CN105194682B - 血栓靶向释放rgdf的抗栓剂聚天冬酰-rgdf的合成和应用 - Google Patents
血栓靶向释放rgdf的抗栓剂聚天冬酰-rgdf的合成和应用 Download PDFInfo
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Abstract
本发明涉及血栓靶向释放RGDF的聚天冬酰‑RGDF,涉及它的制备方法,涉及它在大鼠血栓模型上的靶向抗血栓作用。因而本发明阐明了聚天冬酰‑RGDF作为靶向抗血栓剂的临床应用前景。本发明属于生物医药领域。
Description
发明领域
本发明涉及血栓靶向释放RGDF的聚天冬酰-RGDF,涉及它的制备方法,涉及它在大鼠血栓模型上的靶向抗血栓作用。因而本发明阐明了聚天冬酰-RGDF作为靶向抗血栓剂的临床应用前景。本发明属于生物医药领域。
背景技术
血小板血栓是血栓形成的起始阶段,可以分为血小板粘附、活化和聚集三个过程。在血小板血栓形成过程中,血小板膜上GPIIb/IIIa活化,构象发生改变,然后在Ca2+的参与下,与纤维蛋白原或vWF上的RGD四肽序列特异性结合将血小板和红血球偶联起来,形成血栓。在血小板聚集过程中,纤维蛋白原或vWF上的RGDF与活化的血小板表面受体GP IIb/IIIa结合起至关重要的作用。RGDF与被GP IIb/IIIa识别并竞争性抑制纤维蛋白原或vWF与血小板结合,抑制血小板聚集。血栓形成部位富集了活化的血小板,这些血小板是血栓形成的重要构件。如果能把RGDF定向释放到血栓形成部位,就可以使这些活化的血小板不能参与血栓形成,从而有效地抑制血栓形成。可是,这个目标在本发明申请之前没有达到。根据这些认识,发明人提出了本发明。本发明的突出创造性在于用聚天冬酰-RGDF将RGDF输送到血栓形成部位,然后在血栓中有效地释放RGDF,发挥靶向抗血栓作用。
发明内容
本发明的第一个内容是采用常规的液相合成,逐步接肽制备HCl·Arg(Tos)-Gly-Asp(OBzl)-Phe-OBzl。
本发明的第二个内容是采用标准方法,制备平均分子量为20964,链长为173个天冬氨酸残基的聚天冬氨酸。
本发明的第三个内容是采用EDC法将HCl·Arg(Tos)-Gly-Asp(OBzl)-Phe-OBzl偶联到聚天冬氨酸的羧基上然后脱去RGDF的侧链和羧端保护基,制备聚天冬酰-RGDF。
本发明的第四个内容是测定聚天冬酰-RGDF的抗血小板聚集活性。
本发明的第五个内容是测定聚天冬酰-RGDF的抗血栓活性。
本发明的第六个内容是测定聚天冬酰-RGDF对GPIIb/IIIa的影响。
本发明的第七个内容是测定聚天冬酰-RGDF在血栓中释放RGDF。
本发明中所出现的缩略语的说明:
RGDF Arg-Gly-Asp-Phe
PD 聚天冬氨酸
聚天冬酰-RGDF 聚天冬酰-Arg-Gly-Asp-Phe
THF 四氢呋喃
DCC 二环己基酰亚胺
DCU 二环己基脲
OBzl 苄氧基
Boc 叔丁氧羰基
Tos 对甲苯磺酸基
OMe 甲氧基
HOBt N-羟基苯并三唑
NMM N-甲基吗啉
EDC 1-(3-二甲氨基丙基)-3-乙基碳二亚胺
PAF 血小板活性因子
ADP 腺苷二磷酸
AA 花生四烯酸
TE 凝血酶
附图说明
图1.聚天冬酰-RGDF的合成路线.i)85%H3PO4,180℃,减压;ii)NaOH,H2O,0℃;iii)EDC/HOBt;iv)DCC,HoBt,0℃;v)NaOH(2N),H2O,0℃;vi)氯化氢的乙酸乙酯溶液(4N);vii)CF3CO2H,CF3SO3H。
