CN1208061C青蒿琥酯和二氢青蒿素在抗血管生成药|CN1208061C Artemether and dihydroartemisinin in anti-angiogenesis drugs.
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CN1208061C青蒿琥酯和二氢青蒿素在抗血管生成药
CN1208061C Artemether and dihydroartemisinin in anti-angiogenesis drugs.
青蒿琥酯和二氢青蒿素在抗血管生成药物中的应用
本发明涉及青蒿琥酯和二氢青蒿素在抗血管生成药物中的应用。该药物制剂主要含有青蒿琥酯或二氢青蒿素，其制剂形式主要为微球注射液。本发明提供的药物制剂在抗肿瘤血管生成方面有活性，可用于肿瘤血管生成及其他与血管生成有关的疾病治疗，还可用于肿瘤化疗和/或辅助化疗方面的治疗。本发明以血管生成理论为背景，研究并阐明中药有效单体成分作用和机制，是发展中医药理论新的重要方向，为发现新的理论和药物作用新的靶点提供重要依据，为青蒿素类药物的新用途开发提供依据。
青蒿琥酯和二氢青蒿素在抗血管生成药物中的应用
技术领域
本发明属药物应用，涉及青蒿素及其衍生物在抗血管生成作用方面的药物用途。
背景技术
血管生成是指已存在的血管(毛细血管和小静脉)通过出芽或分裂的方式产生新的血管。生理与病理条件下，如胚胎发生，女性生殖周期、炎症反应、伤口愈合、肿瘤发生等过程都进行着血管生成。特别是病理条件下，据统计，大约20～40种人类疾病与血管生成的上调或下调有关。1971年，Folkman建立了血管生成与肿瘤生长间联系的学说，提出血管生成不但能为肿瘤细胞提供丰富的营养维持其旺盛的代谢，同时又为肿瘤细胞离开原发病灶通过血液转移创造了有利的条件。许多肿瘤在没有新生血管生成之前只能长到几毫米大小，血管生成被抑制，肿瘤细胞虽仍在高速增殖，但同时进行着快速的细胞凋亡，故能有效地抑制肿瘤的生长和转移。因此，寻找新生血管生长抑制剂已成为国际上近年来抗肿瘤生长和转移治疗的又一重要的方向。抗血管生成药物与其他抗癌药物比较有许多优势，如很少产生耐药性，副作用小，效率高，全球已有40家以上的制药公司在开发研制抗血管生成剂。
自从1988年抗血管生成治疗进入临床试验以来，大约已有20余种血管生成抑制剂进入临床试验，其中有的是针对参与新血管形成的特异分子如干扰素α；有的抑制钙介导的细胞信号转导如钙通道阻滞剂；有的则直接抑制内皮细胞的功能或反应如烟曲霉醇(TNP-470)。综合分析上述作用机制，抑制血管生成的较好靶点是内皮细胞，通过封闭或下调其表面的促血管生长因子受体，抑制内皮细胞的生长，迁移和管腔形成，可高效地破坏新生血管形成。而对于肿瘤血管新生，由于许多恶性瘤细胞本身可分泌促血管生长因子，作用于瘤旁血管内皮细胞的受体，促进肿瘤血管生长。故下调肿瘤细胞内的血管生长因子蛋白表达，抑制其血管生长因子的分泌，也可以达到抗肿瘤血管生长作用。
70年代，我国学者自从中草药黄花蒿中分离出抗疟药青蒿素(artemisinin)后，又陆续合成了蒿甲醚、青蒿琥酯(artesunate)和二氢青蒿素(dihydroartemisinin)等有效衍生物。这类药物的问世，在抗疟药物研究史上树立了新的里程碑。在世界各地上百万起抗疟感染使用中，青蒿素类药物疗效显著，对人体毒副作用小，且迄今尚未见抗药性出现，被世界卫生组织推荐为高效安全的重要抗疟药。各国学者对青蒿素类药物十余年研究中发现，这类药物除了其特殊显著的抗疟疗效外，还具有较强的抗肿瘤作用。
青蒿素(Artemisinin)是我国科学家在1971年首次从菊科植物黄花蒿(Artemisia annua Linn)提出的具有新型结构的倍半萜内酯。它具有十分优良的抗疟作用，包括那些对氯喹有耐药性的恶性疟原虫感染。立即引起世界范围内的重视，由于存在口服活性低，水溶度小，复发率高等缺点，使扩大应用受到限制。因此我国科学家合成了大量的衍生物，根据青蒿素在体内的还原代谢物为二氢青蒿素(Dihydroartemisinin)，将青蒿素C10羰基还原得二氢青蒿素，抗鼠疟(P.berghei)比青蒿素强一倍。将二氢青蒿素进行酯化得青蒿琥酯(Artesunate)，化学名为二氢青蒿素-10-α-丁二酸单酯，其钠盐制成粉针剂，供静注，对疟原虫无性体有较强的杀灭作用。青蒿琥酯在机体内立即转化为还原青蒿素，即二氢青蒿素。由于还原青蒿素不溶于水，对血浆和组织蛋白有较强的结合力，使药物迅速向全身各组织转运并清除。因此，青蒿琥酯具有高效、速效、低毒、不易产生耐受等特点，对间日疟、恶性疟、脑型疟均有效，临床主要适用于脑型疟及各种危重疟疾的治疗。
二氢青蒿素 青蒿琥酯
发明内容
本发明的一个目的是提供青蒿琥酯和/或二氢青蒿素在抗血管生成药物中的应用。
本发明提供的青蒿琥酯或二氢青蒿素，还可在肿瘤化疗和/或辅助化疗方面的应用。
本发明具有以下优点：
(1)青蒿琥酯和二氢青蒿素两者是传统的青蒿类抗疟药中最具有代表性的药物，一直以来用于抗疟治疗，本发明的特点是首次提出青蒿琥酯和二氢青蒿素具有抗血管生成作用，并将他们作为用于肿瘤血管生成及其他血管生成有关的疾病治疗的药物。人类许多疾病与血管生成有关，青蒿素类药物作为血管生成抑制剂将会在这些疾病的治疗中得到重要的应用。
(2)本发明为青蒿素类药物开发成为抗肿瘤药物提供药效学及作用机制的研究依据，具有将青蒿素类药物开发成为肿瘤化疗和/或辅助化疗等类药物的价值。
(3)本发明以血管生成理论为背景，研究并阐明中药有效单体成分作用和机制，是发展中医药理论新的重要方向，为发现新的理论和药物作用新的靶点提供重要依据。
附图说明
图1.为青蒿琥酯对鸡胚尿囊膜血管生成的影响。
图2.1a为HE染色(伊红-苏木素染色)。
