CN116549453A - STAT6磷酸化抑制剂在治疗Graves病中的应用 - Google Patents
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Abstract
本发明公开了STAT6磷酸化抑制剂在治疗Graves病中的应用。本发明通过构建Graves病甲亢小鼠模型，并在构建过程中，根据不同分组，对小鼠进行腹腔内注射STAT6磷酸化抑制剂AS1517499，并进一步进行细胞机制验证。结果提示TRAb通过促进IL‑4/STAT6通路增加钠碘转运蛋白NIS的表达，抑制STAT6磷酸化后，阻断了甲状腺素合成关键蛋白NIS的表达。最终提示STAT6磷酸化抑制剂不仅改善Graves病甲状腺细胞增生而且改善甲状腺功能。因此，本发明提出的通过抑制STAT6磷酸化对于临床上治疗Graves病提供了新的治疗药物，具有重要的临床意义。
Description
技术领域
本发明涉及STAT6磷酸化抑制剂在治疗Graves病中的应用，属于生物医药技术领域。
背景技术
弥漫性毒性甲状腺肿(Graves病)是一种器官特异性自身免疫性甲状腺疾病，好发于20-50岁女性，中国的发病率约1％。Graves病的流行病学受到碘营养状态、性别、基因易感性、吸烟、饮酒、药物等多种因素影响。Graves病发病机制至今尚未完全阐述清楚，目前认为因自身抗体促甲状腺素受体抗体(TRAb)产生，模拟促甲状腺素(TSH)作用，与促甲状腺素受体(TSHR)结合，促进甲状腺激素合成增多。甲状腺激素的合成需要钠碘转运蛋白(NIS)、甲状腺过氧化物酶(TPO)、甲状腺球蛋白(TG)等蛋白参与，其中NIS是甲状腺滤泡上皮细胞上唯一参与甲状腺素原料碘吸收的蛋白，对于甲状腺激素的合成发挥着至关重要的作用。NIS的表达受到多种因素的影响，其中TSH随着浓度的增加先促进NIS的表达，但随着浓度进一步增加，又会抑制NIS的表达。TRAb与受体结合后除了增加甲状腺激素的合成，还能促进甲状腺细胞增生，从而患者出现代谢亢进、甲状腺肿大等表现。根据患者的临床表现、甲状腺功能及甲状腺吸碘率、B超等检查报告，做出正确的诊断，才能为患者提供最佳的治疗方案。而未治疗、治疗不充分或误诊误治的甲状腺毒症与体重减轻、骨质疏松、房颤、栓塞、肌肉无力、心功能衰竭、神经精神症状、粒细胞缺乏、肝功能损害、钙磷代谢紊乱等有关，甚至可能导致甲亢危象、死亡。可以说Gravs病是一个影响人类福祉的健康问题。目前治疗Graves病的口服药物甲巯咪唑(MMI)或丙硫氧嘧啶(PTU)作用靶点是抑制甲状腺内过氧化物酶，从而阻碍吸聚到甲状腺内碘化物的氧化及酪氨酸的偶联，阻碍甲状腺素(T4)和三碘甲状腺原氨酸(T3)的合成，不能从根本上阻断免疫应答和甲状腺激素合成原料碘的摄取。此外药物治疗周期长，国外指南推荐，药物治疗疗程在18-24个月，有的患者甚至需要低剂量维持治疗5-10年。MMI和PTU在治疗过程中可导致粒细胞减少、肝功能不全、过敏甚至粒细胞缺乏和肝功能衰竭等威胁生命的副作用。总之在诊疗过程中，可能根据患者出现的多种病情变化，需要不断调整治疗方案，故Graves病存在治愈率低复发率高，部分患者病情难以控制。因此发现一种新的药物，通过调节免疫和减轻甲状腺增生、从源头上阻碍碘的摄取阻止甲状腺素合成不仅缓解甲亢病情，对难治性甲亢更是具有重大意义。
STAT6属于信号转导子和转录激活子家族成员，STAT6分子介导的活化信号可参与细胞的增殖、分化以及免疫调节等许多重要的生物学效应。AS1517499是有效的、可透过血脑屏障的STAT6磷酸化抑制剂，既往报道其可用于哮喘病的治疗，此外，公开号为CN115364223 A的专利“作用于STAT 6靶点的物质成分在制备治疗慢性瘙痒症的药物中的应用”中公开了AS1517499在治疗慢性瘙痒症中的用途，但是目前还没有关于其用于Graves病及相关疾病的医药用途。
