CN105358582B - 一种硫酸酯化聚古洛糖酸多糖或其可药用盐及其制备方法和用途 - Google Patents
一种硫酸酯化聚古洛糖酸多糖或其可药用盐及其制备方法和用途 Download PDFInfo
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- CN105358582B CN105358582B CN201480038171.1A CN201480038171A CN105358582B CN 105358582 B CN105358582 B CN 105358582B CN 201480038171 A CN201480038171 A CN 201480038171A CN 105358582 B CN105358582 B CN 105358582B
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Abstract
本发明提供了一种硫酸酯化聚古洛糖酸多糖或其可药用盐及其制备方法和在制备肿瘤生长和/或转移抑制剂中的用途。本发明的硫酸酯化聚古洛糖酸多糖或其可药用盐可用于制备肿瘤生长抑制剂、肿瘤转移抑制剂、血管生成抑制剂、乙酰肝素酶抑制剂、C‑Met酶抑制剂、微管聚合抑制剂、肌动蛋白解聚因子活性抑制剂和肌动蛋白聚集抑制剂中的任一种或多种。
Description
技术领域
本发明涉及医药领域,且更具体而言,涉及一种硫酸酯化聚古洛糖酸多糖或其可药用盐及其制备方法和在制备肿瘤生长和/或转移抑制剂中的用途。
背景技术
肿瘤是严重威胁人类生命和健康的疾病。恶性肿瘤已成为城市居民的第一死因,农村居民的第二死因,其死亡率居各种疾病的首位。肿瘤转移是肿瘤的恶性特征之一,恶性肿瘤的转移和复发是导致肿瘤治疗失败的主要原因。因此,寻找能够抑制肿瘤生长和转移的抗肿瘤药物是目前人们关注的焦点。
近年的研究发现,糖类物质不仅是一类重要的结构物质和能量物质,而且还具有重要的生物学功能,其参与细胞间的相互识别及信息传递过程,被认为是生物体内除核酸以外的又一类重要的信息分子,而且由于它们常是细胞表面信号识别、抗原抗体反应、细胞间信息传递和感受的关键因子,因此,具有生物活性的活性多糖的研究日益受到重视。但由于糖类物质结构复杂,分离及结构鉴定困难,到目前为止,只有云芝多糖、猪苓多糖、香菇多糖、裂褶多糖、茯苓多糖等用于临床,本领域中需要更多具有生物活性的多糖。
发明内容
针对糖类物质结构解析及制备困难,及天然糖活性往往不理想的问题,结合肿瘤发病形势严峻及治疗困难的现实,本发明人经过广泛深入的研究,最终完成本发明。本发明人将多聚古洛糖醛酸在一定温度下与磺化剂反应一定时间,得到古洛糖醛酸寡糖的硫酸酯化衍生物,再加入还原剂进行还原,制备了硫酸酯化聚古洛糖酸多糖(polygulonic acidsulfate,以下简称“PGAS”),在此基础上完成了本发明。
因此,本发明的一个目的是提供一种硫酸酯化聚古洛糖酸多糖或其可药用盐。本发明的硫酸酯化聚古洛糖酸多糖或其可药用盐具有明显的抑制肿瘤生长和转移的作用,其作用机制同其能够抑制乙酰肝素酶(heparanase)活性、抑制C-Met酶活性、抑制血管生成、抑制微管聚合、抑制肌动蛋白解聚因子等有关。优选地,本发明提供一种硫酸酯化聚古洛糖酸多糖或其可药用盐,其中各个L-古洛糖醛酸单元通过1,4糖苷键连接,其还原端1位为羟基,糖环C-2位完全硫酸酯化。
本发明的另一个目的是提供一种硫酸酯化聚古洛糖酸多糖或其可药用盐的制备方法。本发明的又一个目的是提供一种硫酸酯化聚古洛糖酸多糖或其可药用盐在制备肿瘤生长和/或转移抑制剂中的用途。
本发明的还一个目的是提供一种药物组合物,其包含治疗有效量的本发明的硫酸酯化聚古洛糖酸多糖或其可药用盐。
本发明的再一个目的是提供一种治疗肿瘤的方法。
根据本发明的一个方面,提供了一种结构如下列通式(I)所示的硫酸酯化聚古洛糖酸多糖或其可药用盐:
通式(I)中,n表示0或1-23的整数,R1为SO3H,R2各自独立地为H或SO3H,条件是:硫酸酯化程度换算成硫酸酯化聚古洛糖酸多糖的含硫量,为5-20重量%。
在本发明的由通式(I)表示的硫酸酯化聚古洛糖酸多糖或其可药用盐中,所述古洛糖酸多糖由L-古洛糖醛酸通过1,4糖苷键连接而成,其还原端1位为羟基,糖环C-2位完全硫酸酯化,C-3位部分硫酸酯化。
上述通式(I)中,n为0或1-23的整数,例如0、1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22或23;优选地,n为2-13的整数,更优选n为3、4、5、6、7、8、9或10,最优选n为4、5、6、7或8。优选地,所述含硫量为7-15重量%,更优选9-13重量%。本发明中,四糖至十二糖(特别是六糖至十糖)和/或含硫量为7-15重量%(特别是9-13重量%)时的生物效果较佳,很可能是它们较易被机体细胞识别和接受。
本发明中,所述硫酸酯化聚古洛糖酸多糖的可药用盐,例如可以是这些化合物的钠盐、钾盐、钙盐、镁盐等,其中优选钠盐。所述的可药用盐可用常规方法制得。
根据本发明的另一方面,提供了上述硫酸酯化聚古洛糖酸多糖或其可药用盐的制备方法,其包括:
将下列结构式(II)所示的古洛糖醛酸多糖与磺化试剂反应,再经还原剂还原,形成通式(I)所示的硫酸酯化聚古洛糖酸多糖,
式(II)中,m表示0或1-48的整数;
通式(I)中,n以及R1和R2的定义如上所述。
本发明中,优选地,所述磺化剂可为氯磺酸;优选地,磺化反应温度可为45-85℃,更优选60-75℃,反应时间可为1.5-4.5小时,更优选2-3.5小时,最优选3小时;优选地,所述还原剂可为硼氢化钠、氰基硼氢化钠、镍氢试剂或卤素类还原剂等等。
根据本发明的又一个方面,提供了一种硫酸酯化聚古洛糖酸多糖或其可药用盐在制备肿瘤生长和/或转移抑制剂中的用途。
本发明中,术语“肿瘤”可指包括恶性肿瘤在内的任何形式的肿瘤,例如,所述的肿瘤可为肝癌、胃癌、结直肠癌、肺癌、乳腺癌、胰腺癌、肾癌、膀胱癌、前列腺癌、黑色素瘤、脑瘤等。
本发明中,优选地,所述硫酸酯化聚古洛糖酸多糖或其可药用盐可被用作肿瘤生长抑制剂、肿瘤转移抑制剂、血管生成抑制剂、乙酰肝素酶抑制剂、C-Met酶抑制剂、微管聚合抑制剂、肌动蛋白解聚因子活性抑制剂、和/或肌动蛋白聚集抑制剂。
根据本发明的还一个方面,提供了一种药物组合物,其包含治疗有效量的本发明的硫酸酯化聚古洛糖酸多糖或其可药用盐。优选地,所述药物组合物中的活性成分由一种或多种本发明的硫酸酯化聚古洛糖酸多糖或其可药用盐组成。在另一实施方案中,本发明的药物组合物中除了本发明的硫酸酯化聚古洛糖酸多糖或其可药用盐外,还可以含有一种或多种抗肿瘤药物或抗肿瘤辅助药物作为或活性成分。
本发明中,优选地,所述药物组合物可以进一步包含可药用载体。所述的可药用载体可为本领域中通常使用的。
本发明中,优选地,所述药物组合物可以用作肿瘤生长抑制剂。
本发明中,优选地,所述药物组合物可以用作肿瘤转移抑制剂。
此外,优选地,所述药物组合物可以用作血管生成抑制剂、乙酰肝素酶抑制剂及C-Met酶抑制剂、微管聚合抑制剂、肌动蛋白解聚因子活性抑制剂、和/或肌动蛋白聚集抑制剂。
根据本发明的再一个方面,提供了一种治疗肿瘤的方法,其包括向需要治疗的对象施用治疗有效量的本发明的硫酸酯化聚古洛糖酸多糖或其可药用盐。
本发明中,术语“有效量”可指为实现预期的效果所需的剂量和时段的有效的量。此有效量可能因某些因子而产生不同的变化,如疾病的种类或治疗时疾病的病症、被施用的特定标的器官的构造、病人个体大小、或疾病或症状的严重性。本领域具有通常知识者不需要过度实验即可凭经验决定特定化合物的有效量。
因此,将本发明的硫酸酯化聚古洛糖酸多糖或其可药用盐用于制备肿瘤治疗药物和肿瘤转移治疗药物,对于解决当前肿瘤治疗缺乏有效药物的困难,具有特别重要的意义。
附图说明
图1示出本发明的硫酸酯化聚古洛糖酸多糖各组分的柱分离图。
图2示出本发明的硫酸酯化聚古洛糖酸多糖对人乳腺癌MDA-MB-435原位移植瘤的生长抑制作用。
