CN115487190A - 丙酮酸激酶m2激活剂在制备治疗系统性红斑狼疮的药物中的应用 - Google Patents
丙酮酸激酶m2激活剂在制备治疗系统性红斑狼疮的药物中的应用 Download PDFInfo
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Abstract
本发明公开了丙酮酸激酶M2激活剂在制备治疗系统性红斑狼疮的药物中的应用，特别是丙酮酸激酶M2激活剂TEPP‑46作为唯一活性成分用于制备治疗系统性红斑狼疮的药物，TEPP‑46的CAS号为1221186‑53‑3，分子式为C17H16N4O2S2，结构式为
本发明通过动物实验将TEPP‑46以50mg/kg剂量用于治疗MRL/lpr狼疮小鼠，治疗后症状减轻，得到有效缓解，证实药物靶向丙酮酸激酶M2可作为一种控制T细胞介导的炎症和自身免疫的有效方法，提示TEPP‑46可用于治疗系统性红斑狼疮方面。
Description
技术领域
本发明属于生物医药领域，具体涉及丙酮酸激酶M2激活剂在制备治疗系统性红斑狼疮的药物中的应用。
背景技术
系统性红斑狼疮(SLE)是一种自身免疫性疾病，其特点是免疫系统过度激活，累及肾、脑等多种器官，是多种遗传和环境因素复杂相互作用的结果。既往研究指出，大约70％的患者遵循复发-缓解过程，其余患者平均分为长期缓解和持续活动性疾病。尽管治疗取得了进步，但由于免疫抑制治疗导致疾病活动或感染控制不佳，SLE患者的标准化死亡率比一般人群高出三倍。以高热量、高盐和过量糖摄入为特征的营养模式与代谢和炎症性疾病相关，被认为是自身免疫的危险因素，过量消耗葡萄糖或会直接促进免疫系统中特定细胞的致病性功能，反之，热量减少的饮食或会对机体的免疫性疾病具有一定的有益效应。
丙酮酸激酶(PK)在糖酵解过程中催化磷酸烯醇式丙酮酸(PEP)转化为丙酮酸，在糖酵解中起着至关重要的作用。在哺乳动物中，鉴定出四种不同的PK同工型，但免疫细胞优先表达同工型丙酮酸激酶M1(PKM1)和丙酮酸激酶M2(PKM2)，PKM1作为四聚体蛋白存在于细胞中，它具有高酶活性，可有效地将PEP转化为丙酮酸，并且通常在终末分化组织中表达；PKM2主要以单体/二聚体形式存在于具有合成代谢功能的组织中，包括增殖细胞和癌细胞，并受到复杂的变构调节以控制其酶活性。单体/二聚体PKM2的酶活性低于四聚体异构体，它的表达对于增殖细胞将糖酵解中间体转移到诸如磷酸戊糖途径等途径以合成细胞活化和增殖所必需的核苷酸至关重要。
TEPP-46(ML-265)是一种有效选择性PKM2激活剂，它诱导四聚体PKM2，阻断PKM2的核转位，影响体外和体内T细胞的激活和致病性。CD4+T细胞代表着一种免疫细胞群，对于清除机体内的病原体和长期免疫来说必不可少，研究表明，针对PK的活性特别是其同种异形体PKM2的活性，可以限制CD4+T细胞的过度激活，防止自身免疫的发展。SLE患者的T细胞异常如表型和功能改变与SLE发病机制紧密相关，如Tfh细胞不受控制的扩增。研究发现SLE患者和lupus小鼠中发现慢性活性T细胞糖酵解率增加，这导致mTORC1信号增加并促进自身免疫。研究表明，TEPP-46限制Th17和Th1细胞在体外的发展，并抑制实验性自身免疫性脑脊髓炎(EAE)的发展，但是尚没有其用于治疗SLE的应用报道。
发明内容
本发明提供丙酮酸激酶M2(PKM2)激活剂在制备治疗自身免疫性疾病的药物中的应用，所述自身免疫性疾病包括但不限于系统性红斑狼疮、皮肌炎、硬皮病和银屑病。
作为优选，所述PKM2激活剂为TEPP-46，其CAS号为1221186-53-3，分子式为C17H16N4O2S2，结构式为
作为优选，所述药物以所述PKM2激活剂为唯一活性成分，还包括药学上可接受的辅料。