发明内容
本发明的目的是为解决如何治疗Graves病的技术问题。
为达到解决上述问题的目的，本发明所采取的技术方案是提供STAT6磷酸化抑制剂AS1517499在制备Graves病的药物中的应用，其特征在于，所述AS1517499通过抑制甲状腺滤泡上皮细胞上参与甲状腺素合成的关键蛋白钠碘转运蛋白来改善甲状腺功能状态，所述AS1517499的CAS No.:919486-40-1，化学结构式为：
优选地，所述药物的剂型包括注射剂、片剂、粉剂、混悬剂、胶囊剂、丸剂或糖浆。
与现有技术相比，本发明的有益效果在于：
本发明通过动物实验表明AS1517499能明显改善Graves病小鼠甲状腺体重、甲状腺功能，进一步细胞机制验证中，发现AS1517499主要通过抑制甲状腺滤泡上皮细胞上参与甲状腺素合成的关键蛋白钠碘转运蛋白NIS来改善甲状腺功能状态，因此，本发明为临床上治疗Graves病提供了新的治疗药物，具有重要的临床意义，同时，本发明为AS1517499提供了新的医药用途，拓展了其临床应用方向。
附图说明
图1为重组腺病毒-TSHR289诱导的Graves病小鼠模型与对照组及与重组腺病毒-TSHR289联合AS1517499诱导的小鼠甲状腺重量、甲状腺致病性抗体TRAb及甲状腺功能对比；
图2为重组腺病毒-TSHR289诱导的Graves病小鼠模型与对照组甲状腺滤泡上皮细胞上NIS表达对比；
图3为TSH、TRAb促进甲状腺滤泡上皮细胞膜表面蛋白NIS的表达，抑制STAT6磷酸化后，TSH和TRAb对NIS的刺激作用得到抑制。
具体实施方式
为使本发明更明显易懂，兹以优选实施例，并配合附图作详细说明如下。
下述实施例中所使用的试验方法如无特殊说明，均为常规方法；所使用的材料、试剂等，如无特殊说明，为可从商业途径得到的试剂和材料。
实施例
本实施例首先予Ad-TSHR289肌肉注射BALB/c构建Graves病小鼠模型，在构建过程中，以STAT6磷酸化抑制剂AS1517499腹腔注射(注射剂量为10mg/kg/d)来研究STAT6磷酸化抑制后小鼠的疾病转归。结果表明AS1517499能明显改善Graves病小鼠甲状腺体重、甲状腺功能。进一步细胞机制验证中，发现AS1517499主要通过抑制甲状腺滤泡上皮细胞上参与甲状腺素合成的关键蛋白钠碘转运蛋白来改善甲状腺功能状态。具体实验方法和结果如下所述。
(一)实验方法
1.Graves病造模方法：
以重组腺病毒Ad-TSHR289免疫诱导Graves病小鼠模型(5-10×1010病毒颗粒溶于100μL PBS)。选取雌性6-7周BALB/c小鼠，取25μL腺病毒配置液每隔3周进行肌肉注射免疫1次，共免疫3次，后对小鼠进行处死。造模期间，观察小鼠皮毛、活动、进食，记录小鼠体重变化。根据分组，造模开始，0、21、31、42和52天腹腔内注射STAT6磷酸化抑制剂AS1517499(注射剂量为10mg/kg/d)。
2.动物实验分组方法：
第一组:对照腺病毒Ad-null(组1)，第二组：重组腺病毒Ad-TSHR289(组2)，第三组：重组腺病毒Ad-TSHR289+AS1517499。
3.AS1517499溶液配置：
实验前称取小鼠重量，计算取AS1517499所需重量，以DMSO溶解，然后加入PBS定容成小鼠所需的注射计量10mg/kg/d。在STAT6磷酸化抑制组，每只小鼠腹腔注射100μL的AS1517499。
4.腹腔注射：