图2.聚天冬酰-RGDF治疗的大鼠血液提取物和血栓提取物的FT-MS谱。
具体实施方式
为了进一步阐述本发明,下面给出一系列实施例。这些实施例完全是例证性的,它们仅用来对本发明进行具体描述,不应当理解为对本发明的限制。
实施例1 制备HCl·Arg(Tos)-Gly-Asp(OBzl)-Phe-OBzl
1)制备Boc-Arg(Tos)-Gly-OBzl
将4.986g(11.64mmol)Boc-Arg(Tos),1.310g(9.7mmol)HOBt溶于30mL无水THF,往得到的溶液中加2.398g(11.64mmol)DCC的无水THF溶液,冰浴搅拌20min,得到反应液I。将3.273g(9.7mmol)Tos·Gly-OBzl与30mL无水THF的溶液用NMM调pH9,得到反应液II。将反应液II加到反应液I中,室温反应12小时。TLC(丙酮∶石油醚=2∶1)显示反应完成。过滤除去DCU沉淀,滤液减压浓缩,残留物用150mL乙酸乙酯溶解,过滤除去DCU沉淀。滤液分别用饱和NaHCO3(50mL×1),5%NaHCO3(50mL×2),饱和NaCl(50mL×3),5%KHSO4(50mL×3),饱和NaCl(50mL×3),5%NaHCO3(50mL×3),饱和NaCl(50mL×3)洗,加无水NaSO4干燥。滤出干燥后的乙酸乙酯溶液,减压浓缩得到的5.339g淡黄色固体粗品用硅胶柱纯化(丙酮∶石油醚=1∶4),得到4.93g(88%)纯品,为无色固体。
2)制备Boc-Arg(Tos)-Gly
在冰浴下将5.291g(9.19mmol)Boc-Arg(Tos)-Gly-OBzl溶于20mL甲醇,往得到的溶液中加14mLNaOH水溶液(2N),搅拌1小时,TLC(丙酮∶石油醚=1∶1)显示反应完成。反应液用饱和KHSO4调pH7,减压浓缩。残留液先用NaOH水溶液(2N)调pH9,再用乙醚萃取。水层用饱和KHSO4调pH2,用50mL乙酸乙酯萃取3次,合并的乙酸乙酯萃取液加无水NaSO4干燥。滤出干燥后的乙酸乙酯溶液,减压浓缩得到3.782g(84%)Boc-Arg(Tos)-Gly,为无色固体。ESI-MS(m/e):377[M+H]+。
3)制备Boc-Asp(OBzl)-Phe-OBzl
按照制备Boc-Arg(Tos)-Gly-OBzl的方法,从2.716g(8.4mmol)Boc-Asp(OBzl)和2.993g(7mmol)Tos·Phe-OBzl,得到2.823g(79%)Boc-Asp(OBzl)-Phe-OBzl,为无色固体。
4)制备HCl·Asp(OBzl)-Phe-OBzl
在冰浴下将1.1g(1.9mmol)Boc-Asp(OBzl)-Phe-OBzl溶于6mL乙酸乙酯中,往得到的溶液中加入20mL氯化氢的乙酸乙酯溶液,搅拌4小时,TLC(丙酮∶石油醚=1∶5)显示反应完成,减压抽去乙酸乙酯。残留物加20mL无水乙醚溶解,溶液减压浓缩。该操作反复5次,以除去产品中的氯化氢。残留物最后加5mL无水乙醚研磨,得到0.787g(90%)产品,为无色粉末,直接用于下一步反应。ESI-MS(m/e):461[M+H]+。
5)制备Boc-Arg(Tos)-Gly-Asp(OBzl)-Phe-OBzl