图3示出本发明的硫酸酯化聚古洛糖酸多糖对人乳腺癌MDA-MB-435原位移植瘤肺转移的抑制作用。其中,A为H&E染色显示肺上转移灶的典型照片(放大倍数:200×);B为PGAS对MDA-MB-435原位移植瘤肺转移抑制作用的定量图。图中数据表示为一次典型实验的均值±SD。*P<0.05,**P<0.01,治疗组与对照组进行比较。
图4示出本发明的硫酸酯化聚古洛糖酸多糖对人乳腺癌MDA-MB-435原位移植瘤血管生成的抑制作用。其中,A为CD31染色的典型照片,箭头指向阳性处(放大倍数:200×)(图A中:“C”为对照组,“P”为PGAS处理组);B是PGAS对人乳腺癌MDA-MB-435原位移植瘤血管生成抑制作用的定量图。图中数据表示为一次实验的均值±SD。**P<0.01,治疗组与对照组进行比较。
图5示出本发明的硫酸酯化聚古洛糖酸多糖对鼠黑色素瘤细胞B16F10实验性肺转移的抑制作用。其中,A为肺上转移灶的典型照片;B是PGAS对B16F10实验性肺转移抑制作用的定量图。图中数据表示为一次典型实验的均值±SD。相似的结果可以从至少两次独立实验获得。*P<0.05,**P<0.01,治疗组与对照组进行比较。
图6示出本发明的硫酸酯化聚古洛糖酸多糖抑制鸡胚尿囊膜(CAM)新生血管生成。其中,A是溶媒对照组;B是200μg/egg的PGAS组;C为400μg/egg的PGAS组;D是800μg/egg的PGAS(放大倍数:40倍)。
图7示出本发明的硫酸酯化聚古洛糖酸多糖抑制乙酰肝素酶活性的图谱。其中,A示出本发明的硫酸酯化聚古洛糖酸多糖抑制乙酰肝素酶活性的HPLC图谱;B示出根据A图结果计算的本发明的硫酸酯化聚古洛糖酸多糖对乙酰肝素酶活性的抑制率。
图8示出本发明的硫酸酯化聚古洛糖酸多糖对无细胞体系微管蛋白聚合的抑制作用。其中,A示出时间依赖关系,B示出剂量依赖关系。
图9示出本发明的硫酸酯化聚古洛糖酸多糖对无细胞体系肌动蛋白解聚的抑制作用。
图10示出本发明的硫酸酯化聚古洛糖酸多糖抑制肌动蛋白解聚因子对肌动蛋白的解聚/剪切活性。其中,##P<0.01Cofilin组与对照组比较;**P<0.01药物组与Cofilin组比较。
图11示出本发明的硫酸酯化聚古洛糖酸多糖各糖组分抑制肿瘤转移的活性。
具体实施方式
在下文中,将通过实施例更加详细地阐明本发明。但是,提供下列实施例仅仅是出于说明目的,本发明的范围不限于此。
主要药品与试剂
古洛糖醛酸多糖(polyguluronic acid),购自中国海洋大学兰太药业有限责任公司,相对于右旋糖酐的重均分子量为10000Da;甲酰胺,氯磺酸等均由国药集团化学试剂公司提供,均为分析纯试剂;右旋糖酐分子量标准品购自Fluka公司;阿霉素注射液(Adriamycin,ADM),浙江海门制药厂生产,浙卫药准字(1996)第135501号,含量:10mg/支,溶剂:生理盐水,配置方法:每次给药前用生理盐水稀释到所需浓度;TSK gel2000SWXL,TSKgel G3000SWXL色谱柱,日本TOSOH公司;Bio-Gel-P6,Bio-Gel-P10,Bio-Rad公司产品;Sephadex G-10,Sepharose CL-4B,Pharmacia公司产品;微管蛋白(tubulin),肌动蛋白(actin),均购自SIGMA公司。
实验仪器
NEXUS-470智能型红外光谱仪,NICOLET公司产品;DPX-300型核磁共振波谱仪,Bruker公司产品;凝胶渗透色谱(GPC),北京龙智达有限公司产品;UV-2102紫外可见分光光度计,美国尢尼科公司产品。
实验动物
裸小鼠,BALB/cA,18-22g,由中国科学院上海药物研究所提供;
C57BL/6小鼠,6-7周龄,由中国科学院上海动物中心提供;
新鲜种蛋,购自上海申宝养鸡场。
实施例1 硫酸酯化聚古洛糖酸多糖(PGAS)的制备
