CN112294958A - 一种含有萘醌和菲醌结构的组织蛋白酶k抑制剂及其组合物和应用 - Google Patents
一种含有萘醌和菲醌结构的组织蛋白酶k抑制剂及其组合物和应用 Download PDFInfo
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Abstract
本发明提供了一种含有萘醌和菲醌结构的组织蛋白酶K抑制剂及其组合物和用途,所述抑制剂用于制备以组织蛋白酶K为靶标的疾病的药物,所述化合物作用于组织蛋白酶K活性位点,具有显著抑制酶底物胶原降解活性或抑制弹性纤维蛋白降解活性或抑制甲状腺球蛋白降解活性,可用于制备治疗以组织蛋白酶K异常表达或活化为特征的疾病,包括甲状腺疾病、心血管疾病和骨疾病,具体为甲状腺功能亢进、骨质疏松、牙龈病(牙龈炎和牙周炎)、类风湿性关节炎等。研究证实β‑拉帕醌显著抑制组织蛋白酶K底物胶原、弹性纤维和甲状腺球蛋白的降解;四烯甲萘醌和七烯甲萘醌与组织蛋白酶K分子作用强,能显著抑制组织蛋白酶K与底物胶原纤维的结合,抑制胶原降解。
Description
技术领域
本发明属于医药技术领域,具体涉及一种含有萘醌和菲醌结构的组织蛋白酶K抑制剂及其组合物和应用。
背景技术
组织蛋白酶K属于半胱氨酸蛋白酶家族,与骨质疏松、关节炎、甲状腺异常等多种重大疾病密切相关,是近年来备受关注的靶标蛋白酶。组织蛋白酶K不仅特征性存在于破骨细胞,还存在于皮肤、心脏、骨骼肌、肺、甲状腺等组织,在体内主要发挥降解胶原蛋白、弹性纤维蛋白和甲状腺球蛋白的功能。组织蛋白酶K基因敲除小鼠,除了骨密度显著增高,还表现肺气道损伤,肺或皮肤发生纤维化,甲状腺球蛋白(Tg)释放甲状腺素功能障碍等症状。
β-拉帕醌是一种天然的萘醌类化合物,具有抗肿瘤作用,通过诱导肿瘤细胞内产生大量ROS,诱导肿瘤细胞凋亡,尚无报道具有抑制组织蛋白酶K的作用。维生素K2除了是促进血液正常凝固的重要维生素,现代研究发现它能多方面改善骨质疏松症患者骨量丢失症状,维护健康人群骨骼骨稳态,还被临床用于解痉止痛、毛细支气管炎、小儿肺炎、中毒物质解救等疾病。经过研究显示β-拉帕醌和维生素K2能显著抑制组织蛋白酶K活性,具有显著的抑制胶原降解、弹性纤维降解或甲状腺球蛋白降解作用。
发明内容
本发明的目的是提供了一种含有萘醌和菲醌结构的组织蛋白酶K抑制剂及其组合物和应用。
为了实现上述发明目的,本发明采用以下技术方案予以实现:
本发明提供了一种含有萘醌和菲醌结构的组织蛋白酶K抑制剂,所述组织蛋白酶K抑制剂为母核为式I或式II所示结构的化合物,所述式I、式II结构式如下:
所述式I中A环存在间位双酮,B环为呋喃环、吡喃环、二氢取代呋喃环或二氢取代吡喃环中的一种,C环选自苯环或环己基环,所述式II中n=4~12。
进一步的,所述C环选自甲基苯环或二甲基环己基环。
进一步的,所述化合物为通过在A环或C环上取代形成的可药用的酯、酰胺和盐中的至少一种。
进一步的,所述组织蛋白酶K抑制剂为β-拉帕醌和维生素K2。
进一步的,所述维生素K2包括四烯甲萘醌和七烯甲萘醌。
本发明还提供了含有所述的组织蛋白酶K抑制剂的组合物。
本发明还提供了所述组织蛋白酶K抑制剂在制备用于治疗以组织蛋白酶K为靶向的疾病的药物中的应用。
进一步的,所述蛋白酶K抑制剂作用于组织蛋白酶K活性位点。
进一步的,所述以组织蛋白酶K为靶向的疾病包括甲状腺疾病、心血管疾病和骨疾病。
进一步的,所述甲状腺疾病、心血管疾病和骨疾病包括为甲状腺功能亢进、骨质疏松、牙龈炎、牙周炎、类风湿性关节炎。
进一步的,所述组织蛋白酶K抑制剂,主要抑制组织蛋白酶胶原降解、弹性纤维降解或甲状腺球蛋白降解作用。
