CN108938638A - Zinc62678696在制备抑制肝纤维化药物的用途 - Google Patents
Zinc62678696在制备抑制肝纤维化药物的用途 Download PDFInfo
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- A—HUMAN NECESSITIES
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- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
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Abstract
本发明公开了ZINC62678696在制备抑制肝纤维化药物的用途。本发明提供了能够降低GPR65活性的物质——抑制剂ZINC62678696在制备能够治疗肝纤维化的产品中的应用。本发明通过研究肝纤维化过程中GPR65的作用及机制，发现抑制巨噬细胞中GPR65的活性能够减轻肝纤维化的病情进展。本发明对于肝纤维化的发病机制的研究以及未来治疗策略的选择具有重要参考意义。
Description
技术领域
本发明涉及靶向GPR65的抑制剂ZINC62678696用于制备抑制肝纤维化药物的用途。
背景技术
肝纤维化是多种慢性肝脏疾病向肝硬化发展过程中的必经阶段，是各种病因如病毒性肝炎、酗酒、寄生虫、自身免疫性疾病和营养不良等导致炎症、坏死等组织损伤的修复反应。若肝纤维化进程得不到阻抑或逆转则可能进展为失代偿期肝硬化，并出现各种终末期肝病并发症，严重影响患者生存质量和预后。相关研究报道肝硬化影响着全球1％到2％的人口，并导致全球每年超过100万人死亡，而我国是全球肝纤维化及肝硬化发病率、死亡率最高的国家之一，每年有超过10万人死于肝硬化。但遗憾的是，目前我们对于肝纤维这个世界性的医疗堡垒的干预依旧缺乏力量，寻找能够有效阻止或者逆转肝纤维化的治疗方法已经成为当务之急。
G蛋白偶联受体(GPCRs)，又称七个α螺旋跨膜蛋白受体(7TM receptors)，是目前已知最大的一类细胞表面受体超家族，占人类基因组编码的蛋白质总数的2％左右。它们可以被细胞外包括激素、细胞因子、脂类、神经递质、气味、光线、离子等在内的各种信号激活，并将这些信息传送到细胞内，进而调控一系列的生理进程。由于其数量庞大、种类众多且分布非常广泛，因此GPCRs与其介导的信号通路的改变会严重影响机体的生命活动。GPCRs与糖尿病、肥胖症、心血管疾病、癌症以及炎症有着重大的联系，这也使得GPCRs成为近几十年来世界各国公认的极具吸引力的药物治疗靶点。据统计，以GPCRs为靶点的药物占整个FDA批准药物总数的34％左右，且大部分主要的药物公司正在开展基于GPCRs的创新药物研发项目。GPCRs中除了趋化因子受体如CCR2、CCR5、CXCR9等在肝纤维化发生、发展和修复过程中研究较多外，其他的GPCRs，特别是孤儿GPCRs在肝纤维化中的研究并不是非常多，因此，进一步寻找在肝纤维化发生过程中起重要作用的GPCRs并探明其作用机理可以为肝纤维化的治疗提供新靶标。
GPCRs中包括T细胞死亡偶联基因8(TDAG8，又名GPR65)、卵巢癌G蛋白偶联受体1(OGR1，又名GPR68)、GPR4和诱导细胞停滞于G2/M期的G蛋白偶联受体G2A(又名GPR132)等能够感知细胞外质子改变，统称为OGR1亚家族受体。其中，GPR65最早被发现在免疫系统中高表达，并影响胸腺细胞凋亡。GPR65参与了多种疾病的发生，包括肿瘤、骨质疏松、哮喘、结肠炎等，且GPR65在肿瘤中既能发挥促肿瘤作用，又能发挥抑制肿瘤的作用。研究发现，过表达GPR65能够促进非小细胞肺癌细胞的增殖以及增强肿瘤细胞对酸性环境的抵抗力，而敲低GPR65可逆转此类表型，体内实验结果也进一步验证了GPR65有促进肿瘤生长的作用。目前已在结肠癌、乳腺癌等多种恶性肿瘤中发现GPR65高表达，而在恶性血液病中GPR65的表达量显著减少。研究表达GPR65在恶性血液病中发挥肿瘤抑制特性主要是通过抑制癌基因c-Myc的表达。此外，GPR65还被报道参与MMPs的表达调节。然而，有关GPR65在肝纤维化过程中的表达、功能及作用机制尚未有报道。