图2.1b为血管CD31染色。
图2.2.为青蒿琥酯对VEGF表达的影响。
图2.3.为青蒿琥酯对KDR/flk-1受体表达的影响。
图3.为用药期间裸鼠移植瘤体积变化。
图4为青蒿琥酯和二氢青蒿素对血管内皮细胞增殖的影响。
图5为青蒿琥酯和二氢青蒿素对血管内皮细胞迁移的影响。
图6为青蒿琥酯和二氢青蒿素对血管内皮细胞小管成型的影响。
图7.1为二氢青蒿素对血管内皮细胞迁移的影响。
图7.2为二氢青蒿素对血管内皮细胞小管成型的影响。
具体实施方式
本发明结合具体实施例和附图作进一步说明。应理解，这些实施例仅用于说明目的，而不用于限制本发明范围。
实施例1：制备青蒿琥酯微球注射液的一种方法
用缓释聚合物(Polycaprolactone，PCL)研磨青蒿琥酯，制备成的熔融混合液用生物相容性和生物降解性聚合物制成微球注射液。所用的聚合物释药后水解为对人体无害的降解物(水和二氧化碳)。该微球注射液肌注后，在给药部位缓慢释放药物与吸收，使作用时间延长，可用于肿瘤及其他血管生成有关的疾病治疗。
实施例2：青蒿琥酯微球注射液整体抑制血管生成实验
(1)抑制鸡胚尿囊膜血管生成
实验材料：青蒿琥酯(原料药由广西桂林第二制药厂提供)采用本发明的微球注射液；氢化可的松(浙江仙居制药厂产品)；种蛋(购于杭州四季青名鸡养殖场)。
方法：将7-8天的鸡胚开窗，分别加入青蒿琥酯、阳性对照品(氢化可的松)及溶剂对照品(空白微球注射液)，孵化24h后，用甲醇、丙酮1∶1混合液固定鸡胚尿囊膜，制成标本，在解剖显微镜下计数血管数。
结果：
血管生成抑制率 青蒿琥酯在剂量15、30、60、80μg/胚时，对鸡胚尿囊膜血管生成抑制率分别为50％、55.6％、87.5％及100％。阳性对照组氢化可的松30μg/胚的抑制率66％，溶剂对照组等容积空白微球注射液/胚的抑制率0％。每胚给药容积100μl。结果见表1。
表1青蒿琥酯对鸡胚尿囊膜血管生成的抑制作用
剂量 血管膜阳性数 血管膜 血管膜 抑制率
药品 (μg/胚) + ++ 阴性数 存活数 ％
空白微球液 30 0 0 10 10 0
青蒿琥酯 15 9 0 9 18 50.0
青蒿琥酯 30 8 2 8 18 55.6
青蒿琥酯 60 3 11 2 16 87.5
青蒿琥酯 80 0 10 0 10 100.0
氢化可的松 30 3 5 4 12 66.7
血管计数 溶剂对照组的血管密度、分支明显多于给药组，且管径较粗，参见图1，其中：(a)溶剂对照、(b)青蒿琥酯30μg/胚、(c)氢化可的松30μg/胚。高浓度给药时，只有极少数管径细小的血管、无明显分支状，甚至未见血管，出现溶血现象，个别鸡胚死亡。在青蒿琥酯剂量60、30、15μg/胚时，血管计数分别为3.8、9.7、27.1条，阳性对照组19.5条，溶剂对照组40.5条。与溶剂对照组比较，各组数据呈显著性差异(P＜0.01)。结果见表2。
表2青蒿琥酯对鸡胚尿囊膜血管生成的影响
药品 剂量(μg/胚) n数 血管数( x±s)
空白微球液 30 15 40.6±2.3
青蒿琥酯 15 15 27.1±8.5*
青蒿琥酯 30 15 9.7±2.7*
青蒿琥酯 60 15 3.8±1.6*
氢化可的松 30 15 19.5±5.6*
*P＜0.01vs3％空白微球液，t test
(2)抑制人卵巢癌裸鼠移植瘤血管生成
实验材料：青蒿琥酯(原料药由广西桂林第二制药厂提供)采用本发明的微球注射液。HO-8910人卵巢癌细胞株(由浙江省肿瘤研究所提供)，BALB/C裸小鼠32只，体重(20±2)g，雄性，6～8周龄(由上海市肿瘤研究所提供)。含10％小牛血清的RPMI-1640细胞培养液。血管内皮生长因子(VEGF)多克隆抗体(兔抗人，美国Santa Cruz公司)，VEGFR-2(KDR/flk-1受体)多克隆抗体(兔抗人，美国Santa Cruz公司)，CD31因子单克隆抗体(大鼠抗小鼠，美国Santa Cruz公司)。
方法：
抑制人卵巢癌裸鼠移植瘤模型建立及给药方案 用RPMI-1640加10％小牛血清培养HO-8910人卵巢癌细胞。细胞按1×107/ml悬浮于无血清培养液中，每只小鼠左腋皮下接种0.2ml。约4天左右可形成肉眼可见的瘤块，成瘤率100％。成瘤后第3天开始给药，随机分成4组：青蒿琥酯高剂量组100mg/(kg·d)，中剂量组50mg/(kg·d)，低剂量组10mg/(kg·d)和空白微球注射液组。每组8只，肌肉注射给药，每天1次，连续给药10天，对照组给予等容量的空白微球注射液。末次给药24小时后，处死动物，取出瘤块。
肿瘤体积变化及药物的毒副作用 给药期间观察裸小鼠的一般情况及移植瘤生长情况，每3天测定一次肿瘤体积。测量方法为：用游标卡尺测量肿瘤长轴(a)，短轴(b)，根据公式V＝0.5ab2计算肿瘤体积，绘制肿瘤体积增长曲线。同时测定裸鼠体重。
HE染色和免疫组化 上述肿瘤组织标本经常规处理后制成5μM厚石蜡切片，HE染色，参见图2.1a，常规指标观察。用SABC免疫组化法，采用鼠抗鼠CD31因子抗体，兔抗人VEGF抗体和兔抗人VEGFR-2抗体检测肿瘤标本中血管生成，VEGF和KDR/flk-1受体表达，用BPS代替一抗作阴性。①血管计数：每张切片高倍镜(×200)下随机选择6个视野观察血管并计数，取平均值为每例的最终值。②VEGF及KDR/flk-1受体表达：每张切片高倍镜(×400)下随机选择6个视野，每个视野中计数200个细胞中VEGF与KDR/flk-1受体阳性的细胞个数，小于10％阳性细胞为阴性(-)，10％-30％为弱阳性(+)，30％-70％为中阳性(++)，70％-100％为强阳性(+++)。VEGF在肿瘤细胞浆中表达，KDR/flk-1受体在肿瘤细胞和血管内皮细胞中均有表达，表达部位免疫组化染色呈棕黄色。
结果：