本发明另一方面提供了一种药物组合物在制备以组织蛋白酶K为靶标的疾病药中的应用,所述药物组合物包含组织蛋白酶K抑制剂单个或配伍应用。组织蛋白酶K抑制剂选自:化学式母核为式I和式II所示结构的化合物,所述化合物为通过在A环或C环上取代形成的可药用的酯、酰胺和盐,和包含所述酯或酰胺的盐或组合物。
与现有技术相比,本发明的优点和技术效果在于:本发明的组织蛋白酶K抑制剂集合位点为人体组织蛋白酶K活性位点,具有显著抑制酶底物胶原降解活性或抑制弹性纤维蛋白降解活性或抑制甲状腺球蛋白降解活性。药效学研究表明,能显著抑制人破骨细胞活性,改善D-半乳糖诱导老年性骨质疏松小鼠骨结构特征和生理状态,包括提高骨密度、改善骨微细结构和提高骨组织的生物力学等,且对大脑学习记忆能力无影响,为抗骨质疏松药物研发提供了新的思路。
该组织蛋白酶K抑制剂可用于制备治疗以组织蛋白酶K异常表达或活化为特征的疾病,包括甲状腺疾病、心血管疾病和骨疾病,具体为甲状腺功能亢进、骨质疏松、牙龈病(牙龈炎和牙周炎)、类风湿性关节炎等。研究证实β-拉帕醌显著抑制组织蛋白酶K底物胶原、弹性纤维和甲状腺球蛋白的降解;四烯甲萘醌和七烯甲萘醌与组织蛋白酶K分子作用强,能显著抑制组织蛋白酶K与底物胶原纤维的结合,抑制胶原降解。
附图说明
图1是四烯甲萘醌与人组织蛋白酶K分子对接图。
图2七烯甲萘醌与人组织蛋白酶K分子对接图。
图3组织蛋白酶抑制剂对破骨细胞活力和TRAP活性。
图4组织蛋白酶合成和分泌相关蛋白和基因表达:A)OPG和RANKL的Western blot图,B)RANKL蛋白相对表达量,C)AP-1,TRAF6和MITF的Western blot图和D)CN,NFATC1,TRACP,CTSK,c-fos和MMP9的基因表达。
具体实施方式
下面结合具体实施例,进一步阐述本发明。这些实施例应理解为仅用于说明本发明而不用于限制本发明的保护范围。在阅读了本发明记载的内容之后,本领域技术人员可以对本发明作各种改动或修改,这些等效变化和修饰同样落入本发明权利要求所限定的范围。
本发明提供的一种组织蛋白酶抑制剂(Cathepsin K inhibitor),该化合物母核结构为式I或II所示:
所述式I中A环存在间位双酮,B环为呋喃环、吡喃环、二氢取代呋喃环或二氢取代吡喃环中的一种,C环选自苯环或环己基环,所述式II中n=4~12。
优选地:所述式I中A环存在间位双酮,B环为呋喃环、C环选自甲基苯环和二甲基环己基环。
本发明揭露较佳结构为β-拉帕醌,但并不以此为限。所述式II中n=4~12,本发明揭露较佳结构为四烯甲萘醌(n=4),但并不以此为限。
以下通过实验具体说明本发明应用于改善组织蛋白酶K异常为特征疾病应用。
实施例1:组织蛋白酶K抑制剂初步筛选
组织蛋白酶K抑制剂的鉴定通过考察化合物抑制组织蛋白酶K与荧光标记人工合成底物Z-FR-MCA的结合能力。
1实验材料
单体化合物β-拉帕醌,购买自merck公司;四烯甲萘醌和七烯甲萘醌购自Sigma公司,底物Z-FR-MCA购自日本WAKO公司。
2实验方法
抑制剂用100mM醋酸钠缓冲液(pH5.5,包含2.5mM DTT和2.5mM EDTA)稀释至浓度为25μM,加入96孔板,最终反应容量为200uL。加入final 100,75,25,15,5μM)终浓度为5nM的CatK孵育5分钟,再加入5μL底物1mM的Z-FR-MCA溶液开始反应,检测荧光信号(发散光波长460nm,吸收光波长355nm)。计算公式:抑制率%=100-(1-Vi/V0)。Vi和V0分别表示存在和不存在抑制剂情况下的荧光信号。
3实验结果
表1不同化合物的组织蛋白酶K的Z-FR-MCA活性