按照制备Boc-Arg(Tos)-Gly-OBzl的方法,从1398mg(2.9mmol)Boc-Arg(Tos)-Gly和1192mg(2.4mmol)HCl·Asp(OBzl)-Phe-OBzl得到1355mg(61%)Boc-Arg(Tos)-Gly-Asp(OBzl)-Phe-OBzl。ESI-MS(m/e):928[M+H]+.1H NMR(500MHz,CDCl3)d/ppm=7.73(d,2H),7.66(s,1H),7.31(m,9H),7.17(m,7H),7.04(m,2H),6.43(s,2H),5.70(s,1H),5.03(m,4H),4.82(d,J=7.0Hz,1H),4.73(m,1H),3.93(m,1H),3.75(d,J=3.76Hz,1H),3.16(s,1H),3.03(m,2H),2.85(m,1H),2.75(m,1H),2.34(d,3H),1.99(s,1H),1.78(s,1H),1.57(m,3H),1.40(m,10H),1.25(m,1H).13C NMR(125MHz,CDCl3)d/ppm=173.46,171.34,171.22,171.05,170.19,169.55,157.08,157.04,156.00,142.08,140.70,135.98,135.44,135.16,129.23,128.54,128.51,128.37,128.28,128.16,126.94,126.00,80.15,67.15,66.83,60.33,53.95,52.22,49.41,43.26,41.10,40.28,37.43,35.72,29.66,28.36,25.30,21.35,20.97,14.17。
6)制备HCl·Arg(Tos)-Gly-Asp(OBzl)-Phe-OBzl
按照制备HCl·Asp(OBzl)-Val-OBzl的方法,从3.36g(3.62mmol)Boc-Arg(Tos)-Gly-Asp(OBzl)-Phe-OBzl得2.85g(91%)HCl·Arg(Tos)-Gly-Asp(OBzl)-Phe-OBzl固体,直接用于下一步反应。ESI-MS(m/z)817[M+H]+。
实施例2 制备聚天冬氨酸
1)制备聚丁二酰亚胺
将20.0g D,L-Asp和10.4g H3PO4(85%)混匀并在180℃减压(0.1MPa)反应3.5小时。反应物趁热产物用80mL DMF溶解,得到的溶液滴入400mL水中。生成的沉淀用砂芯漏斗抽滤,滤饼磨碎成粉末,用水洗至中性,35℃减压干燥至恒重,得到14.4g(98.8%)聚丁二酰亚胺,为淡灰色粉末。1H NMR(500MHz,CDCl3)d/ppm:5.294(丁二酰亚胺的H-3),3.233(丁二酰亚胺的H-4).13C NMR(125MHz,CDCl3)d/ppm:173.96(丁二酰亚胺的C-3),172.69(丁二酰亚胺的C-1),47.82(丁二酰亚胺的C-4),33.10(丁二酰亚胺的C-5).IR δ/cm-1:1711和1801(丁二酰亚胺的C=O),1160和1213(丁二酰亚胺的C-N)。
2)制备聚天冬氨酸
冰浴下将3g聚丁二酰亚胺和1.4g NaOH用20mL水溶解,搅拌1小时,用浓度为35%的盐酸调pH1,得到的溶液倒入300mL甲醇中。滤出生成的沉淀在40℃真空干燥,得3.2g聚天冬氨酸,为淡黄色固体。采用凝胶渗透色谱-多角激光光散射法测得聚天冬氨酸的数均分子量(Mn)为20964g/mol,重均分子量(Mw)为30476g/mol,分子量分布(d)为1.4537,数均聚合度为173。[α]D 25=1.21(C=0.01,H2O)。13C NMR(125MHz,CDCl3)d/ppm=177.87,173.41,172.75,172.05,171.89,51.76,51.60,39.12,37.61。
实施例3 制备聚天冬酰-Arg(Tos)-Gly-Asp-(OBzl)-Phe-OBzl
