRNA的产生的核酸药物 - Google Patents
抑制肌肉生长抑制素基因的mRNA的产生的核酸药物 Download PDFInfo
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- CN114080238A CN114080238A CN202080045447.4A CN202080045447A CN114080238A CN 114080238 A CN114080238 A CN 114080238A CN 202080045447 A CN202080045447 A CN 202080045447A CN 114080238 A CN114080238 A CN 114080238A
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Abstract
本发明提供新的肌肉生长抑制素抑制法。碱基长度为15~30的反义寡核苷酸、其盐或溶剂合物,该反义寡核苷酸具有与肌肉生长抑制素基因的外显子1的靶位点互补的核苷酸序列,可抑制肌肉生长抑制素基因的mRNA的产生。含有上述反义寡核苷酸、其盐或溶剂合物的药物、食品、饲料、用于促进肌细胞的增殖和/或肥大的药剂、用于增加肌肉量和/或抑制肌减少的药剂、抑制肌肉生长抑制素基因的mRNA的产生的药剂和肌肉生长抑制素的功能抑制剂。
Description
技术领域
本发明涉及抑制肌肉生长抑制素基因的mRNA的产生的核酸药物。
背景技术
肌肉生长抑制素是TGF-β型细胞增殖因子之一,是由肌肉生长抑制素基因编码的蛋白。肌肉生长抑制素具有抑制肌细胞的增殖/肥大的作用。因此,抑制肌肉生长抑制素的功能会促进肌细胞的增殖/肥大,故作为肌萎缩的治疗目标而受到关注。实际上,开发了抗肌肉生长抑制素抗体或反义寡核苷酸(AO)等,该反义寡核苷酸控制肌肉生长抑制素基因的剪接以产生功能丧失型mRNA (非专利文献1、2)。然而,临床上有效的肌肉生长抑制素抑制法尚未确立。
现有技术文献
非专利文献
非专利文献1:St Andre等人. Skeletal Muscle 2017, 7; 25;
非专利文献2:Kang等人. Mol Ther 2011, 19; 159-64。
发明内容
发明所要解决的课题
本发明的目的在于:提供新的肌肉生长抑制素抑制法。
用于解决课题的手段
肌肉生长抑制素(MSTN)基因是3个外显子的简单结构。本发明人的目标是开发以MSTN基因的外显子1为靶的反义寡核苷酸(AO)。因此,合成了以与外显子内的剪接因子结合位点等的序列互补的ENA为单体的各种AO。将各合成AO引入(导入)到横纹肌肉瘤细胞中,利用RT-PCR法对在细胞内表达的MSTN mRNA进行半定量。然后,鉴定了使mRNA的表达降低的AO(图3)。进一步判明了:通过引入该AO,细胞的肌肉生长抑制素信号传递活性降低(图8)。这些结果显示:所鉴定的AO抑制肌肉生长抑制素的表达,其结果是具有引起肌肉生长抑制素信号传递的降低的作用。本发明是根据这些见解完成的。
本发明的宗旨如下。
(1) 碱基长度为15~30的反义寡核苷酸、其盐或溶剂合物,该反义寡核苷酸具有与肌肉生长抑制素基因的外显子1的靶位点互补的核苷酸序列(碱基序列),可抑制肌肉生长抑制素基因的mRNA的产生。
(2) (1)所述的反义寡核苷酸、其盐或溶剂合物,其中,肌肉生长抑制素基因的外显子1的核苷酸序列为SEQ ID NO: 1的核苷酸序列,肌肉生长抑制素基因的外显子1的靶位点存在于SEQ ID NO: 1的核苷酸序列的碱基编号22~420的区域内。
(3) (1)或(2)所述的反义寡核苷酸、其盐或溶剂合物,其中,反义寡核苷酸的核苷酸序列包含由SEQ ID NO: 2~25中的任一个核苷酸序列(其中,序列中的t可以是u,而u可以是t)中的连续的至少15个碱基构成的序列。
(4) (1)~(3)中任一项所述的反义寡核苷酸、其盐或溶剂合物,其中,反义寡核苷酸的碱基长度为18。
(5) (4)所述的反义寡核苷酸、其盐或溶剂合物,其中,反义寡核苷酸的核苷酸序列为SEQ ID NO: 2~25中的任一个核苷酸序列(其中,序列中的t可以是u,而u可以是t)。
(6) (1)~(5)中任一项所述的反义寡核苷酸、其盐或溶剂合物,其中,至少1个核苷酸被修饰。
(7) (6)所述的反义寡核苷酸、其盐或溶剂合物,其中,构成修饰核苷酸的糖为D-呋喃核糖,D-呋喃核糖的2’位的羟基被修饰。
(8) (7)所述的反义寡核苷酸、其盐或溶剂合物,其中,D-呋喃核糖被2’-O-烷基化和/或2’-O, 4’-C-亚烷基化。
(9) 药物,该药物含有(1)~(8)中任一项所述的反义寡核苷酸、其药学上可接受的盐或溶剂合物。
(10) (9)所述的药物,该药物用于预防和/或治疗肌肉生长抑制素参与的病症和/或疾病。
(11) (10)所述的药物,其中,肌肉生长抑制素参与的病症和/或疾病为肌萎缩。
(12) (11)所述的药物,其中,肌萎缩为选自肌营养不良症、肌病、脊髄性肌萎缩症、肌少症和废用性肌萎缩的至少1种。
(13) (10)所述的药物,其中,肌肉生长抑制素参与的病症和/或疾病为通过恢复肌肉量而产生治疗效果的病症和/或疾病。
(14) (13)所述的药物,其中,通过恢复肌肉量而产生治疗效果的病症和/或疾病为选自癌症恶液质(癌症恶病质)、糖尿病、循环系统疾病、肾病和骨病的至少1种。
(15) (14)所述的药物,其中,循环系统疾病为心功能不全和/或动脉硬化,肾病为慢性肾功能不全,骨病为炎症性关节炎。
(16) 食品,该食品含有(1)~(8)中任一项所述的反义寡核苷酸、其食品上可接受的盐或溶剂合物。
(17) 饲料,该饲料含有(1)~(8)中任一项所述的反义寡核苷酸、其饲料上可接受的盐或溶剂合物。
(18) 用于促进肌细胞的增殖和/或肥大的药剂,该药剂含有(1)~(8)中任一项所述的反义寡核苷酸、其盐或溶剂合物。
(19) 用于增加肌肉量和/或抑制肌减少的药剂,该药剂含有(1)~(8)中任一项所述的反义寡核苷酸、其盐或溶剂合物。
(20) 抑制肌肉生长抑制素基因的mRNA的产生的药剂,该药物含有(1)~(8)中任一项所述的反义寡核苷酸、其盐或溶剂合物。
(21) 肌肉生长抑制素的功能抑制剂,其含有(1)~(8)中任一项所述的反义寡核苷酸、其盐或溶剂合物。
(22) 预防和/或治疗肌肉生长抑制素参与的疾病的方法,该方法包括:对受试者给予有效量的(1)~(8)中任一项所述的反义寡核苷酸、其盐或溶剂合物。
(23) 促进肌细胞的增殖和/或肥大的方法,该方法包括:对受试者给予有效量的(1)~(8)中任一项所述的反义寡核苷酸、其盐或溶剂合物。
(24) 增加肌肉量和/或抑制肌减少的方法,该方法包括:对受试者给予有效量的(1)~(8)中任一项所述的反义寡核苷酸、其盐或溶剂合物。
(25) 抑制肌肉生长抑制素基因的mRNA的产生的方法,该方法包括:对受试者给予有效量的(1)~(8)中任一项所述的反义寡核苷酸、其盐或溶剂合物。
(26) 抑制肌肉生长抑制素的功能的方法,该方法包括:对受试者给予有效量的(1)~(8)中任一项所述的反义寡核苷酸、其盐或溶剂合物。
(27) (1)~(8)中任一项所述的反义寡核苷酸、其盐或溶剂合物,其用于在预防和/或治疗肌肉生长抑制素参与的疾病的方法中应用。
(28) (1)~(8)中任一项所述的反义寡核苷酸、其盐或溶剂合物,其用于在促进肌细胞的增殖和/或肥大的方法中应用。
(29) (1)~(8)中任一项所述的反义寡核苷酸、其盐或溶剂合物,其用于在增加肌肉量和/或抑制肌减少的方法中应用。
(30) (1)~(8)中任一项所述的反义寡核苷酸、其盐或溶剂合物,其用于在抑制肌肉生长抑制素基因的mRNA的产生的方法中应用。
(31) (1)~(8)中任一项所述的反义寡核苷酸、其盐或溶剂合物,其用于在抑制肌肉生长抑制素的功能的方法中应用。
发明效果
利用本发明的AO,可抑制肌肉生长抑制素的表达,降低肌肉生长抑制素信号传递。
本说明书包含作为本申请的优先权基础的日本专利申请、日本特愿2019-118446的说明书和/或附图中记载的内容。
附图说明
[图1] MSTN的pre-mRNA和AO的靶位点。由MSTN基因转录而产生的MSTN pre-mRNA由外显子1、外显子2、外显子3构成。制作了与MSTN pre-mRNA的外显子1内的序列互补的AO(AO1、AO2、AO3)。