结果显示(表1),β-拉帕醌显示显著的抑制组织蛋白酶K与Z-FR-MCA底物的结合率,IC50值为1.3±0.2μM,维生素K2抑制组织蛋白酶K的Z-FR-MCA活性IC50值分别为16.6±1.9和52.8±5.5μM。
实施例2:维生素K2与组织蛋白酶K分子对接
用Discovery studio 2016软件中的libdock分子对接方法考察化合物和组织蛋白酶K(1ATK)蛋白进行刚性对接。
1实验方法:
利用LibDock得分来作为表征化合物最佳结合位置的核心指标。LibDock得分分数越高,预测小分子与受体结合的活性越高。另外通过DS2016软件分析得到配体与受体的氢键数,找到对配体结合有重要贡献的氨基酸残基。
2实验结果
Discovery studio 2016软件中的libdock分子对接,维生素K2对接得分分别MK4为128,MK7为119,体现较强的对接效果。
由二维平面图1可知,MK4环上醛基与GLNAA:19残基和TRPA:184残基存在明显的氢键作用,氢键距离分别为和侧链羰基与HIS A:162残基之间具有明显的相互作用,证明共轭双键促进化合物与周围氨基酸形成氢键和其他相互作用。此外,MK4长侧链与多个氨基酸残基的包围中,存在多个烷基化作用,证明是因为这些残基与配体的相互作用增强了配体与受体的结合能力,从而使该配体化合物活性提高。
图2显示MK7环上醛基与多个残基存在明显的Pi-氢键作用。苯环与ASPA:61残基具有明显的Pi-Anion相互作用,共轭双键促进MK7与周围氨基酸形成氢键。此外,MK7长侧链与META:68,ALAA:163,LEUA:209氨基酸残基,存在多个烷基化作用,是因为这些残基与配体的相互作用增强了配体与受体的结合能力,从而使该配体化合物活性提高。
实施例3:抑制剂对组织蛋白酶K的活性影响
组织蛋白酶K抑制剂抑制组织蛋白酶K活性,主要通过降解天然底物胶原纤维、弹性纤维、甲状腺球蛋白的活性的能力。
1实验材料
单体化合物β-拉帕醌,购买自merck公司;四烯甲萘醌和七烯甲萘醌购自Sigma公司;硫酸软骨素A(C4-S),E-64和胃蛋白酶购自美国Sigma公司;I型胶原购自美国Affymetrix公司;微孔过滤滤膜离心管购自Amico Millipore公司。
2实验方法
2.1胶原纤维降解活性
采用凝胶电泳法(SDS-PAGE),可溶性I型胶原溶解于100mM醋酸钠缓冲液(pH5.5,包含2.5mM DTT和2.5mM EDTA)中,最终浓度为0.6mg/ml,依次将CatK (最终浓度400nM)、C4-S(最终浓度200nM)和化合物单体(不同浓度抑制剂)加入到含有I型胶原蛋白的反应液中,使总反应液容量为50μL。混匀,28℃孵育4小时,取出后加入1μL的100μM E64终止反应。采用10%的SDS-PAGE凝胶电泳分离,卡马斯亮蓝染色20分钟,用乙酸-甲醇(4∶1)脱色。I型胶原的α1条带的灰度用GeneSnap(SyngeneInc.Frederick,MD)软件进行定量分析。非活性位点和活性位点抑制剂均能抑制I型胶原的α1条带降解。
2.2弹性纤维降解活性
称取1mg刚果红弹性纤维蛋白(Congo-Red elastin),置于100μL的100mM醋酸钠缓冲液中(pH5.5,包含2.5mM DTT和2.5mM EDTA),加入终浓度为1μM的CatK和100μM和10μM抑制剂,37℃,200转/min摇床,孵育16小时。样品取出,8000转/min,离心5min,取上清液90μL,490nm波长下检测荧光信号。计算公式:抑制率%=100-(1-Vi/V0)。Vi和V0分别表示存在和不存在抑制剂情况下的荧光信号。