保持在5℃以下,向10ml甲酰胺中逐滴加入3ml氯磺酸,反应20分钟后,加入1g古洛糖醛酸多糖,在65-70℃反应3小时,向反应产物中加入2倍体积的80%乙醇溶液,反复搅拌,得到粘稠物质。再加入80%乙醇溶液,重复以上步骤2次。倾去溶液,加水得到粘稠物质,用1%Na2CO3溶液调节pH为7.0,用2倍体积的95%乙醇溶液醇沉,得到的沉淀物在50-60℃烘干,得到古洛糖醛酸多糖的硫酸酯化衍生物,将该衍生物配成4mg/ml的醋酸钠溶液(pH7.0),加入硼氢化钠,使成50mM,30-40℃反应30min,冰浴终止反应,以0.1M醋酸调节pH值,释放未反应的硼氢化钠,调节pH值中性,乙醇反复沉淀洗涤,干燥,即得硫酸酯化聚古洛糖酸多糖(PGAS)粗品。将PGAS粗品配成10%的溶液,用95%的乙醇溶液进行沉淀,将沉淀用无水乙醇洗涤,干燥后,配成5%溶液,用3μm膜过滤除杂质,在Sephadex G-10柱(15×100cm)上进行脱盐,流动相为水,分步收集,洗脱液用硫酸-咔唑法检测,合并含糖组分,减压浓缩并除盐,冷冻干燥,即得精制的硫酸酯化聚古洛糖酸多糖。
采用氧瓶燃烧法测定上述制备的硫酸酯化聚古洛糖酸多糖的含硫量。取约25mg试样,精密称定,按照氧瓶燃烧法进行有机破坏,选用1000ml燃烧瓶,以浓过氧化氢溶液0.1ml与水10ml为吸收液,待生成的烟雾完全吸收后,置冰浴中15分钟,加热缓缓煮沸2分钟,冷却,加乙醇-醋酸铵缓冲液(pH3.7)50ml,乙醇30ml,0.1%茜素红溶液0.3ml为指示液,用高氯酸钡滴定液(0.05mol/L)滴定至淡橙红色。每1ml高氯酸钡滴定液(0.05mol/L)相当于1.603mg的S。测定结果发现,按干燥品计算,硫酸酯化聚古洛糖酸多糖的含硫量为11.2重量%。
实施例2 硫酸酯化聚古洛糖酸多糖(PGAS)的结构鉴定
对上述硫酸酯化聚古洛糖酸多糖的制备中得到的流分所含糖组分进行结构鉴定。
1.紫外吸收图谱
将上述硫酸酯化聚古洛糖酸多糖稀释至合适浓度,以蒸馏水作为空白,用UV-2102紫外可见分光光度计在190nm-400nm间扫描,发现该流分在紫外区无特异性吸收峰,说明结构中无共轭双键结构。但是在190-200nm处有非特异性吸收。
2.红外光谱测定
取0.5mg的PGAS,用KBr压片,通过NEXUS-470智能型红外光谱仪进行红外光谱测定。结果发现,在3219.53cm-1处有羟基的对称伸缩振动,1612.58cm-1有羧酸盐的羰基对称伸缩振动,1414.33cm-1有羟基的剪式振动,1103.97cm-1有羧基碳氧键的对称伸缩振动,823.30cm-1有C-O-S的伸缩振动峰;1274.62cm-1有硫酸化后硫酸酯基中的S=O伸缩振动峰,表明该化合物含有羧基、羟基和磺酸基的骨架结构。
3.PGAS核磁共振波谱测定
采用Bruker Auance DPX-300型核磁共振波谱仪测定了PGAS核磁共振碳谱(13C-NMR)。结果发现,谱图中基本看不到未磺化的C-2信号峰,而仍然存在未磺化的C-3信号峰。表明C-2位羟基硫酸酯化比较完全,C-3位羟基只有部分被硫酸酯化。
4.PGAS的分子量与分子量分布
采用GPC方法对PGAS的分子量进行测定。使用的分子量标准品为Fluka公司的右旋糖酐,色谱柱为TSK gel2000SWXL柱,流动相为含0.2%叠氮钠和2.84%Na2SO4的水溶液,流速为0.5ml/min,测定温度在35℃,进样量为25μl,使用Refractive Index Detector检测器进行检测。结果发现,PGAS相对于右旋糖酐的重均分子量为2513Da,对多批PGAS样品测定的结果表明,其相对于右旋糖酐的重均分子量在1500-8500Da。
综合以上检测结果,确定上述流分为还原端1位为羟基的聚古洛糖醛酸的C-2位完全硫酸酯化、C-3位部分硫酸酯化得到的硫酸酯化聚古洛糖酸多糖,具有下列化学结构式(I):
通式(I)中,n以及R1和R2的定义如上所述。
实施例3 PGAS各组分的分离制备