左手提起并固定小鼠，使鼠腹部朝上，酒精棉球消毒酒精进针部位，右手持注射器，针头在下腹部进针，刺入皮肤3mm，接着成45°缓慢刺入腹膜腔。固定针头，保持针尖不动，回抽无血、肠液、尿液等，再注射药物。对照组注射DMSO PBS溶液。
5.动物实验终点处理方法：
造模结束后，采用CO2窒息，处死小鼠。通过眼静脉采血，留取血样本于1.5mLEP管中，4℃静置2小时后，2000rpm离心30min，留取血清。送检甲状腺功能和抗体(TT3、TT4、TRAb)。暴露小鼠颈部，轻轻分离出甲状腺，进行称重后放入多聚甲醛固定，梯度酒精脱水，石蜡包埋，后续进行免疫组化实验。
6.细胞实验：
胰酶消化制备细胞悬液，4℃离心3000rmp，5min。弃上清。流式染色缓冲液重悬细胞并进行计数1×107/mL，取出100μL进行后续流式染色。加入1μL NIS抗体，孵育30min，加入2mL流式染色缓冲液重悬细胞，离心弃上清，重复2次。加入100μL流式缓冲液和1μL流式荧光二抗进行孵育，再次进行冲洗。最后加入200μL流式染色缓冲液重悬细胞进行检测。
7.细胞实验分组方法：
实验分5组：空白组、TSH、TSH+AS1517499、TRAb、TRAb+AS1517499。其中TSH为阳性对照组，既往研究证实TSH在50mU/L能显著增加NIS的表达。
(二)实验结果
1.动物实验结果：
造模结束后检测各组TT3、TT4、TRAb水平，取各组甲状腺标本称重，石蜡包埋后HE染色。结果发现，抑制STAT6磷酸化后能显著改善Graves病甲状腺重量、甲状腺功能和TRAb水平。Graves病小鼠模型，甲状腺功能和TRAb水平明显增加，对照组小鼠TRAb低于检测值下限0.8。STAT6磷酸化抑制后，甲状腺功能和TRAb水平也有所改善(图1)。免疫组化显示，Graves病小鼠甲状腺细胞增生排列拥挤，膜表面NIS表达显著增加，提示NIS对Graves病甲状腺功能亢进起着重要的作用(图2)。
2.细胞实验结果：
和TSH一样，TRAb也可促进NIS表达，而且刺激作用比TSH作用更强。抑制STAT6磷酸后，TRAb、TSH对NIS的正调控作用均受到明显抑制。NIS是膜表面蛋白，参与甲状腺素合成原料碘的摄取，也是甲状腺滤泡上皮上唯一参与碘摄取的蛋白。我们看到，TRAb、TSH刺激后，甲状腺滤泡上皮细胞膜上NIS表达增加，抑制STAT6磷酸化后，NIS显著下降(图3)。可见，AS1517499作用于NIS，减少NIS的表达，从而起到治疗作用，是一种潜在的Graves病治疗药物。
上述实施例仅为本发明的优选实施例，并非对本发明任何形式上和实质上的限制，应当指出，对于本技术领域的普通技术人员，在不脱离本发明的前提下，还将可以做出若干改进和补充，这些改进和补充也应视为本发明的保护范围。
Claims (2)
1.STAT6磷酸化抑制剂AS1517499在制备Graves病的药物中的应用，其特征在于，所述AS1517499通过抑制甲状腺滤泡上皮细胞上参与甲状腺素合成的关键蛋白钠碘转运蛋白来改善甲状腺功能状态，所述AS1517499的CASNo.:919486-40-1，化学结构式为：
2.如权利要求1所述的应用，所述药物的剂型包括注射剂、片剂、粉剂、混悬剂、胶囊剂、丸剂或糖浆。
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| WO2013103783A1 (en) * | 2012-01-04 | 2013-07-11 | Sanofi Us | Murine il-13 antibodies |
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