CN104619326A - 骨系统疾病治疗药及其用途 - Google Patents
骨系统疾病治疗药及其用途 Download PDFInfo
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Abstract
本申请发明的课题在于提供对FGFR3过度活化所致的骨系统疾病尤其是软骨发育不全、软骨发育低下的治疗效果优异的新的治疗战略。本申请提供含有美克利嗪或其药学上可接受的盐作为有效成分的骨系统疾病的治疗药。
Description
技术领域
本发明涉及一种骨系统疾病治疗药及其用途。本发明的骨系统疾病治疗药能够用于软骨发育不全、软骨发育低下、致死性骨发育不全、克鲁宗氏病、肢端肢中部畸形性发育不良、严重软骨发育不良伴发育迟缓和黑棘皮症(SADDAN)等的治疗。本申请主张基于2013年3月10日提出申请的日本国专利申请第2013-47426号的优先权,该专利申请的全部内容通过参照引入本申请。
背景技术
软骨发育不全、软骨发育低下是由于作为骨伸展的负调节物的成纤维细胞生长因子受体3(fibroblast growth factor receptor 3(FGFR3))的持续活化型突变而发病。此外,包括特发性矮小症在内的低身高也显示与FGFR3相关。软骨发育不全中,由于骨伸展被抑制,不仅发生低身高,而且发生脊柱管狭窄症、枕骨大孔狭窄等严重的并发症。没有抑制软骨发育不全(低下)症中FGFR3的活性的根本的治疗法,作为针对低身高的对症性治疗,可以进行内科的生长激素治疗,外科的骨延长术。
最近确定了抑制FGFR3信号的低分子化合物。但是,这些化合物的毒理学特征基本未知(非专利文献1~3)。另一方面,C型利钠利尿肽(CNP)是针对FGFR3信号的强力拮抗剂,通过抑制FGFR3-MAPK路径来减轻软骨发育不全(低下)症的表型(短肢)(非专利文献4、5)。CNP的半衰期极短,体内实验中需要持续的静脉注射(非专利文献6)。报告了开发出半衰期长的作为CNP类似物的BMN 111,通过对软骨发育不全(低下)症小鼠进行皮下注射,会显著改善软骨发育不全(低下)症小鼠的骨生长(非专利文献7)。
应予说明,报告了几种针对软骨发育不全的新的治疗战略(例如,参见专利文献1~3)。
现有技术文献
专利文献
专利文献1:日本特表平11-507828号公报
专利文献2:日本特表2011-504506号公报
专利文献3:日本特开2003-104908号公报
非专利文献
非专利文献1:Krejci,P.,Murakami,S.,Prochazkova,J.,Trantirek,L.,Chlebova,K.,Ouyang,Z.,Aklian,A.,Smutny,J.,Bryja,V.,Kozubik,A.et al.(2010)NF449is a novel inhibitor of fibroblast growth factorreceptor 3(FGFR3)signaling active in chondrocytes and multiplemyeloma cells.J.Biol.Chem.,285,20644-20653.
非专利文献2:Jin,M.,Yu,Y.,Qi,H.,Xie,Y.,Su,N.,Wang,X.,Tan,Q.,Luo,F.,Zhu,Y.,Wang,Q.et al.(2012)A novel FGFR3-bindingpeptide inhibits FGFR3signaling and reverses the lethal phenotype ofmice mimicking human thanatophoric dysplasia.Hum.Mol.Genet.,21,5443-5455.
非专利文献3:Jonquoy,A.,Mugniery,E.,Benoist-Lasselin,C.,Kaci,N.,Le Corre,L.,Barbault,F.,Girard,A.L.,Le Merrer,Y.,Busca,P.,Schibler,L.et al.(2012)A novel tyrosine kinase inhibitor restoreschondrocyte differentiation and promotes bone growth in again-of-function Fgfr3mouse model.Hum.Mol.Genet.,21,841-851.
非专利文献4:Krejci,P.,Masri,B.,Fontaine,V.,Mekikian,P.B.,Weis,M.,Prats,H.and Wilcox,W.R.(2005)Interaction of fibroblastgrowth factor and C-natriuretic peptide signaling in regulation ofchondrocyte proliferation and extracellular matrix homeostasis.J.CellSci.,118,5089-5100.
非专利文献5:Yasoda,A.,Komatsu,Y.,Chusho,H.,Miyazawa,T.,Ozasa,A.,Miura,M.,Kurihara,T.,Rogi,T.,Tanaka,S.,Suda,M.et al.(2004)Overexpression of CNP in chondrocytes rescues achondroplasiathrough a MAPK-dependent pathway.Nat.Med.,10,80-86.
非专利文献6:Yasoda,A.,Kitamura,H.,Fujii,T.,Kondo,E.,Murao,N.,Miura,M.,Kanamoto,N.,Komatsu,Y.,Arai,H.and Nakao,K.(2009)Systemic administration of C-type natriuretic peptide as anovel therapeutic strategy for skeletal dysplasias.Endocrinology,150,3138-3144.
非专利文献7:Lorget,F.,Kaci,N.,Peng,J.,Benoist-Lasselin,C.,Mugniery,E.,Oppeneer,T.,Wendt,D.J.,Bell,S.M.,Bullens,S.,Bunting,S.et al.(2012)Evaluation of the therapeutic potential of a CNPanalog in a Fgfr3mouse model recapitulating achondroplasia.Am JHum Genet,91,1108-1114.
