CN104342427B - 用于药物代谢及药效、毒性评价的代谢酶‑水凝胶体系 - Google Patents
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Abstract
本发明属医药技术领域,涉及一种用于药物代谢及药效、毒性评价的代谢酶‑水凝胶体系;该体系由药物代谢酶和水凝胶载体组成;其中,药物代谢酶由微粒体、重组酶等具有代谢功能的蛋白质大分子构成;水凝胶载体由普郎尼克、丙烯酰胺和双丙烯酰胺通过过硫酸铵、N,N,N',N'‑四甲基二乙胺的催化,聚合而成;交联后的水凝胶载体在清洗、冻干等预处理后,吸附可控浓度的药物代谢酶,形成代谢酶‑水凝胶体系。该代谢酶‑水凝胶体系机械强度适宜、无毒、重现性好、操作简便、且具有温敏性,可全面预测药物及其代谢物对细胞的作用,进而进行药效或毒性的准确评价。
Description
技术领域
本发明属医药技术领域,涉及药物代谢、药效及毒性的评价,具体涉及一种用于药物代谢及药效、毒性评价的代谢酶-水凝胶体系;该体系可用于预测药物代谢、药效、毒性评价。
背景技术
细胞培养是重要的体外药学研究手段;由于绝大多数细胞,即使是肝脏细胞,通常不表达或低表达很多重要的药物代谢酶,因此该方法尚不能全面模拟真实的体内环境。药学研究中忽视了药物代谢的生物转化过程,将导致不准确、不全面甚至是错误的研究结果;如解热镇痛抗炎药对乙酰氨基酚,只有在肝脏经过细胞色素酶CYP 2E1转化成活性中间体NAPQI后,未被谷胱甘肽结合的、过量的NAPQI才会攻击大分子蛋白质或核酸,从而诱发肝损伤;此外,一些前药如环磷酰胺,只有经CYP 3A代谢后转化为活性代谢物氮芥,才会发挥其抗肿瘤作用;因此,不管对于药效评价、毒性预测,代谢的过程都起着非常重要的作用;而且,将代谢的过程融入传统的细胞培养对于该种手段本身是一种改进和革新。
基于上述理念,目前已陆续开发了一些新型的评价手段,如转基因细胞、共培养技术、3D细胞培养以及一些高通量代谢预测装置。虽然上述新模型、新方法在一定程度用于药物代谢、药效及毒性的预测,但均存在着其各自的缺点;如在转基因细胞中,只能同时表达一个或几个药物代谢酶,而不能同时稳定表达所有代谢酶;3D细胞培养法虽可一定程度的恢复肝细胞的代谢功能,但相关的预测或研究只适用于肝细胞,而对于其他本身不表达代谢酶的细胞或组织并不可行;对于高通量装置,虽检测可实现高通量,但其制备及操作过程极为繁琐,且就目前的研究状况,很多可能影响结果的因素并未详尽考察。
虽然,所述药物代谢酶如肝微粒体、重组酶等大分子蛋白已作为一种成熟的代谢研究工具广泛使用,但鲜有报道将其直接用于药效、毒性的预测;究其主要原因,是代谢酶的直接引入,会很大程度影响整个细胞培养体系,甚至造成细胞对于该纳米级大小的蛋白的摄取而引起细胞死亡。目前已知,普郎尼克共聚物为一种具有温敏性的高分子材料,由于其具有较好的生物相容性、氮氧通透性、空间微孔结构,而广泛应用于细胞培养、组织工程、药物递送等领域;此外,以化学交联为主要聚合方式的聚丙烯酰胺凝胶,具有制备方法简单、交联程度可控、机械强度较好等特点,通常用于蛋白质电泳分离。
迄今为止,尚未见有关将上述两种材料同时使用,制成一种兼具两种材料优点的新型聚合物,作为药物代谢酶的载体,包裹药物代谢酶后,形成代谢酶-水凝胶体系,进而通过该体系研究药物代谢、药效及毒性的报道。
发明内容
本发明的目的在于提供一种用于药物代谢及药效、毒性评价的代谢酶-水凝胶体系;该体系简便、全面、重现性好、经济适用,可用于评价药物代谢、药效及毒性。
本发明所述的代谢酶-水凝胶体系由药物代谢酶和水凝胶载体组成;其中,所述药物代谢酶用于执行代谢功能,所述水凝胶载体用于承载代谢酶;
本发明中,所述药物代谢酶选自具有代谢功能的大分子蛋白质,如微粒体、重组酶、S9混合物等;
本发明中,所述水凝胶载体由两部分组成:(1)两端修饰有双键结构的具有温敏特性的普郎尼克(Pluronic) 系列三嵌段共聚物,共聚物可优选为两端带有双键的F127;(2)聚丙烯酰胺混合物,即同时包含丙烯酰胺与双丙烯酰胺分子单体的混合溶液;其中,该混合物比例优选为丙烯酰胺:双丙烯酰胺为29:1;
上述两部分材料通过化学交联方式进行聚合形成水凝胶载体,交联剂优选过硫酸铵,交联加速剂优选N,N,N',N'-四甲基二乙胺;