既往研究表明GPR65在被细胞外H+激活后引起细胞内腺苷酸环化酶(cAMP)的蓄积。Im等曾报道神经鞘氨醇半乳糖苷(Psychosine，PSY)可以激活GPR65，然而他们并没有明确的证据证明PSY和GPR65特异性结合。而且后续的多项研究表明GPR65并不参与PSY引起的多核细胞形成。为了寻找到GPR65更加特异性的配体，Oda等通过文库筛选发现一种名为“BTB09089”的化合物是GPR65的特异性激动剂。Roth及同事最近的研究利用基于酵母的筛选方法在310万个分子中进一步确定“BTB09089”化合物是GPR65的一种强效激动剂，而且识别出名为“ZINC62678696”的一种新化合物是GPR65的一种强效特异性抑制剂。然而，这种新化合物对肝脏疾病特别是肝纤维化中的作用还未有报道。
发明内容
为了解决现有技术中的问题，本发明提供ZINC62678696在制备抑制肝纤维化药物的用途，解决现有技术中没有特效药物有效阻止或者逆转肝纤维化的问题。
本发明的技术方案是：ZINC62678696在制备抑制肝纤维化药物的用途，其活性成分为ZINC62678696，加入药学可接收的辅料按常规方法制成各种剂型，所述ZINC62678696化学式为：
本发明的有益效果是：针对GPR65的抑制剂ZINC62678696体内外实验均能显著抑制小鼠肝纤维化的进展；而GPCRs成为近几十年来世界各国公认的极具吸引力的药物治疗靶点。因此，ZINC62678696在制备抑制肝纤维化药物上具有良好的前景。
附图说明
图1：基因芯片技术筛选出GPR65在纤维化肝组织中高表达；(A)基因芯片检测对照小鼠与肝纤维化小鼠mRNAs的热点图；(B)qRT-PCR技术在扩大样本的正常和纤维化Balb/c小鼠肝组织中验证mRNAs的差异，*p<0.05；
图2：GPR65在小鼠纤维化的肝组织中表达增多；(A)腹腔注射CCl4后第0、2、4、6、8、10周后提取肝组织的总RNA，qRT-PCR检测α-SMA和GPR65的表达变化；(B)胆管结扎(BDL)后第0天、第3天、第14天及第21天提取肝组织的总RNA，qRT-PCR检测α-SMA和GPR65的表达改变，*p<0.05；
图3：GPR65在纤维化的肝组织中表达增多；免疫组织化学染色(IHC)检测正常人和肝硬化患者肝组织中以及正常小鼠和纤维化小鼠的肝组织中GPR65的表达和定位；
图4：GPR65的表达分析；(A)从正常小鼠肝脏中分离原代肝星状细胞(HSCs)、肝细胞(HCs)和肝巨噬细胞(HMs)，qRT-PCR技术检测GPR65的表达水平；(B)分别提取正常小鼠及肝纤维化小鼠的肝巨噬细胞，qRT-PCR检测IL-1β、TNF-α、MCP1和GPR65的表达水平，*p<0.05；
图5：体外实验证实抑制GPR65下调促炎症基因及M1型巨噬细胞marker基因表达；(A)从小鼠骨髓中提取单核细胞，用100ng/ml M-CSF培养7天后添加30μM GPR65特异性抑制剂ZINC62678696处理24小时，随后qPCR技术检测IL-1β、CXCL5、CCR2、iNOS和LY6C的表达变化；(B)30μM GPR65特异性抑制剂ZINC62678696处理RAW264.7细胞24小时后，qPCR技术检测IL-1β、CXCL5、CCR2、iNOS和LY6C的表达变化，*p<0.05；
图6：体内实验证实抑制GPR65缓解CCl4诱导的小鼠肝纤维化；(A)四组小鼠(腹腔注射橄榄油+注射生理盐水组；腹腔注射CCl4+注射生理盐水组；腹腔注射橄榄油+注射GPR65抑制剂组；腹腔注射CCl4+注射GPR65抑制剂组)肝组织羟脯氨酸含量测定；(B)四组小鼠的肝组织切片HE染色、天狼猩红染色及Masson染色的比较，*/#p<0.05，*p<0.05vs对照.#p<0.05vs对照+CCl4。