[图2] AO1、AO2、AO3的有效性比较。向人横纹肌肉瘤细胞中引入AO,显示其MSTNmRNA的RT-PCR的结果。a) □内显示AO1、AO2、AO3的互补序列。核苷酸序列的数字是以MSTN的外显子1的5’端的碱基为1时的碱基编号。b) 显示人横纹肌肉瘤细胞的MSTN mRNA、GAPDHmRNA的RT-PCR的结果。w/o表示AO未处理。c) 显示以AO未处理的MSTN/GAPDH为1时的相对值。*P<0.05、***P<0.001。
[图3] 在AO2周边设计的AO的有效性比较。向人横纹肌肉瘤细胞中引入AO,显示其MSTN mRNA的RT-PCR的结果。a) AO用-表示,□内显示AO2的互补序列。在核苷酸序列的上段,显示相对于AO2的序列向5’侧、3’侧各错开3个碱基的AO。在核苷酸序列的下段,显示相对于AO2的序列向3’侧各错开1个碱基的AO。核苷酸序列的数字是以MSTN的外显子1的5’端的碱基为1时的碱基编号。b、d) 显示人横纹肌肉瘤细胞的MSTN mRNA、GAPDH mRNA的RT-PCR的结果。w/o表示AO未处理。c、e) 显示以AO未处理的MSTN/GAPDH为1时的相对值。*P<0.05、***P<0.001。
[图4a] AO2+1与在其他区域设计的AO的有效性比较。向人横纹肌肉瘤细胞中引入AO,显示其MSTN mRNA的RT-PCR的结果。显示外显子1中的AO的互补序列。AO用-表示,配置在互补序列的下方。核苷酸序列的数字是以MSTN的外显子1的5’端的碱基为1时的碱基编号。
[图4bc] b) 显示人横纹肌肉瘤细胞的MSTN mRNA、GAPDH mRNA的RT-PCR的结果。w/o表示AO未处理。c) 显示以AO未处理的MSTN/GAPDH为1时的相对值。*P<0.05、**P<0.01、***P<0.001。
[图5ab] a) AO2+1在人成肌细胞中的有效性的检验。向人成肌细胞中引入AO,显示其MSTN mRNA的RT-PCR的结果。b) 显示人成肌细胞的MSTN mRNA、GAPDH mRNA的RT-PCR的结果。显示以AO 0nM处理的MSTN/GAPDH为1时的相对值。**P<0.01、***P<0.001。
[图6ab] a) AO2+1对GDF11表达的影响的检验。向人横纹肌肉瘤细胞中引入AO,显示其MSTN mRNA和GDF11 mRNA的RT-PCR的结果。b) 显示人横纹肌肉瘤细胞的MSTN mRNA、GDF11 mRNA、GAPDH mRNA的RT-PCR的结果。显示以AO未处理(w/o)的MSTN/GAPDH、GDF11/GAPDH为1时的相对值。***P<0.001。
[图7] 肌肉生长抑制素信号。肌肉生长抑制素通过与细胞表层的受体结合来激活下游的信号传递。若成熟肌肉生长抑制素与受体结合,则Smad2/3发生磷酸化,磷酸化Smad2/3转移到核内。磷酸化Smad2/3与靶基因的启动子内存在的Smad结合区域结合,从而诱导靶基因的表达。
[图8ab] 由AO2+1引起的肌肉生长抑制素信号传递的降低。显示人横纹肌肉瘤细胞中的AO处理时的肌肉生长抑制素信号传递的检验结果。
a) 显示AO2+1处理时对体外肌肉生长抑制素转录活性测定系统的作用的示意图。肌肉生长抑制素信号分析是通过在AO2+1处理、AO未处理时诱导表达的荧光素酶的活性来进行评价。
b) 以AO未处理(w/o)的结果为1,用相对值表示。***P<0.001。
[图9] 由AO2+1引起的肌肉生长抑制素信号传递的降低。显示人成肌细胞中的AO处理时的肌肉生长抑制素信号传递的研究结果。以AO 0nM处理的结果为1,用相对值表示。*P<0.05、**P<0.01、***P<0.001。
[图10] 由AO2+1引起的人成肌细胞的增殖促进。显示人成肌细胞中的AO处理和未处理(w/o)的细胞增殖的研究结果。*P<0.05、***P<0.001。
[图11] 跨物种保守的AO2+1的靶序列。显示人(Homo sapiens)、牛(Bos Taurus)、猪(Sus scrofa)、狗(Canis Lupus familiaris)的MSTN外显子1的氨基酸编码区的核酸序列的比对(不包括5’非翻译区)。□内显示AO2+1的靶序列。
具体实施方式
以下,更详细地对本发明的实施方式进行说明。
本发明提供:碱基长度为15~30的反义寡核苷酸、其盐或溶剂合物,该反义寡核苷酸具有与肌肉生长抑制素基因的外显子1的靶位点互补的核苷酸序列,可抑制肌肉生长抑制素基因的mRNA的产生。
人肌肉生长抑制素基因的外显子1的核苷酸序列见SEQ ID NO: 1。本发明中,在肌肉生长抑制素基因的外显子1的核苷酸序列为SEQ ID NO: 1的核苷酸序列的情况下,肌肉生长抑制素基因的外显子1的靶位点可存在于SEQ ID NO: 1的核苷酸序列的碱基编号22~420的区域内。
而且,在本发明中,反义寡核苷酸的核苷酸序列可包含由SEQ ID NO: 2~25中的任一个核苷酸序列(其中,序列中的t可以是u,而u可以是t)中的连续的至少15个碱基构成的序列。
反义寡核苷酸的碱基长度可以是18,反义寡核苷酸的核苷酸序列可以是SEQ IDNO: 2~25中的任一个核苷酸序列(其中,序列中的t可以是u,而u可以是t)。
构成反义寡核苷酸的核苷酸可以是天然型DNA、天然型RNA、它们的修饰物中的任一者,优选至少1个为修饰核苷酸。
作为修饰核苷酸,可示例:糖被修饰的核苷酸(例如,D-呋喃核糖被2’-O-烷基化的核苷酸、D-呋喃核糖被2’-O,4’-C-亚烷基化的核苷酸等D-呋喃核糖的2’位的羟基被修饰的核苷酸)、磷酸二酯键被修饰(例如,硫代化)的核苷酸、碱基被修饰的核苷酸、将它们组合而成的核苷酸等。构成反义寡核苷酸的至少1个D-呋喃核糖被2’-O-烷基化的核苷酸或被2’-O,4’-C-亚烷基化的核苷酸因对RNA的结合力高、对核酸酶的耐性高,故可期待较天然型核苷酸(即,寡DNA、寡RNA)高的治疗效果。另外,构成寡核苷酸的至少1个磷酸二酯键被硫代化的核苷酸也因对核酸酶的耐性高,故可期待较天然型核苷酸(即,寡DNA、寡RNA)高的治疗效果。包含如上所述的修饰的糖和修饰的磷酸两者的寡核苷酸因对核酸酶的耐性更高,故可期待更高的治疗效果。
关于反义寡核苷酸,作为糖的修饰的例子,可列举:D-呋喃核糖的2’-O-烷基化(例如,2’-O-甲基化、2’-O-氨基乙基化、2’-O-丙基化、2’-O-烯丙基化、2’-O-甲氧基乙基化、2’-O-丁基化、2’-O-戊基化、2’-O-炔丙基化等)、D-呋喃核糖的2’-O,4’-C-亚烷基化(例如,2’-O,4’-C-亚乙基化、2’-O,4’-C-亚甲基化、2’-O,4’-C-亚丙基化、2’-O,4’-C-四亚甲基化、2’-O,4’-C-五亚甲基化等)、3’-脱氧-3’-氨基-2’-脱氧-D-呋喃核糖、3’-脱氧-3’-氨基-2’-脱氧-2’-氟-D-呋喃核糖等。
关于反义寡核苷酸,作为磷酸二酯键的修饰的例子,可列举:硫代磷酸酯键、甲基膦酸酯键、甲基硫代膦酸酯键、二硫代磷酸酯键、氨基磷酸酯键等。
关于反义寡核苷酸,作为碱基的修饰的例子,可列举:胞嘧啶的5-甲基化、5-氟化、5-溴化、5-碘化、N4-甲基化、胸苷的5-脱甲基化(尿嘧啶)、5-氟化、5-溴化、5-碘化、腺嘌呤的N6-甲基化、8-溴化、鸟嘌呤的N2-甲基化、8-溴化等。
本发明的反义寡核苷酸可以是盐的形式。在将本发明的反义寡核苷酸用于药物的情况下,盐可以是药学上可接受的盐,作为这样的盐,可列举:钠盐、钾盐、锂盐这样的碱金属盐、钙盐、镁盐这样的碱土金属盐、铝盐、铁盐、锌盐、铜盐、镍盐、钴盐等金属盐;铵盐这样的无机盐、叔辛胺盐、二苄胺盐、吗啉盐、葡糖胺盐、苯基甘氨酸烷基酯盐、乙二胺盐、N-甲基葡萄糖胺盐、胍盐、二乙胺盐、三乙胺盐、二环己胺盐、N,N’-二苄基乙二胺盐、氯普鲁卡因盐、普鲁卡因盐、二乙醇胺盐、N-苄基-苯乙基胺盐、哌嗪盐、四甲基铵盐、三(羟甲基)氨基甲烷盐这样的有机盐等胺盐;氢氟酸盐、盐酸盐、氢溴酸盐、氢碘酸盐这样的氢卤酸盐、硝酸盐、高氯酸盐、硫酸盐、磷酸盐等无机酸盐;甲磺酸盐、三氟甲磺酸盐、乙磺酸盐这样的低级烷烃磺酸盐、苯磺酸盐、对甲苯磺酸盐这样的芳基磺酸盐、乙酸盐、苹果酸盐、富马酸盐、琥珀酸盐、枸橼酸盐、酒石酸盐、草酸盐、马来酸盐等有机酸盐;甘氨酸盐、赖氨酸盐、精氨酸盐、鸟氨酸盐、谷氨酸盐、天冬氨酸盐这样的氨基酸盐等。这些盐可通过已知的方法进行制造。
另外,反义寡核苷酸有时还以溶剂合物(例如,水合物)的形式存在,可以是这样的溶剂合物。
而且,反义寡核苷酸可按前药的形式给药,作为前药,可列举:酰胺、酯、氨基甲酸盐、碳酸盐、酰脲、磷酸盐等。这些前药可通过已知的方法进行制造。