取上述制备的PGAS样品,配成5%溶液,用3μm膜过滤除杂质,在Bio-Gel-P6凝胶柱(1.6×180cm)上进行分离,流动相为0.2mol/L NH4HCO3,分步收集,洗脱液用硫酸-咔唑法检测,收集各含糖组分,外水体积组分继续上Bio-Gel-P10凝胶柱(1.6×180cm)分离,冷冻干燥,得系列PGAS糖组分,经质谱鉴定,制备了PGAS的2、3、4、5、6、7、8、9、10、11及大于11糖的组分(图1)。
本发明下述各实验中,除实施例12和13使用上述实施例3制备的产品外,其余实施例4-11均使用上述实施例1制备的产品。
实施例4 硫酸酯化聚古洛糖酸多糖(PGAS)抑制肿瘤生长的疗效评价
将处于对数生长期的浓度为2.5×107细胞/毫升的人乳腺癌MDA-MB-435细胞(来源于美国典型培养物保藏中心(American Type Culture Collection,ATCC,Rockville,MD,USA)),接种于4-5周龄的雌性裸小鼠(BALB/cA,由中国科学院上海药物研究所提供)左侧第二乳头脂肪垫。当肿瘤体积生长到100-200mm3时,将动物按瘤体积平均分为阴性对照组、阿霉素注射液(Adriamycin,ADM)给药组(阳性对照组)和PGAS 5mg/kg及20mg/kg给药组(PGAS实验组),每组20只,PGAS实验组和阳性对照组每周静脉注射给药一次,连续给药7周,阴性对照组给以等量生理盐水。实验采用游标卡尺测量瘤径,每周二次,肿瘤体积(V)按以下公式计算:
V=1/2×a×b2
其中,a和b分别表示肿瘤的长和宽。根据测量的结果计算出相对肿瘤体积(relative tumor volume,RTV),计算公式为:RTVt=Vt/V0。其中,V0为分笼给药时(即d0)测量所得肿瘤体积,Vt为测量当天的肿瘤体积。抗肿瘤活性的药效学评价指标为相对肿瘤增殖率T/C(%),计算公式如下:
TRTV:治疗组RTV;CRTV:阴性对照组RTV。疗效评价标准:T/C%>60%为无效;T/C%≤60%,并经统计学处理,P<0.05为有效。
停止给药1周后处死动物。将肺组织固定于Bouin’s液(饱和苦味酸∶甲醛∶冰醋酸=75∶25∶5)24小时以上,再用无水乙醇浸泡至肺组织转移灶呈现为白色结节,肺组织恢复为正常颜色。在解剖显微镜下观测记录每个肺脏的转移结节数。部分原位瘤组织置于液氮中冻存,以供抽提总RNA和蛋白质。部分肺脏、原位瘤组织采用10%福尔马林固定液固定,以H&E染色和免疫组化测定肿瘤组织中血管的生成情况。
结果发现,接种肿瘤后15-20天,人乳腺癌MDA-MB-435移植瘤瘤体积生长至100-200mm3左右,接种成活率在100%;接种后第8周至第9周荷瘤裸小鼠肺组织出现大量转移灶,转移率为100%,此时,处死动物并进行抗肿瘤药效学评价。PGAS按每周一次静脉注射,连续7周的治疗方案,PGAS 5mg/kg处理组即显示抑制原位瘤生长的能力,但抑瘤效果不明显,T/C值为67.3%;PGAS 20mg/kg处理组则显示能够显著抑制人乳腺癌MDA-MB-435裸小鼠原位移植瘤的生长,T/C值达37.6%。阳性对照药ADM显示能够显著抑制人乳腺癌MDA-MB-435原位瘤的生长,ADM 5mg/kg处理组的T/C值为21.8%。由此说明,PGAS能够显著抑制人乳腺癌MDA-MB-435原位移植瘤的生长(图2)。PGAS在5mg/kg、20mg/kg每周静脉注射一次连续给药七周的剂量下,对人乳腺癌MDA-MB-435原位移植瘤肺转移的抑制率分别为60.2%、88.4%。阿霉素(5mg/kg)对肺转移抑制率为89.8%。说明PGAS能够显著抑制人乳腺癌MDA-MB-435原位移植瘤的肺转移(图3)。进一步以免疫组织化学染色评价了PGAS对体内血管生成的影响。内皮细胞特异性标记物CD31检测表明人乳腺癌MDA-MB-435原位移植瘤内有大量小血管生成。PGAS 5mg/kg处理组与对照组相比移植瘤内小血管数无明显变化,PGAS 20mg/kg处理组与对照组相比移植瘤内小血管数明显减少,抑制率达42.1%。说明PGAS可显著抑制人乳腺癌MDA-MB-435原位移植瘤内血管生成(图4)。
