CN113456627A - 青蒿素二倍体衍生物复合物、药物组合物及应用 - Google Patents
青蒿素二倍体衍生物复合物、药物组合物及应用 Download PDFInfo
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- CN113456627A CN113456627A CN202110738310.3A CN202110738310A CN113456627A CN 113456627 A CN113456627 A CN 113456627A CN 202110738310 A CN202110738310 A CN 202110738310A CN 113456627 A CN113456627 A CN 113456627A
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- artemisinin
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Images
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Abstract
本发明公开了一种青蒿素二倍体衍生物复合物、药物组合物及应用，该复合物为由式(1)所示的青蒿素二倍体衍生物自组装胶束为核与阴离子型聚合物为壳复合而成，其粒径为10纳米～1000纳米。该复合物化学稳定性及血液相容性良好，对细胞及生物体毒性低，体内外抗疟效果优异。该复合物与药学上可接受的载体进一步形成药物组合物。上述复合物及药物组合物可制备为抗寄生虫病药物，特别适合用于制备高效治疗疟疾的相关药物。
Description
技术领域
本发明涉及一种青蒿素二倍体衍生物复合物、药物组合物及应用，尤其涉及一种低毒高效的青蒿素二倍体衍生物复合物、药物组合物及应用。
背景技术
寄生虫病是寄生虫侵入人体而引起的疾病。因虫种和寄生部位不同，引起的病理变化和临床表现各异。本类疾病分布广泛，世界各地均可见到，以贫穷落后、卫生条件差的地区多见，热带和亚热带地区更多，感染的人群主要是接触疫源较多的劳动人民及免疫力较低的儿童。
疟疾是热带地区最重要的寄生虫疾病，也是疟原虫寄生于人体所引起的一种传染病。患者经疟蚊叮咬或输入带疟原虫者的血液而感染，不同的疟原虫分别引起间日疟、三日疟、恶性疟及卵圆疟。疟疾主要表现为周期性规律发作，全身发冷、发热、多汗、长期多次发作后，可引起贫血和脾肿大。在严重的病例中会引起黄疸、癫痫发作、昏迷或死亡。奎宁最早用于治疗疟疾，氯喹也是非常有效的抗疟疾药物。20世纪中期以后出现了一些新的药物，中国科学家研制的青蒿素、双氢青蒿素等有很好的抗疟疾效果，但是青蒿素、双氢青蒿素(DHA)、青蒿琥酯的水溶性均非常差。
由于青蒿素及其衍生物均为速效药物，体内稳定性差、半衰期较短。此外，大多数青蒿素衍生物溶解性较低，制剂剂型相对比较局限。目前临床可用注射剂仅青蒿琥酯(ARS)一种，使用时需要临时用碳酸氢钠溶液配制，操作复杂。一旦进入血液，青蒿琥酯会在几分钟内转化为活跃的代谢物DHA。而生成的DHA循环的半衰期有限。由于半衰期较短，青蒿琥酯需要多次大剂量给药，这就带来了产生青蒿素耐药性的风险。尽管青蒿琥酯单一疗法仍被用于严重疾病的初期管理，但世卫组织已建议在全球范围内完全停用基于青蒿琥酯的单一疗法来治疗无并发症疟疾。
目前疟疾逐渐对于几种药物发展出抗药性，例如：具有氯喹抗药性的恶性疟已经遍及世界各地。对两种药物磺胺多辛/乙胺嘧啶联合使用的抗药性在南亚和南美用氯喹难治的地带也已经扩散。另外，青蒿素的抗药性问题在部分东南亚地区日益严重。近年来，柬埔寨西部和北部地区、泰国、越南和缅甸东部地区，疟原虫已普遍发展出对最有效的抗疟药物青蒿素的抗药性；在缅甸中部地区、老挝南部地区和柬埔寨东北部地区，疟原虫也开始出现对青蒿素的抗药性。鉴于已出现的对现有多种抗疟疾药物的抗药性，目前疟疾的建议治疗方法是并用青蒿素及另一种抗疟疾药物，包括甲氟喹、苯芴醇或磺胺多辛/乙胺嘧啶。