对反义寡核苷酸的合成方法没有特别限定,可采用以往已知的方法。上述合成方法例如可列举:基于基因工程学方法的合成法、化学合成法等。基因工程学方法例如可列举:体外转录合成法、使用载体的方法、基于PCR盒的方法。对上述载体没有特别限定,可列举:质粒等非病毒载体、病毒载体等。对上述化学合成法没有特别限定,例如可列举:亚磷酰胺法(phosphoramidite method)和H-膦酸酯法(H-phosphonate method)等。上述化学合成法例如可使用市售的自动核酸合成仪。上述化学合成法通常使用亚酰胺(amidite,亚磷酰胺)。对上述亚酰胺(amidite)没有特别限定,在后述的实施例中,反义寡核苷酸是使用ENA-2CE亚磷酰胺和2’OMe-2CE亚磷酰胺通过亚磷酰胺法进行合成。
关于天然型核苷和2’-O-甲基核苷(即,2’-O-甲基鸟苷、2’-O-甲基腺苷、2’-O-甲基胞嘧啶、2’-O-甲基尿苷),亚磷酰胺试剂可使用市售的试剂。关于烷基的碳原子数为2~6个的2’-O-烷基鸟苷、腺苷、胞嘧啶和尿苷如下所述。
2’-O-氨基乙基鸟苷、腺苷、胞嘧啶、尿苷可按照文献(Blommers等人.Biochemistry (1998), 37, 17714-17725.)进行合成。
2’-O-丙基鸟苷、腺苷、胞嘧啶、尿苷可按照文献(Lesnik, E. A.等人.Biochemistry (1993), 32, 7832-7838.)进行合成。
2’-O-烯丙基鸟苷、腺苷、胞嘧啶、尿苷可使用市售的试剂。
2’-O-甲氧基乙基鸟苷、腺苷、胞嘧啶、尿苷可按照专利(US6261840)或文献(Martin, P. Helv. Chim. Acta. (1995)78, 486-504.进行合成。
2’-O-丁基鸟苷、腺苷、胞嘧啶、尿苷可按照文献(Lesnik, E. A.等人.Biochemistry (1993), 32, 7832-7838.)进行合成。
2’-O-戊基鸟苷、腺苷、胞嘧啶、尿苷可按照文献(Lesnik, E. A.等人.Biochemistry (1993), 32, 7832-7838.)进行合成。
2’-O-炔丙基鸟苷、腺苷、胞嘧啶、尿苷可使用市售的试剂。
关于2’-O, 4’-C-亚甲基鸟苷、腺苷、5-甲基胞嘧啶和胸苷,可按照WO99/14226中记载的方法进行制造,关于亚烷基的碳原子数为2~5个的2’-O, 4’-C-亚烷基鸟苷、腺苷、5-甲基胞嘧啶和胸苷,可按照WO00/47599中记载的方法进行制造。
将亚磷酰胺试剂偶联后,与硫、二硫化四乙基秋兰姆(TETD、Applied Biosystems公司)、Beaucage试剂(Glen Research公司)、或氢化黄原素等试剂反应,从而可合成具有硫代磷酸酯键的反义寡核苷酸(Tetrahedron Letters, 32, 3005 (1991), J. Am. Chem.Soc. 112, 1253 (1990), PCT/WO98/54198)。
作为合成仪中使用的可控孔度玻璃(CPG),结合有2’-O-甲基核苷的CPG可使用市售品。另外,关于2’-O, 4’-C-亚甲基鸟苷、腺苷、5-甲基胞嘧啶和胸苷,可使按照WO99/14226中记载的方法制造的核苷依据文献(Oligonucleotide Synthesis, Edited by M.J. Gait, Oxford University Press, 1984)与CPG结合,而关于亚烷基的碳原子数为2~5个的2’-O, 4’-C-亚烷基鸟苷、腺苷、5-甲基胞嘧啶和胸苷,可使按照WO00/47599中记载的方法制造的核苷依据上述同一文献与CPG结合。通过使用经修饰的CPG (记载于日本特开平7-87982的实施例12b),可合成在3’末端结合有2-羟乙基磷酸基的寡核苷酸。另外,如果使用3’-氨基-修饰剂C3 CPG、3’-氨基-修饰剂C7 CPG、甘油基CPG、(Glen Research)、3’-specer C3 SynBase CPG 1000、3’-specer C9 SynBase CPG 1000 (link technologies),则可合成在3’末端结合有羟基烷基磷酸基、或氨基烷基磷酸基的寡核苷酸。
本发明的反义寡核苷酸可用于药物。在用作药物的情况下,反义寡核苷酸可以是其药学上可接受的盐、溶剂合物或前药的形式。因此,本发明提供药物,该药物包含上述的反义寡核苷酸、其药学上可接受的盐或溶剂合物。药物可以是用于预防和/或治疗肌肉生长抑制素参与的病症和/或疾病的药物,作为肌肉生长抑制素参与的病症和/或疾病,可列举:肌萎缩(例如,肌萎缩为肌营养不良症、肌病、脊髄性肌萎缩症、肌少症、废用性肌萎缩等)、通过恢复肌肉量而产生治疗效果的病症和/或疾病(例如,癌症恶液质、糖尿病、循环系统疾病(心功能不全、动脉硬化等)、肾病(慢性肾功能不全等)、骨病(炎症性关节炎等)等),但并不限于这些。从肌肉生长抑制素的抑制可使骨骼肌量增加的角度考虑,认为无论肌萎缩的原因如何,皆可用于治疗所有的呈现肌萎缩的疾病。骨骼肌量的增加可谋求运动量的增加,还有助于改善全身代谢。另外,还可期待对心肌起作用以恢复其功能。另一方面,肌肉生长抑制素的抑制还被期待:对破骨细胞起作用以抑制骨破坏;激活血管内皮细胞的恒定性维持能力;诱导细胞凋亡;提高胰岛素的敏感性等。
本发明的反义寡核苷酸、其药学上可接受的盐、溶剂合物或前药(以下,记作“有效成分”。)可单独、或者与药理学上可接受的载体、稀释剂或赋形剂一起以适当剂型的制剂形式,对哺乳动物(例如,人、兔、狗、猫、大鼠、小鼠)进行口服或胃肠外给药。给药量还根据给药对象、对象疾病、症状、给药路径等而不同,例如,在用于预防/治疗肌萎缩性疾病(例如,肌营养不良症)的情况下,作为有效成分的1次剂量,通常可将0.1~50mg/kg体重左右、优选0.5mg/kg体重左右以每周1次~每月1次左右的频率、优选以每年1次左右的频率,通过口服/肌肉内注射/皮下注射/静脉注射进行给药(优选连续或隔日给药)。在其他胃肠外给药和口服给药的情况下也可按此量给药。在症状特别严重的情况下,可根据其症状增量。关于其他疾病,也可参考上述的给药量,以适当增减的剂量进行给药。
作为用于口服给药的制剂,可列举:固体剂型或液体剂型,具体而言,有片剂(包括糖衣片、薄膜包衣片)、丸剂、颗粒剂、散剂、胶囊剂(包括软胶囊剂)、糖浆剂、乳剂、悬浮剂等。所述制剂可利用常规方法进行制造,可含有制剂领域通常使用的载体、稀释剂或赋形剂。例如,作为片剂用的载体、赋形剂,可列举:乳糖、淀粉、蔗糖、硬脂酸镁等。
作为用于胃肠外给药的制剂,例如可列举:注射剂、栓剂等,注射剂可以是静脉注射剂、皮下注射剂、皮内注射剂、肌肉注射剂、点滴注射剂等剂型。这样的注射剂通过常规方法、即通过将有效成分溶解、悬浮或乳化于通常用于注射剂的无菌的水性或油性液来调制。作为注射用水性液,可列举:生理盐水、包含葡萄糖或其他辅药的等渗液等,可与适当的助溶剂、例如醇(例如,乙醇)、多元醇(例如,丙二醇、聚乙二醇)、非离子表面活性剂(例如,聚山梨酯80、HCO-50 (氢化蓖麻油的聚氧乙烯(50mol)加成物))等并用。作为油性液,可列举:芝麻油、大豆油等,可并用苯甲酸苄酯、苄醇等作为助溶剂。所调制的注射液通常填充在适当的安瓿中。用于直肠给药的栓剂可通过将有效成分混合在通常的栓剂用基质中来调制。
上述的口服用或胃肠外用药物制剂可调制成适合于有效成分的给药量这样的给药单位的剂型。作为这样的给药单位的剂型,可列举:片剂、丸剂、胶囊剂、注射剂(安瓿)、栓剂等,各自的每个给药单位剂型通常优选含有0.1~1.000mg的有效成分。
本发明的反义寡核苷酸还可用于食品、饲料。例如,使用本发明的反义寡核苷酸作为食品或饲料的添加剂、或者作为人或动物用的补充剂。本发明的反义寡核苷酸可以是作为食品或饲料可接受的盐、溶剂合物或前药的形式。因此,本发明提供食品,该食品含有上述的反义寡核苷酸、其食品上可接受的盐或溶剂合物。另外,本发明还提供饲料,该饲料含有上述的反义寡核苷酸、其饲料上可接受的盐或溶剂合物。作为食品或饲料可接受的盐、溶剂合物或前药的一例,可列举:药学上可接受的盐、溶剂合物或前药,关于它们见上述。