实施例5 硫酸酯化聚古洛糖酸多糖(PGAS)抑制肿瘤转移的疗效评价
将6-7周龄C57BL/6小鼠(由中国科学院上海动物中心提供)随机分为生理盐水阴性对照组、PGAS 5mg/kg及20mg/kg给药组,每组各10只动物。将处于对数生长期的浓度为2.5×106细胞/毫升鼠黑色素瘤细胞B16F10(来源于美国典型培养物保藏中心(American TypeCulture Collection,ATCC,Rockville,MD,USA))接种于小鼠尾静脉。给细胞前20分钟,PGAS组腹腔注射给药一次,阴性对照组给以等量生理盐水。11天后取肺,将肺组织固定于Bouin’s液(饱和苦味酸∶甲醛∶冰醋酸=75∶25∶5)24小时以上,在解剖显微镜下观测记录每个肺脏的转移结节数。或再于无水乙醇中浸泡两天后,待肺组织恢复为正常颜色再观察。结果发现接种后11天,小鼠肺组织出现大量转移灶,转移率为100%,而按腹腔注射一次给药的方案,PGAS 5mg/kg处理组即显示明显的抑制肿瘤转移的能力,抑制率达40.81%,PGAS20mg/kg处理组则显示能极显著抑制鼠黑色素瘤细胞B16F10的肺转移,抑制率达82.20%。结果显示PGAS能显著抑制鼠黑色素瘤细胞B16F10的肺转移(图5)。
本发明人还对PGAS的作用机制进行了研究。
实施例6 PGAS抑制血管生成试验
将新鲜种蛋(购自上海申宝养鸡场)置于39℃、湿度为50%的孵化器Roll-X base(Lyon Electric Company,CA,USA)中(气室端向上)。连续孵育7天后,先用光照鉴定鸡胚是否存活,并确定尿囊膜所在位置,作好标记,在鸡蛋气囊所在一端扎一个小孔,横放鸡胚(尿囊膜朝上),用洗耳球轻吸气囊,使尿囊膜塌陷与蛋壳分开,静置一段时间,然后在鸡胚上方划一1cm2见方的开窗位置,用剪刀磨切开窗,吹去蛋壳上磨切的粉尘,揭去开窗处的蛋壳,尿囊膜清晰可见。将最终作用浓度为200、400、800μg/egg的PGAS 10μl加在0.25×0.25×0.25(长×宽×高)cm3明胶海绵上,同时设置溶媒对照,然后将海绵轻轻地放在尿囊膜无大血管的部位。用灭菌透明胶布封好小窗,继续孵育48小时,揭开胶布进行观察,拍照(放大倍数:40×),评价PGAS对鸡胚尿囊膜血管生成的抑制作用。结果发现(图6),在含有PGAS海绵旁边的尿囊膜,其血管密度明显下降,并且呈浓度依赖性的抑制趋势。表明PGAS具有抑制血管生成的作用。
实施例7 PGAS抑制乙酰肝素酶(heparanase)活性试验
利用从人胎盘中得到的人乙酰肝素酶的cDNA,通过PCR扩增和基因重组,构建了乙酰肝素酶的无血清昆虫表达系统和亲和柱纯化系统,获得了95%以上纯度的高活性乙酰肝素酶。在150μl反应缓冲液(50mM醋酸钠,pH4.2)中,加入0.5μg FITC-HS(异硫氰酸荧光素标记的乙酰肝素)和终浓度为25ng/ml的乙酰肝素酶,同时加入不同浓度的PGAS,在37℃反应3h。100℃5min后10000rpm离心20min以沉淀不溶物。上清液用0.45μm的滤膜过滤后注射到TSK gel G3000SWXL柱上,上样量为20μl,缓冲液为50mM Tris/150mM NaCl,pH7.5,流速为0.8ml/min。用荧光检测器检测FITC-HS产物的荧光强度(Ex:485nm,Em:538nm)。酶的相对活性通过完整FITC-HS前半峰面积的下降来评价,从而确定PGAS对乙酰肝素酶活性的影响。结果发现,PGAS呈剂量依赖性地抑制乙酰肝素酶活性,IC50为6.55ng/ml,当剂量为40ng/ml时,其活性优于阳性对照物肝素(heparin)(图7)。
实施例8 PGAS抑制C-Met酶活性试验