发明内容
发明目的：本发明的第一目的是提供一种青蒿素二倍体衍生物复合物，第二目的是提供一种含有上述青蒿素二倍体衍生物复合物的药物组合物，第三目的是提供上述复合物及药物组合物的应用。
技术方案：本发明的青蒿素二倍体衍生物复合物由式(1)所示的青蒿素二倍体衍生物自组装胶束为核与阴离子型聚合物为壳复合而成，所述复合物的粒径为10纳米～1000纳米：
式(1)中，x为2～10的整数。
本发明的青蒿素二倍体衍生物自组装胶束为核，表面覆盖阴离子型聚合物，成为纳米复合物，具有良好的水溶性，稳定性优异，体内循环时间长，无毒性，可用作液体制剂、固体制剂，可在水、磷酸缓冲液、柠檬酸缓冲液等水相体系下形成纳米颗粒溶液，直接用于注射治疗。
其中，所述青蒿素二倍体衍生物自组装胶束与阴离子型聚合物的质量比为1∶2～1∶10。
所述阴离子聚合物为透明质酸钠、硫酸软骨素或肝素钠。
具体地，所述透明质酸的分子量为10kDa～200kDa，所述硫酸软骨素的分子量为10kDa～200kDa，所述肝素钠的分子量为5kDa～15kDa。透明质酸、硫酸软骨素和肝素钠均具有大量带负电荷的羧基官能团，上述青蒿素二倍体自组装胶束具有带正电荷的季铵盐官能团；在形成的复合物中，蒿素二倍体自组装胶束作为核，阴离子型聚合物作为壳。
本发明还公开了一种青蒿素二倍体衍生物药物组合物，所述药物组合物包含如前所述的复合物和药学上可接受的载体。
具体地，所述复合物为纳米颗粒，所述纳米颗粒的粒径为10纳米～1000纳米。
更具体地，所述药物组合物为颗粒剂、凝胶剂、注射剂、缓释制剂或控释制剂。
本发明的药物组合物可根据本领域公知的方法制备。用于此目的时，如果需要，可将本发明复合物或本发明的药物组合物与一种或多种固体或液体药物赋形剂和/或辅剂结合，制成可作为人药使用的适当的施用形式或剂量形式。
本发明的药物组合物的给药途径可为注射给药，包括静脉注射、肌肉注射、皮下注射、皮内注射和穴位注射等；给药剂型可以是液体剂型、固体剂型。
上述复合物或药物组合物可应用于制备抗寄生虫病药物。更具体应用制备选自如下疾病药物：疟疾、血吸虫病、弓形虫病、利什曼病、丝虫病或钩虫病。
从活性筛选来看，本发明的复合物表现良好的杀灭疟原虫作用。试验表明本发明的复合物的无明显体内毒性。因此可作为抗疟疾药物用于动物，优选用于哺乳动物，特别是人。
有益效果：与现有技术相比，本发明具有如下显著优点：
(1)制备的青蒿素二倍体衍生物复合物具有良好的水溶性、稳定性高(24小时后依然稳定，无明显降解)，体内循环时间长，与血液相容性好，对细胞和生物体低毒，体内外抗疟活性优异(优于青蒿琥酯)；
(2)上述复合物可制备为多种形式的药物组合物，应用广泛，特别适用于人用药物。
附图说明
图1为二青蒿琥酯-3-季铵盐-1,2-丙二醇酯(dACC)的合成路线；
图2a为二青蒿琥酯-3-二甲胺基-1,2-丙二醇酯的质谱图；
图2b为二青蒿琥酯-3-二甲胺基-1,2-丙二醇酯的核磁谱图；
图3a为二青蒿琥酯-3-季铵盐-1,2-丙二醇酯(dACC)的质谱图；
图3b为二青蒿琥酯-3-季铵盐-1,2-丙二醇酯(dACC)的核磁谱图；
图4a为dACC的临界聚集浓度(CAC)曲线；
图4b为dACC组装体透射电镜图(TEM)；
图4c为dACC组装体加入透明质酸(HA)后的透射电镜图；
图5a为双氢青蒿素(DHA)、青蒿琥酯(ARS)和dACC在PBS中孵育0、3、24小时后孵育液的高效液相色谱(HPLC)流出曲线；
图5b为DHA、ARS和dACC在PBS孵育液中，HPLC流出曲线积分面积计算的剩余的DHA与dAPC百分比；
图6a为ARS、dACC和dACC/HA体外溶血实验测试；
图6b为ARS、dACC和dACC/HA体外细胞毒性测试；
图6c为ARS、dACC和dACC/HA体内半致死量测试；
图7a为ARS、dACC和dACC/HA对恶性疟原虫3D7标准株的药效；