可使用本发明的反义寡核苷酸来促进肌细胞的增殖和/或肥大。肌细胞是形成人或动物的肌肉组织的具收缩性的细胞,包括骨骼肌细胞、平滑肌细胞、心肌细胞等。本发明的反义寡核苷酸的肌肉生长抑制素抑制在成肌细胞中具有效果,通过诱导成肌细胞的增殖促进/分化促进,结果可引起肌细胞的增殖/肥大。肌细胞还包括成肌细胞这样的前体细胞。本发明的反义寡核苷酸可以是盐、溶剂合物或前药的形式。作为盐、溶剂合物或前药的一例,可列举:药学上可接受的盐、溶剂合物或前药,关于这些见上述。动物只要是表达肌肉生长抑制素的动物即可,可示例:猫、狗、绵羊、猪、牛、鸡、火鸡等哺乳动物、鲑鱼、鳟鱼、鳕鱼、金枪鱼、黄尾鱼等鱼类等役用、食用的饲养动物。因此,本发明提供:用于促进肌细胞的增殖和/或肥大的药剂,该药剂包含上述的反义寡核苷酸、其盐或溶剂合物。另外,本发明还提供:促进肌细胞的增殖和/或肥大的方法,该方法包括:对受试者给予有效量的上述的反义寡核苷酸、其盐或溶剂合物。受试者可以是人或动物。关于动物见上述。药剂的剂型、给药量、给药路径、给药频率等以上述的药物为基准即可,可适当变更以获得所期望的效果。
另外,本发明的反义寡核苷酸可用于促进人或动物的肌肉形成或抑制肌减少。本发明的反义寡核苷酸可以是盐、溶剂合物或前药的形式。作为盐、溶剂合物或前药的一例,可列举:药学上可接受的盐、溶剂合物或前药,关于这些见上述。动物只要是表达肌肉生长抑制素的动物即可,可示例:猫、狗、绵羊、猪、牛、鸡、火鸡等哺乳动物、鲑鱼、鳟鱼、鳕鱼、金枪鱼、黄尾鱼等鱼类等役用、食用的饲养动物。因此,本发明提供用于促进肌肉形成和/或抑制肌减少的药剂,该药剂包含上述的反义寡核苷酸、其盐或溶剂合物。另外,本发明还提供促进肌肉形成和/或抑制肌减少的方法,该方法包括:对受试者给予有效量的上述的反义寡核苷酸、其盐或溶剂合物。受试者可以是人或动物。关于动物见上述。药剂的剂型、给药量、给药路径、给药频率等以上述的药物为基准即可,可适当变更以获得所期望的效果。
添加本发明的反义寡核苷酸、其食品上可接受的盐、溶剂合物或前药的食品可以是植物性食品、动物性食品、真菌类食品、生鲜食品、加工食品、定向食品、烹饪/调味用材料、饮料、健康食品、航天食品、宠物食品等任何食品。
在本发明的食品中,可添加:蛋白质、脂质、碳水化合物、钠等普通成分;钾、钙、镁、磷等矿物质类;铁、锌、铜、硒、铬等微量元素;维生素A、β-胡萝卜素、维生素B1、维生素B2、维生素B6、维生素B12、维生素C、烟酸、叶酸、维生素D3、维生素E、生物素、泛酸等维生素类;辅酶Q10、α-硫辛酸、半乳寡糖、食物纤维、赋形剂(水、羧甲基纤维素、乳糖等)、甜味剂、矫味剂(苹果酸、枸橼酸、氨基酸等)、香料等。在将本发明的食品制成液体制剂的情况下,作为分散或溶解食品成分的液体,可使用水、生理盐水、汤、牛奶、果汁等。本发明的食品可制成粉末、颗粒、片剂、液体制剂等形状。为了使病人或老年人可容易地摄取,优选制成果冻等凝胶状制品。
添加本发明的反义寡核苷酸、其饲料上可接受的盐、溶剂合物或前药的饲料可以是谷类、植物性油粕类、糠麸类、制造粕类、动物性饲料等单一饲料、掺混有多种饲料原料或饲料添加剂的掺混饲料等任何饲料。
在本发明的饲料中,可添加:抗氧化剂(乙氧基喹啉、二丁基羟基甲苯、丁基羟基苯甲醚等)、防霉剂(丙酸、丙酸钙、丙酸钠等)、粘合剂(海藻酸钠、酪蛋白钠、羧甲基纤维素钠、丙二醇、聚丙烯酸钠等)、乳化剂(脂肪酸甘油酯、蔗糖脂肪酸酯、脱水山梨糖醇脂肪酸酯、聚氧乙烯脱水山梨糖醇脂肪酸酯、聚氧乙烯甘油脂肪酸酯等)、调节剂(甲酸等)、氨基酸等(氨基乙酸、DL-丙氨酸、L-精氨酸、盐酸L-赖氨酸、左旋肉碱、胍基乙酸、L-谷氨酸钠、牛磺酸、2-脱氨基-2-羟基甲硫氨酸、DL-色氨酸、L-色氨酸、L-苏氨酸、L-缬氨酸、DL-甲硫氨酸、硫酸L-赖氨酸等)、维生素(L-抗坏血酸、L-抗坏血酸钙、L-抗坏血酸钠、L-抗坏血酸-2-磷酸酯钠钙、L-抗坏血酸-2-磷酸酯镁、乙酰甲萘醌、肌醇、盐酸二苯甲酰硫胺素、麦角钙化醇、氯化胆碱、盐酸硫胺素、盐酸吡哆醇、β-胡萝卜素、胆钙化醇、乙酸dl-α-生育酚、氰钴胺、硝酸硫胺素、烟酸、烟酸酰胺、对氨基苯甲酸、D-泛酸钙、DL-泛酸钙、d-生物素、维生素A粉末、维生素A油、维生素D粉末、维生素D3油、维生素E粉末、25-羟基胆钙化醇、二甲基嘧啶醇亚硫酸氢甲萘醌、亚硫酸氢钠甲萘醌、叶酸、核黄素、核黄素丁酸酯等)、矿物质(氯化钾、枸橼酸铁、葡萄糖酸钙、琥珀酸枸橼酸铁钠、氧化镁、氢氧化铝、碳酸锌、碳酸钴、碳酸氢钠、碳酸镁、碳酸锰、2-脱氨基-2-羟基甲硫氨酸锌、DL-苏氨酸铁、乳酸钙、富马酸亚铁、肽锌、肽铁、肽铜、肽锰、碘化钾、碘酸钾、碘酸钙、硫酸锌(干燥)、硫酸锌(晶体)、硫酸锌甲硫氨酸、硫酸钠(干燥)、硫酸镁(干燥)、硫酸镁(晶体)、硫酸钴(干燥)、硫酸钴(晶体)、硫酸铁(干燥)、硫酸铜(干燥)、硫酸铜(晶体)、硫酸锰、磷酸一氢钾(干燥)、磷酸一氢钠(干燥)、磷酸二氢钾(干燥)、磷酸二氢钠(干燥)、磷酸二氢钠(晶体)等)、色素、合成抗菌剂、抗生素、着香料、呈味剂、酶、益生素、有机酸等。
食品或饲料的摄取可按照确认到所期望的效果(例如,促进肌肉形成)的摄取量和频率、摄取期间来进行。
本发明的反义寡核苷酸可抑制肌肉生长抑制素基因的mRNA的产生。因此,本发明提供:抑制肌肉生长抑制素基因的mRNA的产生的药剂,该药剂含有上述的反义寡核苷酸。本发明的药剂可用作人或动物用的药物、食品或饲料用的添加剂或补充剂、动物的生长促进剂、或者实验用试剂。本发明的反义寡核苷酸可以是盐、溶剂合物或前药的形式。作为盐、溶剂合物或前药的一例,可列举:药学上可接受的盐、溶剂合物或前药,关于这些见上述。
本发明的反义寡核苷酸可抑制肌肉生长抑制素的功能。因此,提供肌肉生长抑制素的功能抑制剂,其包含本发明的上述反义寡核苷酸。本发明的肌肉生长抑制素功能抑制剂可用作人或动物用的药物、食品或饲料用的添加剂或补充剂、动物的生长促进剂、或者实验用试剂。反义寡核苷酸可以是盐、溶剂合物或前药的形式。作为盐、溶剂合物或前药的一例,可列举:药学上可接受的盐、溶剂合物或前药,关于这些见上述。
在用作实验用试剂的情况下,通过使用本发明的反义寡核苷酸、其盐或溶剂合物来处理表达肌肉生长抑制素的细胞、组织或器官,可抑制肌肉生长抑制素的表达。本发明的反义寡核苷酸、其盐和溶剂合物可以以有效抑制肌肉生长抑制素表达的量使用。作为表达肌肉生长抑制素的细胞,可示例:肌细胞、肌肉瘤细胞、消化器官/肺/食道等的癌细胞等。另外,除了来自天然的细胞以外,还可示例:引入了肌肉生长抑制素基因的重组细胞。作为表达肌肉生长抑制素的组织和器官,可示例:骨骼肌、心肌、血管、肾脏、消化道、子宫、肝脏、胰脏、肺等。肌肉生长抑制素的表达可通过利用RT-PCR分析样品中的肌肉生长抑制素mRNA、或者利用蛋白质印迹法检测样品中的肌肉生长抑制素蛋白、或者利用质谱法进行检测来分析。
实施例
以下,根据实施例来详细地说明本发明,但本发明并不限于这些实施例。
[实施例1~24] 反义寡核苷酸(AO)的合成
合成了表1所示的反义寡核苷酸(AO)。与MSTN pre-mRNA互补的AO的序列位置见图1~4。向AO的序列中的C (胞嘧啶)和T (胸腺嘧啶)中引入修饰核酸的ENA (注册商标)(2’-O,4’-C-亚乙基桥联核酸),提高亲和性和稳定性。
CaCuggaCCaGCaaCaau的合成 (MSTN_Ex1_AO1)的合成(实施例1)
使用核酸自动合成仪(日本Techno Service公司制造的DNA/RNA合成装置 NTS H-6),以1μmol的规模进行。各合成循环中的溶剂、试剂、亚磷酰胺的浓度与合成天然寡核苷酸的情况相同,试剂、2’-O-甲基核苷的亚磷酰胺(腺苷体产品编号10-3100-10、鸟苷体产品编号10-3121-10)使用Glen Research公司制造的产品。溶剂使用和光纯药工业的产品。非天然型的亚磷酰胺使用日本特开2000-297097的实施例22 (5’-O-二甲氧基三苯甲基-2’-O,4’-C-亚乙基-4-N-苯甲酰基-5-甲基胞苷-3’-O-(2-氰基乙基N,N-二异丙基)亚磷酰胺)、实施例9 (5’-O-二甲氧基三苯甲基-2’-O,4’-C-亚乙基-5-甲基尿苷-3’-O-(2-氰基乙基N,N-二异丙基)亚磷酰胺)的化合物。使用通用可控孔度玻璃(CPG) (Glen Research公司制造、产品编号25-5040)作为固相载体,合成了标题化合物。其中,亚酰胺的缩合所需的时间为15分钟。
通过将具有靶序列的受保护的寡核苷酸类似物在浓氨水中进行加热处理(55℃、8小时),从支撑体上切取寡聚物,同时去除磷原子上的保护基氰基乙基和核酸碱基上的保护基。使用Glen-Pak DNA纯化盒(Glen Research公司制造、产品编号60-5100),按照GlenResearch推荐的方法,在盒内对该氨溶液进行脱DMT,减压馏去所回收的溶液,残余物通过反相HPLC (岛津制作所制造的LC-2a、柱(YMC制造的Triart C18 (10×150mm))、A溶液:0.1M的乙酸三乙胺水溶液(TEAA)、 pH7.0;B溶液:乙腈、B%:10%→25% (30分钟、线性梯度);50°C;4.7mL/分钟;280nm)进行纯化。馏去溶剂后,溶解于10mM的NaOH溶液中,使用Microsep离心过滤装置(日本Pall公司制造、产品编号MCP003C),通过超滤进行纯水置换,冷冻干燥后,得到了目标化合物。
