CN113577289A - Rxfp1/3抑制剂在制备预防或治疗青少年特发性脊柱侧凸疾病的药物中的用途 - Google Patents
Rxfp1/3抑制剂在制备预防或治疗青少年特发性脊柱侧凸疾病的药物中的用途 Download PDFInfo
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- CN113577289A CN113577289A CN202111086176.XA CN202111086176A CN113577289A CN 113577289 A CN113577289 A CN 113577289A CN 202111086176 A CN202111086176 A CN 202111086176A CN 113577289 A CN113577289 A CN 113577289A
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Abstract
本申请公开了AIS患者的血清中的RLN3(Relaxin3,松弛素3)水平显著高于正常对照的水平，持续注射RLN3或给予RXFP3(Relaxin Family Peptide Receptor 3)抑制剂可显著改变小鼠动物模型脊柱侧凸发病率，提示RLN3在AIS发病中发挥重要作用。由此，本申请提供了RXFP1/3抑制剂在制备预防和/或治疗青少年特发性脊柱侧凸的药物的用途，以及用于预防和/或治疗青少年特发性脊柱侧凸的包含RXFP1/3抑制剂的药物组合物，检测RLN3水平的试剂在制备用于诊断受试者中青少年特发性脊柱侧凸水平的诊断试剂中的用途以及用于诊断青少年特发性脊柱侧凸或其患病风险的试剂盒。
Description
本申请是申请日为2021年1月11日、发明名称为“RXFP1/3抑制剂在制备预防或治疗青少年特发性脊柱侧凸疾病的药物中的用途”的中国专利申请号202110029072.9的分案申请。
技术领域
本发明属于生物技术领域。更具体地，本发明涉及防治青少年特发性脊柱侧凸疾病的方法和试剂。
背景技术
青少年特发性脊柱侧凸(Adolescent Idiopathic Scoliosis,AIS)是重要的临床疾病，其发生发展的分子调控机制目前受广泛关注：脊柱侧凸是一种古老的疾病。脊柱冠状面侧方弯曲，按Cobb法测量超过10度，且排除其他继发性病因(如先天性、退变性，神经肌肉型等)，称为特发性脊柱侧凸，其中以青少年时期发生的脊柱侧凸最为多见，称为青少年特发性脊柱侧凸。持续进展的AIS患者常伴有肺功能下降和持续腰背痛，甚至继发心肺功能障碍，需手术矫正畸形。脊柱侧凸矫形手术是脊柱外科中较复杂、风险较高且并发症最为严重的手术之一，且手术费用昂贵。鉴于中国庞大的人口基数，AIS患病数量巨大，给患者、家庭和社会带来沉重的经济负担。AIS发生发展的分子调控机制是重要的科学问题(KikanlooSR,Tarpada SP,Cho W.Etiology of Adolescent Idiopathic Scoliosis:A LiteratureReview.Asian Spine J.2019；13(3):519-26.)，目前认为其发生发展是由多种细胞和基因共同参与的疾病(孙峰原,李宗远,何希,杨宗德.有限元分析在脊柱侧凸生物力学研究中的应用及进展[J].中国组织工程研究,2019,23(32):522；Zongde Y,Zhigang Y,Qiulin Z,Jingfeng L,Kai C,Yingchuan Z,Chuanfeng W,Dewei H,Xiaodong Z,MingL.Identification of the relationships between sagittal plane and coronalplane curvature in guppy models[J].International Journal of Surgery,2014,12(1):41-44；Yilin Y,Mingyuan Y,Zongde Y,Kai C,Jinyi B,Jian Z,Haijian N,ChangweiY and Ming L.Postoperative neck tilt in Lenke 1and 2AIS patients aftercorrection surgery:a novel predictive index.BMC Musculoskeletal Disorders,2019,20:405)。
AIS的病因尚不明确，研究者提出了很多假说如基因遗传学异常假说，神经内分泌异常假说、骨成骨方式异常假说、骨生长不平衡假说等等，但上述假说均不能完全解释AIS的病因(Kikanloo SR,Tarpada SP,Cho W.Etiology of Adolescent IdiopathicScoliosis:A Literature Review.Asian Spine J.2019；13(3):519-26.)。
除了手术矫正畸形，目前AIS的治疗还包括医疗器械治疗，如中国专利申请CN111053657A公开了一种新型脊柱侧凸牵引轮椅，CN111035495A公开了一种行走式脊柱侧凸牵引装置。北京协和医院的研究团队发现了AIS患者骨髓的间充质干细胞(MSC)显示出降低的成骨分化能力，经提出了将NF90(白细胞介素增强子结合因子3蛋白家族最主要的蛋白异构体之一)制备调控骨髓的间充质干细胞成骨分化的生物制剂，以及将NF90在制备用于预防和/或治疗青少年特发性脊柱侧凸的药物组合物中的应用(CN109568565A)。由于手术成本高昂且风险和副作用很大，本领域仍然迫切需要能够预防、治疗AIS以及防止其恶化的药物。
发明内容