图7b为ARS、dACC和dACC/HA在不同培养基中孵育3、24h后的药效。
具体实施方式
下面结合实施例对本发明的技术方案作进一步说明。
以下符号代表的化合物：
ARS：青蒿琥酯，HA：透明质酸，dACC：二青蒿琥酯-3-季铵-1,2-丙二醇酯盐，DHA：双氢青蒿素。
实施例1：二青蒿琥酯-3-二甲胺基-1,2-丙二醇酯
按照图1路线合成二青蒿琥酯-3-二甲胺基-1,2-丙二醇酯。
称取3.072g青蒿琥酯(ARS)置于100mL的烧瓶中，并量取40mL的二氯甲烷作为反应溶剂加入烧瓶，向其中加入1.296g的N,N-羰基二咪唑，于室温下搅拌3小时以活化羧基。活化后向烧瓶中加入0.488g的4-二甲氨基吡啶与0.476g的3-二甲胺基-1,2-丙二醇继续在室温下搅拌，反应过夜。期间可以使用薄层色谱点板检测反应进行的程度，薄层色谱展开剂为乙酸乙酯。反应结束后，将反应溶液使用旋转蒸发器在40℃温度下减压浓缩，得粗产物，为淡黄色固体。用石油醚和乙酸乙酯(1:1，v/v)为流动相通过硅胶柱层析进行洗脱，可根据点板情况与出点速度适当调整洗脱剂极性，分离纯化得到中间体，为白色固体。经质谱(MS)和核磁共振谱(1H-NMR)验证得到的化合物为预期产物二青蒿琥酯-3-二甲胺基-1,2-丙二醇酯，见图2。
实施例2：二青蒿琥酯-3-季铵-1,2-丙二醇酯盐(dACC)
按照图1路线合成二青蒿琥酯-3-季铵-1,2-丙二醇酯盐。
称取0.302g的中间体二青蒿琥酯-3-二甲胺基-1,2-丙二醇酯于30mL烧瓶中，用10mL二氯甲烷溶解，于室温下量取0.5mL碘甲烷加入溶液反应并进行搅拌，使用薄层色谱点板监测反应进程。1小时后停止反应将反应液使用旋转蒸发器在40℃温度下减压浓缩，得目标产物0.296g，为白色固体(于空气中氧化后产物会变黄)。将产物通过Amberlite-400(Cl)离子交换树脂进行碘离子与氯离子交换，得到最终产物二青蒿琥酯-3-季铵-1,2-丙二醇酯盐。经MS和1H-NMR验证得到的化合物为预期产物，见图3。
实施例3：二青蒿琥酯-3-季铵-1,2-丙二醇酯盐(dACC)临界聚集浓度(CAC)检测
dACC的临界聚集浓度(CAC)通过芘荧光探针法测量，将芘溶于浓度为6.08mg/L的丙酮中作为原液。制备浓度为500μg/mL的dacac溶液，用去离子水稀释至125、62.5、31.25、15.62、7.81、3.91、1.95、0.97、0.49、0.24和0.12μg/mL。然后将10mL悬液转移到不同的离心管中，加入0.02mL的芘溶液。所有样品在50℃振荡4h后，用荧光分光光度计检测，激发波长为334nm。记录了发射光谱中荧光强度I3(384nm)/I1(373nm)峰值比。I3/I1值用于在对数尺度上绘制浓度对比图，CAC结果如图4a所示为27μg/mL，显示dACC有自组装能力。
实施例4：二青蒿琥酯-3-季铵盐-1,2-丙二醇酯(dACC)自组装胶束与透明质酸(HA)的纳米复合物dACC/HA的制备
将dACC粉末(20mg)溶于10mL PBS(pH 7.4)中，室温振荡30min，然后加入100mg HA到dACC自组装胶束溶液中，制备得到dACC自组装胶束为核HA为壳的dACC/HA纳米复合物，用于以下实施例5-10。
dACC自组装胶束和dACC/HA纳米复合物的形貌通过透射电镜(TEM)分析，在PBS(pH7.4)中浓度为1mg/mL的dACC/HA纳米复合物的形态。溶液置于200目碳膜铜网上，风干，用2％(w/v)的磷钼酸负染。用JEM-2100系统(JEOL，日本)在200kV下记录TEM显微图像。显然，dACC组装体呈现典型球状结构(图4b)，其粒径约为10nm左右。图4c显示，dACC自组装胶束表面复合透明质酸(HA)后仍为球状结构，粒径约为20nm左右，该复合物在水中可以更加稳定地存在。
实施例5：二青蒿琥酯-3-季铵盐-1,2-丙二醇酯(dACC)的自组装胶束及dACC/HA纳米复合物的化学稳定性评价