作为优选，所述药物剂型选自注射剂、输液剂、散剂、片剂、胶囊剂或膏剂。
作为优选，所述药物剂型为注射剂，采用腹腔注射方式给药，给药剂量为1-100mg/kg。
作为更优选，所述药物的给药剂量为50mg/kg。
本发明还提供一种用于治疗自身免疫性疾病的药物组合物，包含丙酮酸激酶M2激活剂TEPP-46且以其作为唯一活性成分，TEPP-46的CAS号为1221186-53-3，分子式为C17H16N4O2S2，结构式为
其中所述自身免疫性疾病包括但不限于系统性红斑狼疮、皮肌炎、硬皮病和银屑病。
本发明通过动物实验将PKM2激活剂TEPP-46以50mg/kg剂量用于治疗MRL/lpr狼疮小鼠，在TEPP-46 50mg/kg药物治疗后症状减轻，得到有效缓解，证实药物靶向PKM2可作为一种控制T细胞介导的炎症和自身免疫的有效方法，提示TEPP-46在治疗系统性红斑狼疮制药应用潜能。
附图说明
图1是实施例中RL/MPJ鼠、MRL/lpr对照鼠和MRL/lpr药物干预鼠在治疗14天后体重、面部和背部皮肤、脾脏、淋巴结的变化；A：面部和背部典型皮损；B：脾脏和淋巴结；C：面部和皮肤和背部皮肤HE染色(100X)；D：为面部皮损面积统计图，三独立样本均数间的比较采用Ordinary one-way ANOVA，****表示P＜0.0001；E：脾脏指数统计图，三独立样本均数间的比较采用Ordinary one-way ANOVA，****表示P＜0.0001；F：体重变化统计图，三独立样本均数间的比较采用Ordinary one-way ANOVA，**表示P＜0.01。
图2是实施例中MRL/MPJ鼠、MRL/lpr对照鼠和MRL/lpr药物干预鼠在治疗14天后肾脏免疫荧光IgG沉积、免疫组化HE染色和PAS染色、尿蛋白和尿肌酐、血清抗dsDNA抗体、ANA抗体水平变化；A：肾小球免疫荧光染色IgG沉积情况(630X)；B：肾脏HE染色和PAS染色(400X)；C：IgG平均荧光强度统计图，三独立样本均数间的比较采用Ordinary one-wayANOVA，****表示P＜0.0001；D：肾小球评分统计图，三独立样本均数间的比较采用Ordinaryone-way ANOVA，**表示P＜0.01；E：24小时尿蛋白和尿肌酐统计图，三独立样本均数间的比较采用Ordinary one-way ANOVA，***表示P＜0.001，**表示P＜0.01；F：血清抗dsDNA抗体、ANA抗体水平统计图，****表示P＜0.0001。
图3是实施例中MRL/MPJ鼠、MRL/lpr对照鼠和MRL/lpr药物干预鼠在治疗14天脾脏和淋巴结内CD4+CXCR5+Pd1+Tfh细胞、CD4+Foxp3+T细胞和CD4-IFNγ细胞变化；A：脾脏内CD4+CXCR5+Pd1+Tfh细胞、CD4+Foxp3+T细胞和CD4-IFNγ细胞比例；B：淋巴结内CD4+CXCR5+Pd1+Tfh细胞、CD4+Foxp3+T细胞和CD4-IFNγ细胞比例；C：脾脏内CD4+CXCR5+Pd1+Tfh细胞、CD4+Foxp3+T细胞和CD4-IFNγ细胞比例统计图，三独立样本均数间的比较采用Ordinaryone-way ANOVA，**表示P＜0.01，****表示P＜0.0001；D：淋巴结内CD4+CXCR5+Pd1+Tfh细胞、CD4+Foxp3+T细胞和CD4-IFNγ细胞比例统计图，三独立样本均数间的比较采用Ordinary one-way ANOVA，****表示P＜0.0001。
具体实施方式
下面结合具体实施例进一步阐述本发明，这些实施例仅用于说明本发明而不用于限制本发明的范围。