augCaTTaCaCagCCCCu (MSTN_Ex1_AO2)的合成(实施例2)
按照与实施例1的化合物同样的方式,合成了具有靶序列的实施例2的化合物。
该化合物通过负离子MALDI-TOFMS来鉴定(计算值:6395.311、测定值:6392.585)。
该化合物的核苷酸序列是与人肌肉生长抑制素(MSTN), 2号染色体上的RefSeqGene (LRG_200) (基因库登录号NG_009800.1)的核苷酸编号240-257互补的序列。
gauuuaguguuuuguCuC (MSTN_Ex1_AO3)的合成(实施例3)
按照与实施例1的化合物同样的方式,合成了具有靶序列的实施例3的化合物。
该化合物通过负离子MALDI-TOFMS来鉴定(计算值:6226.921、测定值:6220.691)。
该化合物的核苷酸序列是与人肌肉生长抑制素(MSTN), 2号染色体上的RefSeqGene (LRG_200) (基因库登录号NG_009800.1)的核苷酸编号265-282互补的序列。
aagTaCaTgCaTTaCaCa (MSTN_Ex1_AO2-6)的合成(实施例4)
按照与实施例1的化合物同样的方式,合成了具有靶序列的实施例4的化合物。
该化合物通过负离子MALDI-TOFMS来鉴定(计算值:6441.351、测定值:6435.8281)。
该化合物的核苷酸序列是与人肌肉生长抑制素(MSTN), 2号染色体上的RefSeqGene(LRG_200) (基因库登录号NG_009800.1)的核苷酸编号246-263互补的序列。
TaCaTgCaTTaCaCagCC (MSTN_Ex1_AO2-3)的合成(实施例5)
按照与实施例1的化合物同样的方式,合成了具有靶序列的实施例5的化合物。
该化合物通过负离子MALDI-TOFMS来鉴定(计算值:6445.375、测定值:6439.7319)。
该化合物的核苷酸序列是与人肌肉生长抑制素(MSTN), 2号染色体上的RefSeqGene (LRG_200) (基因库登录号NG_009800.1)的核苷酸编号243-260互补的序列。
CaTTaCaCagCCCCTCTT (MSTN_Ex1_AO2+3)的合成(实施例6)
按照与实施例1的化合物同样的方式,合成了具有靶序列的实施例6的化合物。
该化合物通过负离子MALDI-TOFMS来鉴定(计算值:6436.396、测定值:6430.9077)。
该化合物的核苷酸序列是与人肌肉生长抑制素(MSTN), 2号染色体上的RefSeqGene (LRG_200) (基因库登录号NG_009800.1)的核苷酸编号237-254互补的序列。
TaCaCagCCCCTCTTTTT (MSTN_Ex1_AO2+6)的合成(实施例7)
按照与实施例1的化合物同样的方式,合成了具有靶序列的实施例7的化合物。
该化合物通过负离子MALDI-TOFMS来鉴定(计算值:6440.376、测定值:6435.562)。
该化合物的核苷酸序列是与人肌肉生长抑制素(MSTN), 2号染色体上的RefSeqGene (LRG_200) (基因库登录号NG_009800.1)的核苷酸编号234-251互补的序列。
aCagCCCCTCTTTTTCCa (MSTN_Ex1_AO2+9)的合成(实施例8)
按照与实施例1的化合物同样的方式,合成了具有靶序列的实施例8的化合物。
该化合物通过负离子MALDI-TOFMS来鉴定(计算值:6439.392、测定值:6434.6094)。
该化合物的核苷酸序列是与人肌肉生长抑制素(MSTN), 2号染色体上的RefSeqGene (LRG_200) (基因库登录号NG_009800.1)的核苷酸编号231-248互补的序列。
TgCaTTaCaCagCCCCTC (MSTN_Ex1_AO2+1)的合成(实施例9)
按照与实施例1的化合物同样的方式,合成了具有靶序列的实施例9的化合物。
该化合物通过负离子MALDI-TOFMS来鉴定(计算值:6449.399、测定值:6443.8228)。
该化合物的核苷酸序列是与人肌肉生长抑制素(MSTN), 2号染色体上的RefSeqGene (LRG_200) (基因库登录号NG_009800.1)的核苷酸编号239-256互补的序列。
gCaTTaCaCagCCCCTCT (MSTN_Ex1_AO2+2)的合成(实施例10)
按照与实施例1的化合物同样的方式,合成了具有靶序列的实施例10的化合物。
该化合物通过负离子MALDI-TOFMS来鉴定(计算值:6449.399、测定值:6443.707)。
该化合物的核苷酸序列是与人肌肉生长抑制素(MSTN), 2号染色体上的RefSeqGene (LRG_200) (基因库登录号NG_009800.1)的核苷酸编号238-255互补的序列。
aTTaCaCagCCCCTCTTT (MSTN_Ex1_AO2+4)的合成(实施例11)
按照与实施例1的化合物同样的方式,合成了具有靶序列的实施例11的化合物。
该化合物通过负离子MALDI-TOFMS来鉴定(计算值:6437.38、测定值:6431.9209)。
该化合物的核苷酸序列是与人肌肉生长抑制素(MSTN), 2号染色体上的RefSeqGene (LRG_200) (基因库登录号NG_009800.1)的核苷酸编号236-253互补的序列。
TTaCaCagCCCCTCTTTT (MSTN_Ex1_AO2+5)的合成(实施例12)
按照与实施例1的化合物同样的方式,合成了具有靶序列的实施例12的化合物。
该化合物通过负离子MALDI-TOFMS来鉴定(计算值:6440.376、测定值:6434.8232)。
该化合物的核苷酸序列是与人肌肉生长抑制素(MSTN), 2号染色体上的RefSeqGene (LRG_200) (基因库登录号NG_009800.1)的核苷酸编号235-252互补的序列。
TgTaCagTCTgagagaCa (MSTN_Ex1_AO4)的合成(实施例13)
按照与实施例1的化合物同样的方式,合成了具有靶序列的实施例13的化合物。
该化合物通过负离子MALDI-TOFMS来鉴定(计算值:6487.336、测定值:6481.8237)。
该化合物的核苷酸序列是与人肌肉生长抑制素(MSTN), 2号染色体上的RefSeqGene (LRG_200) (基因库登录号NG_009800.1)的核苷酸编号22-39互补的序列。
aaTgCaTgTaCagTCTga (MSTN_Ex1_AO4-6)的合成(实施例14)
按照与实施例1的化合物同样的方式,合成了具有靶序列的实施例14的化合物。
该化合物通过负离子MALDI-TOFMS来鉴定(计算值:6474.333、测定值:6468.584)。
该化合物的核苷酸序列是与人肌肉生长抑制素(MSTN), 2号染色体上的RefSeqGene (LRG_200) (基因库登录号NG_009800.1)的核苷酸编号28-45互补的序列。
TTgCTTTTgagTaaTgCC (MSTN_Ex1_AO5)的合成(实施例15)
按照与实施例1的化合物同样的方式,合成了具有靶序列的实施例15的化合物。
该化合物通过负离子MALDI-TOFMS来鉴定(计算值:6483.321、测定值:6477.7007)。
该化合物的核苷酸序列是与人肌肉生长抑制素(MSTN), 2号染色体上的RefSeqGene (LRG_200) (基因库登录号NG_009800.1)的核苷酸编号58-75互补的序列。
aaTCaaTaTaaTCTTTTT (MSTN_Ex1_AO6)的合成(实施例16)
按照与实施例1的化合物同样的方式,合成了具有靶序列的实施例16的化合物。
该化合物通过负离子MALDI-TOFMS来鉴定(计算值:6420.309、测定值:6414.5815)。
该化合物的核苷酸序列是与人肌肉生长抑制素(MSTN), 2号染色体上的RefSeqGene (LRG_200) (基因库登录号NG_009800.1)的核苷酸编号108-125互补的序列。
TTgCTCaCTgTTCTCaTT (MSTN_Ex1_AO7)的合成(实施例17)
按照与实施例1的化合物同样的方式,合成了具有靶序列的实施例17的化合物。