将酶反应底物Poly(Glu,Tyr)包被酶标板,加入ATP溶液及一定浓度的硫酸酯化聚古洛糖酸多糖和c-Met酶液,37℃摇床反应1小时后,加入含BSA5mg/ml的T-PBS稀释PY99抗体,37℃摇床反应0.5小时,再加入辣根过氧化物酶标记羊抗鼠的IgG,37℃摇床继续反应0.5小时,最后加入2mg/ml的OPD显色液,25℃避光反应1-10分钟后,加入2M H2SO4中止反应,酶标仪测490hm处的吸光度值,计算酶活抑制率:
结果发现,硫酸酯化聚古洛糖酸多糖在10μg/ml浓度下对c-Met的酶活抑制率为71.1%,表明硫酸酯化聚古洛糖酸多糖具有抑制c-Met酶活的作用。
实施例9 PGAS抑制微管蛋白(tubulin)聚合试验
96孔板中加入10μl不同浓度的PGAS,在37℃中预热10min。微管蛋白冻干粉用PEM缓冲液(100mM PIPES,1mM MgCl2,1mM EGTA),另加1mM GTP,5%甘油稀释成浓度12μM,加90μl于96孔板中,开启酶标仪,设置温度为37℃,检测波长为340nm,每分钟混匀读取一次,连续测定30min。结果表明,PGAS能明显抑制无细胞体系微管蛋白的聚合,当PGAS的浓度从2.5μM增加到40μM时,其抑制率从18.26%增加到73.44%,呈剂量依赖性,其IC50为8.58μM(图8)。
实施例10 PGAS抑制肌动蛋白(actin)解聚
先将Pyrenyl标记的肌动蛋白加入聚集缓冲液(100mM Tris-HCl,20mM MgCl2,500mM KCl,2mM CaCl2,pH 7.5)37℃孵育30min,使其聚集成纤丝,然后加入肌动蛋白解聚缓冲液(10mM Tris-HCl,0.2mM CaCl2,0.2mM ATP,pH 8.0)及不同浓度的PGAS,将其稀释使其解聚,设置荧光酶标仪,使发射光波长为360nm,吸收光波长为410nm,温度37℃,每分钟混匀读取一次,连续测定30min。结果表明,PGAS能显著地抑制无细胞体系肌动蛋白的解聚过程,且呈剂量依赖性(IC50为10.6μM)(图9)。
实施例11 PGAS抑制cofilin解聚/剪切肌动蛋白的活性
将硫酸酯化聚古洛糖酸多糖与Sepharose CL-4B进行偶联,制备了硫酸酯化聚古洛糖酸多糖的亲和层析柱,利用该层细柱,对硫酸酯化聚古洛糖酸多糖在肺癌细胞株A549上的结合蛋白进行了提取、分离,通过质谱鉴定发现cofilin是与硫酸酯化聚古洛糖酸多糖结合较强的蛋白之一,进一步的研究发现硫酸酯化聚古洛糖酸多糖能明显抑制cofilin对肌动蛋白的解聚/剪切活性(图10)。说明硫酸酯化聚古洛糖酸多糖能与cofilin结合,抑制cofilin的解聚/剪切肌动蛋白的活性,从而发挥抑制肿瘤细胞细胞迁移的作用。
实施例12 PGAS各糖组分的活性测定
对实施例3中制备的各PGAS糖组分的抗肿瘤活性进行了测试,结果发现,PGAS4-12糖(通式(I)中n=2-10)组分在静脉注射剂量小于15mg/kg时对人乳腺癌MDA-MB-435裸小鼠原位移植瘤生长的T/C值达30%以下,对其肺转移的抑制率达95%以上,6-10糖(通式(I)中n=4-8)组分在剂量小于10mg/kg时对人乳腺癌MDA-MB-435裸小鼠原位移植瘤生长的T/C值达30%以下,对其肺转移的抑制率达95%以上,并以含硫量在7-15重量%时效果更好,9-13重量%最好。
实施例13 PGAS各糖组分抑制肿瘤转移活性测定
用Transwell小室实验检测了实施例3中分离得到的PGAS不同糖组分对肿瘤转移的影响。胰酶消化体外传代对数期生长的乳腺癌MDA-MB-435细胞,用无血清培液洗涤细胞三次并稀释细胞至2×106/ml。Transwell小室中每孔上室加入100μl的细胞稀释液,下室加入600μl含10%FBS的培液,上下室均加入100μg/ml的PGAS不同糖组分。在含5%CO2的培养箱中37℃孵育12h后弃去培养液,90%酒精溶液固定细胞30min。用0.1%结晶紫溶液(0.1M硼酸、0.1%(w/v)结晶紫、2%乙醇)于常温染色10min,清水漂净,用棉签拭去上层未迁移的细胞,显微镜下观察和拍照并记录;最后用10%乙酸溶液100μl/孔抽提10min,595nm测定OD值,计算各糖组分对乳腺癌细胞的迁移抑制率。