发明内容
生长激素是利用胰岛素样生长因子1(insulin-like growth factor-1(IGF-1))对骨骺软骨的伸展作用的治疗,但是,由于软骨发育不全中通常没有IGF-1的活性降低,因此得不到期待的效果。此外,仅在刚刚治疗开始时发现效果,但是效果逐渐地减弱。另一方面,骨延长术是使通过截骨产生的假骨质缓慢伸展的骨再生方法,但是存在延长的假骨质成熟需要长时间,与其相伴针脚感染、关节挛缩等并发症多发成为问题。因此,本发明的课题在于提供对FGFR3的过度活化所致的骨系统疾病(特别是软骨发育不全、软骨发育低下)的治疗效果优异的新的治疗战略。
本发明人等着眼于FGFR3信号的活化,尝试从已批准的药品中寻找特异性抑制FGFR3信号活化的低分子化合物。首先,向大鼠软骨肉瘤(rat chondrosarcoma(RCS))细胞添加成纤维细胞生长因子2(fibroblast growth factor 2(FGF2))时FGFR3信号被活化,在利用产生细胞增殖的抑制和细胞外基质的丧失的实验系统中实施筛选。具体而言,RCS细胞中添加FGF2之后,添加1186种已批准的药品(PrestwickChemicals公司),考察其效果。详细研究的结果,令人吃惊的是,具有抗组胺作用、作为晕车药频繁使用的美克利嗪(meclizine)能够浓度依赖性地补偿细胞增殖的抑制和细胞外基质的丧失。此外,在作为软骨系细胞的人软骨肉瘤(human chondrosarcoma)细胞和ATDC5细胞中导入FGFR3突变型时产生细胞增殖的抑制和细胞外基质的丧失,而美克利嗪也会补偿这些作用。进而,在胎生16.5日的小鼠胫骨添加FGF2,进行器官培养时,骨的长轴生长被抑制,但是美克利嗪阻碍了长轴生长抑制效果。
如上文所述,通过大量利用软骨系细胞和骨的器官培养的实验,能够证明美克利嗪的有效性,即,美克利嗪抑制FGFR3信号,具有药效。使用美克利嗪时,能够确立针对以FGFR3的过度活化为主因或部分病因的各种骨系统疾病(特别是软骨发育不全、软骨发育低下、致死性骨发育不全、克鲁宗氏病、肢端肢中部畸形性发育不良、严重软骨发育不良伴发育迟缓和黑棘皮症(SADDAN)等呈现低身高的疾病)的根本的治疗法。另一方面,由于在正常的骨骺软骨伸展中FGFR3作为负调节物也经常表达,因此作为健康人的身高促进剂美克利嗪也是有效的。实际上,通过对正常小鼠经口给予美克利嗪,发现了身长和四肢的伸长效果。即,通过动物实验也能确认美克利嗪的伸长效果。基于这一结果,可以说对于不伴随骨骺线闭合的脑垂体性矮小症、Turner综合症的低身高、Prader-Willi综合症的低身高、生长激素分泌不全性低身高症(GHD)、宫内发育迟缓(SGA)性低身高症等的治疗/预防,美克利嗪也是有效的。
以下示出的本申请发明,主要是基于上述见解和研究。
[1]一种成纤维细胞生长因子受体3(FGFR3)的过度活化所致的骨系统疾病的治疗药,其特征在于,含有美克利嗪或其药学上可接受的盐作为有效成分。
[2]根据[1]记载的治疗药,其中,骨系统疾病是选自软骨发育不全、软骨发育低下、致死性骨发育不全、克鲁宗氏病、肢端肢中部畸形性发育不良和严重软骨发育不良伴发育迟缓和黑棘皮症(SADDAN)中的疾病。
[3]根据[1]或[2]记载的治疗药,其中,有效成分是盐酸美克利嗪。
[4]一种骨系统疾病的治疗法,其中,包括以下步骤:对成纤维细胞生长因子受体3(FGFR3)的过度活化所致的骨系统疾病的患者给予治疗上有效量的美克利嗪或其药学上可接受的盐。
[5]一种身高促进剂,含有美克利嗪或其药学上可接受的盐作为有效成分。
[6]一种身高促进用组合物,含有[5]记载的身高促进剂。
[7]根据[6]记载的身高促进用组合物,其是药品、准药品或食品。
附图说明
图1:美克利嗪补偿RCS细胞中FGF2所致的增殖抑制和细胞外基质丧失。(A)对RCS细胞同时添加FGF2(5ng/ml)和美克利嗪,48小时后通过MTS测定评价细胞增殖能力。图是以vehicle比计算490nm吸光度,表示平均值和SD值(n=8)。美克利嗪浓度依存性地补偿了FGF2所致的RCS细胞的增殖抑制,但是50μM出现毒性。(B)美克利嗪补偿了FGF2所致的细胞形态的变化和细胞外基质丧失。对RCS单独添加FGF2(5ng/ml)或者同时添加CNP(0.2μM)或美克利嗪(20μM),72小时后对细胞外基质进行了阿尔新蓝染色。RCS细胞在FGF2不存在下培养时,形成呈现球形形态的丰富的细胞外基质。这样的软骨样的表型因FGF2而丧失。对RCS细胞同时添加FGF2与CNP或美克利嗪时,细胞形态保持球形,且保持了阿尔新蓝的染色性。(C)美克利嗪抑制了RCS细胞中FGF2所致的细胞外基质分解酶的表达。对RCS细胞同时添加FGF2与CNP或美克利嗪,4小时后通过实时RT-PCR对mRNA进行定量。图是以vehicle比计算Mmp10、Mmp13、Adamts1的表达量,表示平均值和SD值(n=3)。FGF2使Mmp10、Mmp13、Adamts1的表达上升,CNP和美克利嗪抑制了表达上升。