该化合物通过负离子MALDI-TOFMS来鉴定(计算值:6458.343、测定值:6452.9326)。
该化合物的核苷酸序列是与人肌肉生长抑制素(MSTN), 2号染色体上的RefSeqGene (LRG_200) (基因库登录号NG_009800.1)的核苷酸编号203-220互补的序列。
CTTgaagaTTTagTgTTT (MSTN_Ex1_AO3-6)的合成(实施例18)
按照与实施例1的化合物同样的方式,合成了具有靶序列的实施例18的化合物。
该化合物通过负离子MALDI-TOFMS来鉴定(计算值:6482.293、测定值:6476.7026)。
该化合物的核苷酸序列是与人肌肉生长抑制素(MSTN), 2号染色体上的RefSeqGene (LRG_200) (基因库登录号NG_009800.1)的核苷酸编号271-288互补的序列。
TCTaTTCTTgaagaTTTa (MSTN_Ex1_AO3-12)的合成(实施例19)
按照与实施例1的化合物同样的方式,合成了具有靶序列的实施例19的化合物。
该化合物通过负离子MALDI-TOFMS来鉴定(计算值:6452.307、测定值:6446.8232)。
该化合物的核苷酸序列是与人肌肉生长抑制素(MSTN), 2号染色体上的RefSeqGene (LRG_200) (基因库登录号NG_009800.1)的核苷酸编号277-294互补的序列。
TaCTgaggaTTTgTaTCT (MSTN_Ex1_AO8)的合成(实施例20)
按照与实施例1的化合物同样的方式,合成了具有靶序列的实施例20的化合物。
该化合物通过负离子MALDI-TOFMS来鉴定(计算值:6481.309、测定值:6475.8228)。
该化合物的核苷酸序列是与人肌肉生长抑制素(MSTN), 2号染色体上的RefSeqGene (LRG_200) (基因库登录号NG_009800.1)的核苷酸编号303-320互补的序列。
CaTCTTTgCTgaTgTTag (MSTN_Ex1_AO9)的合成(实施例21)
按照与实施例1的化合物同样的方式,合成了具有靶序列的实施例21的化合物。
该化合物通过负离子MALDI-TOFMS来鉴定(计算值:6483.321、测定值:6477.6953)。
该化合物的核苷酸序列是与人肌肉生长抑制素(MSTN), 2号染色体上的RefSeqGene (LRG_200) (基因库登录号NG_009800.1)的核苷酸编号342-359互补的序列。
gTTgTCTTaTaaCaTCTT (MSTN_Ex1_AO9-12)的合成(实施例22)
按照与实施例1的化合物同样的方式,合成了具有靶序列的实施例22的化合物。
该化合物通过负离子MALDI-TOFMS来鉴定(计算值:6454.319、测定值:6448.7021)。
该化合物的核苷酸序列是与人肌肉生长抑制素(MSTN), 2号染色体上的RefSeqGene (LRG_200) (基因库登录号NG_009800.1)的核苷酸编号354-371互补的序列。
TgaTCaaTCagTTCCCgg (MSTN_Ex1_AO10)的合成(实施例23)
按照与实施例1的化合物同样的方式,合成了具有靶序列的实施例23的化合物。
该化合物通过负离子MALDI-TOFMS来鉴定(计算值:6478.357、测定值:6472.9458)。
该化合物的核苷酸序列是与人肌肉生长抑制素(MSTN), 2号染色体上的RefSeqGene (LRG_200) (基因库登录号NG_009800.1)的核苷酸编号394-411互补的序列。
aCaTCaTaCTgaTCaaTC (MSTN_Ex1_AO10-9)的合成(实施例24)
按照与实施例1的化合物同样的方式,合成了具有靶序列的实施例24的化合物。
该化合物通过负离子MALDI-TOFMS来鉴定(计算值:6430.36、测定值:6424.8286)。
该化合物的核苷酸序列是与人肌肉生长抑制素(MSTN), 2号染色体上的RefSeqGene (LRG_200) (基因库登录号NG_009800.1)的核苷酸编号403-420互补的序列。
(表1)
[试验例]
实验方法
1. MSTN mRNA量的评价
使用人横纹肌肉瘤细胞(CRL-2016, ATCC)、人成肌细胞(Wada等人. Development2002,129; 2987-2995),通过RT-PCR评价由AO引起的MSTN mRNA的表达变化。
AO转染
1) 在100μl Opti-MEM培养基(31985070, Thermo Fisher Scientific)中分别混合2μl AO (用MilliQ灭菌水调制成50pmol/μl的溶液)。AO未处理是混合2μl MilliQ灭菌水;
2) 使用另一个管,在100μl Opti-MEM培养基(31985070, Thermo FisherScientific)中混合4μl LipofectamineTM 2000转染试剂(11668019, Thermo FisherScientific);
3) 混合1)液和2)液,在室温下放置20分钟;
4) 将在12-孔板中培养的人横纹肌肉瘤细胞用PBS洗涤1次后,向孔中加入800μlOpti-MEM培养基(31985070, Thermo Fisher Scientific);
5) 将3)液添加到4)中(AO最终浓度为100nM),在37℃、5%CO2下培养3小时后,将培养基交换成含有10%FBS (10270-106, gibco)的RPMI培养基(22400-089, gibco),再继续进行培养;
6) 人成肌细胞的培养在含有20%FBS (10270-106, gibco)、2%Ultroser G的DMEM培养基(043-30085, Wako)中进行,AO以最终浓度0、100、200、400nM进行转染。
RNA的调制
1) 将转染了各AO的细胞培养24小时后,用PBS洗涤1次,向细胞中添加300μl高纯度RNA分离试剂盒(#11828665001, Roche Life Science)的RNA提取试剂;
2) 在室温下放置5分钟后,将孔内的RNA提取试剂回收到管中;
3) 按照高纯度RNA分离试剂盒(#11828665001, Roche Life Science)的方案提取RNA,最终得到了50μl的RNA溶解液。
逆转录反应
1) 在500ng RNA中加入随机引物(#48190011, Thermo Fisher Scientific)、dNTPs(Takara),在65℃下培养5分钟、在25℃下培养10分钟;
2) 在1)液中加入M-MLV逆转录酶(#28025013, Thermo Fisher Scientific)、RNaseOUTTM重组核糖核酸酶抑制剂(#10777-019, Thermo Fisher Scientific)、DTT (附属于MLVRT)、缓冲液(附属于MLVRT),在37℃下培养55分钟、在70℃下培养10分钟,得到了cDNA。
PCR反应和反应产物的确认
1) 相对于所得的2μl cDNA,添加1μl引物MSTN Ex1_F1 (5’-agattcactggtgtggcaag-3’:SEQ ID NO: 26)、1μl MSTN R2 (5’-tgcatgacatgtctttgtgc-3’:SEQ ID NO: 27)、0.1μl TaKaRa Ex Taq (注册商标)DNA聚合酶(#RR001A, Takara)、1.6μl dNTPs (附属于TaKaRa Ex Taq (注册商标))、2μl缓冲液(10×)、12.3μl MilliQ灭菌水;
2) 在94℃下加热3分钟;
3) 进行30个循环的94℃ 0.5分钟/60℃ 0.5分钟/72℃ 1.5分钟的处理;
4) 在72℃下加热3分钟;
5) 对于PCR反应的反应产物,添加Midri Green Direct DNA着色剂(NE-MG06, 日本Genetics)和加样缓冲液(Takara),使用2%琼脂糖凝胶进行电泳后,使用凝胶摄影装置将其可视化。另外,使用Agilent 2100生物分析仪电泳系统(安捷伦科技株式会社),进行PCR反应产物的电泳、定量;
6) 使用引物GAPDH H_F (5’-cccttcattgacctcaac-3’:SEQ ID NO: 28)、GAPDHH_R (5’-ttcacacccatgacgaac-3’:SEQ ID NO: 29),对GAPDH进行上述的1)~5);