2.3甲状腺球蛋白降解
采用凝胶电泳法(SDS-PAGE),100μg的牛甲状腺球蛋白(Tg)溶解在100μL的100mM醋酸钠缓冲液(PH5.5,包含2.5mMDTT,和2.5mM EDTA),加入不同终浓度的100nM的CatK,37℃培养箱中,共同孵育1h后,加入100ug/mLE64终止反应,12000g/min离心10min,取50uL上清液,进行LC-MS检测不同TH,取15uL进行SDS-PAGE凝胶电泳检测。卡马斯亮蓝染色20分钟,用乙酸-甲醇(4∶1)脱色。
3实验结果
表2潜在抑制剂化合物对组织蛋白酶K活性的影响
由表2可得,10μMβ-拉帕醌具有90.3%抑制胶原纤维降解活性,65.4%抑制弹性纤维降解活性,74.3%的抑制甲状腺球蛋白降解活性,接近目前临床效果最佳的,处于III期临床研究的组织蛋白酶K抑制剂Odanacatib,表明β-拉帕醌为较理想的活性位点抑制剂。四烯甲萘醌显示在25uM具有58.9%的抑制胶原降解活性和40.2%的弹性纤维降解活性。七烯甲萘醌在25uM只显示9.4%抑制胶原作用。
实施例4:抑制剂对破骨细胞中组织蛋白酶K合成和活性的影响
破骨细胞表达丰富的胞浆内酶系统,在破骨细胞前体细胞向成熟破骨细胞分化的过程中有一系列的标志性蛋白质产生,可以作为破骨细胞的识别及其分化阶段的标志。其中,分化后的破骨细胞前体细胞所表达的抗酒石酸酸性磷酸酶(tartrate-resistant acidphosphatase,TRAP被认为是破骨细胞的标志酶。本实验采用RANKL和M-CSF骨髓诱导破骨细胞模型,考察抑制剂对破骨细胞分化,TRAP活性的影响。
1实验材料
1.1药材与试剂
动物:新生1-2天的同窝SD小鼠5只,雌雄不限,体重7-8g,由第二军医大学实验动物中心提供。
单体化合物β-拉帕醌,购买自merck公司;四烯甲萘醌和七烯甲萘醌购自Sigma公司。
2实验方法
2.1破骨细胞培养
取新生3天的SD小鼠胫骨,培养基冲洗骨髓腔以收集骨髓细胞,加入等量Ficoll试剂分离骨髓单核细胞,用含有25ng/mL M-CSF、25ng/mL RANKL的细胞因子和10%胎牛血清的α-MEM培养基进行培养,每3天换液1次,6天后破骨细胞分化成熟。
2.2破骨细胞活性研究
(1)破骨细胞活力;细胞成熟后,以1×105/mL接种于96孔板,提前置入牛股骨骨片,加入具有潜在抑制CatK活性的0.1,1和10μM β-拉帕醌和维生素K2。继续培养48h后,取100μL培养基,加入20μL CellTriter-blue试剂,轻轻晃10s混匀,37℃孵育30min,荧光分光光度计检测(发散光波长560nm,吸收光波长590nm)。选择对细胞活力无毒性的浓度范围进行破骨细胞活性测试。
(2)TRACP活性;成熟破骨细胞以1×105/mL接种于96孔板内,加入0.1,1和10μM β-拉帕醌和维生素K2的破骨细胞诱导培养基,提前培养48h后,移出培养基至另一孔板待测TRACP活性。依据TRACP染色试剂盒对破骨细胞细胞核染色,计算TRACP阳性破骨细胞数目。
(3)破骨细胞组织蛋白酶K合成和活性受很多细胞因子和蛋白的调控,本发明采用RT-PCR考察抑制剂对CN,NFATC1,TRACP,CTSK,c-fos和MMP9的基因表达考察,采用westernblot考察对OPG和RANKL、AP-1,TRAF6和MITF等关键蛋白表达的影响。
3实验结果