先将83mg(0.004mmol)聚天冬氨酸溶于1mL无水DMF,冰浴下加入97mg(0.72mmol)HOBt和360mg(1.87mmol)EDC,0.5小时后加入588mg(0.72mmol)HCl·Arg(Tos)-Gly-Asp(OBzl)-Phe-OBzl,NMM调pH9。室温搅拌24小时,TLC(二氯甲烷∶甲醇,5∶1)显示HCl·Arg(Tos)-Gly-Asp(OBzl)-Phe-OBzl消失。反应混合物减压浓缩至干,残留物依次用乙醚(20mL×3),20mL KHSO4水溶液(5%)和H2O(20mL×3)研磨,得到的固体经37℃真空干燥48小时重599mg,为淡黄色。直接用于下一步反应。
实施例4 制备聚天冬酰-Arg-Gly-Asp-Phe(聚天冬酰-RGDF)
冰浴下将599mg聚天冬酰-Arg(Tos)-Gly-Asp-(OBzl)-Phe-OBzl溶于5mL三氟乙酸,往得到的溶液中加入2mL三氟甲磺酸,搅拌90分钟。反应混合物用乙醚洗(150mL×3),残留物溶于50mL水,用NaOH水溶液(2N)调pH7。得到的混浊液离心30分钟,上层清液用孔径为5000的透析膜透析72小时,截留的残留物冷冻干燥,得174mg(43%)聚天冬酰-RGDF,为乳白色固体。[α]D 25=-21.6(C=0.01,H2O)。13C NMR(125MHz,CDCl3)d/ppm=177.33,171.92,170.50,170.07,156.79,138.48,137.48,129.79,129.43,128.56,126.84,60.52,56.21,53.66,53.09,51.25,42.43,40.58,38.83,37.63,27.96,24.32。按照数均聚合度为173计算,聚天冬酰-RGDF的分子量为96231。
实施例5 测定聚天冬酰-RGDF的抗血小板聚集活性
1)制备2×108血小板/mL血浆
用1%的硅醚溶液将采血容器硅烷化。在硅烷化的容器中收集猪血,按照猪血∶抗凝剂9∶1(体积比)加入枸橼酸钠(3.8%)抗凝,水平缓慢晃匀。将抗凝的猪血在120g离心10分钟,得到的上清液即是富血小板血浆(PRP)。残留的猪血继续在1500g离心10分钟,得到的上清液即是贫血小板血浆(PPP)。光学显微镜下观察,用PPP调PRP使血小板数目为2×108血小板/mL血浆。
2)测定聚天冬酰-RGDF的抗血小板聚集活性(IC50)
在血小板聚集分析仪测量管内加入240μL PRP,轻微搅拌下37℃温育10分钟,调零。大约1分钟后加入5μL生理盐水(空白对照)或西洛他唑的生理盐水溶液(阳性对照,终浓度为1mM)或聚天冬酰-RGDF的生理盐水溶液(终浓度为0.01,0.1,1.0,10,100μM)。大约1分钟后加入5μL ADP的生理盐水溶液(终浓度为500μM)或PAF的生理盐水溶液(终浓度为50μM)或AA的生理盐水溶液(终浓度为7.5mg/mL)或TE的生理盐水溶液(终浓度为50U/mL)。待浊度从100下降进入平台期,记录约6分钟的稳定曲线为最大聚集率,计算各种浓度下聚天冬酰-RGDF对血小板聚集的抑制率。每个浓度平行测6次,求平均值。计算式为抑制率%=(生理盐水存在下的血小板聚集率-聚天冬酰-RGDF存在下的血小板聚集率)/生理盐水存在下的血小板聚集率。按照5种浓度聚天冬酰-RGDF存在下血小板聚集的抑制率,计算IC50值。用IC50表示活性,结果见表1。
表1 聚天冬酰-RGDF对ADP,PAF,AA和TH诱导的血小板聚集的影响(n=6)*
*聚天冬氨酸未测出抗血小板聚集活性.