申请人发现，AIS患者的血清中的RLN3(Relaxin3，松弛素3)水平显著高于正常对照的水平，持续注射RLN3或给予RXFP3(Relaxin Family Peptide Receptor 3)抑制剂可显著改变小鼠动物模型脊柱侧凸发病率，提示RLN3在AIS发病中发挥重要作用。申请人确定了脊柱韧带成纤维细胞中RLN3-RXFP1/3-ERK1/2信号轴在促进AIS发病中的作用。申请人由此提出通过RLN3-RXFP1/3-ERK1/2信号轴中相关分子的抑制剂，特别是RXFP1/3抑制剂，可以实现预防或治疗AIS的发生和发展，从而完成了本发明。
具体而言，申请人通过以下技术方案实现了本发明。
1.一种RXFP1/3抑制剂在制备预防和/或治疗青少年特发性脊柱侧凸的药物的用途。
2.项目1的用途，其中所述RXFP3抑制剂在使用Linda M.Haugaard-Kedstrom所述的方法的测定中与RXFP3竞争性结合的表观平衡常数(pKi)为<9，优选为5至9。
3.项目1或2任一项的用途，其中所述抑制剂为小分子化合物如肽，和抗体或适体。
4.项目1或2任一项的用途，其中所述RXFP3抑制剂选自R3，R3/I5，R3(B△23-27)R/I5,R3(B1-22R)(或R3 B1-22R)，R3 B1-22R酸,R3 B1-22R C10/22A,R3 B2-22R，R3 B3-22R，R3 B4-22R，R3 B5-22R，R3 B6-22R，R3 B1-22R二聚体，INSL5，和最小化的松弛素3-类似物3，优选R3(B1-22)R，或其药学上可接受的盐。
5.一种用于预防和/或治疗青少年特发性脊柱侧凸的药物组合物，其包含RXFP1/3抑制剂和药学上可接收的载体。
6.项目5所述的药物组合物，其中所述RXFP3抑制剂在使用Linda M.Haugaard-Kedstrom所述的方法的测定中与RXFP3竞争性结合的表观平衡常数(pKi)为<9，优选为5至9。
7.项目5或6所述的药物组合物，其中所述抑制剂为小分子化合物如肽，和抗体或适体。
8.项目5或6所述的药物组合物，其中所述RXFP3抑制剂选自R3，R3/I5，R3(B△23-27)R/I5,R3(B1-22R)(或R3 B1-22R)，R3 B1-22R酸,R3 B1-22R C10/22A,R3 B2-22R，R3B3-22R，R3 B4-22R，R3 B5-22R，R3 B6-22R，R3 B1-22R二聚体，INSL5，和最小化的松弛素3-类似物3，优选R3(B1-22)R，或其药学上可接受的盐。
9.检测RLN3水平的试剂在制备用于诊断受试者中青少年特发性脊柱侧凸水平的诊断试剂中的用途，其中所述诊断试剂用于测定受试者样品的RLN3水平，其中所述RLN3水平高于健康对照组样品中RLN3水平表明所述受试者患有或者有风险患有青少年特发性脊柱侧凸。
10.项目9的用途，其中所述检测RLN3水平的试剂选自用于测定RLN3蛋白质水平的试剂，如抗RLN3抗体、用于测定编码RLN3的mRNA水平的试剂，如引物或探针，和特异性结合RLN3蛋白质的核酸如适体。
11.一种用于诊断青少年特发性脊柱侧凸或其患病风险的试剂盒，其中所述试剂盒包含用于检测RLN3水平的诊断试剂。
12.项目11的试剂盒，其中所述检测RLN3水平的试剂选自用于测定RLN3蛋白质水平的试剂，如抗RLN3抗体、用于测定编码RLN3的mRNA水平的试剂，如引物或探针，和特异性结合RLN3蛋白质的核酸如适体。
如本发明所用，术语“个体”、“受试者”或“患者”包括但不限于人和其他灵长类(例如黑猩猩以及其他猿和猴类)。在一些实施方案中，所述受试者或患者是人。
本文所述的“药学上可接受”是指以通常的医药服用量利用时避免相当大的毒性效果，并能够获得或已获得政府或许可此的监管机构的许可或列在药典上或公知的一般的药典，从而能够在动物、更具体为人体上使用。
本文所述的药学上可接收的载体包括在药物使用中但不引起明显副作用的各种赋形剂、稀释剂和佐剂，包括但不限于：纯化水、生理盐水、缓冲液、葡萄糖、水、甘油、甘露醇、乙醇、表面活性剂和盐如氯化钠、EDTA钠等。
本文所述的“药学上可接受的盐”是指本领域公知的，并且包括但不限于：(1)由如盐酸、氢溴酸、硫酸、硝酸、磷酸等无机酸形成的盐；或者由如乙酸、丙酸、己酸、环戊烷丙酸、乙醇酸、丙酮酸、乳酸、丙二酸、琥珀酸、苹果酸、马来酸、富马酸、酒石酸、柠檬酸、苯甲酸、3-(4-羟基苯甲酰基)苯甲酸、肉桂酸、扁桃酸、甲磺酸、乙磺酸、1，2-乙烷-二磺酸、2-羟基乙磺酸、苯磺酸、4-氯苯磺酸、2-萘磺酸、4-甲苯磺酸、樟脑磺酸、4-甲基双环[2,2,2]-辛-2-烯-1-羧酸、葡庚糖酸、3-苯基丙酸、三甲基乙酸、叔丁基乙酸、月桂基硫酸、葡糖酸、谷氨酸、羟基萘甲酸、水杨酸、硬脂酸、己二烯二酸等有机酸形成的酸加成盐；或者(2)取代存在于母体化合物中的酸性质子时形成的盐。
本文所述的RXFP1/3抑制剂表示RXFP1和/或RXFP3的抑制剂，所述抑制剂能够与RXFP1和/或RXFP3结合并阻断松弛素3(RLN3)对RXFP1和/或RXFP3的激动作用。本文所述的RXFP1/3抑制剂包括RXFP1/3的拮抗剂。在一个实施方案中，本文所述的RXFP1/3抑制剂只与RXFP1和/或RXFP3结合但是不与RXFP2和/或RXFP4结合。