7) 使用引物GDF11Ex1F4 (5’-ctgcagcagatcctggacct-3’:SEQ ID NO: 34)、GDF11Ex1R4 (5’-catgaacatgtactcgcact-3’:SEQ ID NO: 35),对GDF11进行上述的1)~5);
8) 将通过琼脂糖电泳分离的PCR扩增产物用Image J进行半定量,以AO未处理的MSTN/GAPDH为1进行相对比较。同样,在使用Agilent 2100生物分析仪电泳系统定量的PCR扩增产物中,也以AO未处理的MSTN/GAPDH、GDF11/GAPDH为1,进行相对比较。
2. 肌肉生长抑制素信号传递的评价
肌肉生长抑制素信号是通过向人横纹肌肉瘤细胞(CRL-2061, ATCC)、人成肌细胞(Wada et al. Development 2002, 129; 2987-2995)中引入报道基因(SBE4-Luc质粒, #16495, Addgene),测定所表达诱导的荧光素酶的发光来进行评价。
AO转染
1) 在100μl Opti-MEM培养基(31985070, Thermo Fisher Scientific)中混合2μl AO2+1 (用MilliQ灭菌水调制成50pmol/μl的溶液);
2) 使用另一个管,在100μl Opti-MEM培养基(31985070, Thermo FisherScientific)中混合3μg SBE4-Luc质粒、1μg pSV-β-半乳糖苷酶对照载体(E108A,Promega)和4μl LipofectamineTM 3000转染试剂(11668019, Thermo FisherScientific)、8μl P3000试剂(11668019, Thermo Fisher Scientific);
3) 混合1)液和2)液,在室温下放置15分钟;
4) 将12-孔板中培养的人横纹肌肉瘤细胞用PBS洗涤1次后,在孔中加入800μlOpti-MEM培养基(31985070, Thermo Fisher Scientific);
5) 将3)液添加到4)中(AO最终浓度为100nM),在37℃、5%CO2下培养3小时后,将培养基交换成含有10%FBS (10270-106, gibco)的RPMI培养基(22400-089, gibco),再继续进行培养;
6) 人成肌细胞的培养在含有20%FBS (10270-106, gibco)、2%Ultroser G的DMEM培养基(043-30085, Wako)中进行,转染AO使最终浓度为0、50、100、150、200、300 400nM。
细胞提取液的调制
1) 将从转染起24小时后的细胞用PBS洗涤1次后,在孔中添加带有报道基因裂解缓冲液的荧光素酶测定系统(Luciferase Assay System with Reporter Lysis Buffer,E4030, Promega)的250μl报道基因裂解缓冲液;
2) 将1)的细胞破碎液在15000rpm、4℃下离心10分钟,得到上清;
3) 细胞提取液中的蛋白定量使用Qubit (注册商标)蛋白测定试剂盒(ProteinAssay Kit, #Q33211, Thermo Fisher Scientific)依方案进行。
荧光素酶的活性测定
1) 在96孔板上混合100μl细胞提取液和100μl荧光素酶底物(带有报道基因裂解缓冲液的荧光素酶测定系统, #E4030, Promega);
2) 使用多标签板读取器ARVOTMX3 (PerkinElmer)测定荧光素酶发光信号。
β-半乳糖苷酶的活性测定
1) 在96孔板上混合100μl细胞提取液和100μl β-半乳糖苷酶底物(带有报道基因裂解缓冲液的β-半乳糖苷酶酶测定系统, #E2000, Promega),在37℃下培养1小时;
2) 在孔中添加100μl 1M的碳酸钠以停止反应,之后使用多标签板读取器ARVOTMX3(PerkinElmer)测定420nm的吸光。
活性评价
通过用β-半乳糖苷酶活性值除以荧光素酶活性值进行标准化后,测定来自AO未处理细胞的提取液,以所得结果为1,根据相对值进行评价。
3. 细胞增殖的测定
细胞增殖是使用细胞计数试剂盒-8 (347-07621, 同仁化学研究所)对人成肌细胞(Wada等人. Development 2002, 129; 2987-2995)的增殖进行评价。
AO转染
1) 在50μl Opti-MEM培养基(31985070, Thermo Fisher Scientific)中混合0.4μl AO2+1 (用MilliQ灭菌水调制成50pmol/μl的溶液);
2) 使用另一个管,在50μl Opti-MEM培养基(31985070, Thermo FisherScientific)中混合0.4μl LipofectamineTM 3000转染试剂(11668019, Thermo FisherScientific);
3) 混合1)液和2)液,在室温下放置15分钟;
4) 将在96-孔板中培养的人成肌细胞用PBS洗涤1次后,在孔中添加3)液(AO最终浓度为200nM),在37℃、5%CO2下培养3小时后,将培养基交换成含有20%FBS (10270-106,gibco)、2%Ultroser G的DMEM培养基(043-30085, Wako),再继续进行培养。
活细胞的测量
在刚刚转染AO后(第0天)、2天后、3天后向各孔中添加10μl细胞计数试剂盒-8(347-07621, 同仁化学研究所),在37℃、5%CO2下培养1小时后,测定450nm的吸光度,对细胞数进行评价。
基于比对的核酸序列比较
进行了人肌肉生长抑制素(NM_005259.2)、牛肌肉生长抑制素(NC_037329.1)、猪肌肉生长抑制素(NC_010457.5)、狗肌肉生长抑制素(NM_001002959.1)的外显子1的氨基酸编码区的核酸序列的比对。在比对中使用EMBL-EBI的多序列比对程序Clustal Omega(https://www.ebi.ac.uk/Tools/msa/clustalo/)。用于人、牛、猪、狗的肌肉生长抑制素外显子1核酸序列的比对比较的序列见SEQ ID NO: 30~33。
实验结果
为了肌肉生长抑制素(MSTN)的表达抑制,制作了3种具有与MSTN pre-mRNA的外显子1互补的序列的18个碱基的AO (图1、图2a)。各AO是根据MSTN pre-mRNA中的剪接因子的结合预测制作的。将人横纹肌肉瘤细胞用各自的AO处理后,通过RT-PCR检验MSTN mRNA量时观察到:在处理24小时后经AO1、AO2处理MSTN mRNA明显减少(图2)。
由于AO2较AO1更使MSTN mRNA减少,所以以AO2为中心进行最适AO序列的筛选。首先,制作在AO2的序列前后各错开3个碱基的AO,进行了以MSTN外显子1 (SEQ ID NO: 1)的第231位~第263位碱基为靶的6条AO的检验(图3a)。人横纹肌肉瘤细胞中的检验结果,在AO2-3、AO2、AO2+3、AO2+6、AO2+9处理24小时观察到MSTN mRNA明显减少(图3b、c)。其中,AO2+3显示最大的MSTN mRNA的减少效果,因此制作以AO2+3为中心各错开1个碱基的AO,进行以MSTN外显子1 (SEQ ID NO: 1)的第234位~第257位碱基为靶的7条AO的检验(图3a)。人横纹肌肉瘤细胞中的检验结果,在AO2、AO2+1、AO2+2、AO2+3、AO2+4、AO2+5、AO2+6处理24小时观察到MSTN mRNA的明显减少(图3d、e)。通过上述的筛选,得到了AO2+1作为对MSTN表达抑制最有效的AO。
由于利用以MSTN的外显子1为靶的AO可抑制MSTN表达,所以除AO2+1周边序列以外还制作了AO,与AO2+1进行有效性比较。制作了12条以MSTN外显子1 (SEQ ID NO: 1)的第22位~第420位碱基的范围为靶的AO,将加上AO2+1在内的13条分别在人横纹肌肉瘤细胞中处理24小时,结果见到任一的AO均有抑制MSTN mRNA表达的倾向(图4)。特别是AO6、AO7、AO3-6、AO3-12、AO8、AO9、AO9-12、AO10、AO10-9使MSTN mRNA明显减少。在比较这些MSTN mRNA的减少率时,显示AO2+1最有效。而且,AO2+1的效果在人成肌细胞中也有显示(图5)。另一方面,AO2+1对GDF11的表达没有影响(图6)。