血管计数高剂量组(100mg kg-1d-1)，中剂量组(50mg kg-1d-1)和低剂量组(10mg kg-1d-1)血管CD31染色，结果参见图2.1b。血管计数分别为11.61±4.32，27.75±8.04，47.92±11.49，空白微球注射液组为48.98±9.40，与空白微球注射液对照组相比较高、中剂量组血管计数均明显减少(P＜0.01)，低剂量组无明显差异(P＞0.05)。参见表3。
表3 青蒿琥酯对裸鼠移植瘤血管密度的影响
组别(mg/kg·d) 动物数(n) 血管密度( x±s)
空白微球(10ml/kg.d) 8 48.98±9.40
低剂量10 8 47.92±11.49
中剂量50 8 27.75±8.04**
高剂量100 8 11.61±4.32**
*P＜0.05，**P＜0.01vs空白微球组，t检验
VEGF，KDR/flk-1蛋白表达 治疗组与空白微球注射液组VEGF，KDR/flk-1受体在肿瘤细胞和血管细胞中表达阳性率参见图2.2、图2.3，图2.2中：(a)空白微球注射液组、(b)青蒿琥酯中剂量组、(c)阴性对照组，图2.3中：(a)空白微球注射液组、(b)青蒿琥酯中剂量组、(c)青蒿琥酯高剂量组。高剂量组(100mg/kg·d)，中剂量组(50mg/kg·d)和低剂量组(10mg/kg·d)VEGF，flt-1免疫组化染色阳性率与对照组相比较均有明显差异(P＜0.05)。见表4。
表4青蒿琥酯对肿瘤组织VEGF和 KDR/flt-1表达的影响
组别 动物数 VEGF表达 KDR/flk-1表达
(mg/kg·d) (n) 肿瘤细胞 肿瘤细胞 血管内皮细胞
空白微球 8 +++(8) +++(7)++(1) +++(8)
10 8 +++(4)++(4) +++(3)++(5) +++(2)++(6)*
50 8 ++(1)+(4)-(3)** ++(4)+(3)-(1)** ++(2)+(4)-(2)**
100 8 ++(1)+(2)-(5)** ++(2)+(2)-(4)** ++(1)+(2)-(5)**
*P＜0.05，**P＜0.01vs空白微球组，x2检验
肿瘤体积变化及药物的毒副作用 移植瘤生长及体积变化参见图3，成瘤时(接种4天后)，治疗组与空白微球注射液组瘤体大小差异无显著性(P＞0.05)。用药过程中，治疗组肿瘤生长较慢，用药第1，4，7，10天测定肿瘤体积，高，中剂量组肿瘤体积均小于对照组，有显著性差异(P＜0.05)。用药期间，四组荷瘤裸鼠活动良好，未见特殊不良反应，无一例死亡。用药10天后对照组及高，中，低治疗组平均去瘤鼠重(g)分别为22.1±1.86，22.62±2.4，21.83±2.1，22.25±2.36，治疗组与空白微球注射液对照组比较无显著差异(P＞0.05)。
实施例3：青蒿琥酯和二氢青蒿素体外抑制血管生成实验
实验材料：青蒿琥酯、二氢青蒿素(原料药由广西桂林第二制药厂提供)
方法：
(1)人脐静脉内皮细胞(HUVEC)培养
取新鲜健康的胎儿脐带，灌入5％的胶原酶，37℃水浴15～20min，用PBS液冲洗脐静脉，离心，弃上清液，用培养液悬浮细胞，置培养瓶中培养，隔天换液一次，培养液为DMEM含15％的小牛血清和10ng/ml的VEGF。用兔抗人VIII因子抗体行免疫组化鉴定后取第2～3代细胞供研究用。
(2)HUVEC增殖抑制
将HUVEC以5×104个/ml密度接种于24孔培养板中，置孵箱5％CO2，37℃孵育24小时待完全贴壁后，加入药物(青蒿琥酯或二氢青蒿素)，并设生理盐水和DMSO对照组。每组设3个复孔，作用48小时后，用MTT法检测细胞存活率。
(3)HUVEC迁移抑制
用刀片损伤生长在35mm塑料培养皿中已融合为单层的HUVEC，用PBS液冲洗3次，加入含15％小牛血清的DMEM培养液，然后加入测试药物(青蒿琥酯或二氢青蒿素)，置5％CO2，37℃培养箱中，24小时后从受伤边缘起连续计数(500×250)μM区段中迁移的HUVEC数，数值表示10个随机视野内的细胞数均值。
(4)HUVEC小管形成抑制
I型胶原(Sigma，Bomem Belgium)，DMEM(×10)，0.05M NaOH+0.2MHEPES+0.26MNaHCO2以体积比8∶1∶1：于冰浴下迅速混合，涂布于24孔培养板底部，置37℃形成胶原凝胶，将HUVEC以5×104个/ml的密度悬浮于10％小牛血清DMEM培养液中，每孔加入0.5ml，所用的药物(青蒿琥酯或二氢青蒿素)也在此时以一定的浓度加入，在5％CO2，37℃培养箱中培育48小时，于倒置显微镜下(×200)用曲尺随机测定10个视野中小管的总长度。
结果：
(1)对HUVEC增殖的影响
生理盐水对照组和DMSO对照组OD值为0.71±0.05和0.72±0.05。与相应的溶剂对照组比较，药物浓度为0.5μmol/L时，对HUVEC增殖抑制作用均不明显，青蒿琥酯和二氢青蒿素的OD值为0.70±0.04和0.69±0.07(P＞0.05)；浓度为2.5μmol/L时，二氢青蒿素的OD值为0.66±0.04(P＜0.05)，对HUVEC的增殖有抑制作用；当浓度为12.5和50μmol/L时，二种药物对HUVEC均有明显的增殖抑制作用(P＜0.01)，OD值分别为0.59±0.04，0.42±0.03(青蒿琥酯)和0.50±0.04，0.36±0.03(二氢青蒿素)；OD值下降有剂量依赖性，各用药组间差异有显著性(P＜0.05，n＝3)。药物浓度(μmol/L)与细胞存活率(％)的关系见参图4(n＝3)，其中(▲)青蒿琥酯、(■)二氢青蒿素。
(2)对HUVEC迁移的影响
细胞迁移实验结果表明，在青蒿琥酯和二氢青蒿素的作用下，HUVEC迁移进入受伤裸露区的数量明显减少，参见图5、图7.1，图5中：(▲)青蒿琥酯、(■)二氢青蒿素，图7.1中：(a)溶剂对照、(b)2.5μM二氢青蒿素、(c)50μM二氢青蒿素。