具体实施方式
实施例1
ZINC62678696加入药学可接收的辅料按常规方法制成各种剂型，如各种规格的液体注射剂，粉针注射剂，注射用乳剂，片剂，丸剂，胶囊剂，膏剂，霜剂，贴剂，擦剂，粉剂，喷雾剂，植入剂，滴剂，栓剂，软膏剂，糖果剂等。
ZINC62678696的给药途径包括各种给药途径：口服给药，注射给药，植入给药，腔内给药，舌下给药，肛门给药，透皮给药，内外敷等。
实验例1：基因芯片筛选与验证
通过基因芯片对纤维化肝组织与正常肝组织中的差异mRNAs进行筛选。该芯片是目前最强大的全转录组研究工具，设计有550万个探针，其中基本包括所有已知的经典转录本以及预测出的所有可能基因转录形式。此外该芯片针对每条序列都设计了多条探针，增加了信号的可靠度。我们提取5个纤维化肝组织和5个正常肝组织的总RNA，质检合格后通过高通量芯片筛选，并按照纤维化肝组织与正常肝组织表达差异在1.6倍以上并且P值小于等于0.05的标准，发现在纤维化肝组织与正常肝组织差异表达的mRNAs有1526个，其中在纤维化肝组织中过表达的mRNAs有1007个，下调表达的mRNAs是有519个。在扩大样本的正常和纤维化Balb/c小鼠肝组织中验证芯片筛选的差异GPCRs的表达，发现GPR65在纤维化的肝组织中高表达。
如图1所示：(A)基因芯片检测对照小鼠与肝纤维化小鼠mRNAs的热点图；结果证实在纤维化肝组织与正常肝组织差异表达的mRNAs有1526个；(B)qRT-PCR技术在扩大样本的正常和纤维化Balb/c小鼠肝组织中验证mRNAs的差异，证实芯片结果准确可靠，GPR65在纤维化的肝组织中高表达。*p<0.05。
实验例2：构建CCl4及胆管结扎(BDL)诱导的小鼠肝纤维化模型
CCl4诱导的小鼠肝纤维化模型：购买36只balb/c雄性小鼠，正常喂食一周后，随机平均分为6组，每组6只：对照组(注射橄榄油)和CCl4 2、4、6、8、10周组。CCl4组小鼠为腹腔注射0.3ml/kg CCl4(5％，v/v,溶于橄榄油；)，共8周每周两次。处理8周后利用水合氯醛麻醉处死对照组和CCl4组小鼠，记录肝脏外观变化及拍照，并取肝组织分别固定以及冻存用于后续试验。
胆管结扎(BDL)诱导的小鼠肝纤维化模型：
(1)购买24只balb/c雄性小鼠正常喂食一周后，随机平均分为四组，每组6只：对照组(只开腹缝合不进行手术)和BDL(胆管结扎)3、14、21天组。
(2)手术前一晚禁止小鼠饮水吃食，手术必须在无菌间进行，术后恢复期注意止痛、保暖、无菌。所有手术器材均需高压灭菌后使用。
(3)备皮后腹腔注射水合氯醛麻醉；
(4)开腹：手术部位铺一张洞巾暴露腹部，依次剪开皮肤和筋膜(开口约2cm)，(手术剪11.5cm)后放入扩胸器，沿腹中线剪开腹膜，然后将缝合线穿入胸骨适当拉升并固定；
(5)往腹腔滴加生理盐水，用湿棉签轻移肠道器官以暴露胆总管；用弯头镊将胆总管完全游离；
(6)用两道缝合线结扎胆总管，各打三个结并剪断多余长度；轻轻撤掉扩胸器；
(7)向腹腔中滴加生理盐水并使器官回位，依次缝合腹腔及表皮，间隔适宜；
(8)将小鼠转移至温暖的环境使其自然清醒，腹部每天用碘伏进行消毒，密切观察小鼠状态；
(9)21d后注射过量水合氯醛麻醉处死小鼠，记录肝脏外观变化及拍照，取肝组织分别固定以及冻存用于后续试验。
如图2所示：GPR65在纤维化的肝组织中表达增多。
(A)腹腔注射CCl4后第0、2、4、6、8、10周后提取肝组织的总RNA，qRT-PCR检测α-SMA和GPR65的表达变化；结果证实α-SMA和GPR65在纤维化的肝组织中表达量逐渐增多；(B)胆管结扎(BDL)后第0天、第3天、第14天及第21天提取肝组织的总RNA，qRT-PCR检测α-SMA和GPR65的表达改变；结果证实α-SMA和GPR65在纤维化的肝组织中表达量逐渐增多。*p<0.05。