表1的数据说明,与RGDF相比聚天冬酰-RGDF抑制ADP,PAF,AA和TE诱导的血小板聚集的活性分别高7,408,80和80倍。可见将RGDF连接到聚天冬氨酸上,大大提高了RGDF的抗血小板聚集活性。
实施例6 测定聚天冬酰-RGDF对GPIIb/IIIa的抑制作用
1)制备聚天冬酰-RGDF处理的活化血小板样品
雄性SD大鼠(220~230g)按1200mg/kg剂量腹腔注射乌拉坦水溶液使麻醉。在硅烷化的容器中收集大鼠动脉血,加1/9体积的枸橼酸钠(3.8%)抗凝,水平缓慢晃匀,120g离心10分钟,取上清液,得到PRP血浆。向96μL血浆中加入2μL聚天冬酰-RGDF的生理盐水溶液(终浓度50nM),孵育5分钟,加2μL AA的生理盐水溶液(终浓度7.5mg/mL),37℃温育3分钟。
2)测定聚天冬酰-RGDF对GPIIb/IIIa抑制作用
按照GPIIb/IIIa ELISA试剂盒(上海源叶生物科技有限公司)的标准方法,96孔板室温平衡20分钟,设置标准品孔和聚天冬酰-RGDF孔。绘制标准曲线用的标准品孔先加50μL试剂盒的标准溶液(终浓度为1600,800,400,200,100和0ng/mL),再加100μL辣根过氧化物酶标记的检测抗体。聚天冬酰-RGDV孔先加96μL PRP血浆和10μL聚天冬酰-RGDV的生理盐水溶液(终浓度50nM),37℃孵育5分钟,再加2μL AA的生理盐水溶液(终浓度7.5mg/mL),37℃温育3分钟,后加40μL试剂盒的样本稀释液。RGDF孔先加96μL PRP血浆和10μL RGDF的生理盐水溶液(终浓度25mM),37℃孵育5分钟,再加2μL AA的生理盐水溶液(终浓度7.5mg/mL),37℃温育3分钟,后加40μL试剂盒的样本稀释液。空白孔孔先加96μL PRP血浆和10μL生理盐水,37℃孵育5分钟,再加2μL AA的生理盐水溶液(终浓度7.5mg/mL),37℃温育3分钟,再加40μL试剂盒的样本稀释液,后加100μL试剂盒的辣根过氧化物酶标记的检测抗体。96孔板用封板膜封孔,37℃孵育60分钟。每个孔重复3次。
3)洗板/显色和测定
各孔弃去溶液后在吸水纸上拍干,加满试剂盒的洗涤液,静置1分钟。甩去洗涤液后在吸水纸上拍干,如此重复洗板5次。各孔加入50μL试剂盒的底物A和50μL试剂盒的底物B,37℃避光孵育15分钟。向各孔加50μL试剂盒的终止液,15分钟内在450nm波长处测定各孔的OD值。根据标准曲线计算GPIIb/IIIa的量,结果见表2。可见将RGDF连接到聚天冬氨酸上,大大提高了RGDF抑制GPIIb/IIIa的活性。
表2 聚天冬酰-RGDF对GPIIb/IIIa抑制作用*
*聚天冬酰-RGDF的浓度为1μM,RGDF的浓度为2.5mM,n=3.a)与NS组比p<0.05.