迁移抑制率(%)=(1-OD处理组/OD对照组)×100%
结果发现,对照组的MDA-MB-435细胞在12h之内可以沿FBS的浓度梯度自由的从上室迁移到下室,从结晶紫染色后照相的结果可以看出,PGAS各糖组分(从4糖(通式I中,n=2)至11糖(通式I中,n=9)及大于11糖组分)可以明显抑制肿瘤细胞迁移,2糖组分(通式I中,n=0)、3糖组分(通式I中,n=1)抑制率较低(图11),说明PGAS各糖组分(特别是,从4糖至11糖及大于11糖组分)均具有抑制肿瘤细胞转移的作用。
统计学处理
以上数据应用Statview统计处理软件进行统计分析,结果以“均值±标准误”(Mean values±SE)表示,采用方差分析(ANOVA)进行比较。
根据上述药理结果,用常规的制剂手段将有效量的本发明的硫酸酯化聚古洛糖酸多糖与药用载体混合,可以制备药学组合物。所述药学组合物可以是肿瘤治疗药物和肿瘤转移治疗药物,也可以是血管生成抑制剂、乙酰肝素酶抑制剂、C-Met酶抑制剂及微管聚合抑制剂和肌动蛋白解聚抑制剂。将本发明的硫酸酯化聚古洛糖酸多糖用于制备肿瘤治疗药物和肿瘤转移治疗药物具有特别重要的意义。
Claims (15)
1.一种硫酸酯化聚古洛糖酸或其可药用盐,其中各个L-古洛糖醛酸单元通过1,4糖苷键连接,其还原端1位为羟基,糖环C-2位完全硫酸酯化,其中所述硫酸酯化聚古洛糖酸具有如下通式(I)的结构:
其中,n表示0或1-23的整数,R1为SO3H,R2各自独立地为H或SO3H,条件是:硫酸酯化程度换算成硫酸酯化聚古洛糖酸的含硫量,为5-20重量%。
2.根据权利要求1所述的硫酸酯化聚古洛糖酸或其可药用盐,其中,通式(I)中,n为2-13的整数。
3.根据权利要求1所述的硫酸酯化聚古洛糖酸或其可药用盐,其中,n选自3、4、5、6、7或8。
4.根据权利要求1-3之一所述的硫酸酯化聚古洛糖酸或其可药用盐,其中,所述含硫量为7-15重量%。
5.根据权利要求1-3之一所述的硫酸酯化聚古洛糖酸或其可药用盐,其中,所述含硫量为9-13重量%。
6.权利要求1所述的硫酸酯化聚古洛糖酸或其可药用盐的制备方法,其包括:
将下列结构式(II)所示的聚古洛糖醛酸与磺化试剂反应,再经还原剂还原,形成通式(I)所示的硫酸酯化聚古洛糖酸,
结构式(II)中,m表示0或1-48的整数;
通式(I)中,n以及R1和R2的定义如权利要求1所述。
7.根据权利要求6所述的制备方法,其中,所述磺化剂为氯磺酸;磺化反应温度为45-85℃;反应时间为1.5-4.5小时;和/或,所述还原剂为硼氢化钠、氰基硼氢化钠、镍氢试剂或卤素类还原剂。
8.根据权利要求7所述的制备方法,其中,所述磺化反应温度为60-75℃,反应时间为2-3.5小时。
9.根据权利要求7所述的制备方法,其中反应时间为3小时。
10.权利要求1-5中任一项所述的硫酸酯化聚古洛糖酸或其可药用盐在制备肿瘤生长和/或转移抑制剂中的用途。
11.根据权利要求10所述用途,其中,所述肿瘤选自肝癌、胃癌、结直肠癌、肺癌、乳腺癌、胰腺癌、肾癌、膀胱癌、前列腺癌、黑色素瘤、脑瘤。
12.权利要求1-5中任一项所述的硫酸酯化聚古洛糖酸或其可药用盐在制备肿瘤生长抑制剂、肿瘤转移抑制剂、血管生成抑制剂、乙酰肝素酶抑制剂、C-Met酶抑制剂、微管聚合抑制剂、肌动蛋白解聚因子活性抑制剂、和/或肌动蛋白聚集抑制剂中的用途。
13.一种药物组合物,其包含治疗有效量的权利要求1-5任一项所述的硫酸酯化聚古洛糖酸或其可药用盐。
14.根据权利要求13所述的药物组合物在制备肿瘤生长和/或转移抑制剂中的用途。
15.根据权利要求13所述的药物组合物在制备肿瘤生长抑制剂、肿瘤转移抑制剂、血管生成抑制剂、乙酰肝素酶抑制剂、C-Met酶抑制剂、微管聚合抑制剂、肌动蛋白解聚因子活性抑制剂、和/或肌动蛋白聚集抑制剂中的用途。
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