CN114105916B - 一种RyR2稳定剂及其制备方法、相应的药用组合物和应用 - Google Patents
一种RyR2稳定剂及其制备方法、相应的药用组合物和应用 Download PDFInfo
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Abstract
本发明提供了一种RyR2稳定剂及其制备方法、相应的药用组合物和应用,涉及化学药物技术领域,所述的RyR2稳定剂,为FornicinA或其药学上的盐。FornicinA能够作为RyR2稳定剂,通过与兰尼碱受体2(RyR2)相互作用,抑制心肌细胞钙漏流,进而能够治疗罕见病CPVT。尤其是(‑)‑FornicinA,对RyR2‑R2474S转基因小鼠原代心肌细胞钙火花释放具有抑制作用,且具有剂量依赖关系;能够减少RyR2‑R2474S转基因小鼠原代心肌细胞的起搏后起搏后钙释放异常事件;对WT小鼠的心电图并无影响;预防性给药能够显著降低室速的发作率;治疗性给药能够在30min内恢复窦性心律。
Description
技术领域
本发明涉及化学药物技术领域,尤其是指一种RyR2稳定剂及其制备方法、相应的药用组合物和应用。
背景技术
RyR2是Ryanodine(兰尼碱)的一种受体,是存在于心肌细胞(CMs)肌浆网(SR)上的一种钙离子释放通道,在控制心肌兴奋-收缩偶联中起重要作用。儿茶酚胺敏感型多形性室速(CPVT)的发生常与RyR2基因突变有关,研究认为RyR2基因突变在儿茶酚胺释放时通过RyR2通道导致舒张期Ca2+释放异常增强,异常增强的肌浆网Ca2+泄漏又反过来驱动Na+/Ca2+交换器的活动,进而产生去极化Na+电流(INa),触发动作电位异常、去极化后早期(EAD)或去极化后延迟(DAD),导致CPVT。《本草纲目》中记载灵芝具有“补中益气,增智慧,好颜色,久食轻身不老,延年神仙”的功效。反柄紫芝在民间常作为紫芝使用,研究发现反柄紫芝中的酚性杂萜类化合物亚型丰富,且具有多种药理活性,如肾保护、抗肿瘤、抗炎等,但在治疗罕见病CPVT中并未见相关报道。
发明内容
本发明所要解决的技术问题是:寻找能够有效治疗罕见病CPVT的药物成分。
为了解决上述技术问题,本发明采用的技术方案为:
一种RyR2稳定剂,为Fornicin A或其药学上的盐。
进一步地,所述Fornicin A为负旋光化合物,结构式为:后续中,Fornicin A的负旋光化合物简称(–)-Fornicin A,正旋光化合物简称(+)-Fornicin A,其中,(+)-Fornicin A的结构式为:
一种药用组合物,包含上述所述的RyR2稳定剂。
进一步地,还包括至少一种药用载体或赋形剂。
一种应用,根据上述所述的RyR2稳定剂或根据上述所述的药用组合物,制备用于治疗CPVT相关疾病的药物或制备保健品。
进一步地,上述应用中,Fornicin A与兰尼碱受体2(RyR2)相互作用,抑制心肌细胞钙漏流。
一种RyR2稳定剂的制备方法,包括依次执行的以下步骤:
S1:使用乙酸乙酯萃取灵芝的醇溶提取物,取乙酸乙酯部分,得到酯溶混合物;
S2:所述酯溶混合物先经过硅胶柱进行第一梯度洗脱,经薄层色谱(TLC)检测,合并相同流分,得到第一洗脱液;所述第一梯度洗脱的洗脱液为石油醚-丙酮的混合物,且石油醚:丙酮的梯度依次为:91:9,120L;88:12,120L;85:15,150L;80:20,150L;70:30,150L;60:40,150L;50:50,150L;0:100,100L;
S3:所述第一洗脱液经过第一MCI gel CHP 20P柱进行第二梯度洗脱,经薄层色谱(TLC)检测,合并相同流分,得到第二洗脱液;所述第二梯度洗脱的洗脱液为第一甲醇水溶液,且甲醇:水的梯度依次为:60:40,5L;65:35,10L;70:30,8L;75:25,10L;80:20,28L;85:15,18L;90:10,18L;95:5,10L;100:0,10L;