图2:美克利嗪补偿导入了FGFR3突变型(K650E、K650M)的HCS-2/8细胞的增殖抑制。(A)播种FGFR3的野生型或突变型(K650E)进行表达的HCS-2/8细胞,48小时后通过MTS测定对细胞增殖能力进行定量。突变型(K650E)表达的HCS-2/8细胞的增殖能力与野生型比较,显著受到抑制(p<0.001)。图表示平均值和SD值。(B)对突变型(K650E或K650M)进行表达的HCS-2/8细胞添加美克利嗪,48小时后通过MTS测定评价增殖能力。图表示vehicle比的平均值和SD值(n=12)。美克利嗪补偿了突变型(K650E、K650M)的导入所致的增殖抑制。(C)评价由慢病毒导入HCS-2/8细胞的Venus的荧光强度时,显示突变型(K650E)所致的增殖抑制被CNP和美克利嗪补偿。由上段板可知转染效率是90%以上。通过CNP和美克利嗪的给予,Venus的荧光强度增强。图是计算vehicle比,表示平均值和SD值(n=3)。
图3:美克利嗪补偿导入了FGFR3突变型(G380R)的ATDC5细胞的分化抑制。对ATDC5细胞的微团培养(micromass culture)添加CNP或美克利嗪,培养6日,进行阿尔新蓝染色。染色后,添加6M盐酸胍200μl,进行610nm的吸光度测定。图表示平均值和SD值(n=6)。CNP和美克利嗪使导入了突变型(G380R)的ATDC5细胞的阿尔新蓝染色性增强。
图4:无论有无添加FGF2,美克利嗪均在胎鼠胫骨的器官培养中促进骨的长轴生长。培养6天器官后,将胎鼠胫骨的切片用于HE染色和阿尔新蓝染色。将同一个体的两侧胫骨相邻摆放。骨的长轴方向的长度是计算相对侧的长度的比,图表示平均值和SD值。FGF2抑制骨的长轴生长,对FGF2添加CNP或美克利嗪时长轴生长有意义增大(P<0.01,t-test)。即便是在FGF2不存在下,美克利嗪也会使骨的长轴生长有意义增大(P<0.05,t-test)。
图5:美克利嗪抑制RCS细胞中FGF2所致的ERK磷酸化。(A)对RCS细胞添加FGF2(5ng/ml)的30分钟前给予美克利嗪,通过WesternBlotting法评价ERK和MEK的磷酸化水平。将ERK和MEK作为内参。美克利嗪在FGF2添加5分后抑制了ERK的磷酸化,但是没有抑制MEK的磷酸化。(B)使用慢病毒,将持续活化的ERK、MEK、RAF的各个突变型导入RCS细胞。美克利嗪添加后48小时后通过MTS测定对细胞增殖能力进行定量。以vehicle比计算490nm吸光度,示出平均值和SD值(n=3)。美克利嗪补偿了持续活化的MEK和RAF所致的增殖抑制,但是对于ERK所致的增殖抑制没有效果。
图6:软骨中的FGFR3信号转导系统和FGFR3抑制剂的机理。MAPK路径和STAT路径在软骨细胞的增殖和分化中发挥抑制作用。MAPK路径中,RAS、RAF、MEK、ERK的信号级联放大阶段性活化。CNP与natriuretic peptide receptor-B结合时,细胞内cGMP上升,介由PKG的活化抑制RAF。NF449(参考文献14)、A31(参考文献16)、P3(参考文献15)是近年被确定的FGFR3抑制剂。NF449和A31抑制FGFR3激酶活性。P3与FGFR3胞外域结合。美克利嗪抑制ERK的磷酸化。
图7:美克利嗪给予实验的结果。对正常妊娠小鼠,从妊娠第14日开始内服给予美克利嗪,评价出生后第5日的仔小鼠的体长、四肢长。
图8:美克利嗪给予实验的结果。对正常小鼠从出生后2周开始内服给予美克利嗪,在第5周评价体长、四肢长。
具体实施方式
本发明的第1方面涉及骨系统疾病的治疗药(为了说明的方便,以下称为“本发明药品”)及其用途。“治疗药”是指对目标疾病或病理状态具有治疗或预防性效果的药品。治疗的效果包括缓和目标疾病/病理状态中特征性的症状或随伴症状(轻症化)、阻止或延迟症状的恶化等。关于后者,在预防重症化这一点上,可以作为预防的效果之一来掌握。因此,治疗的效果和预防的效果是部分重叠的概念,将其明确地区分掌握是困难的,而且将其区分的实际意义少。应予说明,预防的效果的典型的例子是,阻止或延迟目标疾病/病理状态中特征性的症状的复发。应予说明,对于目标疾病/病理状态,只要具有某种的治疗的效果或预防的效果、或者这两者,就属于针对目标疾病/病理状态的治疗药。
本发明是在发现美克利嗪的新的药理作用,即抑制FGFR3信号的活性的成果的基础上而完成的发明。本发明药品是利用该药理作用的药物,其用于FGFR3的过度活化所致的骨系统疾病的治疗或预防。不拘泥于理论,本发明药品能够抑制或阻碍FGFR3的过度活化(换而言之持续活化),发挥治疗效果。如后述的实施例所示,发现美克利嗪在经典的RAS/MAPK级联放大的下游(从MEK向ERK的信号传递)具有抑制活性(图6)。这一事实提示美克利嗪是特异性优异、副作用少的药剂。
作为治疗或预防对象的“骨系统疾病”是FGFR3的过度活化所致的疾病即可,没有特别限制。本说明书中,“FGFR3的过度活化所致”是指FGFR3的过度活化是主因或病因的至少一部分。例示所述的“骨系统疾病”时,是软骨发育不全(Achondroplasia)、软骨发育低下(Hypochondroplasia)、致死性骨发育不全(Thanatophoric dysplasia)、克鲁宗氏病(Crouzon disease)、肢端肢中部畸形性发育不良(Acromesomelic dysplasias)、严重软骨发育不良伴发育迟缓和黑棘皮症(SADDAN)。优选的一个实施方式中,本发明药品适用于软骨发育不全或软骨发育低下的治疗或预防。软骨发育不全是呈现近位四肢缩短型的低身高的代表性疾病,频度也高。在大多数患者中,发现第4染色体短臂上存在FGFR3的G380R点突变(第380位甘氨酸被精氨酸置换的突变)。由于突变,FGFR3被持续活化,软骨细胞的增殖和分化被抑制。其结果是,通过软骨内骨化,长骨向长轴方向的生长被显著阻碍。软骨发育低下也是呈现近位四肢缩短型的低身高的疾病。软骨发育低下的问题基因也是FGFR3,在约半数的患者中,发现N540K点突变(第540位的天冬酰胺被赖氨酸置换的突变)。应予说明,骨系统疾病国际分类中,将软骨发育不全和软骨发育低下分类为同一组。