本发明中,所述水凝胶载体中,修饰有双键结构的Pluronic 系列三嵌段共聚物为5wt%-30wt%;聚丙烯酰胺混合物为0.1wt%-3wt%;交联剂为0.5‰-5‰;交联加速剂为0.5‰-5‰;其优选为:10wt%的Pluronic、0.3%的聚丙烯酰胺混合物(29:1)、1‰的交联剂、0.5‰的交联加速剂,混合后,室温或37℃聚合为水凝胶载体,具体水凝胶载体性状、大小可通过承载溶胶的容器形状及使用溶胶的量控制,具体可优选为96孔板中加入50ul或100ul溶胶进行交联;
本发明中,所述水凝胶载体的杂质部分可通过缓冲清洗溶液进行震荡清洗,除去未反应的单体以及有毒的交联剂、交联加速剂;所述缓冲清洗溶液优选为甘露醇溶液;所述溶液亦可作为冻干时的塑形剂;
本发明中,所述水凝胶载体需冻干后除去水分,以冻干胶的形式备用;即清洗好的水凝胶载体需进行冻干处理,将水凝胶无干扰的整齐排列后,于-80℃预冻30min,再将其置于冻干机,进行冻干;
本发明中,所述水凝胶载体对于代谢酶的包裹采用4℃低温时吸附的方式,且低温可保持代谢酶活性;本发明的实验结果显示,代谢酶如微粒体放置于4℃保存条件,48 h内对于其活性基本无影响;因此,将冻干的水凝胶载体浸泡于设定浓度的代谢酶溶液,通过溶胀吸附的方式将代谢酶包裹于水凝胶载体中,48 h后,即得微粒体-水凝胶体系;结果表明,冻干好的水凝胶载体可通过浸泡吸附的方式包裹设定浓度的代谢酶;
本发明中,所述代谢酶-水凝胶体系发挥作用的温度为37℃;37℃时,该体系处于收缩状态,可锁紧代谢酶不致泄露,且该温度适于代谢反应的进行。
本发明所述代谢酶-水凝胶体系与现有技术相比较,具有以下优点:
(1)温敏性:4℃时,该代谢酶-水凝胶体系中大分子单体处于链舒张状态,易于代谢酶的吸附,且低温可保持代谢酶活性;37℃时,该体系处于收缩状态,可锁紧代谢酶不致泄露,且高温适于代谢反应的进行;
(2)空间结构适宜:优选的10wt% Pluronic及0.3wt% 聚丙烯酰胺混合物(29:1)的水凝胶体系适于代谢酶的包裹及药物等小分子底物的进出,均有利于代谢反应的发生及药效、毒性的预测;
(3)无毒安全:反复震荡清洗可将交联剂等影响代谢酶活性及细胞活力的有毒物质及时清除,所述代谢酶-水凝胶体系保留了大部分代谢酶的活性且对细胞活力无影响;
(4)简单易行:使用所述代谢酶-水凝胶体系时,只需将其放入普通细胞培养的体系中,即可全面、客观的评价药物及其代谢物对细胞的作用,进而预测药物代谢、药效及毒性。
本发明所述代谢酶-水凝胶体系与底物共同孵育,可预测与代谢相关的参数;与普通培养的细胞共同孵育,可全面评价药物及药物代谢物所产生的药效或毒性;该代谢酶-水凝胶体系机械强度适宜、无毒、重现性好、操作简便、且具有温敏性,可用于任何传统细胞培养方式中,且不限细胞的种类、培养条件;将本代谢酶-水凝胶体系加入细胞培养体系中,可全面预测药物及其代谢物对细胞的作用,进而进行药效或毒性的准确评价,并适于药物代谢及药效、毒性的相关深入研究。
附图说明
图1 为实施例1所述代谢酶-水凝胶体系的构建路线。
图2显示了实施例2所述代谢酶-水凝胶体系代谢能力。
图3显示了实施例3所述代谢酶-水凝胶体系预测药效及毒性能力。
具体实施方式
下面结合具体实施方式对本发明进行进一步的详细描述,给出的实施例仅为了阐明本发明,而不是为了限制本发明的范围。
为了具体阐明本发明,以微粒体作为药物代谢酶;F127、聚丙烯酰胺混合物制备的水凝胶作为载体进行后续实施例。
实施例1 构建代谢酶-水凝胶体系
制备步骤如图1所示,
(1)通过丙烯酰氯与F127在碱性条件下发生的缩合反应,以酯键成功修饰F127,使其两端带有双键;将该大分子单体溶于去离子水中,配制成30%浓度的母液,4℃保存;
(2)为配制100 ul水凝胶载体,吸取33 ul双键F127母液,1 ul 购得的30%丙烯酰胺:双丙烯酰胺(29:1)混合溶液,1 ul 10% 过硫酸铵溶液,0.5 ul TEMED,其余以64.5 ulPBS磷酸缓冲液补齐;将该混合物转移至96孔板中,室温或37℃进行交联,约30 min后即得水凝胶;
(3)将上述步骤(2) 所得水凝胶置于5%甘露醇溶液中反复震荡清洗,约5次,每次20 min;