本申请发明人通过在动物实验中进行用受体RXFP1或RXFP3抑制剂阻断RXFP1或RXFP3与松弛素3(RLN3)的结合，发现实验组中脊柱侧凸发生率显著小于对照组，从而提出通过RLN3-RXFP1/3-ERK1/2信号轴中相关分子的抑制剂，特别是RXFP1或RXFP3(RXFP1/3)抑制剂的施用，可以实现预防或治疗AIS的发生和发展。在一个实施方案中，本文RXFP3抑制剂是使用Linda M.Haugaard-Kedstrom等所述的方法测定的与RXFP3竞争性结合的表观平衡常数(pKi)为<9，优选为5至9的抑制剂。在一个实施方案中，本文RXFP3抑制剂(或拮抗剂)包括但不限于R3，R3/I5，R3(B△23-27)R/I5,R3(B1-22R)(或R3 B1-22R)，R3 B1-22R酸,R3B1-22R C10/22A,R3 B2-22R，R3 B3-22R，R3 B4-22R，R3 B5-22R，R3 B6-22R，R3 B1-22R二聚体，INSL5，最小化的松弛素3-类似物3(minimized relaxin-3 analog3等)(见LindaM.Haugaard-Kedstrom等,Design,Synthesis,and Characterization of a Single-ChaiPeptide Antagonist for the Relaxin-3 Receptor RXFP3,Journal of the AmericanChemical Society,2011,133,4965-4967的表1)。在本发明的一个优选的实施方案中，RXFP3抑制剂是R3(B1-22)R(其结构式为Arg-Ala-Ala-Pro-Tyr-Gly-Val-Arg-Leu-Ser-Gly-Arg-Glu-Phe-Ile-Arg-Ala-Val-Ile-Phe-Thr-Ser-Arg-NH2)。筛选RXFP1和/或RXFP3的抑制剂的方法是本领域已知的(见例如，Michelle L,Halls等人，International Unionof Basic and Clinical Pharmacology.XCV.Recent Advances in the Understandingof the Pharmacology and Biological Roles of Relaxin Family Peptide Receptors1-4,the Receptors for Relaxin Family Peptides,Pharmacol Rev 67:389-440,April2015,和Linda M.Haugaard-Kedstrom等,Design，Synthesis，and Characterization of aSingle-Chai Peptide Antagonist for the Relaxin-3 Receptor RXFP3,Journal ofthe American Chemical Society,2011,133,4965-4967,将其全文并入本文作为参考)。
在一些实施方案中，本发明的RXFP1或RXFP3的抑制剂是抗RXFP1或RXFP3的抗体。本文所述的抗体可以是单克隆抗体或多克隆抗体，优选地单克隆抗体。在一个实施方案中，本发明中所用的抗体是单克隆抗体，包括嵌合、人源化或人抗体。在一个实施方案中，本发明中所用的抗体是抗体片段，例如，Fv，Fab，Fab'，scFv，双抗体或F(ab')2片段。在另一个实施方案中，所述抗体是全长抗体，例如，完整的IgG1，IgG2，IgG3或IgG4抗体或本文中限定的其他抗体类别或同种型。在一些实施方案中，本发明的抗体特异性结合RXFP1和/或RXFP3并阻断阻断松弛素3(RLN3)对RXFP1和/或RXFP3的激动作用。本发明的抗体可以通过本领域公知的技术制备，如杂交瘤技术等。
本发明的药物组合物可以包括经典的药物配制品。可以通过任意常规途径施用根据本发明的药物组合物，只要靶组织通过该途径可以获得即可，例如，静脉内、病灶内，鞘内，皮内，皮下，肌内，口服等等。
如本文中使用的，“治疗”是指尝试改变受治疗的个体的自然病程的临床干预，并且可以为了预防或在临床病理的病程期间进行。治疗的理想效果包括但不限于防止疾病发生或复发，减轻症状，消除疾病的任何直接或间接的病理结果，防止转移，降低疾病进展的速率，改善或减轻疾病状态，以及消除或改善预后。
在一些实施方案中，本发明的RXFP1或RXFP3抑制剂是适体。如本文中使用的，“适体”是指核酸适体(Aptamer)，也被称作适配体、适体、适配子等，是一类单链RNA分子或单链DNA分子，通常由几十到一百多个碱基组成，是从人工合成的RNA或DNA分子随机文库中筛选分离出能与目标物质进行高特异性、高灵敏度结合的单链寡核苷酸。虽然适体的结合方式与抗体较为相似，但核酸适体特有的性质在很多方面都优于抗体。与抗体相比，核酸适体可以通过体外筛选获得，在生产成本、稳定性、修饰难易程度和组织穿透力等方面具有明显优势。通常，核酸适体是通过指数级富集配体系统进化技术(Systematic evolution ofligands by exponential enrichment，SELEX)筛选得到的。以DNA适体为例，SELEX的基本步骤是将包含大容量的随机寡核苷酸序列的DNA分子随机文库溶液与靶分子共同孵育，并通过各种分离手段将结合靶分子的DNA分子与未结合的DNA分子分离，并对这些能够结合的DNA分子通过聚合酶链式反应进行扩增，经分离为单链后，生成次级文库，再用于下轮筛选，如此重复数轮后，挑选出富集的DNA分子，并进行测序即获得特异性识别靶分子的寡核苷酸分子，即核酸适体。大容量的分子文库能够涵盖丰富多样的立体构象，理论上可以针对任何靶分子进行筛选，得到其特异性识别的核酸适体。在一些实施方案中，本发明的适体能够特异性结合RXFP1和/或RXFP3并阻断松弛素3(RLN3)对RXFP1和/或RXFP3的激动作用。
在一些实施方案中，本发明的RXFP1或RXFP3抑制剂是小分子化合物抑制剂，例如肽。
在本发明的一些实施方案中，所述药物组合物还任选地包含一种或多种其他对治疗AIS有效的药物，这些药物是本领域技术人员所熟知的。本发明的药物组合物可以与其他治疗手段联合施用，用于AIS的预防和/或治疗。
用于所述药物组合物中的RXFP1或RXFP3抑制剂的量是用于AIS的预防和/或治疗的有效量。本领域技术人员可以根据本领域公知的方法确定所述有效量。
本发明的药物组合物可以以合适的剂量适用于受试者，例如人。
附图说明
图1：AIS患者松弛素(RLN)血清学水平检测结果提示RLN3血清水平在AIS患者中明显升高。A-C，检测AIS组和对照组之间血浆RLN1、RLN2和RLN3水平。D，AIS组关节活动度与RLN3血清学水平的相关分析。