若成熟肌肉生长抑制素与细胞膜上的受体作用,则转录因子Smad2/3被磷酸化、并转移到核内,诱导靶基因的表达(图7)。使用体外肌肉生长抑制素转录活性测定系统评价该肌肉生长抑制素信号的活化(图8)。在体外肌肉生长抑制素转录活性测定系统中,使用在Smad结合启动子下游配置有荧光素酶基因的质粒,测定由该质粒表达的荧光素酶的活性,从而检验肌肉生长抑制素信号。在人横纹肌肉瘤细胞、人成肌细胞中进行肌肉生长抑制素信号检验的结果,通过AO2+1的处理,荧光素酶活性得到抑制,由此显示出:AO2+1抑制肌肉生长抑制素信号(图8、9)。而且,AO2+1还显示了人成肌细胞的增殖促进效果(图10)。
考察
由于肌肉生长抑制素的功能抑制会促进肌肉形成,因此作为肌萎缩性疾病治疗的方法受到关注。实际上,对于抑制肌肉生长抑制素的功能的抗体或肽,以Duchenne型肌营养不良症(DMD)为代表的肌萎缩性疾病的临床试验正在进行。另一方面,在这些抗体或肽中,以肌肉生长抑制素以外的蛋白为靶的脱靶效应被视为问题。例如,激活肌肉生长抑制素信号的成熟肌肉生长抑制素与相同的TGF-β家族的成熟GDF11具有90%的氨基酸同一性,肌肉生长抑制素抑制肽也抑制GDF11 (Osawa等人, PLoS One, 2015)。相对于此,本研究的AO2+1对GDF11的mRNA的表达没有影响。另外,迄今为止还开发了以MSTN pre-mRNA的外显子2的跳跃为目的的功能丧失型AO,但尚未得到在临床上有效的AO。本发明的AO是以MSTN pre-mRNA的外显子1为靶的表达抑制型,其作用和效果均不同于迄今为止的功能丧失型AO,因此被期待可作为治疗药。
本发明的AO2+1,其有效性特别高,不仅抑制MSTN mRNA的表达,还抑制肌肉生长抑制素信号,促进成肌细胞的增殖。认为肌肉生长抑制素的抑制是有效的疾病有许多,作为药物,可用于肌萎缩性疾病(例如,肌营养不良症、脊髄性肌萎缩症、肌少症、废用性肌萎缩)、循环系统疾病(例如,心功能不全、动脉硬化等)、肾病(例如,慢性肾功能不全等)、骨病(例如,炎症性关节炎等)、癌症或糖尿病的预防和/或治疗。而且,肌肉生长抑制素的抑制还使骨骼肌量增加,谋求运动量的增加,还有助于改善全身的代谢。另外,可期待与心肌作用以恢复其功能。而且,肌肉生长抑制素抑制还被期待:与破骨细胞作用以抑制骨破坏、激活血管内皮细胞的稳态维持能力、诱导细胞凋亡、提高对胰岛素的敏感性等。
本发明的AO2+1的靶序列在牛或猪中也有保守,也可用于促进这些家畜的生长(图11)。通过对家畜给予AO来抑制MSTN表达并不相当于基因重组生物的制作,因此容易应用。另外,认为对狗也同样有效,认为也可用于作为宠物的狗的肌减少(图11)。
本说明书中引用的所有的出版物、专利和专利申请直接作为参考而纳入本说明书中。
产业实用性
本发明可用作抑制肌肉生长抑制素基因的mRNA的产生的核酸药物。
[序列表自由文本]
<SEQ ID NO: 1>显示MSTN外显子1的核苷酸序列信息。
(全部的506个碱基。□内显示起始密码子(atg))
<SEQ ID NO: 2~25>显示实施例中合成的AO的核苷酸序列。构成反义寡核苷酸的核苷酸可以是天然型DNA、天然型RNA、DNA/RNA嵌合物、它们的修饰物中的任一种,另外,至少1个可以是修饰核苷酸。
<SEQ ID NO: 26~29>显示试验例中使用的引物的核苷酸序列。
<SEQ ID NO: 30~33>显示用于人、牛、猪、狗的肌肉生长抑制素外显子1核酸序列的比对比较的序列。比对比较使用EMBL-EBI的多序列比对来进行。仅在外显子1的氨基酸编码区进行比较。()内是在BLAST中的名称。□内的序列是AO2+1的靶序列。
Ex1 (ORF)
<SEQ ID NO: 34和35>显示引物GDF11Ex1F4和GDF11Ex1R4的核苷酸序列。
Claims (31)
1.碱基长度为15~30的反义寡核苷酸、其盐或溶剂合物,该反义寡核苷酸具有与肌肉生长抑制素基因的外显子1的靶位点互补的核苷酸序列,可抑制肌肉生长抑制素基因的mRNA的产生。
2. 权利要求1所述的反义寡核苷酸、其盐或溶剂合物,其中,肌肉生长抑制素基因的外显子1的核苷酸序列为SEQ ID NO: 1的核苷酸序列,肌肉生长抑制素基因的外显子1的靶位点存在于SEQ ID NO: 1的核苷酸序列的碱基编号22~420的区域内。
3. 权利要求1或2所述的反义寡核苷酸、其盐或溶剂合物,其中,反义寡核苷酸的核苷酸序列包含由SEQ ID NO: 2~25中的任一个核苷酸序列中的连续的至少15个碱基构成的序列,其中上述任一个核苷酸序列中的t可以是u、而u可以是t。
4.权利要求1~3中任一项所述的反义寡核苷酸、其盐或溶剂合物,其中,反义寡核苷酸的碱基长度为18。
5. 权利要求4所述的反义寡核苷酸、其盐或溶剂合物,其中,反义寡核苷酸的核苷酸序列为SEQ ID NO: 2~25中的任一个核苷酸序列,其中上述任一个核苷酸序列中的t可以是u、而u可以是t。
6.权利要求1~5中任一项所述的反义寡核苷酸、其盐或溶剂合物,其中,至少1个核苷酸被修饰。
7.权利要求6所述的反义寡核苷酸、其盐或溶剂合物,其中,构成修饰核苷酸的糖为D-呋喃核糖,D-呋喃核糖的2’位的羟基被修饰。
8. 权利要求7所述的反义寡核苷酸、其盐或溶剂合物,其中,D-呋喃核糖被2’-O-烷基化和/或2’-O, 4’-C-亚烷基化。
9.药物,该药物含有权利要求1~8中任一项所述的反义寡核苷酸、其药学上可接受的盐或溶剂合物。
10.权利要求9所述的药物,该药物用于预防和/或治疗肌肉生长抑制素参与的病症和/或疾病。
11.权利要求10所述的药物,其中,肌肉生长抑制素参与的病症和/或疾病为肌萎缩。
12.权利要求11所述的药物,其中,肌萎缩为选自肌营养不良症、肌病、脊髄性肌萎缩症、肌少症和废用性肌萎缩的至少1种。
13.权利要求10所述的药物,其中,肌肉生长抑制素参与的病症和/或疾病为通过恢复肌肉量而产生治疗效果的病症和/或疾病。
14.权利要求13所述的药物,其中,通过恢复肌肉量而产生治疗效果的病症和/或疾病为选自癌症恶液质、糖尿病、循环系统疾病、肾病和骨病的至少1种。
15.权利要求14所述的药物,其中,循环系统疾病为心功能不全和/或动脉硬化,肾病为慢性肾功能不全,骨病为炎症性关节炎。
16.食品,该食品含有权利要求1~8中任一项所述的反义寡核苷酸、其食品上可接受的盐或溶剂合物。
17.饲料,该饲料含有权利要求1~8中任一项所述的反义寡核苷酸、其饲料上可接受的盐或溶剂合物。
18.用于促进肌细胞的增殖和/或肥大的药剂,该药剂含有权利要求1~8中任一项所述的反义寡核苷酸、其盐或溶剂合物。
19.用于增加肌肉量和/或抑制肌减少的药剂,该药剂含有权利要求1~8中任一项所述的反义寡核苷酸、其盐或溶剂合物。
20.抑制肌肉生长抑制素基因的mRNA的产生的药剂,该药剂含有权利要求1~8中任一项所述的反义寡核苷酸、其盐或溶剂合物。
21.肌肉生长抑制素的功能抑制剂,其含有权利要求1~8中任一项所述的反义寡核苷酸、其盐或溶剂合物。
22.预防和/或治疗肌肉生长抑制素参与的疾病的方法,该方法包括:对受试者给予有效量的权利要求1~8中任一项所述的反义寡核苷酸、其盐或溶剂合物。
23.促进肌细胞的增殖和/或肥大的方法,该方法包括:对受试者给予有效量的权利要求1~8中任一项所述的反义寡核苷酸、其盐或溶剂合物。
24.增加肌肉量和/或抑制肌减少的方法,该方法包括:对受试者给予有效量的权利要求1~8中任一项所述的反义寡核苷酸、其盐或溶剂合物。
25.抑制肌肉生长抑制素基因的mRNA的产生的方法,该方法包括:对受试者给予有效量的权利要求1~8中任一项所述的反义寡核苷酸、其盐或溶剂合物。
26.抑制肌肉生长抑制素的功能的方法,该方法包括:对受试者给予有效量的权利要求1~8中任一项所述的反义寡核苷酸、其盐或溶剂合物。
27.权利要求1~8中任一项所述的反义寡核苷酸、其盐或溶剂合物,其用于在预防和/或治疗肌肉生长抑制素参与的疾病的方法中应用。
28.权利要求1~8中任一项所述的反义寡核苷酸、其盐或溶剂合物,其用于在促进肌细胞的增殖和/或肥大的方法中应用。
29.权利要求1~8中任一项所述的反义寡核苷酸、其盐或溶剂合物,其用于在增加肌肉量和/或抑制肌减少的方法中应用。
30.权利要求1~8中任一项所述的反义寡核苷酸、其盐或溶剂合物,其用于在抑制肌肉生长抑制素基因的mRNA的产生的方法中应用。
31.权利要求1~8中任一项所述的反义寡核苷酸、其盐或溶剂合物,其用于在抑制肌肉生长抑制素的功能的方法中应用。
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