(4)将上述步骤(3)所得水凝胶于-80℃预冻30 min,再将其置于冻干机上进行冻干;
(5)将上述步骤(4)所得冻干水凝胶浸泡于设定浓度的微粒体溶液中,4℃存放48h,微粒体即吸附进入水凝胶中,制得微粒体-水凝胶体系。
实施例2 微粒体-水凝胶体系的代谢能力
如图2所示,微粒体中包含了几乎所有一项代谢酶,且一项代谢酶负责绝大多数药物的生物转化过程,因此,选用几个具有代表性的一项代谢酶包括CYP3A、CYP2D6、CYP2E1验证微粒体-水凝胶体系的代谢能力。
选用FDA认证的三种酶的标准底物90μM睾酮(CYP3A)、100μM右美沙芬(CYP2D6)及200μM氯唑沙宗(CYP2E1)在37℃进行孵育;500ul的孵育体系中,包括50ul如上浓度的底物、100ul代谢反应启动体系NADPH(终浓度1mM)、350ul PBS缓冲液及100ul的微粒体-水凝胶;孵育开始后,分别在15 min、30 min、1 h、2 h、4 h、6 h、8 h及24 h吸取50 ul 样品,通过高效液相色谱的方法分析样品中对应底物6β-OH睾酮、去甲右美沙芬及6-OH睾酮的含量,绘制时效曲线;同时,将未被包裹在水凝胶中的微粒体在同样条件下与底物进行孵育,亦绘制时效曲线;比较两条曲线的差别,结果如图2所示,微粒体-水凝胶体系保持了60%、甚至70%以上的底物代谢能力,可较好的用于相关代谢研究的开展。
实施例3 微粒体-水凝胶体系的药效、毒性预测能力
如图3所示,环磷酰胺为经典的抗肿瘤前药,在体内只有经过代谢后才可转化为具有活性的氮芥,从而发挥其抗肿瘤能力。选用环磷酰胺作为模型药物,人乳腺癌MCF-7细胞作为待测细胞,考察微粒体-水凝胶体系的存在与否对于其抗肿瘤药效的影响。
分别以5 mM、10 mM、15 mM及20 mM 的环磷酰胺作用MCF-7细胞24h,培养体系中一组均加入微粒体-水凝胶体系,另设置不加入该体系的对照组,收集细胞,以Annexin V/PI染色,最后通过流式细胞术对其凋亡程度进行分析;结果如图3所示,加入微粒体-水凝胶体系组中的凋亡细胞数量明显高于未加入该体系的组别,且随着环磷酰胺浓度的增加,该差别越来越明显;结果表明,所述微粒体-水凝胶体系可用于药物及其代谢物的药效、毒性的全面、客观、合理的评价。
Claims (8)
1.一种用于药物代谢及药效、毒性评价的代谢酶-水凝胶体系,其特征在于,由药物代谢酶和水凝胶载体组成;其中,药物代谢酶用于执行代谢功能,水凝胶载体用于承载代谢酶;
所述药物代谢酶选自微粒体、重组酶或S9混合物;
所述水凝胶载体由两端修饰有双键结构的具有温敏特性的普郎尼克系列三嵌段共聚物,及聚丙烯酰胺混合物组成,所述共聚物为两端带有双键的F127;所述聚丙烯酰胺混合物中,丙烯酰胺:双丙烯酰胺为29:1。
2.按权利要求1所述的代谢酶-水凝胶体系,其特征在于,所述水凝胶载体中,普郎尼克系列三嵌段共聚物及聚丙烯酰胺混合物通过化学交联方式进行聚合;其中,交联剂为过硫酸铵,交联加速剂为N,N,N',N'-四甲基二乙胺。
3.按权利要求1所述的代谢酶-水凝胶体系,其特征在于,所述水凝胶载体中,修饰有双键结构的普郎尼克系列三嵌段共聚物为5wt%-30wt%、聚丙烯酰胺混合物为0.1wt%-3wt%、交联剂为0.5‰-5‰、交联加速剂为0.5‰-5‰。
4.按权利要求1所述的代谢酶-水凝胶体系,其特征在于,所述水凝胶载体中,普郎尼克系列三嵌段共聚物为10wt%、聚丙烯酰胺混合物为0.3wt%、交联剂为1‰、交联加速剂为0.5‰。
5.按权利要求1所述的代谢酶-水凝胶体系,其特征在于,所述水凝胶载体的杂质部分通过缓冲清洗溶液进行震荡清洗;其中,
所述缓冲清洗溶液为甘露醇溶液;
所述缓冲清洗溶液还作为冻干时的塑形剂。
6.按权利要求1所述的代谢酶-水凝胶体系,其特征在于,所述水凝胶载体对于代谢酶的包裹采用4℃低温时吸附的方式。
7.按权利要求1所述的代谢酶-水凝胶体系,其特征在于,所述代谢酶-水凝胶体系发挥作用的温度为37℃。
8.权利要求1所述的代谢酶-水凝胶体系在体外评价药物代谢及药效、毒性的应用。
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