图2:C57BL/6J小鼠脊柱侧凸动物模型中RLN3血清学水平明显升高。A,C57BL/6J小鼠脊柱侧凸动物模型的X线片检查照片。B,C57BL/6J小鼠脊柱侧凸动物模型的三维CT检查照片。C、C57BL/6J小鼠脊柱侧凸动物模型中侧凸组小鼠(n＝67)和正常小鼠(n＝33)的血清学RLN3水平。
图3:AIS患者韧带成纤维细胞的活化较正常对照受到明显抑制；RLN3通过RXFP3受体抑制了人类脊柱韧带成纤维细胞的活化，同时促进了其MMP2和MMP9的表达。A、TGF-β1(10ng/mL)和RLN3(100ng/mL)处理人脊柱韧带成纤维细胞后α-SMA的免疫荧光图像。B、CCK-8实验证实，RLN3(100ng/mL)可显著抑制TGF-β1(10ng/mL)对人脊柱韧带成纤维细胞的活化作用，**p<0.01。C、TGFβ1及RLN3处理后的韧带成纤维细胞羟脯氨酸含量。D–E，Westernblot分析证实RLN3处理后脊柱韧带成纤维细胞MMP2、MMP9和RXFP3的表达显著增加。
图4RLN3通过RXFP3升高ERK1/2相关信号通路水平，抑制TGF-β/SMAD2信号通路。A-B：RLN3对人脊柱韧带成纤维细胞ERK1/2相关信号通路和TGF-β/SMAD相关信号通路的作用。Western Blot分析证实，RLN3促进ERK1/2磷酸化以及nNOS的表达，并降低SMAD2蛋白的磷酸化。C-D，R3(B1-22)R(RXFP3受体拮抗剂)对人脊柱韧带成纤维细胞信号传导通路的作用。Western-blot检测证实R3(B1-22)R可显著抑制RLN3对ERK1/2及nNOS相关信号通路的活化作用。
图5：R3(B1-22)R可显著降低侧凸小鼠动物模型的侧凸发病率。A-B，R3(B1-22)R处理后的C57BL/6J小鼠脊柱侧凸动物模型的X线片和CT图像。C、R3(B1-22)R处理C57BL/6J小鼠脊柱侧凸动物模型后侧凸发病率变化。
具体实施方式
AIS和韧带松弛度的关系
已有研究表明，AIS和韧带松弛密切相关：①AIS患者关节过度活动的发生率(51％)是正常对照的2倍(21％)(Dariusz C,Tomasz K,Paulina P,et al.Jointhypermobility in children with idiopathic scoliosis:SOSORT award 2011winner[J].Scoliosis,2011,6(1):22)，多项研究进一步支持此研究结果(Hengwei F,Zifang H,Qifei W,Weiqing T,Nali D,Ping Y,Junlin Y.Prevalence of idiopathic scoliosisin Chinese schoolchildren:a large,population-based study.Spine(Phila Pa1976).2016,41(3):259-64)；②芭蕾舞者的AIS发病率(25％)是正常(1-2％)的10倍(Bozkurt S,Kayalar G,Tezel N,et al.Hypermobility Frequency in SchoolChildren:Relationship With Idiopathic Scoliosis,Age,Sex and MusculoskeletalProblems.[J].Arch Rheumatol,2019,34:268-273)，同时其关节活动度高于正常约70％，后续两项研究提示芭蕾舞者的AIS发病率甚至分别达到27％和30％(Longworth B,Fary R,Hopper D.Prevalence and predictors of adolescent idiopathic scoliosis inadolescent ballet dancers.[J].Arch Phys Med Rehabil,2014,95:1725-30)；③韵律操运动员的AIS发病率是27％(Tanchev,Panayot I,Dzherov,Assen D,Parushev,Anton D,etal.Scoliosis in Rhythmic Gymnasts[J].Spine,25(11):1367-1372)，而体操运动员总AIS发病率甚至达到36％(Kakar RS,Simpson KJ,Das BM,et al.Review of PhysicalActivity Benefits and Potential Considerations for Individuals with SurgicalFusion of Spine for Scoliosis[J].International Journal of Exercise Science，2017，10(2):166-177)；④AIS患者存在明显的关节以及足弓站立位塌陷的临床特征(WhyteLF.Adolescent idiopathic scoliosis:The Tethered Spine III:Is fascial spiralthe key？[J].Journal of Bodywork&Movement Therapies,2017,21(4):948)；⑤结缔组织异常相关的疾病如马凡综合症和Ehlers-Danlos综合征脊柱畸形与AIS的弯型异常相似(Giunta C,Baumann M,Fauth C,et al.A cohort of 17patients with kyphoscolioticEhlers–Danlos syndrome caused by biallelic mutations inFKBP14:expansion ofthe clinical and mutational spectrum and description of the natural history:[J].Genetics in Medicine,2018,20(1):42-54)。⑥美国圣地亚哥儿童医学中心的PeterNewton发现通过前方手术栓系可显著纠正AIS，提示脊柱前方韧带松弛可能与AIS发病密切相关(Newton PO，Bartley CE，Bastrom TP,et al.Anterior Spinal Growth Modulationin Skeletally Immature Patients with Idiopathic Scoliosis:A Comparison withPosterior Spinal Fusion at 2to 5Years Postoperatively[J].JBJS,2020)。