S4:所述第二洗脱液经过RP-18柱进行第三梯度洗脱,经薄层色谱(TLC)检测,合并相同流分,得到第三洗脱液;所述第三梯度洗脱的洗脱液为第二甲醇水溶液,且甲醇:水的梯度依次为:40:60,0.5L;45:55,1.3L;50:50,2.4L;55:45,3.6L;60:40,4.2L;65:35,4.2L;70:30,3.6L;75:25,3.0L;80:20,5.4L;85:15,3.0L;95:5,3.0L;100:0,3.0L;
S5:所述第三洗脱液经过第二MCI gel CHP 20P柱进行第四梯度洗脱,经薄层色谱(TLC)检测,合并相同流分,得到第四洗脱液;所述第四梯度洗脱的洗脱液为第三甲醇水溶液,且甲醇:水的梯度依次为:40:60,0.8L;50:50,0.8L;60:40,0.8L;70:30,0.8L;80:20,0.8L;85:150.8L;90:10,0.8L;95:5,1.0L;100:0,1.0L;
S6:所述第四洗脱液依次经过Sephadex LH-20柱、半制备HPLC纯化,得到FornicinA;在Sephadex LH-20柱中使用甲醇洗脱;在半制备HPLC中使用TFA-甲醇溶液洗脱,且甲醇:TFA溶液=75:25;所述TFA溶液为含有0.05%TFA的水溶液。
进一步地,还包括步骤S7:
S7:所述Fornicin A经过手性柱拆分得到负旋光的Fornicin A;在手性柱中使用正己烷-乙醇溶液洗脱,且正己烷:乙醇=88:22。
进一步地,在步骤S1中,酯溶混合物的具体制备步骤为:将灵芝粉碎,用70-90%的乙醇回流提取,提取次数为2-6次,除去乙醇得到醇溶提取物;将醇溶提取物悬浮于30-40℃的水中,用等体积的乙酸乙酯萃取1-5次,合并得到灵芝的酯溶混合物。
进一步地,所述灵芝为反柄紫芝。
本发明的有益效果在于:Fornicin A能够作为RyR2稳定剂,通过与兰尼碱受体2(RyR2)相互作用,抑制心肌细胞钙漏流,进而能够治疗罕见病CPVT。尤其是(–)-FornicinA,对RyR2-R2474S转基因小鼠原代心肌细胞钙火花释放具有抑制作用,且具有剂量依赖关系;能够减少RyR2-R2474S转基因小鼠原代心肌细胞的起搏后起搏后钙释放异常事件;对WT小鼠的心电图并无影响;预防性给药能够显著降低室速的发作率;治疗性给药能够在30min内恢复窦性心律。
附图说明
下面结合附图详述本发明的具体结构
图1为本发明的不同剂量的(–)-Fornicin A对RyR2-R2474S转基因小鼠原代心肌细胞钙火花释放影响结果图,其中,Z16b为(–)-Fornicin A,Vehicle为溶剂对照组;
图2为本发明的不同剂量的(–)-Fornicin A对RyR2-R2474S转基因小鼠原代心肌细胞钙火花释放影响的结果统计图,其中,Z16b为(–)-Fornicin A,Vehicle为溶剂对照组;
图3为本发明的不同剂量的(–)-Fornicin A对RyR2-R2474S转基因小鼠原代心肌细胞的起搏后钙释放的影响结果图,其中,Z16b为(–)-Fornicin A,Vehicle为溶剂对照组;
图4为本发明的不同剂量的(–)-Fornicin A对RyR2-R2474S转基因小鼠原代心肌细胞的起搏后钙释放的影响结果统计图,其中,Z16b为(–)-Fornicin A,Vehicle为溶剂对照组;
图5为本发明的0.5mg/kg剂量的(–)-Fornicin A对小鼠心电图的影响结果图,其中,A为WT小鼠腹腔注射0.5h后的二导联心电图结果,B为心率变化结果,C为PR变化结果,D为QRS变化结果,E为QT变化结果;
图6为本发明的(–)-Fornicin A预防性给药对室速发作率的影响结果图——心电图图谱,其中,Z16b为(–)-Fornicin A,Vehicle为溶剂对照组,Resting为静息状态,Epi+Cafstimulation为肾上腺素+咖啡因刺激;
图7为本发明的(–)-Fornicin A预防性给药对室速发作率的影响结果图——室速发作率统计结果,其中,Z16b为(–)-Fornicin A,Vehicle为溶剂对照组,JTV-519为阳性药;