本发明药品含有美克利嗪(meclizine)或其药学上可接受的盐作为有效成分。已知美克利嗪是物质名称为1-[(4-氯苯基)苯基甲基]-4-[(3-甲基苯基)甲基]哌嗪的化合物,它是抗组胺药的一种。美克利嗪作为晕车药以OTC(over the counter)销售。美克利嗪也被称为美克洛嗪(meclozine)。本说明书中术语“美克利嗪”与术语“美克洛嗪”可以交换使用。
作为本发明药品的有效成分,可以使用美克利嗪的药理学上可接受的盐。市售的美克利嗪制剂中,大多数情况下,以美克利嗪盐酸盐的形式作为处方。因此、本发明中优选使用盐酸盐。但是,“药理学上可接受的盐”不局限于此,可以想到利用各种盐,例如酸加成盐、金属盐、铵盐、有机胺加成盐、氨基酸加成盐等。作为酸加成盐的例子,可举出盐酸盐、硫酸盐、硝酸盐、磷酸盐、氢溴酸盐等无机酸盐、醋酸盐、马来酸盐、富马酸盐、柠檬酸盐、苯磺酸盐、苯甲酸盐、苹果酸盐、草酸盐、甲磺酸盐、酒石酸盐等有机酸盐。作为金属盐的例子,可举出钠盐、钾盐、锂盐等碱金属盐、镁盐、钙盐等碱土金属盐、铝盐、锌盐。作为铵盐的例子可举出铵、四甲基铵等的盐。作为有机胺加成盐的例子,可举出吗啉加成盐、哌啶加成盐。作为氨基酸加成盐的例子,可举出甘氨酸加成盐、苯丙氨酸加成盐、赖氨酸加成盐、天冬氨酸加成盐、谷氨酸加成盐。
本发明药品的制剂化可以按照常用方法进行。制剂化时,可以含有制剂上可接受的其他成分(例如,载体、赋形剂、崩解剂、缓冲剂、乳化剂、混悬剂、无痛化剂、稳定剂、保存剂、防腐剂、生理盐水等)。作为赋形剂可以使用乳糖、淀粉、山梨醇、D-甘露醇、白糖等。作为崩解剂可以使用淀粉、羧甲基纤维素、碳酸钙等。作为缓冲剂可以使用磷酸盐、柠檬酸盐、醋酸盐等。作为乳化剂可以使用阿拉伯胶、精氨酸钠、黄蓍胶等。作为混悬剂可以使用单硬脂酸甘油、单硬脂酸铝、甲基纤维素、羧甲基纤维素、羟甲基纤维素、月桂基硫酸钠等。作为无痛化剂可以使用苄醇、氯丁醇、山梨醇等。作为稳定剂可以使用丙二醇、抗坏血酸等。作为保存剂可以使用苯酚、苯扎氯铵、苄醇、氯丁醇、对羟基苯甲酸甲酯等。作为防腐剂可以使用苯扎氯铵、对羟基苯甲酸、氯丁醇等。
制剂化时的剂型没有特别限制。剂型的例子是片剂、散剂、微粒剂、颗粒剂、胶囊剂、糖浆剂、注射剤、外用剂、和栓剂。本发明药品根据其剂型通过经口给予或非经口给予(静脉内、动脉内、皮下、皮内、肌肉内、或腹腔内注射、经皮、经鼻、经粘膜等)应用于对象。此外,根据对象也可以应用全身给予或局部给予。这些给予途径相互之间并不排斥,也可以并用任意选择的两种以上(例如,经口给予的同时或经过特定时间后进行静脉注射等等)。为了获得期待的治疗效果,本发明药品含有必需量(即,治疗有效量)的有效成分。本发明药品中的有效成分量通常因剂型而不同,但将有效成分量例如设置在约0.1重量%~约99重量%的范围内以实现期望的给予量。
本发明药品的给予量,以获得期待的治疗效果的方式进行设定。治疗上有效的给予量的设定中,通常考虑症状、患者的年龄、性别、和体重等。本领域技术人员考虑到这些事项能够设定适当的给予量。例如,将成人(体重约60kg)作为对象,可以以每日的有效成分量是1mg~500mg,优选为5mg~300mg,特别优选为10mg~200mg的方式设定给予量。作为给予方案可以采用例如1日1次~数次、2日1次、或者3日1次等。给予方案的确定中,可以考虑患者的病状、有效成分的效果持续时间等。关于局部给予,包括以手术时的使用、或者治愈过程的促进为目的的以载体或者相应的剂型在局部注入等方法。
可以与利用本发明药品的治疗平行地进行利用其他药品(例如已有的治疗药)的治疗,也可以对已有的治疗手段组合利用本发明药品的治疗。作为已有的治疗法,可以例示生长激素疗法,作为已有的治疗手段可以例示骨延长术。骨延长术中,可以使用被称为固定装置(内固定型或外固定型)或骨延长器等的专用装置。骨延长术的方法通常由截骨、等待期间、骨延长期间和骨硬化期间的疗程组成。应予说明,关于骨延长术,例如,详细参见ADVANCE SERIES II-9骨延长术:最近的进步(克诚堂出版,波利井清纪监修,杉原平树编著)。
根据以上描述所示,本申请还提供一种治疗法,其特征在于,对FGFR3的过度活化所致的骨系统疾病患者给予治疗有效量的本发明药品。
本发明的其他方面提供含有美克利嗪或其药学上可接受的盐作为有效成分的身高促进剂。应予说明,关于没有特别提及的事项,援引上述方面的相应的说明。
本发明的身高促进剂不仅能够应用于呈现低身高的患者,而且可以应用于健康人。例如,可以将本发明应用于不伴随骨骺线闭合的脑垂体性矮小症、Turner综合症的低身高、Prader-Willi综合症的低身高、生长激素分泌不全性低身高症(GHD)、宫内发育迟缓(SGA)性低身高症等的治疗或预防。本发明的身高促进剂可以以各种组合物(例如,药品组合物、准药品组合物、食品组合物)的形态提供。