通过高效液相色谱(HPLC)研究dACC/HA纳米复合物于37℃下在PBS中孵育0、3和24小时后孵育液的化学组成并与ARS、DHA和dACC自组装胶束进行了比较。将ARS、DHA、dACC胶束和浓度为1mg/mL的dACC/HA纳米复合物溶液(10mL)分别置于透析袋中，于入200mL PBS缓冲液(pH 7.4)中37℃孵育。取样，用高效液相色谱法(Agilent 1100,CA)测定各组分(乙腈/水＝60/40洗脱剂)。根据流出曲线可知dACC孵育3小时后，几乎没有明显的变化，其主峰(滞留时间4.0分钟)积分面积占比88.6％(图5a)。在3小时后，DHA则主要出现了构型的变化，由标样中β-DHA(滞留时间8.3分钟)变为α-DHA(滞留时间6.1分钟)为主(构型的变化并不会影响药效)。在24小时后，DHA出现了明显的降解，α-DHA和β-DHA的总和的降解比例大约50％，该结果与药效稳定性结果相符合。dACC的主峰(滞留时间4.0分钟)也出现了一定的降低(约68％)，然后降解产物主要是更高药效的DHA活性成分(约占比30％)。结果表明，dACC/HA纳米复合物中的dACC的化学稳定性优异。
实施例6：二青蒿琥酯-3-季铵盐-1,2-丙二醇酯(dACC)的自组装胶束及dACC/HA纳米复合物的血液相容性
溶血实验考察dACC自组装胶束和dACC/HA纳米复合物的血液相容性。用小鼠的新鲜红细胞(rbc)孵育dACC自组装胶束和dACC/HA纳米复合物的溶血毒性，以游离ARS为对照。简而言之，取新鲜小鼠全血5mL，1500rpm离心10min，取出上清。将沉淀的红细胞用生理盐水洗涤三次，直到上清变为无色。获得的红细胞与生理盐水混合，制备2％的红细胞悬液。然后将500μL的红细胞悬液分别在ARS等效浓度为200、100和10nM时暴露于dACC自组装胶束、dACC/HA纳米复合物和游离ARS。所有红细胞标本于37℃摇床中孵育3h，10000rpm离心5分钟。收集上清液，用紫外-可见分光光度计在540nm波长下测定血红蛋白的吸光度。由图6a所得，与2％血细胞在37℃共孵育4h后，dACC自组装胶束和dACC/HA纳米复合物溶血率均小于5％，表明它们在体内给药时具有良好的生物相容性。
实施例7：二青蒿琥酯-3-季铵盐-1,2-丙二醇酯(dACC)的自组装胶束及dACC/HA纳米复合物的细胞毒性
细胞毒性能够初步显示出药物药理活性的高低。以游离ARS为对照，采用典型MTT法检测dACC自组装胶束和dACC/HA纳米复合物对L929细胞系的细胞毒性。简而言之，将L929细胞以8×103个/孔的培养强度放在96孔板下培养12h，然后加入含药物的培养基。孵育24h后，每孔加入MTT溶液20μL，继续孵育4h。最后仔细分离上清，加入DMSO 150μL溶解甲瓒晶体。最后，使用分光光度计(Multiskan Go 1510,Thermo Fisher Scientific,Waltham,MA)在570nm波长下测定样品的吸光度。结果表明，dACC/HA纳米复合物的细胞毒性很小，与ARS非常相似。相比之下，未添加HA的dACC自组装胶束毒性更强，推测是由于表面的阳离子导致的(图6b)。
实施例8：二青蒿琥酯-3-季铵-1,2-丙二醇酯盐(dACC)的自组装胶束及dACC/HA纳米复合物的半致死剂量
通过小鼠腹腔注射后的致死剂量来评估dACC自组装胶束和dACC/HA纳米复合物在小鼠体内安全性。该剂量来自AOT425-执行上下法的文件，这是一种替代的急性毒性试验，通过使用连续的给药步骤，提供了一种用较少的试验动物来确定化学品毒性的方法。结果显示，dACC/HA纳米复合物的LD50为1098mg/kg，说明dACC/HA纳米复合物体内毒性很低(图6c)。
实施例9：二青蒿琥酯-3-季铵-1,2-丙二醇酯盐(dACC)的自组装胶束及dACC/HA纳米复合物的体外抗疟效果测定