图8为在室速发作后,本发明的(–)-Fornicin A恢复窦性心律的能力结果图——心电图图谱,其中,Z16b为(–)-Fornicin A,Vehicle为溶剂对照组,Resting为静息状态,Epi+Cafstimulation为肾上腺素+咖啡因刺激;
图9为在室速发作后,本发明的(–)-Fornicin A恢复窦性心律的能力结果图——窦性心律恢复情况统计结果,其中,Z16b为(–)-Fornicin A,Vehicle为溶剂对照组。
具体实施方式
下面将结合本发明实施例中的附图,对本发明实施例中的技术方案进行清楚、完整地描述,显然,所描述的实施例是本发明一部分实施例,而不是全部的实施例。基于本发明中的实施例,本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例,都属于本发明保护的范围。
应当理解,在此本发明实施例说明书中所使用的术语仅仅是出于描述特定实施例的目的而并不意在限制本发明实施例。如在本发明实施例说明书和所附权利要求书中所使用的那样,除非上下文清楚地指明其它情况,否则单数形式的“一”、“一个”及“该”意在包括复数形式。
实施例1:
(–)-Fornicin A的提取分离及结构鉴定
反柄紫芝(Ganoderma cochlear)200kg,粉碎,80%的乙醇回流提取(3×120L,4h,3h,3h),减压回收乙醇,得到浸膏(即醇溶提取物)16.8kg,取出8kg浸膏悬浮于温水,用乙酸乙酯等体积萃取3次,得到乙酸乙酯部分4.0kg。
乙酸酯部分(4.0kg)行硅胶柱层析,石油醚/丙酮(91:9,120L;88:12,120L;85:15,150L;80:20,150L;70:30,150L;60:40,150L;50:50,150L;0:100,100L)室温梯度洗脱,经薄层色谱(TLC)检测,合并相同流分,得4个流分段,分别为Fractions A-D。其中,Fr.B(860.0g)行MCI gel CHP 20P柱层析,甲醇/水(60:40,5L;65:35,10L;70:30,8L;75:25,10L;80:20,28L;85:15,18L;90:10,18L;95:5,10L;100:0,10L)室温梯度洗脱,经TLC检测,合并相同流分,得到6个组分,分别为Fr.B1-B6。Fr.B2(120.0g)经RP-18柱层析,以甲醇/水(40:60,0.5L;45:55,1.3L;50:50,2.4L;55:45,3.6L;60:40,4.2L;65:35,4.2L;70:30,3.6L;75:25,3.0L;80:20,5.4L;85:15,3.0L;95:5,3.0L;100:0,3.0L)室温梯度洗脱,经TLC检测,合并相同流分,得5个部分,分别为Fr.B21-B25。Fr.B22(12.4g)经MCI gel CHP 20P柱层析,甲醇/水(40:60,0.8L;50:50,0.8L;60:40,0.8L;70:30,0.8L;80:20,0.8L;85:150.8L;90:10,0.8L;95:5,1.0L;100:0,1.0L)室温梯度洗脱,经TLC检测,合并相同流分,得到3个部分,分别为Fr.B221-B223。Fr.B223(7.4g)先于室温行Sephadex LH-20柱(甲醇)层析,然后经半制备HPLC(甲醇/水,水中含有0.05%的TFA,75:25,流速3mL/min)室温纯化,得到Fornicin A(350.0mg,tR=8.5min)。Fornicin A再经手性柱拆分,正己烷/乙醇(88:22)洗脱,柱温25℃,流速3mL/min,得具有正旋光的化合物(+)-Fornicin A(162.5mg,tR=10.8min)和具有负旋光的化合物(–)-Fornicin A(166.8mg,tR=12.1min)。
化合物(–)-Fornicin A的结构式为:
(–)-Fornicin A的鉴定结果如下:化合物(–)-Fornicin A,淡黄色固体, (c0.30,MeOH),CD(MeOH)Δε210–0.95,Δε302–19.88,UV(MeOH)λmax(logε)301(3.44)nm;HRESIMSm/z275.1282[M+H]+(calcdforC16H19O4,275.1278);1Hand13CNMRdata见表1。
