CN105148289B - 一种两亲性寡聚多肽药物结合物 - Google Patents
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Abstract
本发明公开了一种两亲性寡聚多肽药物结合物,本发明将药物通过化学结构上的羟基或氨基直接与寡聚多肽C端的羧基缩合形成酯键或酰胺键,或者药物通过化学结构上的羧基与寡聚多肽N端的氨基缩合形成酰胺键,或者药物通过一定的链接分子与寡聚多肽结合。本发明以安全性高的氨基酸制备的寡聚多肽为主要构筑材料,与抗炎药物偶联,制备为寡聚多肽药物结合物,与传统的制剂相比,由于药物与载体材料直接结合,具有安全性好、载药量高的优点,进入体内后可以长时间发挥药效的作用。
Description
技术领域
本发明属于多肽自组装领域,特别涉及一种安全性高的氨基酸制备的寡聚多肽用作主要构筑材料,与抗炎药物偶联,制备为寡聚多肽药物结合物(Amphiphilic peptidedrug conjugate:APD)。
背景技术
骨关节炎(Osteoarthritis,OA)和类风湿性关节炎(Rheumatoid arthritis,RA)是一种慢性炎症和破坏性疾病,是一种累及周围关节为主的多系统炎症性的自身免疫性疾病,该疾病被视为影响人类健康的五大疾病(心血管病、关节炎病、阿尔茨海默氏病、癌症及艾滋病)之一,它使数以百万计的病人丧失生活和工作能力,称为不死的癌症,给病人带来极大的痛苦。据WHO提供的数字,全球有1%的人患有类风湿性关节炎病,仅中国患者就达1000多万。
由于RA发病机理不清,临床上尚缺乏根愈RA的方案和预防的措施。对于RA治疗的总原则是对症治疗,即对于患有RA的患者,采取缓解患者关节处的炎症,从而减轻或消除由于炎症引起的关节肿痛、压痛、晨僵或关节外的症状;同时控制疾病的进一步恶化,防止和减少关节、骨的破坏,尽可能的保持受累关节的功能;并促进已破坏的关节、骨的修复。目前治疗RA的方法通常是口服非甾体抗炎药等,但RA的主要炎症部位在关节腔,为了增加药物在关节腔中的浓度,常常加大给药剂量,导致在提高药效的同时伴随着严重的全身毒副作用和胃肠道毒性。因此,迫切需要进一步改进和提高RA治疗的策略和手段。采用多种途径并用,着眼于提高药物在关节腔中的浓度,减少药物在体内的分布,从而进一步降低药物的毒副作用,对RA的治疗更具潜力。
近年来,关节腔注射给药可以直接将药物输送至炎症部位,改变药物在体内的分布模式,避开药物在体内转运中的生理屏障,以最小的药量发挥最大的药效。关节腔注射治疗RA从1951年开始沿用至今。目前上市的关节腔注射制剂多是以溶液剂形式给药,给药后药物易迅速地渗透进入到体循环,在病变部位存留时间短、药效持续时间短,存在需频繁给药等缺点。
为了延长药物在关节腔内的驻留时间,研究较多的关节腔注射缓释剂型主要包括脂质体、微球及原位凝胶等,脂质体在生理环境下不稳定易产生突释现象,而利用生物可降解材料制备的微球虽然可延长药物在关节腔内的驻留时间,但制备过程较复杂,步骤较多且制备过程中使用有机溶剂;采用加温或减压挥发所得微球球体往往多孔而不致密,影响药物释放,微球的载药量较低,产品批间质量差异大。有研究表明利用以弹性蛋白样多肽为载体的温敏型凝胶与溶液剂关节腔注射给药后,温敏型凝胶制剂的半衰期延长达25倍,大大降低给药次数,延长药效。但弹性蛋白样多肽来源较少,获得的工艺较复杂,不利于其在RA治疗中广泛推广。因此,寻求合理的药物载体进行RA治疗,使药物可以在关节腔内局部注射达到长效缓释效果是目前治疗RA的研究重点。
发明内容
本发明的目的在于解决现有关节腔内注射给药制剂不足的问题,提供一种载药量高、生物相容性好、在病变部位驻留时间长的两亲性寡聚多肽药物结合物,该药物结合物能够在疏水作用、氢键和静电作用的引导下,在溶液中自发形成高度有序的纳米结构,且随着环境的变化可以进一步形成凝胶。
技术方案:为了达到以上目的,本发明采取的技术方案如下:
一种两亲性寡聚多肽药物结合物,药物通过化学结构上的羟基或氨基直接与寡聚多肽C端的羧基缩合形成酯键或酰胺键,或者药物通过化学结构上的羧基与寡聚多肽N端的氨基缩合形成酰胺键,或者药物通过一定的链接分子与寡聚多肽结合。
作为优选方案,以上所述的两亲性寡聚多肽药物结合物,所述的药物为酮洛芬、青藤碱、雷公藤甲素或者它们的衍生物。
作为优选方案,以上所述的两亲性寡聚多肽药物结合物,所述的寡聚多肽由缬氨酸和赖氨酸/或谷氨酸组成的寡聚多肽,氨基酸的数目为2-20。
作为更加优选方案,以上所述的两亲性寡聚多肽药物结合物,所述的寡聚多肽由缬氨酸和赖氨酸/和谷氨酸交替组成,氨基酸数目为4-16。
本发明所述的种两亲性寡聚多肽药物结合物,优选如下结构如:酮洛芬-(VE)n、酮洛芬-(VK)n,可以很好的形成纤维,进一步形成凝胶。
根据权利要求1所述的两亲性寡聚多肽药物结合物,其特征在于,两亲性寡聚多肽药物结合物能够自组装形成纤维或形成凝胶。
一种关节腔注射原位制剂,它含有本发明所述的两亲性寡聚多肽药物结合物。
本发明提供的关节腔注射原位制剂,所述寡聚多肽药物结合物能够在体内发生缓慢降解,释放药物发挥药效。
本发明所述的关节腔注射原位制剂,所述寡聚多肽药物结合物能够用于关节腔注射或者肌肉注射。
本发明的另一目的在于提供本发明所述的两亲性寡聚多肽药物结合物在制备具有抗炎药物中的应用,如用于制备治疗骨关节炎和类风湿性关节炎药物中的应用。
本发明的另一目的在于提供本发明所述的原位凝胶在制备抗炎药物中达到缓释长效治疗关节炎中的应用。
有益效果:
(1)本发明的制备方便快捷、操作简单和产率较高。
(2)本发明以安全性高的氨基酸制备的寡聚多肽为主要构筑材料,与抗炎药物偶联,制备为寡聚多肽药物结合物,具有结构稳定,载药量高,且均匀可控等特点。
(3)本发明提供了一种利用新型多肽药物结合物自组装构建纳米凝胶,丰富了关节腔原位注射的制剂的种类。
附图说明
图1为实施例1制备的keto-VEVE高效液相色谱图;
图2为实施例1制备的keto-VEVE质谱图;
图3为实施例3中keto-VEVE建立的标准曲线;
图4为实施例5中keto-VEVE在不同pH值下自组装的透射电镜显微镜检测图;
图5为实施例7中keto-VEVE在不同离子强度下自组装的透射电镜显微镜检测图。
具体实施方式
实施例1:由缬氨酸和谷氨酸和抗炎药物酮洛芬制备两亲性寡聚多肽药物结合物(keto-VEVE)
1、材料
用芴甲氧羰酰基(Fmoc)保护的缬氨酸(V)、用芴甲氧羰酰基(Fmoc)和叔丁基(But)谷氨酸(E)、N,N'-二异丙基碳二亚胺(DIC、99%)、1-羟基苯并三唑(HOBT、99%)、2-(1H-苯并三唑)-N,N,N’,N’-四甲基脲六氟磷酸盐(HBTU、99%)、N,N-二甲基氨基吡啶(DMAP、99%)、9-芴甲氧羰基(Fmoc、99%)、N,N-二异丙基乙胺(DIEA、99%)、N,N-二甲基甲酰胺(DMF、99%)、吡啶、哌啶、乙酸酐、茚三酮、王树脂,乙腈,三氟乙酸(TFA),甲醇,1%氨水溶液
2、制备方法
利用常规的固相合成方法,进行寡聚多肽的合成,然后再偶联药物。具体如下:
(1)先把谷氨酸(E)接到王树脂上,用HOBT+E+DMAP进行反应DIC作为缩合试剂。反应4个小时,反应结束之后用等量的吡啶和乙酸酐进行反应,用来封闭王树脂上多余的活性基团。反应半小时之后。加入20%的哌啶进行反应脱去FMOC保护,用茚三酮进行检测。
(2)加入HBTU+V用DIEA作为缩合试剂反应35分钟,反应时间到了用DMF洗干净,加入20%的哌啶进行反应脱去FMOC保护,用茚三酮进行检测,用茚三酮进行检测,没有颜色证明反应完全,如果有颜色则说明没反应完全需要重新进行反应。
(3)确认缬氨酸(V)接上之后脱去保护接E方法同接E,再接V。
(4)酮洛芬与上一步产物,用HOBT进行反应DIC做缩合试剂进行反应,反应1个小时进行检测。用茚三酮进行检测,没有颜色证明反应完全,如果有颜色则说明没反应完全需要重新进行反应。