本实验采用骨髓细胞与成骨细胞共培养诱导破骨细胞模型,考察组织蛋白酶K抑制剂对破骨细胞分化和TRAP活性的作用,如表2所示。骨髓细胞经诱导培养8天后发育为成熟的破骨细胞,经0.01-1μM的维生素K2作用48h后,细胞活力均无显著性差异(P>0.05),但1uM的β-拉帕醌可显著影响破骨细胞细胞活力(P<0.01)。抗酒石酸酸性磷酸酶(TRAP)活性在0.01-1μMβ-拉帕醌和四烯甲萘醌(P<0.01),及0.1-1uM七烯甲萘醌显著抑制(P<0.05),见图3。
3.2四烯甲萘醌对破骨细胞组织蛋白酶K合成相关基因和蛋白表达的影响
图4为组织蛋白酶合成和分泌相关蛋白和基因表达A)OPG和RANKL的Western blot图,B)RANKL蛋白相对表达量,C)AP-1,TRAF6和MITF的Westernblot图和D)CN,NFATC1,TRACP,CTSK,c-fos和MMP9的基因表达。
OPG/RANKL通路是骨重建调控成骨细胞和破骨细胞交叉作用的关键纽带,成骨细胞释放RANKL细胞因子是诱导破骨细胞形成的关键因子。RANKL通过刺激NFAT、Mitf、TRAF6以及AP-1等破骨因子的表达,促进破骨细胞形成和骨吸收活性。研究证明四烯甲萘醌在0.1-10μm/L下显著增强OPG表达,降低RANKL表达(Fig.3A,3B)。研究证明四烯甲萘醌能显著降低c-Fos,NFATc1,CTSK和TRAP表达(p<0.05).尤其MK-4抑制(p<0.01)CN,c-Fos,NFATc1,CTSK and TRAP mRNA表达水平(Fig.3D)。
实施例5:四烯甲萘醌对D-半乳糖诱导老年性骨质疏松大鼠的影响
1实验材料
动物选择:本实验选择SD大鼠品系,因该品系对疾病的抵抗力较强,尤其在骨质疏松研究领域应用多,广泛用于药理、毒理、及GLP实验,本实验D-半乳糖诱导骨质疏松症为经典模型,能真实接近反映老年人群骨质疏松特征。动物分笼饲养(3~4只/笼)于空调温室内,温度21±2℃,湿度40~60%;用颗粒饲料喂养,自由饮水。
动物接触的主要器械设备,包括动物饲养笼,代谢笼,麻醉设备和手术台。新桥动物中心均配备专业的、符合标准的设备可满足本实验要求。
2实验方法
2.1造模方法:
在大鼠饲养笼中适应性饲养,每笼3-4只,一周后动物按体重随机分为3组,每组10只,以鼠尾标记各组内大鼠,设置空白对照(N)、D-半乳糖模型组(M)、药物干预组维生素K2(MK4)。每只动物尾巴根部有特制标记,标记组别和个体标识,如空白对照组1号大鼠(N-1)。在整个实验过程中,在得知实验结果时,及时选择动物表现疼痛或痛苦的较早阶段,终止实验,人道终止动物生命,最大限度减少动物痛苦,使动物安静、快速死亡。
2.2样本收集
尿液:末次给药后代谢笼收集(9:00am-17:00pm)8h尿液,大鼠单只置入代谢笼,提供饮用水,但不提供鼠粮,记录尿液量。每只大鼠取澄清尿液10mL,置于样品管中,3000转/min离心10min,加入叠氮化钠,存放于-80℃。
血液:尿液收集后,3%戊巴比妥钠麻醉剂,腹腔注射,剂量40mg/kg麻醉大鼠,腹主动脉取血,收集尽可能多的血液,室温下,3000转/min离心10min,取血清,置于样品管,储存于-80℃冰箱中。采用手术剪,打开胸腔,取出两个股骨组织。
2.3观察指标:
(1)尿液钙、磷和肌酐,及血清组织蛋白酶K、CTX-I和RANKL
腹主动脉取血,置于抗凝剂肝素钠管中,以3000转/min离心10分钟,分离血清,冻于-80℃备用。依据试剂盒方法检测血清中钙、磷、组织蛋白酶K、CTX-I和RANKL。
(2)骨密度和骨组织形态计量学
小鼠处死后,剥离左侧股骨,剔除骨周围附属组织,用生理盐水擦洗干净,用锡箔纸包好,-80℃冻存,用于μCT检测骨组织参数。
(3)行为学和记忆学习能力