以下实施例中以自身免疫性疾病系统性红斑狼疮为例构建动物模型，验证PKM2激活剂TEPP-46在治疗系统性红斑狼疮的效果。
实施例1动物实验
选取11周龄的MRL/MPJ小鼠4只，MRL/lpr小鼠8只随机分成对照组4只和TEPP-46(50mg/kg)干预组4只，从第0天到第14天每隔一天对小鼠进行一次治疗。14天后，收集小鼠24小时尿进行尿蛋白和尿肌酐检测；收集小鼠面部和背部皮肤进行组化HE染色；收集小鼠血清进行ds-DNA和ANA检测；收集小鼠肾脏进行组化HE和PSA检测，进行免疫荧光IgG检测；收集小鼠脾脏进行称重，提取脾脏和淋巴结细胞进行流式检测，具体如下：
MRL/MPJ、MRL/lpr小鼠购于上海吉辉实验动物饲养有限公司，于复旦大学上海医学院动物实验中心SPF级动物房常规饲养，如表1所示。
表1
| 分组 | 小鼠情况 | 注射药物 |
| 小鼠对照组 | MRL/MPJ雌4只11周龄 | 5％DMSO+40％PEG300+5％Tween80 |
| 溶剂对照组 | MRL/lpr雌4只11周龄 | 5％DMSO+40％PEG300+5％Tween80 |
| 药物干预组 | MRL/lpr雌4只11周龄 | 5％DMSO+40％PEG300+5％Tween80+TEPP-46 |
TEPP-46购买于Selleck，药物注射浓度为50mg/kg；注射方式：腹腔注射。
动物饲养及给药：12只鼠按照分组情况饲养在3个笼位，于第0、2、4、6、8、10、12、14日记录体重，按照50mg/kg的浓度用5％DMSO+40％PEG300+5％Tween80配制TEPP-46，于第1、3、5、7、9、11、13日对4只MRL/MPJ小鼠和4只注射5％DMSO+40％PEG300+5％Tween80作为对照组，4只MRL/lpr小鼠注射TEPP-46药物。
药物注射结束后，12只鼠使用代谢笼收集24小时蛋白尿。
尿蛋白检测：尿蛋白定量测试盒(南京建成生物工程研究所)
尿肌酐检测：肌酐测定试剂盒(南京建成生物工程研究所)
记录小鼠的体重，分别眼球取血，将眼球血收集到1.5mL离心管内室温静置2小时后，3000rpm离心10分钟，收集血清。
血清抗dsDNA抗体检测：Mouse Anti-dsDNA IgG ELISA Kit(Alpha DiagnosticIntl)
ANA抗体检测：Mouse Anti-Nuclear Antibodies(ANA)Total Ig ELISA Kit(Alpha Diagnostic Intl)
取小鼠的脾脏进行称重，研磨，裂红，按2.0×106个/mL浓度将细胞用含15％FBS的1640培养基进行培养，加入eBioscience Cell Stimulation Cocktail Plus ProteinTransport Inhibitors，置于37℃、5％ CO2的培养箱中培养，6小时后取出细胞进行固定破膜，流式染色，并使用流式细胞仪BD Aria III检测。
取小鼠的淋巴结，研磨，裂红，按2.0×106个/mL浓度将细胞用含15％FBS的1640培养基进行培养，加入eBioscience Cell Stimulation Cocktail Plus Protein TransportInhibitors，置于37℃，5％ CO2的培养箱中培养，6小时后取出细胞进行固定破膜，流式染色，并使用流式细胞仪BD Aria III检测。
取小鼠的面部和背部皮肤，左肾进行免疫组化染色和免疫荧光染色。
如图1所示，和对照组相比，TEPP-46 50mg/kg药物治疗组的面部和背部皮损减轻，从免疫组化结果来看，狼疮鼠面部的毛囊汗腺附属器结构破坏，角质层松散；背部的皮肤出现颗粒层增厚，角质层破坏。小鼠体重减轻，可以代表水肿减轻；脾脏指数降低，淋巴结数量和体积减少。