松弛素
松弛素(Relaxin，RLN)是一种神经肽，属于胰岛素/松弛素超家族成员，分布广泛，其中人RLN包括7种成员，以RLN1、RLN2、RLN3等最常见(Kaftanovskaya EM,Ng HH,Soula M,et al.Therapeutic effects of a small molecule agonist of the relaxin receptorML290in liver fibrosis.[J].FASEB J.，2019，33:12435-12446)。RLN1分子的生物学功能在人类及灵长类动物中未知。RLN2分子是最早发现的胰岛素/松弛素超家族成员之一，分子量6KDa，主要由卵巢等性器官通过脉冲方式分泌，与雌激素水平变化密切相关，在低等啮齿类动物可通过对成纤维细胞的抗纤维化作用松弛骨盆韧带促进及保护分娩过程(UnemoriE.Serelaxin in clinical development:past,present and future.[J].Br.J.Pharmacol.,2017,174:921-932)。研究发现RLN2可在多种器官中发挥抗纤维化作用，是非常有前景的抗纤维化药物(Summers RJ,Recent progress in the understandingof relaxin family peptides and their receptors.[J].Br.J.Pharmacol.,2017,174:915-920)。RLN3分子量为5KDa，包括2条肽链和3个二硫键，由小脑脑干神经核细胞分泌，在种属间高度保守，几乎所有脊椎动物均存在RLN3分子，被认为是胰岛素/松弛素超家族成员的祖先分子。RLN3在唤醒、促进食欲、调控应激和认知、降低焦虑和抑郁、调节生物节律等生理过程中起重要的作用(de
C,Chometton S,Ma S,Pedersen LT,Timofeeva E,Cifani C,Gundlach AL.Effects of chronic silencing of relaxin-3production innucleus incertus neurons on food intake,body weight,anxiety-like behaviourand limbic brain activity in female rats.Psychopharmacology(Berl).2020Jan3.doi:10.1007/s00213-019-05439-1.[Epub ahead of print])；研究发现RLN3同样可作用于韧带成纤维细胞发挥抗纤维化作用(Zhang X，Fu Y,Li H,Shen L,Chang Q,Pan L,Hong S,Yin X.H3relaxin inhibits the collagen synthesis via ROS-and P2X7R-mediated NLRP3inflammasome activation in cardiac fibroblasts under highglucose.J Cell Mol Med.2018,22(3):1816-1825)。RLN3在糖尿病心肌病大鼠中起抗心脏纤维化等作用(Zhang X,Pan L,Yang K,Fu Y,Liu Y,Chi J,Zhang X,Hong S,Ma X,YinX.H3relaxin protects against myocardial injury in experimental diabeticcardiomyopathy by inhibiting myocardial apoptosis,fibrosis andinflammation.Cell Physiol Biochem.2017,43(4):1311-1324)。RLN3还抑制TGF-β1诱导的大鼠心室成纤维细胞的纤维化、肌成纤维细胞分化和胶原蛋白的沉积。
RXFP
RLN3的受体为松弛素家族肽受体(Relaxin Family Peptide Receptor，RXFP)，包括RXFP1、3、4。虽然偶联G蛋白的RXFP3在各受体中与RLN3的亲和力最强(Ma S,Smith C M,Blasiak A,et al.Distribution,physiology and pharmacology of relaxin-3/RXFP3systems in brain[J].British journal of pharmacology,2017,174(10):1034-1048)，但多项研究提示RLN3也可以通过RXFP1下游信号通路抑制韧带成纤维细胞向肌成纤维细胞分化从而干扰韧带组织的正常纤维化过程。