生理盐水和DMSO对照组HUVEC迁移的细胞数为256±28和258±29。当药物浓度为0.5μmol/L时，迁移抑制作用不明显，青蒿琥酯和二氢青蒿素组细胞迁移个数为252±46和241±23(P＞0.05)；当浓度为2.5，12.5，50μmol/L时，用药组与对照组之间有显著性差异(P＜0.01)，青蒿琥酯和二氢青蒿素组迁移细胞数分别为182.6±25，88.7±11，7.6±6和147±18，45±9，1.5±1。药物浓度(μmol/L)与细胞迁移率(％)的关系参见图5(n＝3)。
(3)青蒿琥酯和二氢青蒿素对HUVEC小管成型的影响
HUVEC在胶原凝胶基质中生长2~3天时，细胞由原来的多角形变成长梭形，并向凝胶基质中延伸生长，呈线形排列而形成管状结构，多个管状结构相互连接形成三维网状结构。参见图6、图7.2，图6中：(▲)青蒿琥酯、(■)二氢青蒿素，图7.2中：(a)溶剂对照、(b)12.5μM二氢青蒿素、(c)50μM二氢青蒿素。
48h后，生理盐水和DMSO对照组每个视野中小管总长度为4.90±0.55mm和5.18±0.65mm；当药物浓度为0.5μmol/L时，用药组小管长度与对照组比较均没有显著差异(P＞0.05)；当药物浓度为2.5μmol/L时，二氢青蒿素组小管总长度为3.85±0.35mm，呈现出明显的抑制作用(P＜0.05)；当浓度为12.5，50μmol/L时，二种药物均有显著的小管抑制作用(P＜0.01)。青蒿琥酯和二氢青蒿素组小管总长度分别为3.47±0.32mm，1.30±0.08mm和1.65±0.73mm，0.42±0.02mm。药物浓度(μmol/L)与小管形成率(％)之间的关系参见图6(n＝3)。
本发明涉及的部分参考文献：
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本发明提及的所有文献都在申请中引用作为参考，就如同每一篇文献都被单独引用作为参考那样。此外应理解，本发明是结合最佳实施例进行描述的，然而在阅读了本发明的上述内容后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。
Artesunate and dihydroarteannuin medicine preparation for proofing formation of blood vessel and use thereof
The present invention relates to an artesunate and dihydroartemisinin medicine preparation for resisting angiogenesis and an application thereof. The medicine preparation principally contains artesunate or dihydroartemisinin, and the dosage form principally is a microsphere injection. The medicine preparation provided by the present invention has activity in resisting tumor angiogenesis and can be used for treating tumor angiogenesis and other diseases relevant to angiogenesis, and for therapies, such as tumour chemotherapies and/or adjuvant chemotherapies. The preparation slowly releases medicine and absorbs in administration sites and prolongs medicine action time. The present invention uses an angiogenesis theory as a background, researches and states traditional Chinese medicine effective monomer ingredient action and mechanisms, is new and important direction for developing Chinese medicine theories, provides important reference for discovering new theories and medicine action new targets and provides reference for the development of the new applications of artemisinin medicine.
The application in anti-angiogenic medicaments of artesunate and dihydroartemisinine
Technical field
The invention belongs to medicinal application, relate to arteannuin and derivant thereof the medicinal usage aspect blood vessel formation against function.