表2的数据说明,聚天冬酰-RGDF可有效地抑制GPIIb/IIIa。与RGDF相比聚天冬酰-RGDF抑制AA活性的血小板上GPIIb/IIIa的活性是RGDF的2500倍。可见将RGDF连接到聚天冬氨酸上,大大提高了RGDF对GPIIb/IIIa的抑制活性。
实施例7 测定聚天冬酰-RGDV的抗血栓活性
1)给药
雄性SD大鼠(220~230g)分别口服生理盐水(3ml/kg),阿斯匹林的生理盐水溶液(167μmol/kg),聚天冬酰-RGDF的生理盐水溶液(1nmol/kg),以及聚天冬氨酸(1nmol/kg)和RGDF(1nmol/kg)的混合物的生理盐水溶液。各10只大鼠,30分钟之后进行下面的手术。
2)大鼠手术与插管
雄性SD大鼠(220~230g)按1200mg/kg剂量腹腔注射乌拉坦溶液进行麻醉。麻醉大鼠仰卧位固定,分离右颈总动脉,于近心端夹动脉夹,近心端和远心端分别穿入手术线,将远心端的手术线于皮毛用止血钳夹紧,准备在远心端插管。
插管为硅烷化过的聚乙烯胶管,分三段。中段为聚乙烯胶管,长6cm,内径3.5mm。另外两段聚乙烯管,长10cm,内径1.0mm,外径2.0mm。它们的一端拉成外径为1.0mm的尖管(用于插入大鼠颈动脉或静脉)。将编好号的干净青霉素小瓶中分别装入6cm长的硅烷化黑色手术线,称重,放入中段插管中。
打开大鼠右侧动脉夹,用注射器通过尖管端将管中注满肝素生理盐水溶液(50IU/kg),然后将插管的动脉端插入大鼠右侧颈总动脉,将计算量的肝素缓缓注入大鼠体内。夹闭右侧颈动脉夹,将插管的静脉端插入分离好的大鼠左侧颈静脉,打开动脉夹,使血液开始循环。并同时开始计时15分钟。
3)血栓称重
15分钟后,剪断动静脉插管,停止循环,用眼科镊小心取出丝线,在滤纸上轻轻蘸掉血滴,放入事先称重好的青霉素小瓶中,精确称重并记录,计算的血栓湿重代表活性,结果见表3。
表3 聚天冬酰-RGDF对在大鼠血栓形成的影响*
*n=10;a)与生理盐水和聚天冬氨酸+RGDV比P<0.01,与阿司匹林比p>0.05;b)与生理盐水比p>0.05.
表3的数据说明,聚天冬酰-RGDF可有效地抑制大鼠形成血栓。与阿司匹林相比聚天冬酰-RGDF的活性是阿司匹林的167000倍。可见将RGDF连接到聚天冬氨酸上,大大提高了RGDF对大鼠形成血栓的抑制活性。
实施例7 测定聚天冬酰-RGDF的血栓靶向作用
收集实施例6中接受聚天冬酰-RGDF治疗的大鼠的500mg血液加1mL枸橼酸钠的水溶液(3.8%)抗凝,4℃和3000g离心10分钟,上清液与2mL甲醇充分混合,于4℃和12000g离心10分钟,取10μL进样测定FT-MS。在血液提取物的质谱图中没有发现任何与聚天冬酰-RGDF相关的峰(图2上)。测定结果说明,聚天冬酰-RGDF从血液中快速向血栓形成部位转移、在血液中不释放RGDF、不代谢。
收集实施例6中接受聚天冬酰-RGDF治疗的大鼠插管中的50mg血栓先用2mL高纯水洗,然后用2mL高纯水制备匀浆。得到的匀浆于4℃和3000g离心10分钟,上清液与2mL甲醇充分混合,于4℃和12000g离心10分钟,取10μL进样测定FT-MS。在血栓提取物的质谱图中看到RGDF加H的峰(987.35746,图2左下)和RGDF加Na的峰(1009.87493,图2右下)。测定结果说明,聚天冬酰-RGDF进入血栓之后,释放RGDF,抑制大鼠血栓形成。可见,聚天冬酰-RGDF具有血栓靶向作用。
Claims (4)
1.具有以下结构的聚天冬氨酰-RGDF
。
2.制备权利要求1所述聚天冬氨酰-RGDF的方法,该方法包括下面四个步骤:
(1) 采用液相合成方法逐步接肽, 合成HCl·Arg(Tos)-Gly-Asp(OBzl)-Phe-OBzl;
(2) 制备具有以下结构的聚天冬氨酸
(3) 把HCl·Arg(Tos)-Gly-Asp(OBzl)-Phe-OBzl连接到上面步骤的聚天冬氨酸的侧链羧基上,制备具有以下结构的聚天冬酰-Arg(Tos)-Gly-Asp(OBzl)-Phe-Obzl
(4) 脱去上面步骤的聚天冬氨酰-Arg(Tos)-Gly-Asp(OBzl)-Phe-OBzl的保护基。
3.权利要求1的聚天冬氨酰-RGDF在制备抗血栓药物中的用途。
4.权利要求3的聚天冬氨酰-RGDF在制备靶向抗血栓药物中的用途。
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