实验例3：免疫组化染色步骤
(1)烤片、常规脱蜡、再水合；
(2)1×PBS洗3遍，每次5分钟；
(3)抗原修复：将需要修复的玻片放入抗原修复液中，然后置于高压锅，从常温升至108℃后保持5分钟(不同的抗体修复时间不尽相同)；
(4)取出后自然冷却至室温，倾倒掉修复液；
(5)1×PBS洗3遍，每次5分钟；
(6)H2O2封闭：首先用1×PBS将30％的H2O2稀释至3％，然后用100μl的移液枪滴加到擦去周围水的组织块上，湿盒中避光静置15分钟；
(7)1×PBS洗3遍，每次5分钟；
(8)山羊血清封闭：擦去组织块周围的水，然后将玻片放入湿盒中，用100μl的移液枪滴加山羊血清(1×PBS稀释10倍)室温15分钟(不同抗体封闭时间不尽相同)；
(9)滴加一抗GPR65：弃山羊血清，将组织块周围擦干(否则不能形成表面张力，一抗容易流到一旁，导致干片)，并标记抗体的名称，然后置于湿盒中，100μl的移液枪滴加相应的抗体，4℃冰箱避光过夜(约16小时)；
(10)从4℃冰箱取出湿盒，室温复温15分钟；
(11)将一抗回收到标记(抗体名称、稀释比例、回收日期)好的EP管中；
(12)1×PBS洗3遍，每次5分钟；
(13)滴加二抗：用100μl的移液枪滴加酶标二抗(无菌1×PBS稀释，1:200)，置于湿盒中37℃恒温避光孵育1小时；
(14)1×PBS洗3遍，每次5分钟；
(15)DAB显色：首先取1ml的A液并滴加2滴B液配制成DAB显色液(现配现用)，然后用100μl的移液枪滴加至完全覆盖组织块，显微镜下控制显色时间，蒸馏水终止；
(16)复染：擦干玻片周围的水，置于装有苏木精的染色缸中，染色2分钟；
(17)返蓝：使流水从玻片的一侧缓慢冲洗掉染液，并在自来水中返蓝20分钟；(18)脱水、透明、封片；
(19)待自然风干后置于电子显微镜下观察；
(20)注意以上操作步骤严禁组织块风干。
如图3所示，GPR65在纤维化的肝组织中表达增多
免疫组织化学染色(IHC)检测正常人和肝硬化患者肝组织中以及正常小鼠和纤维化小鼠的肝组织中GPR65的表达和定位；结果证实GPR65在纤维化或硬化的肝组织中高表达，且主要定位在肝巨噬细胞中。
实验例4：GPR65抑制剂ZINC62678696的体外实验：
如图4所示，GPR65的表达分析
(A)从正常小鼠肝脏中分离原代肝星状细胞(HSCs)、肝细胞(HCs)和肝巨噬细胞(HMs)，qRT-PCR技术检测GPR65的表达水平；结果证实GPR65主要存在于肝脏巨噬细胞中；(B)分别提取正常小鼠及肝纤维化小鼠的肝巨噬细胞，qRT-PCR检测IL-1β、TNF-α、MCP1和GPR65的表达水平；结果证实GPR65在纤维化的巨噬细胞中高表达。*p<0.05。
如图5所示，体外实验证实抑制GPR65下调促炎症基因及M1型巨噬细胞marker基因表达
(A)从小鼠骨髓中提取单核细胞，用100ng/ml M-CSF培养7天后添加30μM GPR65特异性抑制剂ZINC62678696处理24小时，随后qPCR技术检测IL-1β、CXCL5、CCR2、iNOS和LY6C的表达变化；结果证实抑制GPR65显著下调IL-1β、CXCL5、CCR2、iNOS和LY6C的表达水平；(B)30μM GPR65特异性抑制剂ZINC62678696处理RAW264.7细胞24小时后，qPCR技术检测IL-1β、CXCL5、CCR2、iNOS和LY6C的表达变化；结果证实抑制GPR65显著下调IL-1β、CXCL5、CCR2、iNOS和LY6C的表达水平。*p<0.05。
实验例5：GPR65抑制剂ZINC62678696的体内实验：