Background technology
Angiogenesis is meant that already present blood vessel (blood capillary and venule) is by sprouting or splitted mode produces new blood vessel.Under physiology and the pathological conditions, take place as the embryo, processes such as female reproduction cycle, inflammatory reaction, wound healing, tumor generation are all being carried out angiogenesis.Particularly under the pathological conditions, according to statistics, the rise of about 20～40 kinds of human diseasess and angiogenesis or reduce relevant.1971, Folkman has set up the theory of getting in touch between angiogenesis and tumor growth, propose angiogenesis and not only can keep its vigorous metabolism, created advantageous conditions for tumor cell leaves primary lesion by blood transfer again simultaneously for tumor cell provides abundant nutrition.Many tumors are not having can only to grow several millimeters sizes before the new vessels generation, and angiogenesis is suppressed, though tumor cell is carrying out apoptosis fast, so can suppress growth of tumor and transfer effectively simultaneously still breeding at a high speed.Therefore, seek the new vessels growth inhibitor and become in the world neoplasm growth and the another important direction that shifts treatment in recent years.Anti-angiogenic medicaments and other cancer therapy drugs relatively have many advantages, and as seldom producing drug resistance, side effect is little, the efficient height, and the whole world above drugmaker of existing 40 families is developing anti-angiogenic agent.
Since angiogenesis inhibitor treatment in 1988 entered clinical trial, approximately kind of an angiogenesis inhibitor entered clinical trial surplus in the of existing 20, wherein had plenty of at the special molecular such as the interferon-ALPHA that participate in neovascularization; The cell signalling such as the calcium channel blocker of the inhibition calcium mediation that has; Function that then directly suppresses endotheliocyte that has or reaction are as aspergillus fumigatus cedrol (TNP-470).The above-mentioned mechanism of action of analysis-by-synthesis, the better target spot that suppresses angiogenesis is an endotheliocyte, by sealing or reduce its surperficial angiogenic growth factor receptor, suppresses the growth of endotheliocyte, migration and tube chamber form, and can destroy new vessels efficiently and form.And,, act on the receptor of the other vascular endothelial cell of tumor because many malignant tumor cells itself can be secreted angiogenic growth factor for neonate tumour blood vessel, promote tumor vascular growth.So the angiogenesis factor protein expression in the downward modulation tumor cell suppresses the secretion of its angiogenesis factor, also can reach antineoplastic vascular growth effect.
The seventies, Chinese scholar is isolated antimalarial arteannuin (artemisinin) in Chinese herbal medicine Hemerocallis citrina Baroni Artemisia after, Artemether, artesunate (artesunate) and dihydroartemisinine effective derivants such as (dihydroartemisinin) have been synthesized again successively.New milestone is set up in the appearance of this class medicine on the antimalarial agent research history.In up to a million malaria infection were used all over the world, artemisinin-based drug is evident in efficacy, and was little to the human body toxic and side effects, and do not see as yet that so far Drug resistance occurs, and is the important antimalarial of highly effective and safe by world health organisation recommendations.The various countries scholar is to finding that this class medicine also has stronger antitumor action except its special significant malaria curative effect in the research in surplus the artemisinin-based drug ten year.
Arteannuin (Artemisinin) is the sesquiterpene lactones with new structure that China scientist proposed from feverfew Herba Artemisiae annuae (Artemisia annua Linn) first in 1971.It has very good malaria effect, comprises that those have chemical sproof falciparum infection to chloroquine.Cause worldwide attention immediately, owing to exist Orally active low, water-soluble degree is little, and shortcomings such as relapse rate height make to enlarge to use to be restricted.Therefore China scientist has synthesized a large amount of derivants, is dihydroartemisinine (Dihydroartemisinin) according to arteannuin reductive metabolites in vivo, with arteannuin C 10Carbonyl reduction gets dihydroartemisinine, and anti-Mus malaria (P.berghei) is stronger one times than arteannuin.Dihydroartemisinine is carried out esterification get artesunate (Artesunate), chemistry dihydroartemisinine by name-10-α-succinate monoester, its sodium salt is made injectable powder, for quiet notes, the plasmodium phorozoon is had stronger killing action.Artesunate is converted into dihydroartemisinine, i.e. dihydroartemisinine immediately in body.Because dihydroartemisinine is water insoluble, and blood plasma and histone are had stronger adhesion, make medicine rapidly to whole body each tissue transhipment and removing.Therefore, that artesunate has is efficient, quick-acting, low toxicity, be difficult for producing characteristics such as tolerance, all effective to tertian malaria, subtertian malaria, cerebral malaria, the clinical treatment that mainly is applicable to cerebral malaria and various critical malaria.
The dihydroartemisinine artesunate
Summary of the invention
An object of the present invention is to provide the application in anti-angiogenic medicaments of artesunate and/or dihydroartemisinine.
Artesunate provided by the invention or dihydroartemisinine also can be in the application aspect chemotherapy of tumors and/or the adjuvant chemotherapy.
The present invention has the following advantages:
(1) artesunate and dihydroartemisinine are the most representative medicines in the traditional Herba Artemisiae Annuae class antimalarial, be used for the malaria treatment all the time, characteristics of the present invention are to propose artesunate first and dihydroartemisinine has blood vessel formation against function, and with their medicine as the disease treatment that is used for tumor-blood-vessel growth and other associated angiogenesis.Human numerous disease and associated angiogenesis, artemisinin-based drug will obtain important use as angiogenesis inhibitor in these treatment of diseases.
(2) the present invention provides pharmacodynamics and Its Mechanisms foundation for the artemisinin-based drug exploitation becomes antitumor drug, has the value with artemisinin-based drug exploitation becoming class medicines such as chemotherapy of tumors and/or adjuvant chemotherapy.
(3) the present invention is a background with the angiogenesis theory, studies and illustrate effect of Chinese medicine effective monomer component and mechanism, is the new important directions of developing Chinese medicine pharmacology opinion, provides important evidence for finding the new target spot of new theory and drug effect.
Description of drawings
Fig. 1. be the influence of artesunate to the chick chorioallantoic membrane angiogenesis.
Fig. 2 .1a is HE dyeing (Yihong-haematoxylin dyeing).
Fig. 2 .1b is blood vessel CD 31Dyeing.