(5)将(3)的产物,加入95%TFA,2%1,2-乙二硫醇,2%异丙醇和水反应2h后,用加入乙醚离心,抽滤分离。即得到粗肽,通过高效液相色谱法(HPLC)纯化,经冷冻干燥即得到本发明所述酮洛芬多肽药物结合物(keto-APD),氨基酸序列为序列表中SEQ ID NO.1所述。其中keto-VEVE的氨基酸序列为SEQ ID NO.1中的第一个。
实施例2:两亲性寡聚多肽药物结合物keto-VEVE的高效液相色谱和质谱检测
将实施例1制备的keto-VEVE利用高效液相色谱(HPLC)检测,参见图1,由图1中的谱峰面积可计算其纯度已达到97%。将实施例1制备的keto-VEVE采用质谱(MS)检测,参见图2,说明其分子量为710是正确的。
实施例3:keto-VEVE的标准曲线的建立
取实施例1制备的keto-VEVE配制成1mg/ml的母液,逐级稀释为10、20、50、100、250、500、1000ug/ml进高效液相(HPLC)记录峰面积,绘制标准曲线,参见图3。
实施例4:keto-VEVE在不同pH值水溶液下平衡溶解度的考察()
取过量实施例1制备的keto-VEVE于pH值为1、3、5、7、9、11的水溶液中,室温放入快速混匀器中振荡0.5h,静置0.5h,重复上述操作3次,离心(14000r/min,10min),吸取上清液于液相小瓶中,注入液相色谱仪,记录峰面积,以实施例3标准曲线,计算在pH不同条件的溶解度。溶解性如下:
实施例5:keto-VEVE在不同pH值下自组装的透射电镜显微镜(TEM)检查
(1)盐酸和氢氧化钠溶液调节水溶液的pH值分别为7、8、9、10,备用。称取实施例1的ket-VEVE适量加入配好的不同pH值的溶液中制成500uM的溶液,静置约24小时。
(2)首先,将配制好的待测样品滴加在400目的铜网上,片刻用,镊子取出铜网,滤纸吸去多余液体,将铜网放在磷钨酸染色剂上染色约90s,用镊子取出铜网后将多余的液体用滤纸吸掉,放置晾干,准备实验,用透射电子显微镜观察其成像的情况,参见图4,从上自下分别为pH 7、8、9、10下的电镜图,观察的结果为在pH小于9时,无法形成纳米结构,当pH大于9时,可以形成形貌稳定的纳米纤维,且随着pH值升高,其形成的纳米结构越稳定和规则。结果显示,pH值的变化改变keto-VEVE中氨基酸的存在形式,从而影响keto-VEVE的自组装的行为。
实施例6:keto-APD中氨基酸数目对其组装的影响
分别取氨基酸数目为4、8、12、16的keto-APD,即ket-(VE)2、ket-(VE)4、ket-(VE)6、ket-(VE)8,测定其临界胶束浓度,比较其形成自组装的能力。发现随着氨基酸数目的增加,keto-APD组装性能增强,在较低浓度的情况下可以形成自组装。
实施例7:自组装keto-VEVE在不同离子强度下用透射电镜显微镜(TEM)检查
称取实施例1的ket-VEVE适量加入不同离子强度的溶液(分别为水、生理盐水、葡萄糖及PBS)配制成500uM的溶液,放置24小时后取样,采用实施例5的透射电镜操作方法观察,观察的结果为在不同离子强度下,展现了不同的组装结构,从上往下:在水和生理盐水下可以形成很好的纳米纤维结构,具有较强的刚性,并且多个纤维可以进一步形成纳米带,在葡萄糖溶液中,形成胶束,而在PBS溶液中,形成的纳米纤维具有较强的柔性,但无法进一步形成纳米带。参见图5。
SEQUENCE LISTING
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Claims (2)
1.一种关节腔注射原位制剂,其特征在于,它含有两亲性寡聚多肽药物结合物;药物通过化学结构上的羧基与寡聚多肽N端的氨基缩合形成酰胺键;
所述的药物为酮洛芬;所述的寡聚多肽由缬氨酸和谷氨酸交替组成,氨基酸数目为4-16;所述的两亲性寡聚多肽药物结合物能够自组装形成纤维或形成凝胶。
2.权利要求1所述的关节腔注射原位制剂在制备治疗骨关节炎和类风湿性关节炎药物中的应用。
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