40只6周龄SPF级雄性Balb/c小鼠正常饲喂一周后，随机平均分为四组(n＝10)：对照组、对照+CCl4组、GPR65抑制剂ZINC62678696组和GPR65抑制剂ZINC62678696+CCl4组。对照组为腹腔注射橄榄油，同时腹腔注射生理盐水；对照+CCl4组为腹腔注射5％CCl4(v/v，溶于橄榄油；0.3ml/kg，每周2次)，同时腹腔注射生理盐水；GPR65抑制剂组为腹腔注射橄榄油，同时腹腔注射GPR65抑制剂ZINC62678696；GPR65抑制剂ZINC62678696+CCl4组为腹腔注射5％CCl4，同时腹腔注射GPR65抑制剂ZINC62678696。CCl4注射4周后，各小鼠开始通过腹腔注射GPR65抑制剂ZINC62678696，抑制剂ZINC62678696的注射量为10mg/kg，每2天一次；抑制剂ZINC62678696用DMSO溶解成30mM，随后缓慢溶解至橄榄油中，注射体积一般不超过200μl。抑制剂ZINC62678696开始注射后CCl4再腹腔注射4周，最后一次注射48小时后所有小鼠麻醉处死。如图6所示，体内实验证实抑制GPR65缓解CCl4诱导的小鼠肝纤维化(A)四组小鼠(腹腔注射橄榄油+注射生理盐水组；腹腔注射CCl4+注射生理盐水组；腹腔注射橄榄油+注射GPR65抑制剂组；腹腔注射CCl4+注射GPR65抑制剂组)肝组织羟脯氨酸含量测定，结果证实对照+CCl4组的肝组织羟脯氨酸含量显著高于CCl4+注射GPR65抑制剂组；(B)四组小鼠的肝组织切片HE染色、天狼猩红染色及Masson染色的比较，结果证实抑制GPR65显著抑制肝纤维化的进展。*/#p<0.05。*p<0.05vs对照.#p<0.05vs对照+CCl4。
综合以上全部结果，可见：对巨噬细胞靶向使用GPR65的抑制剂来控制肝纤维化的病情进展会成为一种可能。抑制巨噬细胞中GPR65的活性能够延缓肝纤维化的病情进展(用于各种慢性肝病特别是肝纤维化的治疗)。
Claims (1)
1.靶向GPR65的抑制剂ZINC62678696在制备抑制肝纤维化药物的用途，其特征在于，其活性成分为ZINC62678696，加入药学可接收的辅料按常规方法制成各种剂型，所述ZINC62678696化学式为：
。
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| WO2022136844A1 (en) * | 2020-12-22 | 2022-06-30 | Pathios Therapeutics Limited | N-(pyridin-2-yl)-6,7,8,9-tetrahydro-5h-5,8-epiminocyclohepta[c]pyridine-10-carboxamide derivatives and similar compounds as gpr65 modulators for the treatment of cancer |
| WO2022229615A1 (en) * | 2021-04-26 | 2022-11-03 | Pathios Therapeutics Limited | Compounds |
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| CN104095832A (zh) * | 2014-07-08 | 2014-10-15 | 华侨大学 | 一种防治肝纤维化药物及其制备方法 |
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| WO2022136844A1 (en) * | 2020-12-22 | 2022-06-30 | Pathios Therapeutics Limited | N-(pyridin-2-yl)-6,7,8,9-tetrahydro-5h-5,8-epiminocyclohepta[c]pyridine-10-carboxamide derivatives and similar compounds as gpr65 modulators for the treatment of cancer |
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