表1.化合物(–)-Fornicin A的1H和13CNMR数据
实施例2:
实施例1中化合物(–)-Fornicin A按常规法加注射用溶媒,精滤,灌封灭菌后可制成注射液。
对比例1:
实施例1中同时分离得到的化合物(+)-Fornicin A的结构式为:
(+)-Fornicin A的NMR鉴定结果与(–)-Fornicin A的鉴定结果相同。
化合物(+)-Fornicin A按常规法加注射用溶媒,精滤,灌封灭菌后可制成注射液。
采用实施例2的注射液和对比例1的注射液进行初步的CPVT的潜在治疗作用研究,发现(+)-Fornicin A具有一定的活性作用,但在治疗CPVT上的活性效果远远不及(–)-Fornicin A的活性效果。故本申请针对(–)-Fornicin A作进一步深入的研究,具体详见实施例6和实施例7。
实施例3:
实施例1中化合物(–)-Fornicin A按常规法配以各种药用辅料可制成片剂。
使用实施例1中化合物(–)-Fornicin A作为药物活性成分,使用几种赋形剂作为制备组合药物片剂的辅料成分,按照一定比例配比制成每片含有药物成分1-100mg的片剂样品。
实施例4:
实施例1中化合物(–)-Fornicin A按常规法配以各种药用辅料可制成胶囊剂:
含有实施例1中化合物(–)-Fornicin A作为有效成分的药物组合胶囊制剂的制备,使用实施例1中化合物(–)-Fornicin A作为药物活性成分、使用几种赋形剂作为制备组合药物胶囊剂的辅料成分,按照一定比例配比制成每粒胶囊中含有化合物成分1-100mg的胶囊制剂。
实施例5:
取实施例1的方法制得的化合物(–)-Fornicin A 1份,10份植脂末,混匀,按照常规方法制成固体饮料。
实施例6:
通过体外药效学检测Ca2+火花频率、对心肌细胞内钙库的影响和钙释放异常事件,研究(–)-Fornicin A在in vitro层面对CPVT的潜在治疗作用。具体步骤如下:
(1)通过激光共聚焦显微镜观测细胞内钙火花的释放,发现(–)-Fornicin A(即:Z16b)对RyR2-R2474S转基因小鼠原代心肌细胞钙火花释放具有抑制作用,且具有剂量依赖关系,具体结果详见图1和图2。
(2)通过测定心肌细胞起搏后钙释放异常事件(post-pacing calcium releaseevents),发现(–)-Fornicin A能显著抑制RyR2-R2474S转基因小鼠原代心肌细胞的起搏后钙释放异常事件频率,具体结果详见图3和图4。
以上体外实验结果支持(–)-Fornicin A具有潜在的治疗RyR2突变引起的心律失常的作用,对因RyR2突变引起的异常心肌细胞钙漏流具有抑制作用。
实施例7:
通过构建CPVT动物模型,检测(–)-Fornicin A对CPVT模型小鼠的治疗作用。并同时检测(–)-Fornicin A对小鼠心电图参数的影响。具体步骤如下:
(1)采用WT小鼠腹腔0.5mg/kg剂量的(–)-Fornicin A。于0.5小时后,检测小鼠心电图,发现对常规心电参数无明显影响,具体结果详见图5A、图5B、图5C、图5D以及图5E。
(2)采用RyR2-R2474S转基因小鼠(雌雄各半)以腹腔注射咖啡因与肾上腺素激发心律失常,结果发现,经0.5小时前预防性给药0.5mg/kg剂量的(–)-Fornicin A显著降低了心律失常发生的几率,具体结果详见图6和图7。
(3)采用RyR2-R2474S转基因小鼠(雌雄各半)以腹腔注射咖啡因与肾上腺素激发心律失常,结果发现,在出现心律失常后的小鼠中,治疗性给药0.5mg/kg剂量的(–)-Fornicin A较溶剂对照组能显著提高30分钟内小鼠恢复窦性心律的比率,具体结果详见图8和图9。