RLN在AIS中的效应及相关性目前还未有报道。发明人在前期研究中对AIS患者血清中RLN1,RLN2,RLN3水平进行检测，发现AIS患者的血清RLN3水平显著高于正常对照组，提示RLN3与AIS发病相关。发明人推测，RLN3通过抑制脊柱周围韧带中成纤维细胞的纤维化，包括降低成纤维细胞的增殖、向肌成纤维细胞的分化和胶原的沉积等，继而改变韧带的特性，诱导AIS的发病。发明人通过在动物实验中进行RLN3的受体RXFP1或RXFP3用其抑制剂抑制RXFP1或RXFP3的活性，发现实验组中脊柱侧凸发生率显著小于对照组，提出通过RLN3-RXFP1/3-ERK1/2信号轴中相关分子的抑制剂，特别是RXFP1和/或RXFP3(RXFP1/3)抑制剂，可以实现预防或治疗AIS的发生和发展。在本发明的一个实施方案中，RXFP3抑制剂是R3(B1-22)R。
实施例
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件如Sambrook等人，分子克隆：实验室指南(New York：Cold Spring Harbor LaboratoryPress，1989)中所述的条件，或按照制造厂商所建议的条件。除非另外说明，否则百分比为质量/体积百分比，份数按重量计算。除非另行定义，文中所使用的所有专业与科学用语与本领域熟练人员所熟悉的意义相同。此外，任何与所记载内容相似或均等的方法及材料皆可应用于本发明中。文中所述的较佳实施方法与材料仅作示范之用。
实施例1 AIS患者中松弛素水平的测定
临床受试者的入组
研究对象为2016年5月至2019年4月在长海医院脊柱外科门诊及住院部的女性AIS患者。根据临床病史和X线片检查进行AIS诊断。排除结缔组织疾病(马凡氏综合症，埃勒斯-丹洛斯综合症等)等非特发性脊柱侧凸。棘上韧带组织是在AIS患者进行后路矫形内固定术中获得。AIS患者血清标本采集自在住院部及门诊就诊进行支具或手术治疗的AIS患者。在长海医院脊柱外科门诊就诊的年龄匹配的女性腰椎间盘突出症(无脊柱侧凸)患者作为对照组。两组之间的体重指数，初潮年龄或Risser分类无显著统计学差异。表1总结了受试者的临床特征。
表1本次研究参与受试者的临床特征总表
BMI:体重指数,VAS:视觉疼痛评分,CRP:C反应蛋白，ESR:血沉,JHM:关节活动度
这项研究是根据《赫尔辛基宣言》的原则进行的，并得到上海长海医院医学伦理委员会的批准。在登记之前，已从所有参与者处获得书面知情同意。
实验方法：
为了确定松弛素在AIS发病机制中的作用，我们比较了AIS患者(n＝30)和健康对照组(n＝30)的松弛素-1(RLN1)、松弛素-2(RLN2)和松弛素-3(RLN3)的血清学水平。对AIS患者进行体检，明确其HJM关节活动度评分。明确AIS及对照组三种RLN水平是否存在统计学差异；明确AIS患者HJM活动度评分与RLN3血清学水平是否存在统计学相关性。
实验步骤：
1、对符合入选条件的AIS患者及正常对照组患者在月经结束2周采集静脉血10ml。离心机((水平/角转高速冷冻离心机Eppendorf 5810R,1000转/分，5分)离心取上清。
2、血清样本通过ELISA检测分析其RLN1(EK1953,Human CHRDL1 ELISA Kit，Boster Biological Technology Co.)，RLN2(EK1428,Human RLN2 ELISA Kit，BosterBiological Technology Co.)，RLN3(EK1407Human Relaxin 3ELISA Kit，BosterBiological Technology Co.)血清学水平，双标准曲线，3复孔。
3、通过HJM评分量表对AIS患者进行关节活动度评分。
4、使用单因素方差分析检测RLN1，RLN2，RLN3血清学水平在AIS组及对照组是否存在统计学差异。通过Pearson检验HJM评分与RLN3血清学水平是否存在相关性。如果存在相关性，则进一步进行线性相关曲线拟合。
实验结果：
单因素方差分析结果提示AIS组和对照组之间RLN1(76.69±50.05ng/L vs.82.29±44.13ng/L；p＝0.696)或RLN2(9.72±11.08ng/L vs.8.40±7.71ng/L；p＝0.649)无显著统计学差异(图1A和1B)。但我们发现AIS组的RLN3血清学水平显著高于正常对照组(84.94±64.48ng/L vs.29.42±14.27ng/L；p<0.001)(图1C)。同时我们发现AIS患者RLN3血清学水平与关节活动度评分(JHM)呈正相关(p＝0.004)。说明RLN3水平升高可能导致韧带松弛。线性相关曲线拟合图(如图1D)所示进一步证实了RLN3与韧带松弛的相关性。
实施例2 AIS脊柱侧凸小鼠动物模型中松弛素水平的测定
1、脊柱侧凸小鼠动物模型的制备
如文献(Machida,M.et al.Experimental scoliosis in melatonin-deficientC57BL/6J mice without pinealectomy.Journal of Pineal Research 41,1-7(2010))报道，C57BL/6J小鼠可通过双前肢切除而无需进行松果体切除术建立小鼠脊柱侧凸动物模型(发病率60-90％)。三周龄的C57BL/6J从Charles River实验室获得(n＝100)，并分组笼养在空调室(22±2℃)中，空调室具有受控照明功能(从07:00开始照明到19:00)。在建模的4个月中，将去除前肢的小鼠放在特制笼子里，逐步增加食物和水放置的高度，以保持小鼠必须维持站立姿势才能进食。通过前后位脊柱X射线检查Cobb角>10°的小鼠可诊断为脊柱侧凸。通过螺旋3D-CT评估椎体旋转度和肋骨畸形程度。
第二军医大学医学动物研究委员会批准了这项研究中的动物工作。实验方案是根据美国国立卫生研究院建立的实验动物的护理和使用指南进行的。
脊柱侧凸小鼠模型和对照小鼠中松弛素3(RLN3)水平的测定
松弛素3水平测定步骤如下：
实验步骤：
1、C57BL/6J小鼠动物模型3周龄引进适应喂养1周，4周龄进行去除前肢手术。
2、4月龄C57BL/6J小鼠动物模型在0.5％水合氯醛麻醉下进行眼眶取血，血液经1000r/min离心(水平/角转高速冷冻离心机Eppendorf 5810R)5min后取上清。