Fig. 2 .2. is the influence of artesunate to vegf expression.
Fig. 2 .3. is the influence of artesunate to the KDR/flk-1 expression of receptor.
Fig. 3. be transplanted tumor in nude mice change in volume during the medication.
Fig. 4 is the influence to vascular endothelial cell proliferation of artesunate and dihydroartemisinine.
Fig. 5 is the influence to migration of vascular endothelial cells of artesunate and dihydroartemisinine.
Fig. 6 is the influence to the molding of vascular endothelial cell tubule of artesunate and dihydroartemisinine.
Fig. 7 .1 is the influence of dihydroartemisinine to migration of vascular endothelial cells.
Fig. 7 .2 is the influence of dihydroartemisinine to the molding of vascular endothelial cell tubule.
The specific embodiment
The present invention is described further with accompanying drawing in conjunction with specific embodiments.Should be understood that these embodiment only are used for illustration purpose, and be not used in the restriction scope of the invention.
Embodiment 1: a kind of method of preparation artesunate microsphere injection liquid
(Polycaprolactone PCL) grinds artesunate, and the melting mixing liquid that is prepared into is made microsphere injection liquid with biocompatibility and Biodegradable polymer with release polymer.Used polymer release posthydrolysis is harmless degradation product (water and carbon dioxide).After this microsphere injection liquid intramuscular injection, slowly discharge medicine and absorption, prolonged action time, can be used for the disease treatment of tumor and other associated angiogenesis at medicine-feeding part.
Embodiment 2: the whole angiogenesis that suppresses of artesunate microsphere injection liquid is tested
(1) suppresses the chick chorioallantoic membrane angiogenesis
Experiment material: artesunate (crude drug is provided by Guilin second pharmaceutical factory) adopts microsphere injection liquid of the present invention; Hydrocortisone (Xianju, Zhejiang pharmaceutical factory product); Hatching egg (purchasing) in Hangzhou Ilex purpurea Hassk.[I.chinensis Sims name chicken plant.
Method: 7-8 days Embryo Gallus domesticus is windowed, add artesunate, positive reference substance (hydrocortisone) and solvent control product (blank microsphere injection liquid) respectively, behind the hatching 24h, with the fixing chick chorioallantoic membrane of 1: 1 mixed liquor of methanol, acetone, make specimen, counting blood vessel number under anatomic microscope.
The result:
The angiogenesis suppression ratioArtesunate is respectively 50%, 55.6%, 87.5% and 100% to chick chorioallantoic membrane angiogenesis suppression ratio when dosage 15,30,60,80 μ g/ embryos.The suppression ratio 66% of positive controls hydrocortisone 30 μ g/ embryos, the suppression ratio 0% of the blank microsphere injection liquid/embryo of solvent control group isometric(al).Every embryo administration volume 100 μ l.The results are shown in Table 1.
Table 1 artesunate is to the inhibitory action of chick chorioallantoic membrane angiogenesis
Dosage The tunica vasculose number positiveTunica vasculose tunica vasculose suppression ratio
Medicine (μ g/ embryo)+++ negative number survival number %
Blank microsphere liquid 30 00 10 10 0
Artesunate 15 909 18 50.0
Artesunate 30 828 18 55.6
Artesunate 60 3 11 2 16 87.5
Artesunate 80 0 10 0 10 100.0
Hydrocortisone 30 354 12 66.7
Vascular countsThe vessel density of solvent control group, branch are obviously more than the administration group, and caliber is thicker, referring to Fig. 1, wherein: (a) solvent control, (b) artesunate 30 μ g/ embryos, (c) hydrocortisone 30 μ g/ embryos.During the high concentration administration, have only the tiny blood vessel of only a few caliber, do not have obvious branch-like, even do not see and haemolysis occurs by blood vessel, indivedual chicken embryo deaths.When artesunate dosage 60,30,15 μ g/ embryos, vascular counts is respectively 3.8,9.7,27.1,19.5 of positive controls, 40.5 of solvent control groups.Compare with the solvent control group, each is organized data and is significant difference (P＜0.01).The results are shown in Table 2.
Table 2 artesunate is to the influence of chick chorioallantoic membrane angiogenesis
Drug dose (μ g/ embryo) n counts blood vessel number (x ± s)
Blank microsphere liquid 30 15 40.6 ± 2.3
Artesunate 15 15 27.1 ± 8.5 *
Artesunate 30 15 9.7 ± 2.7 *
Artesunate 60 15 3.8 ± 1.6 *
*The blank microsphere liquid of P＜0.01vs3%, t test
(2) suppress human ovarian cancer transplanted tumor in nude mice angiogenesis
Experiment material: artesunate (crude drug is provided by Guilin second pharmaceutical factory) adopts microsphere injection liquid of the present invention.HO-8910 human oophoroma cell line (providing) by the institute of oncology, Zhejiang Province, 32 of BALB/C nude mouses, body weight (20 ± 2) g, male, 6～8 ages (providing) in week by the Shanghai Inst. of Tumor.The RPMI-1640 cell culture fluid that contains 10% calf serum.VEGF (VEGF) polyclonal antibody (the anti-people of rabbit, U.S. Santa Cruz company), VEGFR-2 (KDR/flk-1 receptor) polyclonal antibody (the anti-people of rabbit, U.S. Santa Cruz company), CD 31Factor monoclonal antibodies (rat anti-mouse, U.S. Santa Cruz company).
Method:
Suppress human ovarian cancer transplanted tumor in nude mice modelling and dosage regimen Add 10% calf serum with RPMI-1640 and cultivate the HO-8910 Proliferation of Human Ovarian Cell.Cell is by 1 * 10 7/ ml is suspended in the serum-free medium, every mice left side axil subcutaneous vaccination 0.2ml.Can form macroscopic tumor piece, tumor formation rate 100% in about about 4 days.Beginning administration in the 3rd day is divided into 4 groups: artesunate high dose group 100mg/ (kgd), middle dosage group 50mg/ (kgd), low dose group 10mg/ (kgd) and blank microsphere injection liquid group at random after the one-tenth tumor.Every group 8, administered intramuscular, every day 1 time, successive administration 10 days, matched group wait the blank microsphere injection liquid of capacity.After the last administration 24 hours, put to death animal, take out the tumor piece.