本发明的身高促进剂以药品组合物或准药品组合物的形态提供时的制剂化、剂型、给予途径、给予量等,以上述方面为基准。另一方面,食品组合物的情况下,例如,作为营养补充食品(补充剂、营养饮料等)可以以粉末、颗粒末、片、糊、液体等形态提供本发明的身高促进剂。通过以食品组合物的形态提供,日常摄取、持续摄取本发明的身高促进剂变得容易。本发明的食品组合物中优选含有治疗或预防性效果能够期待的量的有效成分。添加量可以考虑作为其使用对象的患者的病状、健康状态、年龄、性别、体重等来进行设定。
实施例
近年,探索已有的药物的新的功能,实现应用扩大,即药物再定位(drug repositioning)战略受到瞩目(参考文献20、21)。这一战略的优点之一是被确定的药剂的最佳服用量、副作用等已经明确,因此能够立即进行临床应用。以下,以确定对软骨发育不全(低下)症、FGFR3参与的其他骨骼发育不良的治疗有效的药剂为目的,筛选了1186种FDA批准的药品。
1.材料和方法
(1)使用大鼠软骨肉瘤(rat chondrosarcoma(RCS))细胞的对1186种FDA批准的药品的筛选
将Pavel Krejci博士(Medical Genetics Institute,Cedars-SinaiMedical Center,LA)提供的RCS细胞,在添加10%胎牛血清(FBS,Thermo Scientific)的Dulbecco改良的Eagle培养基(DMEM,Invitrogen)中培养(参考文献14)。为了RSC增殖测定(growth arrestassay),将约5×103个细胞播种于96孔培养板(Falcon),在10μM的FDA批准的药品(1186种,Prestwick Chemical)和5ng/ml的FGF2(R&D Systems)的存在下,培养48小时。通过MTS测定(Cell 96AQueus One Solution Cell Proliferation Assay,Promega)将细胞的增殖定量。操作按照添付的手册进行。
(2)阿尔新蓝染色
为了阿尔新蓝染色,在12孔板中培养RCS细胞,各孔添加5ng/ml的FGF2和10μM的美克利嗪或0.2μM的CNP(Calbiochem)。72小时后,用甲醇将细胞固定化(-20℃、30分钟),用以1N HCl溶解的0.5%Alcian Blue 8GX(Sigma)进行染色(overnight)。为了定量分析,将用阿尔新蓝染色的细胞,用200μl的6M盐酸胍在室温下提取6小时(参考文献28)。使用PowerScan 4(DS Pharma Biomedical),测定提取的阿尔新蓝的光学浓度(610nm的吸光度)。
(3)总RNA的提取和实时RT-PCR解析
在20μM的美克利嗪或0.2μM的CNP的存在下,使用Trizol从FGF2处理的RSC细胞分离总RNA。使用ReverTra Ace(Toyobo)合成第一链cDNA。利用LightCycler 480Real-Time PCR(Roche)和SYBR Green(Takara),将基质蛋白酶(Mmp10、Mmp13、Adamts1)的mRNA水平定量。应予说明,测定值用Gapdh的mRNA表达水平校正。
(4)载体和转染
表达野生型FGFR3的载体pRK7-FGFR3-WT以及表达突变型FGFR3(参考文献29)的载体pRK7-FGFR3-K650E和pRK7-FGFR3-K650M由Pavel Krejci博士(Medical Genetics Institute,Cedars-Sinai Medical Center,LA)提供。使用QuikChange site-directedmutagenesis kit(Stratagene),制备pRK7-FGFR3-G380R。通过利用限制酶HindIII和BamHI处理,将野生型FGFR3cDNA和突变型FGFR3cDNA从载体切割下来。利用NheI位点和BamHI位点,在慢病毒载体CSII-CMV-MCS-IRES2-Venus中克隆各片段。CSII-CMV-MCS-IRES2-Venus由三好浩之博士(Riken BioResourceCenter,Tsukuba,Japan.)提供。在连接之前,使用Quick Blunting Kit(New England Biolabs)将插入的HindIII位点和载体的NheI位点平滑化。转染的前一天将HEK293细胞播种于150mm培养皿。使用Lipofectamine 2000(Invitrogen),将pLP1质粒、pLP2质粒、pLP/VSVG质粒(ViraPower Packaging Mix,Invitrogen)和CSII-CMV-MCS-IRES2-Venus载体导入HEK293。操作按照添付的手册进行。转染48小时后,使用Millex-HV 0.45μm PVDF过滤器(Millex)将含有病毒粒子的培养液进行过滤处理,利用2步的超速离心处理(Beckman Coulter)将慢病毒精制。将精制的慢病毒添加到HCS-2/8细胞或ATDC5细胞的培养液。48小时后,确认90%以上的细胞是病毒信号阳性。