3、血清样本通过ELISA检测(Relaxin-3试剂盒SAB EK1460)分析其RLN3血清学水平，单标准曲线，双复孔。
4、麻醉状态下进行前后位及侧位X线片检查以及三维CT检查。并对脊柱侧凸发病率进行统计。
3、使用单因素方差分析检测RLN3血清学水平在侧凸组及正常组是否存在统计学差异。
实验结果：
对4月龄C57BL/6J小鼠的X线和三维CT重建检查发现，67％(67/100)的C57BL/6J小鼠出现脊柱侧凸(图2A和2B)。ELISA检测提示脊柱侧凸小鼠血清RLN3水平(8.9±0.7ng/mL；)相比正常C57BL/6J小鼠(7.0±0.6ng/mL)显著升高(p<0.001)(图2C)。进一步证实RLN3与AIS发病密切相关。
实施例3：RLN3受体RXFP3的抑制剂的给药对脊柱侧凸小鼠韧带成纤维细胞生长的影响
实验方法：
1、通过I型胶原酶消化从对照组的正常韧带组织中分离出小鼠韧带成纤维细胞。消化后的细胞在培养箱(37℃，5％CO2)中在补充了胎牛血清(10％)和青霉素/链霉素(1％)的Dulbecco改良的Eagle培养基中进行传代。韧带成纤维细胞通过α-SMA的免疫荧光鉴定。随后的细胞实验是在第3-5代对韧带成纤维细胞进行的。应用TGF-β1(10ng/mL)，RLN3(100ng/mL)和R3(B1-22)R(0.1umol/L)刺激韧带成纤维细胞48小时。
2、免疫荧光测定：对韧带成纤维细胞α-SMA进行免疫荧光测定。将低聚甲醛固定的成纤维细胞的8μm冰冻切片与含0.3％Triton X-100的磷酸盐缓冲液在室温温育20分钟。然后，将细胞用一抗(Anti-αsmooth muscle actin(a-SMA)Antibody(monoclonal,1A4),BM0002,Boster Biological Technology Co.)在4℃温育过夜，然后与荧光素标记的二抗(Goat anti-Mouse IgG(H+L)Secondary Antibody,DyLight 594,#35510,Invitrogen)在37℃温育1小时。细胞核用4'，6-二脒基-2-苯基吲哚(DAPI.Beyotime)染色，然后用荧光显微镜(Nikon，Japan)观察。
3、细胞活力分析：韧带成纤维细胞生长活力使用Cell Counting Kit-8测定法(Beyotime,China)进行检测。将韧带成纤维细胞接种在96孔板中(每组6孔)并孵育过夜，然后进行TGF-β和RLN3处理48小时。加入10μL Cell Counting Kit-8溶液后，将成纤维细胞孵育2小时。使用酶标仪(BioTek，德国)根据使用手册的说明检测生长活力。
4、羟脯氨酸检测：根据制造商的说明书，使用羟脯氨酸分析试剂盒(Sigma，MO，USA)的水解产物测量韧带成纤维细胞中羟脯氨酸的含量。简而言之，将收集的细胞在100升浓盐酸(HCl，12M)中于120℃水解3小时。离心后，将上清液(10μL)转移至96孔板，然后在60℃的烤箱中干燥。在每个样品和标准孔中依次加入氯胺T/氧化缓冲液混合物和稀释的DMAB试剂，然后在60℃温育90分钟。使用酶标仪(Beckman Coulter，CA，USA)在560nm下测量吸光度。
5、Western Blot检测：使用10％SDS-PAGE进行蛋白质电泳分离。将非脂乳封闭的聚偏二氟乙烯膜与一抗(ERK1/2：CST公司，4695；p-ERK1/2：CST公司，4376；nNOS：CST公司，4231；smad2：CST公司，5339；psmad2：CST公司，18338；TGF-β1：Abcam公司，ab92486；MMP2：Abcam公司，ab37150；MMP9：Abcam公司，ab38898)在4℃下孵育过夜，然后与二抗(HRP-labeled Goat Anti-rabbit IgG(H+L)Jackson 111-005-003)在室温下孵育2小时。
使用ECL Plus Western Blotting Substrate(Thermo Scientific,WI,USA)在ChemiDoc MP系统(Bio-Rad,PA,USA)上实现可视化。基于条带灰度值对蛋白质的相对水平进行定量，并使用Image J软件进行分析。β-肌动蛋白用作内部对照。
实验结果：
1、图3A显示了TGF-β1(10ng/mL)和RLN3(100ng/mL)处理人脊柱韧带成纤维细胞后α-SMA的免疫荧光图像。可以看出，TGF-β1可激活人脊髓韧带成纤维细胞α-SMA的表达，RLN3可抑制α-SMA的表达。
2、CCK-8实验证实，RLN3(100ng/mL)可显著抑制TGF-β1(10ng/mL)对人脊柱韧带成纤维细胞生长活力的活化作用(图3B)。
3、图3C显示了TGFβ1及RLN3处理后的韧带成纤维细胞羟脯氨酸含量。TGFβ1处理的韧带成纤维细胞中羟脯氨酸显著增加，而RLN3处理的韧带成纤维细胞中羟脯氨酸显著减小。**p<0.01。
4、Western blot分析证实RLN3处理后脊柱韧带成纤维细胞MMP2、MMP9和RXFP3的表达显著增加。根据相应条带的灰度值评价相对蛋白水平(图3D-E)。**p<0.01。
5、Western blot分析证实RLN3促进ERK1/2磷酸化以及nNOS的表达，并降低SMAD2蛋白的磷酸化。每组设3个生物重复。根据相应条带的灰度值评价相对蛋白水平(图4A-B)。**p<0.01。
6、R3(B1-22)R(一种受体RXFP3拮抗剂)对人脊柱韧带成纤维细胞信号传导通路的作用：Western-blot检测证实R3(B1-22)R可显著抑制RLN3对ERK1/2及nNOS相关信号通路的活化作用(图4C-D)。
上述结果表明,RLN3受体RXFP3的抑制剂(例如R3(B1-22)R)通过显著抑制RLN3对ERK1/2及nNOS相关信号通路的活化作用，从而阻止了RLN3对韧带成纤维细胞活化的抑制，从而促进了韧带成纤维细胞活化为肌成纤维细胞，进而改变了韧带的理化性质。