The toxic and side effects of gross tumor volume variation and medicineObserve ordinary circumstance and the growth of xenografted situation of nude mouse during the administration, gross tumor volume of per 3 days mensuration.Measuring method is: with vernier caliper measurement tumor major axis (a), minor axis (b) is according to formula V=0.5ab 2Calculate gross tumor volume, draw the gross tumor volume growth curve.Measure the nude mice body weight simultaneously.
HE dyeing and SABCAbove-mentioned tumor tissues specimen is made the thick paraffin section of 5 μ M after routine is handled, HE dyeing, and referring to Fig. 2 .1a, conventional index is observed.With SABC SABC method, adopt mouse-anti Mus CD 31Factor antibody, rabbit anti-people VEGF antibody and rabbit human VEGFR-3 resistant-2 antibody test tumor specimen medium vessels generate, and VEGF and KDR/flk-1 expression of receptor replace an anti-feminine gender of doing with BPS.1. vascular counts: select 6 visuals field to observe blood vessels and counting under every section high power lens (* 200) at random, averaging is the end value of every example.2. VEGF and KDR/flk-1 expression of receptor: select 6 visuals field at random under every section high power lens (* 400), the number of cells of VEGF and KDR/flk-1 receptor positive in 200 cells of counting in each visual field, less than 10% positive cell negative (-), 10%-30% is the weak positive (+), 30%-70% is the middle positive (++), and 70%-100% is strong positive (+++).VEGF expresses in the tumor cell slurry, and the KDR/flk-1 receptor all has expression in tumor cell and vascular endothelial cell, and the expressive site immunohistochemical staining is pale brown color.
The result:
Vascular countsHigh dose group (100mg kg -1d -1), middle dosage group (50mg kg -1d -1) and low dose group (10mg kg -1d -1) blood vessel CD 31Dyeing, the result is referring to Fig. 2 .1b.Vascular counts is respectively 11.61 ± 4.32,27.75 ± 8.04,47.92 ± 11.49, blank microsphere injection liquid group is 48.98 ± 9.40, compare higher, middle dosage group vascular counts with blank microsphere injection liquid matched group and all obviously reduce (P＜0.01), low dose group no significant difference (P＞0.05).Referring to table 3.
Table 3 artesunate is to the influence of transplanted tumor in nude mice vessel density
Group (mg/kgd) number of animals (n) vessel density (x ± s)
Blank microsphere (10ml/kg.d) 8 48.98 ± 9.40
Low dosage 10 8 47.92 ± 11.49
*P＜0.05, *The blank microsphere group of P＜0.01vs, the t check
VEGF, the KDR/flk-1 protein expressionTreatment group and blank microsphere injection liquid group VEGF, the KDR/flk-1 receptor in tumor cell and vascular cell The positive expression rate referring to Fig. 2 .2, Fig. 2 .3, among Fig. 2 .2: (a) dosage group, (c) negative control group in blank microsphere injection liquid group, (b) artesunate, among Fig. 2 .3: (a) dosage group, (c) artesunate high dose group in blank microsphere injection liquid group, (b) artesunate.High dose group (100mg/kgd), middle dosage group (50mg/kgd) and low dose group (10mg/kgd) VEGF, flt-1 immunohistochemical staining positive rate compare with matched group all notable difference (P＜0.05).See Table 4.
Table 4 artesunate to tumor tissues VEGF and KDR/The influence that flt-1 expresses
Group number of animals vegf expression KDR/Flk-1 expresses
(mg/kgd) (n) tumor cell tumor cell vascular endothelial cell
Blank microsphere 8 +++(8) +++(7) ++ (1) +++(8)
10 8 +++(4)++(4) +++(3)++(5) +++(2)++(6) *
50 8 ++(1)+(4)-(3) ** ++(4)+(3)-(1) ** ++(2)+(4)-(2) **
100 8 ++(1)+(2)-(5) ** ++(2)+(2)-(4) ** ++(1)+(2)-(5) **
*P＜0.05, *The blank microsphere group of P＜0.01vs, x 2Check
The toxic and side effects of gross tumor volume variation and medicineGrowth of xenografted and change in volume are referring to Fig. 3, and when becoming tumor (inoculating after 4 days), treatment group and blank microsphere injection liquid group tumor body difference in size do not have significance (P＞0.05).In the medication process, treatment group tumor growth is slower, and medication the 1st, 4 was measured gross tumor volume in 7,10 days, and height, middle dosage group gross tumor volume have significant difference (P＜0.05) all less than matched group.During the medication, four groups of tumor bearing nude mices are movable good, do not see special untoward reaction, and none example is dead.Medication is matched group and height after 10 days, in, low treatment group on average goes tumor Mus heavy (g) to be respectively 22.1 ± 1.86,22.62 ± 2.4, and 21.83 ± 2.1,22.25 ± 2.36, treatment group and blank microsphere injection liquid matched group relatively do not have significant difference (P＞0.05).
Embodiment 3: artesunate and the experiment of dihydroartemisinine vitro inhibition angiogenesis
Experiment material: artesunate, dihydroartemisinine (crude drug is provided by Guilin second pharmaceutical factory)
Method:
(1) Human umbilical vein endothelial cells (HUVEC) is cultivated
Get the fetal cord of fresh and healthy, pour into 5% collagenase, 37 ℃ of water-bath 15～20min are with PBS liquid flushing umbilical vein, centrifugal, abandon supernatant, use the culture fluid suspension cell, put in the culture bottle and cultivate, change liquid every other day once, culture fluid is that DMEM contains 15% calf serum and the VEGF of 10ng/ml.Use with getting the cell confession research of the 2nd～3 generation after the evaluation of the anti-people VIII of rabbit factor antibody row SABC.