综上所述,本发明提供的一种RyR2稳定剂及其制备方法、相应的药用组合物和应用,从反柄紫芝中提取得到的(–)-Fornicin A,能够作为RyR2稳定剂,用于治疗RyR2基因突变而导致的CPVT及相关疾病时,通过与兰尼碱受体2(RyR2)相互作用,抑制心肌细胞钙漏流,进而达到治疗罕见病CPVT及相关疾病的目的,具体为:(-)-Fornicin A对RyR2基因变异的原代心肌细胞钙火花释放具有抑制作用,且具有剂量依赖关系;(-)-Fornicin A能够减少RyR2基因变异的原代心肌细胞的起搏后起搏后钙释放异常事件;(-)-Fornicin A对心电图并无影响;预防性给药能够显著降低室速的发作率;治疗性给药能够在30min内恢复窦性心律。
此处第一、第二……只代表其名称的区分,不代表它们的重要程度和位置有什么不同。
以上所述仅为本发明的实施例,并非因此限制本发明的专利范围,凡是利用本发明说明书及附图内容所作的等效结构或等效流程变换,或直接或间接运用在其他相关的技术领域,均同理包括在本发明的专利保护范围内。
Claims (5)
2.如权利要求1所述的应用,其特征在于,所述的负旋光的Fornicin A与兰尼碱受体2相互作用,抑制心肌细胞钙漏流。
3.如权利要求1或2所述的应用,其特征在于,所述负旋光的Fornicin A的制备方法包括依次执行的以下步骤:
S1:使用乙酸乙酯萃取灵芝的醇溶提取物,取乙酸乙酯部分,得到酯溶混合物;
S2:所述酯溶混合物先经过硅胶柱进行第一梯度洗脱,经薄层色谱检测,合并相同流分,得到第一洗脱液;所述第一梯度洗脱的洗脱液为石油醚-丙酮的混合物,且石油醚:丙酮的梯度依次为:91:9,120L;88:12,120L;85:15,150L;80:20,150L;70:30,150L;60:40,150L;50:50,150L;0:100,100L;
S3:所述第一洗脱液经过第一MCI gel CHP 20P柱进行第二梯度洗脱,经薄层色谱检测,合并相同流分,得到第二洗脱液;所述第二梯度洗脱的洗脱液为第一甲醇水溶液,且甲醇:水的梯度依次为:60:40,5L;65:35,10L;70:30,8L;75:25,10L;80:20,28L;85:15,18L;90:10,18L;95:5,10L;100:0,10L;
S4:所述第二洗脱液经过RP-18柱进行第三梯度洗脱,经薄层色谱检测,合并相同流分,得到第三洗脱液;所述第三梯度洗脱的洗脱液为第二甲醇水溶液,且甲醇:水的梯度依次为:40:60,0.5L;45:55,1.3L;50:50,2.4L;55:45,3.6L;60:40,4.2L;65:35,4.2L;70:30,3.6L;75:25,3.0L;80:20,5.4L;85:15,3.0L;95:5,3.0L;100:0,3.0L;
S5:所述第三洗脱液经过第二MCI gel CHP 20P柱进行第四梯度洗脱,经薄层色谱检测,合并相同流分,得到第四洗脱液;所述第四梯度洗脱的洗脱液为第三甲醇水溶液,且甲醇:水的梯度依次为:40:60,0.8L;50:50,0.8L;60:40,0.8L;70:30,0.8L;80:20,0.8L;85:15 0.8L;90:10,0.8L;95:5,1.0L;100:0,1.0L;
S6:所述第四洗脱液依次经过Sephadex LH-20柱、半制备HPLC纯化,得到Fornicin A;在Sephadex LH-20柱中使用甲醇洗脱;在半制备HPLC中使用TFA-甲醇溶液洗脱,且甲醇:TFA溶液=75:25;所述TFA溶液为含有0.05%TFA的水溶液;
S7:所述Fornicin A经过手性柱拆分得到负旋光的Fornicin A;在手性柱中使用正己烷-乙醇溶液洗脱,且正己烷:乙醇=88:22。
4.如权利要求3所述的应用,其特征在于,在步骤S1中,酯溶混合物的具体制备步骤为:将灵芝粉碎,用70-90%的乙醇回流提取,提取次数为2-6次,除去乙醇得到醇溶提取物;将醇溶提取物悬浮于30-40℃的水中,用等体积的乙酸乙酯萃取1-5次,合并得到灵芝的酯溶混合物。
5.如权利要求4所述的应用,其特征在于,所述灵芝为反柄紫芝。
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