如图2所示，和对照组相比，TEPP-46 50mg/kg药物治疗组肾小球IgG沉积减少，免疫组化结果显示狼疮鼠的肾小球增多变大，炎性细胞浸润，治疗后炎症损伤缓解。小鼠尿蛋白和肌酐减少，血清抗dsDNA抗体、ANA抗体水平降低。
如图3所示，和对照组相比，TEPP-46 50mg/kg药物治疗组脾脏和淋巴结内CD4+CXCR5+Pd1+Tfh细胞比例降低、CD4+Foxp3+T细胞比例升高，CD4-IFNγ细胞降低。
总体来看，狼疮小鼠在TEPP-46 50mg/kg药物治疗后症状减轻，得到有效缓解。药物靶向PKM2可能代表一种控制T细胞介导的炎症和自身免疫的有效方法，提示TEPP-46在治疗系统性红斑狼疮制药应用的巨大潜能。
上述对实施例的描述是为了便于该技术领域的普通技术人员能理解和使用本发明。熟悉本领域技术人员显然可以容易的对这些实施例做出各种修改，并把在此说明的一般原理应用到其他实施例中，而不必经过创造性的劳动。因此，本发明不限于上述实施例，本领域技术人员根据本发明的原理，不脱离本发明的范畴所做出的改进和修改都应该在本发明的保护范围之内。
Claims (7)
1.丙酮酸激酶M2激活剂在制备治疗自身免疫性疾病的药物中的应用，所述自身免疫性疾病包括但不限于系统性红斑狼疮、皮肌炎、硬皮病和银屑病。
2.根据权利要求1所述的应用，其特征在于，所述丙酮酸激酶M2激活剂为TEPP-46，其CAS号为1221186-53-3，分子式为C17H16N4O2S2，结构式为
3.根据权利要求1或2所述的应用，其特征在于，所述药物以所述丙酮酸激酶M2激活剂作为唯一活性成分，或者还包括药学上可接受的辅料。
4.根据权利要求1或2所述的应用，其特征在于，所述药物剂型选自注射剂、输液剂、散剂、片剂、胶囊剂或膏剂。
5.根据权利要求4所述的应用，其特征在于，所述药物剂型为注射剂，采用腹腔注射方式给药，给药剂量为1-100mg/kg。
6.根据权利要求5所述的应用，其特征在于，所述药物的给药剂量为50mg/kg。
7.一种用于治疗自身免疫性疾病的药物组合物，包含丙酮酸激酶M2激活剂TEPP-46且以其作为唯一活性成分，TEPP-46的CAS号为1221186-53-3，分子式为C17H16N4O2S2，结构式为
其中所述自身免疫性疾病包括但不限于系统性红斑狼疮、皮肌炎、硬皮病和银屑病。
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| US20110046083A1 (en) * | 2007-08-16 | 2011-02-24 | Beth Israel Deaconess Medical Center | Activators of pyruvate kinase m2 and methods of treating disease |
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| US20220127267A1 (en) * | 2019-02-13 | 2022-04-28 | Agios Pharmaceuticals, Inc. | Thieno[3,2-b] pyrrole[3,2-d]pyridazinone derivatives and their use as pkm2 derivatives for the treatment of cancer, obesity and diabetes related disorders |
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