实施例4 RLN3受体RXFP3的抑制剂(R3(B1-22)R)给药对脊柱侧凸小鼠动物模型中脊柱侧凸发病率的影响
小鼠动物模型的制备同实施例2。
实验步骤：
1、C57BL/6J小鼠动物模型3周龄引进适应喂养1周，4周龄进行去除前肢手术。5周龄开始每月进行0.5％水合氯醛麻醉下皮下R3(B1-22)R渗透泵植入手术。通过皮下渗透泵(Alzet,Durect Corporation,CA,USA)植入缓慢输注R3(B1-22)R(PhoenixPharmaceuticals Inc,CA,USA,036-14 2.5μg/天),或生理盐水(NS)。
2、麻醉状态下进行前后位及侧位X线片检查以及三维CT检查。并对脊柱侧凸发病率进行统计。
抑制剂R3(B1-22)R通过皮下渗透泵给药16周后，对C57BL/6J小鼠脊柱侧凸动物模型进行脊柱X线片及CT检查。
实验结果：
X射线和3D-CT检查(图5A和5B)证实C57BL/6J小鼠脊柱侧凸动物模型中R3(B1-22)R治疗组的脊柱侧凸发生率明显低于NS组(22.0％vs 62.0％，图5C)。上述结果表明,RLN3受体RXFP3的抑制剂(例如R3(B1-22)R)可阻止甚至逆转AIS的发生和发展。
显然，本领域技术人员可知，并非仅仅R3(B1-22)R能够降低脊柱侧凸发生率。实际上，本领域技术人员从上述实施例的结果可以预期，RLN3受体的抑制剂，特别是RXFP1和RXFP3受体的抑制剂，如小分子化合物、抗体、或适体等等。不受特定理论的束缚，发明人认为所述受体RXFP1或RXFP3的抑制剂通过抑制或拮抗受体RXFP1和RXFP3与其配体RLN3的结合，阻止RLN3通过RLN3-RXFP1/3-ERK1/2信号轴对韧带成纤维细胞活化的抑制，从而促进了韧带成纤维细胞生长，进而阻止甚至逆转AIS的发生和发展。因此，本领域技术人员可以得到(例如，通过商业途径获得或根据已知的方法制备或筛选)各种RLN3受体的抑制剂，特别是RXFP1和RXFP3受体的抑制剂，并筛选适用于哺乳动物，特别是人类给药的预防和/或治疗青少年特发性脊柱侧凸的化合物，并将其制备为药物。
统计学分析方法
所有统计学分析均使用SPSS 22.0版进行。使用Fisher精确检验比较定性数据。必要时，使用双因素ANOVA或Kruskal-Wallis检验比较定量数据。RLN3血浆水平与HJM点之间的关系通过Pearson检验进行了分析。脊柱侧凸发生率的比较通过卡方检验进行比较。所有p值均为双侧的，小于0.05的p值被认为是统计学显著的。
以上所述的具体实施例，对本发明的目的、技术方案和有益效果进行了进一步详细说明，应理解的是，以上所述仅为本发明的具体实施例而已，并不用于限制本发明，凡在本发明的精神和原则之内，所做的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。
Claims (12)
1.一种RXFP1/3抑制剂在制备预防和/或治疗青少年特发性脊柱侧凸的药物的用途。
2.权利要求1的用途，其中所述RXFP3抑制剂在使用Linda M.Haugaard-Kedstrom所述的方法的测定中与RXFP3竞争性结合的表观平衡常数(pKi)为<9，优选为5至9。
3.权利要求1或2任一项的用途，其中所述抑制剂为小分子化合物如肽，和抗体或适体。
4.权利要求1或2任一项的用途，其中所述RXFP3抑制剂选自R3，R3/I5，R3(B△23-27)R/I5,R3(B1-22R)(或R3 B1-22R)，R3 B1-22R酸,R3 B1-22R C10/22A,R3 B2-22R，R3 B3-22R，R3 B4-22R，R3 B5-22R，R3 B6-22R，R3 B1-22R二聚体，INSL5，和最小化的松弛素3-类似物3，优选R3(B1-22)R，或其药学上可接受的盐。
5.一种用于预防和/或治疗青少年特发性脊柱侧凸的药物组合物，其包含RXFP1/3抑制剂和药学上可接收的载体。
6.权利要求5所述的药物组合物，其中所述RXFP3抑制剂在使用Linda M.Haugaard-Kedstrom所述的方法的测定中与RXFP3竞争性结合的表观平衡常数(pKi)为<9，优选为5至9。
7.权利要求5或6所述的药物组合物，其中所述抑制剂为小分子化合物如肽，和抗体或适体。
8.权利要求5或6所述的药物组合物，其中所述RXFP3抑制剂选自R3，R3/I5，R3(B△23-27)R/I5,R3(B1-22R)(或R3 B1-22R)，R3 B1-22R酸,R3 B1-22R C10/22A,R3 B2-22R，R3B3-22R，R3 B4-22R，R3 B5-22R，R3 B6-22R，R3 B1-22R二聚体，INSL5，和最小化的松弛素3-类似物3，优选R3(B1-22)R，或其药学上可接受的盐。
9.检测RLN3水平的试剂在制备用于诊断受试者中青少年特发性脊柱侧凸水平的诊断试剂中的用途，其中所述诊断试剂用于测定受试者样品的RLN3水平，其中所述RLN3水平高于健康对照组样品中RLN3水平表明所述受试者患有或者有风险患有青少年特发性脊柱侧凸。
10.权利要求9的用途，其中所述检测RLN3水平的试剂选自用于测定RLN3蛋白质水平的试剂，如抗RLN3抗体、用于测定编码RLN3的mRNA水平的试剂，如引物或探针，和特异性结合RLN3蛋白质的核酸如适体。
11.一种用于诊断青少年特发性脊柱侧凸或其患病风险的试剂盒，其中所述试剂盒包含用于检测RLN3水平的诊断试剂。
12.权利要求11的试剂盒，其中所述检测RLN3水平的试剂选自用于测定RLN3蛋白质水平的试剂，如抗RLN3抗体、用于测定编码RLN3的mRNA水平的试剂，如引物或探针，和特异性结合RLN3蛋白质的核酸如适体。
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