(2) HUVEC propagation suppresses
With HUVEC with 5 * 10 4Individual/ml density is inoculated in 24 well culture plates, puts incubator 5%CO 2, 37 ℃ hatch treated in 24 hours fully adherent after, add medicine (artesunate or dihydroartemisinine), and establish normal saline and DMSO matched group.Establish 3 multiple holes for every group, act on after 48 hours, detect cell survival rate with mtt assay.
(3) the HUVEC migration suppresses
Be grown in the HUVEC that has been fused to monolayer in the 35mm plastic culture dish with the blade damage,, add the DMEM culture fluid that contains 15% calf serum, add testing drug (artesunate or dihydroartemisinine) then, put 5%CO with PBS liquid flushing 3 times 2, in 37 ℃ of incubators, the HUVEC number that from injured edge continuous counter (500 * 250) μ M section, moves after 24 hours, 10 of numeric representations are the cell number average in the visual field at random.
(4) the HUVEC tubule forms and suppresses
Type i collagen (Sigma, Bomem Belgium), DMEM (* 10), 0.05M NaOH+0.2MHEPES+0.26MNaHCO 2With volume ratio 8: 1: 1: mix rapidly down in ice bath, coat 24 well culture plates bottom, put 37 ℃ and form collagen gels, with HUVEC with 5 * 10 4The density of individual/ml is suspended in the 10% calf serum DMEM culture fluid, and every hole adds 0.5ml, and used medicine (artesunate or dihydroartemisinine) also adds with certain concentration at this moment, at 5%CO 2, cultivated 48 hours in 37 ℃ of incubators, (* 200) measure the total length of tubule in 10 visuals field at random with trisquare under inverted microscope.
The result:
(1) influence that HUVEC is bred
Normal saline matched group and DMSO matched group OD value are 0.71 ± 0.05 and 0.72 ± 0.05.Compare with the corresponding solvent matched group, all not obvious to the HUVEC inhibited proliferation when drug level is 0.5 μ mol/L, the OD value of artesunate and dihydroartemisinine is 0.70 ± 0.04 and 0.69 ± 0.07 (P＞0.05); When concentration was 2.5 μ mol/L, the OD value of dihydroartemisinine was 0.66 ± 0.04 (P＜0.05), and the propagation of HUVEC is had inhibitory action; When concentration was 12.5 and 50 μ mol/L, two kinds of medicines all had tangible inhibited proliferation (P＜0.01) to HUVEC, and the OD value is respectively 0.59 ± 0.04,0.42 ± 0.03 (artesunate) and 0.50 ± 0.04,0.36 ± 0.03 (dihydroartemisinine); The OD value descend have dose dependent, each medication group difference have significance (P＜0.05, n=3).Drug level (μ mol/L) is seen ginseng Fig. 4 (n=3), wherein (▲) artesunate, (■) dihydroartemisinine with the relation of cell survival rate (%).
(2) influence that HUVEC is moved
The cell migration experimental result shows, under the effect of artesunate and dihydroartemisinine, the quantity that the HUVEC migration enters injured exposed area obviously reduces, referring to Fig. 5, Fig. 7 .1, among Fig. 5: (▲) artesunate, (■) dihydroartemisinine, among Fig. 7 .1: (a) solvent control, (b) 2.5 μ M dihydroartemisinines, (c) 50 μ M dihydroartemisinines.
The cell number of normal saline and DMSO matched group HUVEC migration is 256 ± 28 and 258 ± 29.When drug level was 0.5 μ mol/L, the migration inhibitory action was not obvious, and artesunate and dihydroartemisinine group cell migration number are 252 ± 46 and 241 ± 23 (P＞0.05); When concentration is 2.5,12.5,50 μ mol/L, significant difference (P＜0.01) is arranged between medication group and the matched group, artesunate and dihydroartemisinine group migrating cell number are respectively 182.6 ± 25,88.7 ± 11,7.6 ± 6 and 147 ± 18,45 ± 9,1.5 ± 1.The relation of drug level (μ mol/L) and cell migration rate (%) is referring to Fig. 5 (n=3).
(3) artesunate and dihydroartemisinine are to the influence of HUVEC tubule molding
HUVEC grew in collagen gel substrate 2 ~ 3 days the time, and cell becomes spindle shape by original polygon, and in gel-type vehicle elongation growth, be linear array and form tubular structure, a plurality of tubular structures are interconnected to form tridimensional network.Referring to Fig. 6, Fig. 7 .2, among Fig. 6: (▲) artesunate, (■) dihydroartemisinine, among Fig. 7 .2: (a) solvent control, (b) 12.5 μ M dihydroartemisinines, (c) 50 μ M dihydroartemisinines.
Behind the 48h, the tubule total length is 4.90 ± 0.55mm and 5.18 ± 0.65mm in normal saline and each visual field of DMSO matched group; When drug level was 0.5 μ mol/L, little length of tube of medication group and matched group did not more all have significant difference (P＞0.05); When drug level was 2.5 μ mol/L, dihydroartemisinine group tubule total length was 3.85 ± 0.35mm, presented obvious suppression effect (P＜0.05); When concentration was 12.5,50 μ mol/L, two kinds of medicines all had significant tubule inhibitory action (P＜0.01).Artesunate and dihydroartemisinine group tubule total length are respectively 3.47 ± 0.32mm, 1.30 ± 0.08mm and 1.65 ± 0.73mm, 0.42 ± 0.02mm.Relation between drug level (μ mol/L) and the tubule formation rate (%) is referring to Fig. 6 (n=3).
The partial reference document that the present invention relates to:
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All documents that the present invention mentions are all quoted in application as a reference, are just all quoted as a reference separately as each piece document.Should understand in addition, the present invention is described in conjunction with most preferred embodiment, yet after having read foregoing of the present invention, those skilled in the art can make various changes or modifications the present invention, and these equivalent form of values fall within the application's appended claims institute restricted portion equally.
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