MAPK/ERK路径中,表达持续活化的突变体的克隆pcDNA4Myc-ERK2(PD)、pcDNA3HA-MEK1(DD)和pcDNA3Flag-C-rafΔN(参考文献30)由武川睦宽博士(Medical ScienceInstitute,Tokyo University,Japan)提供。使用BamHI和XhoI处理插入物,平滑化之后,在CSII-CMV-MCS-IRES2-Venus的BamHI位点进行克隆。利用上述方法制备慢病毒粒子,应用于RCS细胞。
(5)HCS-2/8细胞的增殖测定
使慢病毒(表达FGFR3-WT、FGFR3-K650E或FGFR3-K650M)感染HCS-2/8(参考文献31)细胞,在96孔培养板中播种约5×103个。48小时后,通过MTS测定测定细胞数。此外,向HCS-2/8细胞导入表达FGFR3-K650E的慢病毒,在12孔板播种约1×105个。72小时后,利用ArrayScan VTI HCS Reader(Thermo Scientific)对Venus蛋白质的荧光强度进行定量。
(6)ATDC5细胞的微团培养
使慢病毒(表达FGFR3-WT或FGFR3-G380R)感染小鼠胚性癌瘤来源的ATDC5细胞(参考文献32)。将感染后的细胞用于微团培养(参考文献33)。下面介绍其概要,在含有5%FBS的DMEM/F-12(1:1)培养基(Sigma)中以1×107cells/ml的浓度混悬ATDC5细胞,为了模仿高浓度软骨凝缩,以10μl的液滴进行播种。孵育1小时后,添加添加有1%胰岛素-转铁蛋白-亚硒酸钠(ITS,Sigma)的培养基。在第6日回收细胞前,每隔1日交换培养基。
(7)器官培养
为了器官培养,显微镜下,采取野生型小鼠胎鼠(E16.5)的胫骨,转移至96孔培养板后,在添加0.2%牛血清白蛋白、1mMβ-glycerophosphate和50μg/ml抗坏血酸的α-minimal essential medium(Invitrogen)中培养。接下来,在20μM美克利嗪或0.2μM CNP的存在下和不存在下,利用100ng/ml FGF2处理6日,利用10%甲醛(磷酸缓冲液内)固定。利用0.5M EDTA脱矿,进行石蜡包埋。利用苏木精伊红和阿尔新蓝将切片染色后,利用安装XZ-1数码相机(Olympus)的SZ61显微镜(Olympus)拍照。使用ImageJ(NIH)测定骨的长径的长度(将其定义为关节软骨的基部-前端部之间的长度)。
(8)关于Western Blot和信号路径的研究
利用20μM美克利嗪将RCS细胞处理30分钟(将美克利嗪非处理的细胞作为Vehicle)。添加5ng/ml FGF2,5分钟后,在添加蛋白酶抑制剂的冰冷RIPA Lysis Buffer(Santa Cruz)中将细胞融解。利用SDS-PAGE将全细胞溶解产物分离,转印至硝酸纤维素膜。使用以下抗体、即ERK1/2、磷酸化-ERK1/2(Thr202/Tyr204)、MEK1/2、和磷酸化-MEK1/2(Ser217/221)(Cell Signaling),通过Western Blot确定MAPK路径中各分子的磷酸化水平。
(9)美克利嗪对正常小鼠的内服给予
对正常妊娠小鼠从妊娠第14日开始内服给予美克利嗪(5g饵料中含有美克利嗪2mg)、评价出生后第5日的仔小鼠的身长、四肢长。另一方面,对正常小鼠从出生后2周开始内服给予美克利嗪,在第5周进行同样地评价。
2.结果
(1)美克利嗪补偿RCS细胞中FGF2所致的增殖抑制和细胞外基质丧失
由于大鼠软骨肉瘤(RCS)细胞高表达FGFR,因此添加FGF2时在体外能够再现软骨发育不全(低下)症的发育软骨中看到的特征(参考文献22)。对添加FGF2(5ng/ml)的RCS细胞分别添加1186种FDA批准的药品(Prestwick Chemical)10μM。通过MTS测定将RCS细胞的增殖能力定量,结果表明添加美克利嗪时,与vehicle比较,具有再现性地显示1.4倍以上的细胞增殖能力。另外,添加0、1、2、5、10、20、50μM的美克利嗪时,RCS的细胞增殖能力会浓度依存性增加,而在50μM发现毒性(图1A)。
接下来,将CNP(参考文献6、17)作为阳性对照研究美克利嗪的效果。RCS细胞经过培养产生丰富的软骨性蛋白多糖,具有阿尔新蓝染色性。添加FGF2时,由于蛋白多糖的产生被抑制,而且产生细胞外基质分解酶,因此72小时后基本没有发现阿尔新蓝染色性(参考文献6)。除添加FGF2之外添加美克利嗪时,RCS细胞的阿尔新蓝染色性和软骨细胞样的形态被保持,其效果与CNP是同等的(图1B)。已知对RCS细胞添加FGF2时,4小时后Mmp10、Mmp13、Adamts1的表达上升(参考文献6)。对RCS细胞添加FGF2时,Mmp10、Mmp13、Adamts1的表达上升,而且美克利嗪和CNP抑制了这些细胞外基质分解酶的表达(图1C)。综上所述,可知美克利嗪抑制RCS细胞中FGF2所致的细胞外基质分解酶的表达,抑制蛋白多糖丧失。
(2)美克利嗪补偿导入FGFR3突变型(K650E、K650M)的HCS-2/8的增殖抑制
接下来,在使用慢病毒导入FGFR3突变型(K650E:致死性发育不良,K650M:SADDAN)的人软骨肉瘤(human chondrosarcoma)细胞(HCS-2/8)中,研究美克利嗪所带来的FGFR3信号抑制效果。通过MTS测定,确认了导入K650E的HCS-2/8的细胞增殖能力有意义降低(图2A)。在K650E或K650M表达的任一HCS-2/8中,美克利嗪(20μM)部分抑制增殖抑制(图2B)。进而,通过比较细胞内表达的Venus的荧光强度,可知导入K650E的HCS-2/8被美克利嗪促进细胞增殖(图2C)。