通过药物脉冲试验研究其抗疟疾活性。环期寄生虫用Ht感染红细胞。疟原虫株与Rh阳性O型人红细胞混合培养，红细胞压积(haematocrit，Ht)为0.2％。疟原虫感染率(parasitemia，Pt)通过涂片后经吉姆萨染料染色后再正置光学显微镜下观察、计算。扩增培养过程中，Pt需维持在5％以下。以添加1μM DHA的孔为阳性对照，以未处理的孔为阴性对照。将平板置于37℃的混合气体(1％O2、5％CO2、94％N2)中孵育72h，然后取出每孔中的培养基，加入20μL SYTO61(2μM)。孵育15min后，加入180μL PBS，再孵育30min。流式细胞术分析染色后的寄生虫感染率和抑制率。抑制率计算为：
抑制率(％)＝(1-(Ptwell-Ptpositive)/(Ptnegative-Ptpositive))×100％
其中Ptwell为实验组的Pt，Ptpositive为阳性对照组的Pt，Ptnegative为阴性对照组的Pt。
结果见图7a，dACC自组装胶束及dACC/HA纳米复合物的的体外抗疟活性均显著高于ARS。
实施例10：体外抗疟药效稳定性测定
采用体外标准测定法评价二青蒿琥酯-3-季铵-1,2-丙二醇酯盐(dACC)自组装胶束及dACC/HA纳米复合物在培养基中的疗效稳定性。将样品置于浓度为100μM的PBS缓冲液或PBS缓冲液含10％血清中37℃孵育3和24h，然后采用相同的体外效果标准实验方法分析孵育效果。计算50％抑制浓度(IC50)。
二青蒿琥酯-3-季铵-1,2-丙二醇酯盐(dACC)自组装胶束及dACC/HA纳米复合物的效价相等，其半数有效抑制浓度(IC50)为1.5nM，与DHA(IC50为3nM)相当(图7a)。同时采用体外标准测定法评价dACC和dACC/HA纳米复合物在培养基中的疗效稳定性。将化合物置于PBS缓冲液或PBS缓冲液+10％血清中37℃孵育3小时和24小时。测定IC50值，如图7b所示。在PBS缓冲液中，24h内所有化合物均稳定，IC50相似。与此相反，加入10％的血清后，24h后DHA和ARS的效力显著降低，IC50值分别约为360nM和35nM。相比之下，dACC和dACC/HA纳米复合物在同一时期表现出相似的IC50值。结合HPLC稳定性和体外实验研究，dACC自组装胶束和dACC/HA纳米复合物明显比临床使用的DHA和ARS更稳定。
Claims (10)
1.一种青蒿素二倍体衍生物复合物，其特征在于，所述复合物由式(1)所示的青蒿素二倍体衍生物自组装胶束为核与阴离子型聚合物为壳复合而成，所述复合物的粒径为10纳米～1000纳米：
式(1)中，x为2～10的整数。
2.根据权利要求1所述的复合物，其特征在于，所述青蒿素二倍体衍生物自组装胶束与阴离子型聚合物的质量比为1∶2～1∶10。
3.根据权利要求1所述的复合物，其特征在于，所述阴离子聚合物为透明质酸钠、硫酸软骨素或肝素钠。
4.根据权利要求3所述的复合物，其特征在于，所述透明质酸的分子量为10kDa～200kDa，所述硫酸软骨素的分子量为10kDa～200kDa，所述肝素钠的分子量为5kDa～15kDa。
5.一种青蒿素二倍体衍生物药物组合物，其特征在于，所述药物组合物包含如权利要求1-4任一所述的复合物和药学上可接受的载体。
6.根据权利要求5所述的药物组合物，其特征在于，所述复合物为纳米颗粒，所述纳米颗粒的粒径为10纳米～1000纳米。
7.根据权利要求5所述的药物组合物，其特征在于，所述药物组合物为颗粒剂、凝胶剂、注射剂、缓释制剂或控释制剂。
8.权利要求1-4任一所述的复合物在制备抗寄生虫病药物中的应用。
9.权利要求5-7任一所述的药物组合物在制备抗寄生虫病药物中的应用。
10.根据权利要求8或9所述的应用，其特征在于，所述寄生虫病为疟疾、血吸虫病、弓形虫病、利什曼病、丝虫病或钩虫病。
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