小鼠在处死前,进行旷场实验和水迷宫实验,用于评价大鼠的一般行为和学习记忆能力。
3实验结果
3.1对血清生化指标的影响
RANKL细胞因子参与破骨细胞形成、分化、融合、生存、激活、成熟的全过程,组织蛋白酶K受RANKL调控,对组织蛋白酶K的分泌和合成及活性。CTX-I为I型胶原最终降解产物,直接反映了骨胶原的降解状态。尿液中钙、磷指标为反映骨丢失的重要生化指标。
结果显示(表3),与模型组比较,尿液中尿钙/肌酐和尿磷/肌酐比显著增高(P<0.05),血清中组织蛋白酶K、RANKL和CTX-1显著增高(P<0.05),四烯甲萘醌干预后,血清组织蛋白酶K(P<0.05)、RANKL(P<0.001)和CTX-I水平显著降低(P<0.05)。说明四烯甲萘醌具有较强抑制骨吸收作用。
表3MK4对D-半乳糖骨质疏松大鼠血清生化参数的影响
3.2对骨密度和骨计量学指标影响
与正常组比较,模型组显示,股骨BV/TV比例(BV/TV%)、骨小梁数目(Tb.N)、骨连接密度(Conn.Dn)和骨小梁厚度(Tb.Th)显著降低(P<0.05),骨小梁间隙(Tb.Sp)显著增大(P<0.01)。相对于模型组,四烯甲萘醌的股骨指标,BV/TV比例显著增大(P<0.001),骨小梁数目(Tb.N)显著增多(P<0.05),骨小梁厚度增大(P<0.05),骨小梁间隙(Tb.Sp)显著减小(P<0.05),骨连接密度(Conn.Dn)显著增大(P<0.01),见表4。
表4MK4对D-半乳糖骨质疏松大鼠股骨骨计量参数的影响
本发明通过高通量筛选得到组织蛋白酶K抑制剂β-拉帕醌和维生素K2,确定β-拉帕醌能显著抑制组织蛋白酶K的胶原、弹性纤维和甲状腺球蛋白降解的生理活性。维生素K2显示了较显著的抑制组织蛋白酶K的Z-FR-MCA和胶原降解活性,与组织蛋白酶K对接得分高,作用强。本发明考察了抑制剂对破骨细胞骨吸收活性(破骨细胞生长数量和TRAP活性)的作用,结果确定β-拉帕醌和维生素K2抑制剂在体外具有较好抑制骨吸收作用。
同时,本发明采用D-半乳糖诱导老年性骨质疏松大鼠模型来考察四烯甲萘醌抗骨质疏松作用,结果四烯甲萘醌显示显著的抑制组织蛋白酶K蛋白和基因的表达,发挥显著的抗骨质疏松作用。因此,本发明经过试验证明了所述β-拉帕醌和维生素K2具有进一步开发为以组织蛋白酶K为靶标的疾病药物的潜力和应用前景。
Claims (10)
2.根据权利要求1所述的含有萘醌和菲醌结构的组织蛋白酶K抑制剂,其特征在于,所述C环为甲基苯环或二甲基环己基环。
3.根据权利要求1所述的含有萘醌和菲醌结构的组织蛋白酶K抑制剂,其特征在于,所述化合物为通过在A环或C环上取代形成的可药用的酯、酰胺和盐中的至少一种。
4.根据权利要求1所述的含有萘醌和菲醌结构的组织蛋白酶K抑制剂,其特征在于,所述组织蛋白酶K抑制剂为β-拉帕醌和维生素K2。
5.根据权利要求4所述的含有萘醌和菲醌结构的组织蛋白酶K抑制剂,其特征在于,所述维生素K2包括四烯甲萘醌和七烯甲萘醌。
6.含有权利要求1-5任一项所述的组织蛋白酶K抑制剂的组合物。
7.权利要求1所述组织蛋白酶K抑制剂在制备用于治疗以组织蛋白酶K为靶向的疾病的药物中的应用。
8.根据权利要求7所述的应用,其特征在于,所述蛋白酶K抑制剂作用于组织蛋白酶K活性位点。
9.根据权利要求7所述的应用,其特征在于,所述以组织蛋白酶K为靶向的疾病包括甲状腺疾病、心血管疾病和骨疾病。
10.根据权利要求9所述的应用,其特征在于,所述甲状腺疾病、心血管疾病和骨疾病包括为甲状腺功能亢进、骨质疏松、牙龈炎、牙周炎、类风湿性关节炎。
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