(3)美克利嗪补偿导入FGFR3突变型(G380R)的ATDC5细胞的分化抑制
由于ATDC5细胞具有软骨分化能力,所以通常在体外解析软骨分化过程(参考文献15)中使用。已知软骨发育不全的生长软骨中会引起分化抑制,通过在使用慢病毒导入FGFR3突变型(G380R:软骨发育不全)的ATDC5细胞的微团培养(micromass culture)中添加美克利嗪(20μM)进行分化诱导,研究美克利嗪能否补偿软骨的分化抑制。导入FGFR3野生型的ATDC5细胞中阿尔新蓝的染色性良好,与此相对,导入突变型的ATDC5细胞中染色性降低。对该导入突变型的ATCD5细胞添加美克利嗪时,阿尔新蓝的染色性被补偿。接下来,阿尔新蓝染色后添加盐酸胍,测定610nm的吸光度,进行染色性的定量。发现导入突变型的ATDC5中吸光度降低,但美克利嗪对其进行了补偿(图3)。综上所述,美克利嗪补偿FGFR3突变型的导入所致的软骨分化抑制效果。
(4)美克利嗪无论FGF2添加的有无均在胎鼠胫骨的器官培养中促进骨的长轴生长
已知胎鼠胫骨中添加FGF2进行器官培养时,骨的长轴生长被抑制(参考文献14),但是同时给予FGF2和美克利嗪时,研究了生长抑制是否被补偿。在器官培养液中同时添加FGF2(100ng/ml)和CNP(0.2μM)或美克利嗪(20μM),将骨的长度与同一个体的相对侧进行比较。添加FGF2,进行6日器官培养时,胎鼠胫骨的长轴生长被抑制,CNP和美克利嗪有意义地补偿FGF2所致的骨的生长抑制(图4)。进而,美克利嗪促进了不添加FGF2的胫骨的长轴生长。
(5)美克利嗪抑制RCS中FGF2所致的ERK的磷酸化
使用RCS细胞研究了FGFR3信号中美克利嗪的效果。对RCS细胞添加FGF2的30分钟前给予美克利嗪,通过Western Blotting法评价ERK和MEK的磷酸化水平。美克利嗪抑制了FGF2添加所致的ERK1/2的磷酸化,而没有抑制MEK1/2的磷酸化(图5A)。进而,利用慢病毒向RCS导入持续信号活化的ERK、MEK、RAF各个突变型,通过MTS测定评价了细胞增殖能力。美克利嗪补偿了活化的MEK和RAF所致的增殖抑制,另一方面没有发现抑制ERK所致的增殖抑制的效果(图5B)。综上所述,提示了美克利嗪抑制磷酸化MEK所致的ERK的磷酸化,或抑制ERK的磷酸化酶的活性(图6)。
(6)美克利嗪增大身长和四肢长
对正常妊娠小鼠从妊娠第14日开始内服给予美克利嗪,评价出生后第5日的仔小鼠,结果发现美克利嗪给予组中身长、上肢长和下肢长的增大(图7)。此外,对正常小鼠从出生后2周开始给予美克利嗪,在第5周评价,结果发现美克利嗪给予组中身长和尾长增大(图8)。
3.讨论
Drug Repositioning是发现已有的药物的新的效能的方法,按照阶段性缩小候选药的顺序,具有能够削减研究经费和时间的优点(参考文献20、23)。在有用性和毒性已保证的1186种FDA批准的药品中,美克利嗪是新的FGFR3信号抑制剂,隐含了作为软骨发育不全(低下)症中的低身高的治疗药进行临床应用的可能性。美克利嗪具有抗组胺作用,作为晕车药进行OTC(over the counter)销售,具有安全使用50年以上的实践。因此,最佳服用量·副作用·禁忌等安全性被明确,如果该药剂在可临床应用的浓度中具有FGFR3抑制效果,则能够直接进行临床应用的可能性高。
现在,由于软骨发育不全(低下)症中没有根本的治疗法,因此期待开发出抑制FGFR3信号活性的新的治疗法。Krejci等进行了1120种化合物的筛选,结果报告了NF449在RCS和胎鼠胫骨的器官培养中抑制FGFR3信号。但是,NF449与具有抗癌作用的suramin结构相似(参考文献14)。Jonquoyet等通过in silico解析,研发了具有FGFR3酪氨酸激酶抑制作用的合成化合物(A31)。结果表明A31抑制FGFR3的持续磷酸化,在器官培养中补偿软骨发育不全模型小鼠(Fgfr3Y367C/+)的长骨的生长抑制。进而表明A31补偿Fgfr3Y367C/+的生长软骨的分化抑制(参考文献16)。Jin等进行了肽噬菌体库的筛选,结果P3与FGFR3的胞外域的亲和性最高,从而确定了P3。表明P3在ATDC5细胞中具有增殖·分化促进作用,补偿致死性发育不良模型小鼠(Fgfr3Neo-K644E/+)的长骨的器官培养中骨的生长抑制,进而通过对母体给予,Fgfr3Neo-K644E/+能够长期存活(参考文献15)。但是,这些新的FGFR3抑制剂可能也作用于FGFR3以外的酪氨酸激酶,担心对人给予时的毒性。虽然担心美克利嗪的作用部位同样具有酪氨酸激酶的特异性,但是,由于具有安全使用50年以上的实践,因此认为没有严重的副作用。
CNP具有抑制软骨发育不全中FGFR3信号活性的作用。CNP缺失小鼠的表型是低身高,其特征是生长软骨的增殖软骨细胞层和肥大软骨细胞层狭小化(参考文献24)。acromesomelic dysplasia Maroteaux-type(AMDM)通过作为CNP受体基因的NPR2的功能丧失变为四肢缩短型低身高(参考文献25)。另一方面,通过CNP的过剩表达,介由MAPK信号的抑制,补偿了软骨发育不全中长骨的生长抑制(参考文献17)。八十田等成功实现了通过给予CNP,补偿软骨发育不全模型小鼠(Fgfr3ach)的低身高。但是,由于CNP的半衰期非常短因此需要持续静脉给予(参考文献18)。Lorget等开发了难以被中性肽链内切酶分解的结构的CNP类似物(BMN111),由此延长半衰期,通过皮下注射,能够改善Fgfr3Y367C/+的低身高(参考文献19)。本次研究中,美克利嗪在体外和取自活体(ex vivo)模型中能够与CNP同等地抑制FGFR3信号。此外,由于美克利嗪具有作为晕车药在临床上使用的实践,因此期待将其作为代替CNP或CNP类似物的药剂使用。
软骨细胞的增殖和分化中,MAPK路径是FGFR3信号的主要路径之一。已知软骨细胞中ERK持续活化时,会引起增殖抑制、细胞外基质的分解、细胞形态的变化、分化抑制(参考文献5、6)。CNP介由PKGII的活化来抑制RAF1激酶的磷酸化(参考文献6、17)。本次证明了美克利嗪在软骨细胞中抑制ERK的磷酸化。Gohil等报告美克利嗪除了抗组胺作用、抗毒蕈碱作用之外具有抗氧化性磷酸化作用(参考文献26、27)。表明美克利嗪抑制脑、心脏的缺血所致的细胞损害。本次研究中,在具有抗组胺作用、抗毒蕈碱作用或抗氧化性磷酸化作用的药剂中,仅美克利嗪抑制了FGFR3信号。因此,认为内软骨性骨化中美克利嗪的药理作用机理不是抗组胺作用、抗毒蕈碱作用或抗氧化性磷酸化作用。
本次研究中,证明了美克利嗪补偿RCS细胞中FGF2所致的细胞增殖抑制、细胞外基质的丧失、细胞形态的变化、细胞外基质分解酶的表达。此外,美克利嗪补偿了FGFR3突变型表达的HCS细胞和ATDC5细胞中的增殖抑制和分化抑制。进而,美克利嗪补偿了FGF2所致的胎鼠长骨的生长抑制。美克利嗪能够成为针对软骨发育不全(低下)症有用的治疗方法。
另一方面,动物实验的结果,证明了美克利嗪具有身长和四肢的伸长效果。鉴于这一事实,认为美克利嗪对例如不伴随骨骺线闭合的脑垂体性矮小症、Turner综合症的低身高、Prader-Willi综合症的低身高、生长激素分泌不全性低身高症(GHD)、宫内发育迟缓(SGA)性低身高症也是有效的。
产业上的可利用性
本发明的治疗药通过FGFR3信号的抑制这样的美克利嗪的新的作用而显示药效。美克利嗪作为晕车药进行OTC(over the counter)销售,具有安全使用50年以上的实践,最佳服用量·副作用·禁忌等、安全性被明确。这一事实成为临床应用上的很大的优点。期待将本发明应用于软骨发育不全、软骨发育低下、致死性骨发育不全、克鲁宗氏病、肢端肢中部畸形性发育不良、严重软骨发育不良伴发育迟缓和黑棘皮症(SADDAN)等FGFR3的过度活化所致的各种骨系统疾病的治疗。
本发明不局限于上述发明的实施方式和实施例的说明。在不脱离本发明要求保护的范围的记载而本领域技术人员容易想到的范围内各种变形方式也包含在本发明中。本说明书中明示的论文、公开专利公报、以及专利公报等内容,通过援引而引用其全部内容。
<参考文献>
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Claims (7)
1.一种成纤维细胞生长因子受体3即FGFR3的过度活化所致的骨系统疾病的治疗药,其特征在于,含有美克利嗪或其药学上可接受的盐作为有效成分。
2.根据权利要求1所述的治疗药,其中,骨系统疾病是选自软骨发育不全、软骨发育低下、致死性骨发育不全、克鲁宗氏病、肢端肢中部畸形性发育不良以及严重软骨发育不良伴发育迟缓和黑棘皮症即SADDAN中的疾病。
3.根据权利要求1或2所述的治疗药,其中,有效成分是盐酸美克利嗪。
4.一种骨系统疾病的治疗法,其特征在于,包括以下步骤:对成纤维细胞生长因子受体3即FGFR3的过度活化所致的骨系统疾病的患者给予治疗上有效量的美克利嗪或其药学上可接受的盐。
5.一种身高促进剂,其特征在于,含有美克利嗪或其药学上可接受的盐作为有效成分。
6.一种身高促进用组合物,其特征在于,含有权利要求5所述的身高促进剂。
7.根据权利要求6所述的身高促进用组合物,其是药品、准药品或食品。
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KR20150124941A (ko) | 2015-11-06 |
EP2985024A1 (en) | 2016-02-17 |
MX2015002959A (es) | 2015-06-05 |
US20150216860A1 (en) | 2015-08-06 |
JPWO2014141847A1 (ja) | 2017-02-16 |
CA2882881A1 (en) | 2014-09-18 |
EP2985024A4 (en) | 2016-09-14 |
JP6232630B2 (ja) | 2017-11-22 |
AU2014232030B2 (en) | 2016-05-26 |
AU2014232030A1 (en) | 2015-03-19 |
WO2014141847A1 (ja) | 2014-09-18 |
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